THE LEACHING BEHAVIOUR OF

VARIOUS ZINC SULPHIDE

MINERALS WITH THREE

THIOBACILLUS SPECIES

THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN THE

SCHOOL OF BIOLOGICAL TECHNOLOGY

UNIVERSITY OF NEW SOUTH WALES.

BY

AHMAD MUKHTAR KHALID

JULY, 1978.

ACKNOWLEDGEMENT SUMMARY

1- INTRODUCTION

C O N T E N T S

1. 1- GENERAL INTRODUCTION

1.2- THE THIOBACILLI

1.2.1- Isolation and Taxonomical Characteristics

of Thiobacilli.

1.2.2- Isolation of Thiobacilli.

1

1

4

8

10

1.2.3- Taxonomy of Thiobacilli. 16

1.2.3.1- Morphology. 16

1.2.3.2- Biochemical Characteristics. 18

1.2.3.2- Submicroscopic Organisation of Thiobacilli. 27

1.3-BIODEGRADATION OF MINERALS BY MICROORGANISMS.36

1.3.1- Biodegradation of Minerals by

Heterotrophic Microorganisms. 36

Acidification or Alkalisation. 39

Chelation of Metal ions by Metabolites. 40

1.3.2- Biodegradtion of Minerals by Thiobacilli. 43

1.3.3- Factors Affecting the Microbial Leaching

Processes.

1.3.3.1- Nature of Substrate.

Metal distribution.

Properties of component minerals.

1.3.3.2- Effect of Physico-Chemical Factors.

Particle size.

Surface effects.

Temperature.

Redox potential and pH.

45

45

45

46

47

47

50

51

54

1.3.3.3- Nutritional Requirements of Microflora

Involved in Leaching.

Requirement for carbon.

Nitrogen source.

Phosphate.

Sulphate.

Trace elements.

Effect of dissolved ions on growth and

leaching behaviour of Thiobacilli.

Effect of organic compounds.

1.4-BIOGEOCHEMISTRY OF ZINC SULPHIDE.

1.4.1- Microbiological Oxidation of Zinc Sulphide.

1.4.2- Effect of Crystal and Lattice Structure on

Rates of Leaching of Zinc Sulphide.

1.5- AIMS OF THE CURRENT INVESTIGATION.

2- MATERIALS AND METHODS.

2.1- PREPARATION OF SULPHUR SUBSTRATES.

2.1.1- Polythionates.

2.1.2- Synthetic Zinc Sulphide.

2.1.3- Natural Zinc Sulphide Minerals.

2.1.4- Preparation of Zinc Sulphide Substrates.

2.2-ASSAY PROCEDURES.

2.2.1- Thiosulphate.

2.2.2- Polythionates.

2.2.3- Ferrous & Ferric Ions.

2.2.4- Emission Spectroscopic Analysis of Minerals.

2.2.5- Mineral Analysis by Atomic Absorption

Spectroscopy.

2.2.6- X-ray Diffraction Measurements.

57

57

61

62

63

64

65

70

75

79

85

87

88

88

88

89

89

90 90

92

92

94

94

96

2.3- ISOLATION OF THIOBACILLI.

2.3.1- Isolation of Non-Acidophilic Thiobacilli.

Source samples.

Media.

Isolation procedures.

2.3.2. Isolation of Acidophilic Thiobacilli.

Source samples.

Medium.

Isolation procedures.

2.3.3- Maintenance of cultures.

2.4-TAXONOMICAL INVESTIGATIONS.

2.4.1- Microscopic Examination.

2.4.2- Biochemical Tests.

Growth on nutrient agar.

Growth on different sulphur substrates.

Oxidation of ferrous ion.

Anaerobic growth.

Nitrate reduction.

Determination of DNA base composition.

Determination of FAME profile.

2.5-CULTIVATION OF THIOBACILLI.

2.5.1- Culture Vessels.

2.5.2- Preparation of Meida.

Batch culture.

Continuous culture.

2.5.3- Measurement of Dissolved oxygen.

2.5.4- Measurement of co2 in the Effluent Gas Stream.

2.5.5- Determination of Total Bacterial Population.

3- RESULTS AND DI SC USS I ON.

98

98

98

99

100

101

101

101

102

102

104

104

105

105

103

105

106

106

106

107

109

109

109

109

112

112

113

113

3.1- ISOLATION AND CHARACTERISATION OF TH10BACILLI.

3.1.1- Isolation of Non-:-,acidophilic sulphur oxidising

Bacteria. 115

3.1.2- Isolation of Acidophilic Iron-Oxidising Bacteria. 116

3.1.3- Isolation of Acidophilic Sulphur Oxidising

Bacteria. 117

3.1.4- Taxonomy of Isolates. 117

BJR-451. 117

BJR-Kl 128

BJR-K0l 133

3. 2- OPT IMI SATI ON OF I SO LA TES. 134

3.2.1- Selection of an Appropriate Growth Medium for

Non-acidophilic BJR-451. 135

3.2.2- Batch Growth of BJR-451. 137

3.2.3- Batch Growth of BJR-451 in Fermenter. 137

3.2.4- Determination of the Optimum pH for Growth of

BJR-451. 142

3.2.5- Determination of the Optimum Temperature for

the Growth of BJR-451. 14 7

3.2.6- Growth of BJR-451 in a Chemostat. 153

3.2.7- Effect of Carbon dioxide on Growth of BJR-451. 157

3.2.8- Toxicity of Zinc ions towards Thiobacillus

thioparus, Thiobacillus ferrooxidans & Thiobacillus

thiooxidans. 158.

3.3- LEACHING OF ZINC SULPHIDE BY THIOBACILLUS

FERROOXIDANS, THIOBACILLUS THIOOXIDANS AND

THIOBACILLUS THIOPARUS. 163

3.3.1- Leaching Capabilities of Thiobacillus thioparus,

BJR-451. 164

3.3.2- Leaching of Zinc Sulphides by Thiobacillus

ferrooxidans.

Leaching of marmatite.

Leaching of synthetic zinc sulphide.

Leaching of museum-grade zinc sulphide

minerals.

Discussion.

168

170

170

172

172

3.3.3- Leaching of Zinc Sulphides by Thiobacillus

thiooxidans.

Leaching of marmatite.

Leaching of synthetic zinc sulphide.

Leaching of museum grade zinc sulphide

minerals.

Discussion.

3.3.4- Leaching of Zinc Sulphide by Thiobacillus

176

176

178

178

178

thioparus. 182

Leaching of marmatite. 182

Leaching of synthetic zinc sulphide. 184

Leaching of natural zinc sulphide minerals. 184

Discussion. 184

3.3.5- Influence of Structure of Zinc Sulphide on

Microbial Leaching by Thiobacilli.

Comparative leaching of marmatite.

Comparative leaching of synthetic

zinc sulphide.

Comparative leaching of natural

188

189

192

zinc sulphide minerals. 194

Comparative leaching of natural wurtzite. 196

Discussion. 196.

3.3.6- Effect of Ferrous ion on Leaching of Synthetic

Zinc Sulphide by Thiobacillus ferrooxidans and

Thiobacillus thiooxidans.

Discussion.

4- C ONC LU S IONS

5- REFERENCES

215

218

223

229

6- APPENDICES

6.1- Isolation of Non-Acidophilic Sulphur

Oxidising Bacteria. (i)

6.2- Isolation of Acidophilic Iron Oxidising

Bacteria.

6.3- Isolation of Acidophilic Sulphur

Oxidising Bacteria.

6.4- Calculation of DNA base composition.

(xiv)

(xv)

(xvi)

6.5- Wave length, Slit Width, Lamp Current and

Gas Mixtures Used for Analysis by

Atomic Absorption Spectrophotometer. ( xxi) .

6.6- The Leaching Behaviour of Various Zinc

Sulphide Minerals with Three Thiobacillus

Species.

6.7- Microbial Ecology of Overburden Heaps

From Uranium Mining at Rum Jungle, N. T.

Australia.

A C K N O W L E D G E M E N T

I am greatly indebted to my supervisors, Professor

B. J. Ralph and Associate Professor Dr. P. A. D.

Rickard, for their continued guidance and criticism

throughout the course of this work. I am also

grateful to my consultant supervisors, Mr. B. Harris

(School of Metallurgy) and Professor R. Golding of

Physical Chemistry for their assistance and helpful

suggestions during experimentation.

I have benefited greatly from the advice, criticism,

and encouragement of my colleagues in the School of

Biological Technology.

I am also greatful to the Australian Development

Assistance Bureau of the Commonwealth of Australia,

for financial support.

I am thankful to my parents whose patience and

encouragement have always been a source of comfort.

Last, but not least, I wish to convey my sincere

gratitude and thanks to the Australian People, who

were very kind and friendly throughout my stay in

this country.

S U M M A R Y

Comparatively little data describing the role of the

physical and chemical characteristics of mineral substrates

in biodegradative processes is available. As a contribution

to this relatively unexplored area, the current investigation

was concerned with the characteristics of various forms of

zinc sulphide and their effects on biodegradation by three

different species of ThiobaciZZi namely, ThiobaciZZus ferro­

oxidans ( BJR-Kl ), ThiobaciZZus thiooxidans ( BJR-K0l) & ThiobaciZZus thioparus ( BJR-451 ).

Isolates of the three species were purified and

characterised with respect to their principal biochemical and

physiological attributes. The ThiobaciZZus ferrooxidans strain

was isolated from a water sample from the Mount Lyell Mining and

Railway Company operating at Queenstown, Tasmania;it resembled

in most of its characteristics those ThiobaciZZus ferrooxidans

strains described in the literature.The ThiobaciZZus thiooxidans

strain, an isolate from' a sulphur heap at the Zinc Sulphide

Corporation, Boolaroo, N. S. W., was found to be closely related

to the type species previously described.

An isolate from road side near Tamworth, N. S. W., grew

at comparatively high pH values, ranging from 5.0 to 7.0. As

information regarding mineral leaching at pH values higher than

2.5 is sparse, this bacterium was considered suitable for study­

ing microbial degradation of zinc sulphide at relatively high pH

values, i.e., those appropriate for this organism's prolifera­

tion. It was extensively studied with respect to its biochemi-

cal and physiological characteristics and was found to be

identical to ThiobaciZZus thioparus. The optimum conditions

for its growth were precisely determined using a continuous

flow fermenter; the optimum pH and temperature for growth on

thiosulphate were found to be 5.5 and 30± 0.5° C respectively.

The ThiobaciZZus ferrooxidans strain was found to

tolerate zinc concentrations as high as 20,000 p.p.m.without any

significant deleterious effects on its growth rate being obser­

ved. Concentrations of zinc higher than 15,000 p.p.m.were found

to inhibit the growth of the ThiobaciZZus thiooxidans strain.

The investigation involved the two crystallographic forms

of zinc sulphide, in the form of museum-grade specimens of

sphalerite and wurtzite. In addition, museum-grade marmatite

( a zinc iron sulphide Zn3.5FeS5) and synthetic zinc sulphide

were studied. The minerals were characterised by their X-ray

diffraction patterns and their purity was ascertained by

chemical analysis. Studies on their degradation by the three

pure cultures of ThiobaciZZi strains showed that the sphalerite

was degraded more rapidly and to a greater extent than was the

wurtzite: the latter could not be leached significantly by any

of the ThiobaciZZus species employed. The zinc sulphide with

iron substitution (marmatite) was the most amenable to bacterial

attack.

The ThiobaciZZus thioparus strain was the least active

in degrading any of the minerals and consequently showed the

lowest rates of metal release. The ThiobaciZZus thiooxidans

strain degraded synthetic zinc sulphide more easily than the

iron-oxidising ThiobaciZZus ferrooxidans, strain.

The results obtained indicated that:

1- The degradability of zinc sulphide minerals depends

upon the species of ThiobaciZZus used: usually it

is greater with the acidophilic members of the

genus. The rate and extent of metal release from

natural museum-grade mineral specimens decreased in

the following order :

T. ferrooxidans > T. thiooxidans > T. thioparus

and for the synthetic mineral the capabilities were

( in decreasing order) T. thiooxidans> T. ferroo­

xidans > T. thioparus.

2- Ferrous iron concentrations higher than 100 p.p.m.

were found to inhibit the synthetic zinc sulphide

leaching by Thiobacillus thiooxidans strain.

3- The crytallographic form to which a substrate

belongs is of some importance. In the case of zinc

sulphides, the cubical form (sphalerite) was found

to be more readily leached than was the

form (wurtzite).

hexagonal

4- The release of zinc and iron from marmatite was

found to occur at different rates & the pattern

of release of these metals to be different in the

degradations catalysed by Thiobacillus thiooxidans

and Thiobacillus ferrooxidans. The implications

of these observations for the interpretation of

the release mechanisms is discussed.

'I INTRODUCTION

1

1.1 GENERAL INTRODUCTION

Winogradsky (1887), while working with Beggiatoa, envisaged an

ecological niche where microorganisms could procure all their energy

from the oxidation of inorganic compomds. In 1888, he isolated a

chemolithotrophic bacterium which could fulfil all its energy require­

ments by the oxidation of exogenous iron salts. This discovery

introduced a second group of autotrophic organisms, the chemolithotrophs.

Until then the only autotrophs known were the photoautotrophs, viz.

plants and algae, characterised by their de nova synthesis of cellular

organic materials without requiring any preformed organic compomds.

As a result of the work of Winogradsky and others, it is now known that

both chemoautotrophs and phototrophs will grow on completely inorganic

media with carbon dioxide as the sole carbon source. Chemoautotrophs,

however, unlike phototrophs, obtain their energy for the synthesis of

cellular material, not from the electromagnetic radiations of visible

light, but from the oxidation of simple inorganic compounds. Thus, if

the chemoautotrophic bacterium is one of the nitrifiers, Nitrobaater or

Nitrosomonas, the inorganic compound would be either nitrite or annno­

nilUil ion; if it were one of the non-photosynthetic sulphur bacteria,

the inorganic ion would be either sulphide, sulphur or thiosulphate;

hydrogen gas would be an equally good substrate for the growth of

Hydrogenomonas.

In Table 1.1 some chemoautotrophic bacteria, along with their

substrates, have been listed. It is evident from this table that

a common characteristic of these chemoautotrophic bacteria is the

oxidation of the exogenous inorganic substrates, regardless of their

chemical nature, which supplies the energy to the cellular machinery

for all synthetic activities including the assimilation of carbon

2

l11or~a11ic 1·11erl!,

, 11 I" trait·

II,

S"

F .. •

(:()

ll2 + ~02

TABLE 1.1

CHEMOLIIBOTROPHIC

Formal t'q11atio11 for rt'adio11

BACTERIA

Badni11111

---+;\;02 - + H20 + 2H· N11,·01orruHw,

,:l.(; 0 = -oo kcal

---+ :'\i O:, ,\' Urobnrtn

~(; 0 = -17 .. 5 kl'al

---+ H,<> H_wlrof!nwmona, ~( ;o = -.57 kl'al

S0 + ;o, + I 1,0 -----+H,SO. ThwhanJ/u., ~(; 0 = -118 kl'al

F,,2 --.F .. 3 ' +, T. ferrooxulans ~(; 0 = - 11 kcal

co+ (o, --+(:<>, Carho.,·wiomon"' ,l(;'' = -oo kl'al

Taken from: Doetsch & Cook- 1973. Introduction to Bacteria & Their Ecobiology.

Family

THIOBACTERIACEAE

BEOOIATOACl!AB

ACHROMATACEAE

TABLE 1. 2

THE LIIBOTROPHIC SULPHUR MICROORGANISMS

General characteristics Habitat

CHEMOLITHOTROPHS colourless, coccoid, straight or curved rod

shaped bacteria; polar flagellate when motile. Oxidi.te sulphur compounds and usually depo,it sulphur granules within or without the cells

colourless cells occurring in 1 rid1romcs with­in which they a,e arr.in!'cd in chains. The trichromcs shnw a gliding motion when in contact with a substrate. When grown in the presence or hydrogen sulphide the trichromes contain sulphur globules

large spherical, ovoid or short cylindric_al cells containing sulphur granules and sometimes inclusions of calcium car­bonate

a wide variety of fresh-water and marine environments containing H,S. T/Jiobacil!us found in soil, mine waste-waters, sewage, industrial effluents

fresh water and marine environments con­taining H,S

fresh water and brackish mud containing H,S

Genera

Thiobacterium Macromonas Thio1•11/111n Thiospira Thiobacil/11s Beggiatoa Thiospiril/opsis Thioplaca Thiothrix

.Achromatium

Taken from: Roy & Trudinger - 1970. The Biochemistry of Inorganic Compounds of Sulphur.

3

dioxide.

Although a number of heterotrophs have also been found to oxidise

reduced sulphur compounds to sulphate in soils or other natural milieu,

the roles played by these transformations in the physiology of the micro­

organisms are not yet fully understood. Nevertheless, most chemolitho­

trophs of sulphur utilise the energy released by the oxidation of the

reduced compounds of sulphur for their synthetic activities. Table 1.2

lists the main groups of these chemolithotrophic bacteria of sulphur

and their main characteristics.

The not insignificant problems involved in the study of these

organisms are aggravated by the slow growth rates exhibited by some

of these genera. Therefore, for this reason very little information

pertaining to them is available at present. ThiobaciZZi, however, due

to their comparatively faster growth rates and the role played by them

in the mineral degradation and oxidation of various sulphur compounds,

have been far more extensively studied. They have been frequently

employed in various mineral mop-up operations for extracting metal

values from low grade ores and, due to their unique physiological

attributes, have found some novel applications, for example, the

removing of rust microbiologically (Iida et al., 1975) and the

recovery of hydrocarbons by the degradation of oil shales (Findley et

al., 1974; Meyer & Yen, 1976).

Since this study is mainly concerned with the degradative

activities of ThiobaciZZi on various zinc sulphide minerals, an

account of their principal characteristics follows.

4

1.2 THE THIOBACILLI

The credit for discovery of the ThiobaciZZi is attributed to

Nathansohn, who in 1902 succeeded in isolating small, Gram negative,

motile rods, which were capable of oxidising thiosulphate (Nathansohn,

1902). Since no carbon source was present in the medium, it was

correctly assumed that this bacterium could fix atmospheric carbon

dioxide, at the expense of energy obtained by the oxidation of thio­

sulphate, thereby confirming the definition of a chemoautotroph, as

proposed by Winogradsky (1887). This bacterium was named ThiobaciZZus

thioparus a type species of the genus ThiobaciZZus.

ThiobaciZZi are Gram negative rods, generally motile, occurring

in various natural milieus, including mine waters, sewage water and

soils. Their common characteristic is the the capability of oxidising

elemental sulphur. They are usually aerobic, the exception being one

species, ThiobaciZZus denitrificans, which can proliferate anaerobically,

using nitrate instead of molecular oxygen as an electron acceptor.

The classification bases for the ThiobaciZZi have been bio­

chemical and metabolic ones, like the oxidation of various compounds

of sulphur, thiocyanates, ferrous sulphate and the growth conditions

required. These bases of classification are fully debatable, particu­

larly now that evidence for the existence of intermingled properties

in this genus has been amply provided. For example, when ThiobaciZZus

denitrificans, an anaerobe, is repeatedly plated in normal atmospheric

conditions, it loses its anaerobic trait and exhibits the characteris­

tics of ThiobaciZZus thiopaPUs (Baalsrud & Baalsrud, 1954; Vishniac &

Santer, 1957). Similarly, ThiobaciZZus denitrificans & ThiobaciZZus

thiocyanoxidans were considered by Beijerinck (1904) and Happold et

5

al. (1954) to metabolise nitrate and thiocyanate respectively, and

were mainly distinguished from each other and from Thiobacillus thio­

parus by these metabolic differences. With the accumulation of more

information, it has been noted that these metabolic characteristics are

commonly met in the genus Thiobacillus and therefore should no longer

be regarded as the basis of distinctionbetween the various species of

this genus ( Van der Walt & De Kruyff, 1955; De Kruyff et al., 1957;

Happold et al., 1958; Woolley et al., 1962).

Another interesting situation is the taxonomical position of the

bacteria oxidising ferrous ions. In the literature one generally comes

across three different names of microorganisms capable of oxidising

ferrous to ferric ions. These are Thiobacillus ferrooxidan,s, Ferro­

bacillus ferrooxidans and Ferrobacillus sulfooxidan,s, which suggests

two different genera to be involved in this transformation. Leathen

et al. (1956) reported that the main difference between Thiobacillus

ferrooxidans and Ferrobacillus sulfooxidans was the capability of the

former to oxidise both sulphur as well as ferrous ions, whereas Fer­

robooillus sulfooxidans could not oxidise sulphur at all when it was

present as the sole oxidisable substrate. However, Silverman & Lund­

gren (1959-a) observed a slow but significant oxidation of elemental

sulphur by their strain of Ferrobacillus ferrooxidans, and Unz and

Lundgren (1961), studying the comparative nutritional patterns of

Ferrobooillus ferrooxidans, Thiobacillus ferrooxidan,s & Thiobacillus

thiooxidans, could not confirm Leathen's observations. Recently,

Silver (1970) has conclusively demonstrated that Ferrobacillus ferr­

ooxidans can oxidise sulphur compounds concomitantly with fixing

carbon dioxide.

This leads to an important question, viz.,"Are the species of

ThiobaciUus distinct or simple variants of one another"? The use of

6

biochemical characteristics for the characterisation of the different

members of the

difficulties.

Thiobacillus group is not without some taxonomic

For example the variant components of some described

isolates in respect to the oxidation of iron, the adaptation of iso­

lates to different substrates, the varying degrees of persistence to

the presence of organic matter and to heavy metals have led to some

taxonomic confusion. Some important points remain to be solved,

for example, the validity or otherwise of the novel species Thiobaci­

llus acidophilus (Guay & Silver, 1975) with its capacity for the

oxidation of elemental sulphur and glucose but inability to oxidise

ferrous ion, even though de.rived by stepwise subcul turing from an

authentic Thiobacillus ferrooxida:ns strain. These continuing un­

resolved problems are referred to in greater detail later in this

introduction.

I f

TABLE 1. 3

HABITATS OF T!-IIOBACILLI

HABITAT SPECIES ISOLATED REF. HABITAT SPECIES ISOLATED

~ SOILS T. novellus 33 ACIDIC LEACHING F. ferrooxidans

I T. traut-weinii 33 WATER

T. thiopar-us 33 ACIDIC COPPER T. ferrooxidans

T. thiooxidans 33 LEACHING WATER

T. denitrificans 34,35

PEAT SOILS Sulphur bacteria 15 MINE WATERS & F. f er'Y'ooxidans

MARSH T. thiopar-us 15 ORES (Uranium, F. sulfooxidans

T. denitrificans 15 Copper & Zinc) T. f errooxidans

T. f errooxidans

HOT SPRINGS T. thermophilica 5,6, F. ferrooxidans

& 41

ThermophiZic 40 COAL WATERS T. thiooxidans

thiobacilZus? T. f errooxidans

PONDS, LAKES T. thiopar-us 2 LIMESTONE & T. thiooxidans

T. thiooxidans 2 SULPHUR T. thiopar-us

T. denitrificans 2

OIL BRH!ES T. thiopo.rus 30

SERGIEV SPRINGS T. thiopar-us 16 BITUMINOUS T. thiooxidans

V[;-;ERA SULPHUR T. neapo Zi tanus 7 COAL SPRH;GS T. oonoretivorus 7 MINES T. ferrooxidans

1- Baalsrud & Baalsrud, 1954. 2- Barvenik & Jones, 1969. 3- Beck & Elsden, 1958. 6- Brierley & Brierley, 1973. 7- Cocuzza & Nicoletti,1961. 8- Colmer et al.1950. 11-Egoroya & Deryugina, 1963. 12- Forrester, 1959. 12- Gilchrist, 1953. 16-Ivanov, 1957. 17- Jones & Carrington, 1972. 18- Karavaiko, 1959. 21-Kinsel, 1960. 22- Marchlewitz & Schwartzl96~23- Mutze & Engel, 1960 26-Parker, 1945. 27- Razzell & Trussell,1963. 28- Rogoff et al, 1960. 31-Sol:olova & Karavaiko, 1962. 32- Sokolova& Karavaiko,1963. 33- Starkey, 1935. 36-Terr.ple & Delchamps, 1953. 37- Tilton et al., 1967. 38- •romizuka & Takahara,1972. 41-Zavarzin & Zhilina, 1964.

REF. HABITAT SPECIES ISOLA'fED ~!:F.

"'-J

3 COAL (PYRITE) F. fer'Y'ooxidans s T. thiooxidans 28

27

ACTIVATED T. thio?a:r>uB 17

SLUDGE PLANTS T. thiooyanoxidans 14

3r, & SEWAGE T. f errooxidans 9

38 SEWER T. concretivorus 26,12

38 T. thiopar-us 13

39

21 SEA WATER T. thiopar-us 24,37

& 4

22 MARINE MUD T. denitrificans 1

22 RIVER WATERS T. ferrooxidans 25

T. thiooxidans 25

18 T. thiooxidans 23

19 T. denitrificans 23

MARINE MUD T. feY'!'ooxidans 10

SULPHUR DEPOSITS T. thiooxidans 20

36 T. thiopar-us 29

T. denitrificans 29

8 T. thiopa:,'ur; 32

4- Beijernick, 1904. 5- Brierley, 1966. 9- Corrans, 1970. 10- Corrans et al. 1972.

14- Happold et al. 1954. 15- Ilyaletdinov & Kanatchinova,l964, 19- Karavaiko et al. 1962. 20- Karavaiko & Pivovaroa, 1973. 24- Nath6nsohn, 1902. 25- Niemela & Tuovinen, 1972. 29- Rogovskaya & Lazareva,1961.30- Sokolova, 1960. 34- Taylor & Hoare, 1969. 35- Taylor et al., 1971. 39- Torma & Legault, 1971. 40- Williams & Hoare, 1972.

3

1.2.1 ISOLATION AND TAXONOMICAL CHARACTERISTICS OF THIOBACILLI

Thiobcwilli are ubiquitous in nature, occurring in a wide

variety of natural environments including sewage water, hot springs

and soils. The acidophilic members of the genus have been detected in

acid mine waters, sewage holes, sulphur dumps and natural waters. A

large number of Thiobaeilli capable of growing at neutral pH have been

isolated from soils. In Table 1.3 the various habitats of Thiobaoilli

so far reported are summarised. The first detected member of the genus,

Thiobaoillus thioparus, was isolated from marine mud by Nathansohn

(1902) and has been frequently re-isolated from fresh waters and garden

soils. Starkey (1935) noticed that only 2 out of 29 soil samples, when

tested for the presence of the acidophilic Thiobaoillus thiooxidans,

showed positive results. However, he was able to isolate other species

of this genus from all these samples and concluded that the acidophilic

members occur sparsely in the soils. The isolation of Thiobaoillus

ferrooxidans, by Colmer et al. (1950), from bituminous coal mines.drainage

provided a ground to study the role of these microorganisms in mineral

leaching. It is evident from the Table 1.3 that most of the Thiobaoi-

llus ferrooxidans and Thiobaoillus thiooxidans strains have been iso­

lated from the acidic waters of copper, uranium and bituminous coal

mines. The capability of these species to degrade sulphide minerals

and tolerate relatively high hydrogen ion concentrations suggests sul­

phide ores as suitable preferred milieus for their proliferations.

Various workers have reported the isolation of Thiobaoillus ferrooxidans

and Thiobaoillus thiooxidans strains from the sulphide ores of uranium,

copper and nickel.

Thermophilic members of the genus Thiobaoillus have also been

isolated from hot springs containing sulphur by groups of scientists

working in Russia, United States of America & United Kingdom (Kaplan,

- I.O

'Tj -C") ;1~ 0 • ~ t--' (D

{ 1 THIOBACILLUS THIOOXIDANS

I THIOBACILUS FERROOXIDANS ] Ul

"O I-'• ::::: 5. 0 I THIOBACILLUS ACIDOPHILUS

·I -I-'· -0 () '~

I"'! Ill I -(D rt ~ '"O (D ;

~ I\) w ~ 0, 0) ....... 0) <O 0 I"'! ' (D '"O j::t> Ul ::i:: z (D '0

. ·o :I:

::, I"'! rt Ill :;i::,

::, ;:t> '"O IQ z ::i:: (D C")

,m 0 Hl

I THIOBACILLUS INTERMEDIUS I fTHIOBACILLUS DENITRIFICANS

'"O 0 'T1 rt I"'! 0 I-'· 8 IQ

:;i::, T. RUBELLUS Ill 11 C") . 0 :;i::, ....., ( 0

T. DELICATUS

rt ~ ::r :i Ill ::, 0 0, 'T1

Ul ~ 0 .... 8 I-'• 0, ~ .... ~ I-'• ::, t--< (D t--< Ul 1--.t

I THIOBACILLUS NEAPOLITANUS I I ] THIOBACILLUS NOVELLUS

- [ THIOBACILLUS PEROMETABOLIS I [ THIOBACILLUS THIOPARUS

J.O

1956; Schwartz & Schwartz, 1965; Williams & Hoare, 1972; Le Roux et al.

1977). Egorova & Deryugina (1963) were among the early investigators

to report the isolation from a hot spring of a spore forming, thermo­

philic Thiobacillus, Thiobacillus thermophilica Imchenestskii, its

temperature optimum for growth being between 55° and 66°C. Recently

these thermophilic thionic bacteria have been studied for their role

in the leaching of sulphide minerals (Brierley & Murr, 1973).

1.2.2 ISOLATION OF THIOBACILLI

The Thiobacilli have been isolated from their environments by

enrichment techniques, employing suitable media and appropriate cultu­

ral conditions.

The genus Thiobacillus includes a variety of microorganisms which

vary considerably in their pH optima for growth (Fig. 1.1). Thus,

ThiobaciUus thiooxidans & ThiobaciUus ferrooxidans grow luxuriantly

at pH 2.5, or even less, whereas Thiobacillus thiopa:r>us, Thiobacillus

denitrifioo.ns, and other species prefer a higher pH for their proli­

feration. The Thiobacilli are pH-sensitive and are easily killed by

unfavourable pH environmental conditions. This characteristic of

these bacteria makes it very important that their growth media should

be properly buffered.

Vishniac & Santer (1957) in their review have listed a number of

media used for isolation of the various species of Thiobacilli and

Tables 1.4A & 1.4B describe the composition of the different media

usually employed for the isolation and growth of the individual species

of Thiobacilli.

Since these bacteria are autotrophic, bubbling Co2 through the

medium usually enhances the growth processes. Generally, K2HP04 and

KH2P04 , combined in buffers, control the pH and fulfil the phosphate

TABLE 1. 4 A- MEDIA FOR NON-ACIDOPHILIC THIOBCILLI

T. thioparus T. n,.apoZitanuo T. dc,dt.rificans 1', r.ovcUuo T. iritn,rtcdiuo T, DCT'C"netaboU/J T. ·thfo "!i1GWJ:rf_ d.:-:.,,r:

NO MEDIA Grams per litre Grams per litre Grams per litre Grams per Ji tn• Grnms per Ii trc Grams per litre Grains per litre

CONSTITU~NTS l I 2 l 3 I 4 s I 14 6 I 7 I I 9 10 I I 11

I 12 8 2 I 14 13 I 14 15 I 16 I 16

1--'

N,i/11'04 7.9 1.0 l. 0 I-'

2 KH2Po4 4.0 4.0 0.2 4.0 5,0 2,0 2,0 l.S 4,0 4.0 0.4 0.4 1.0 0.4 0.6 3.5 3.S 3 K2HP04 4.0 4.0 · 4.0 0.2 4.0 s.o 2.0 2.0 4.0 4.0 0.6 0.6 0.6 4 MgS04• 7H20 0.8 0.1 0.5 - 0.8 0.5 0.8 0.1 0.1 0.8 o.s 0.3 o.s 0.02 0,001 0.001 5 MgCJ 2• 6 H2o 0.5 0,5 0.1 0.5 o.s 6 Nli4CJ 0.4 0.1 1.0 1.0 o.s 1,0 0.30 0.5 1.0 1.0 1.0 1.0 0.1 C. I

7 (NH4)2S04 0.1 0.4 0.1 8 CaC03 9 KC!

10 KN03 2.0 2.0 2.0 5.0 I 2.0 11 KCNS - - 0.2 0. 25 12 CaCJ2.2 H2o 0.1 0.2S T - 0,1 - 0,01 0.01 13 Ca(NO3) 2 14 KOH - - 1.4 1.4

15 Nar1C03 - 1,0 1,0 2.0 1,0@ 2.0. 16 FeCJ 3. 6H20 0.02 0,001 0.02 - 0.02 0.02 0.02 0.02 0.002 0.002 17 FeS04. 7H20 - 0.5 - 0.01 0.01 0.02 18 Na2S203. 5 H2o 10 10 5 10 10 10 5 5 s s s 10 8 10 5 - 10

19 Ynast ext~act. 5 20 Trace metal

solution ml# 10 JO l 5 JO 21 Trace metal

solution ml• 3 3 20 3 22 MnS04• 4H20 0.02 0,2 0.002 o.oc~ 23 Agar s 15 20 24 pH 7.0 6.6 7.0 S.6to 6.6 6.8 ND 6.9 7.0 ND 8.5 6.6 ND 6.8 6.8 6.9 6.8 7.0 ND ND

7.2 25 Adjust with 2N ND ND ND SO% Nb 1-!D I\ ND ND 10\ ND ND ND Na2co3 KOH ND ND ND ND

HCl K2C03 NaHC03 NallC:03 26 Solvent ow ND DW DW ND ND ND ow DW ND DW DW D• SW OW DW ND TW ND DW

SW= Sea water DW= Distilled water ND= Not described. Tc Traces. @=Used during anaerobic cultivation only.

TW = Tap W!ltcr 4. Tilton & Johnson,1967 . .S- Mayeux et al. 1967. 6- Baalsrud & Baalsrud, 1954. 1-Vishniac & Santer, 1957, 2- Starkey, 1934-a, 3- Starkey, 1935.

JO- Taylor & Hoare, 1969. 11- Santer et al. 1959. 12- Matin & Rittcnberg, 1970. 7-Sargeant et al. 1966. 8- Taylor et al. 1971. 9- Lieske, 1912. 13-London & Rittenberg, 1967. 14- Matin & Rittenberg, 1971.

15- Youatt, 1954. 16- Happold et al. 1958.

f Trace metal solution of Vishniac & Santer, 1957. • • Pfenning's medium (Postgate, 1966).

12

demand at the same time. Ammonium salts provide the necessary nitrogen

for growth; either ammonium chloride or ammonium sulphate can be safely

used. ThiobaciZZus denitrificans, an anaerobic member of the genus,

requires nitrate as an electron acceptor. ThiobaciZZi can oxidise a

number of reduced sulphur compounds but sodium thiosulphate is most

commonly employed as the energy source.

Like all soil bacteria, they each have specific demands for the

trace minerals. For example, ThiobaciZZus thioparus has been shown

to require iron and manganese (Starkey, 1934) and Baalsrud & Baalsrud

(1954) reported augmentation in the growth rate of ThiobaciZZus

denitrificans in the presence of traces of ferrous ions. Neverthe~

less, a trace metal solution as described by Vishniac & Santer (1957)

meets the demands for trace elements for most species.

ThiobaciZZus thiocyanoxidans, which utilises CNS as a carbon and

sulphur source can also oxidise sodium thiosulphate in the presence

of ammonium ions plus carbon dioxide.

compounds, facultatively autotrophic

In addition to reduced sulphur

ThiobaciZZi can also obtain

their energy from organic compounds such as D-glucose, D-glactose,

sodium citrate, DL-aspartic acid and yeast extract (London, 1963;

London & Rittenberg, 1966; Guay & Silver, 1975; Mizoguchi et al.,

1976).

The acidophilic members of the genus do not require high concen­

trations of phosphate and trace elements (Manning, 1975). They require

a low pH ( 2.0 to 2.5) and can oxidise either ferrous ions, elementary

sulphur or thiosulphate and other reduced sulphur compounds to obtain

their energy. Due to the lack of knowledge of appropriate growth media

and harvesting techniques, the work on the acidophilic ThiobaciZZi

was hindered until Silverman and Lundgren (1959) devised their, now

most widely used, medium known as '9K'. The composition of this medium

is shown in Table 1.4B.

Ti\11 LE 1 :4B •

MEDIA FOR J\CJDOPIIILJC Tl!IOBACILLI

-T. f en•ooxidane I 'I'. thloc,xidane f T. conr:r•e t ivoi•u;,

No I MEDIA I Grams per litre I Grams per litre I Grams per litre ......

IT, (.-.)

CONSTITUENTS

I 3 I 4 I 5 I 6 I 7 111 111 I 3 I 8 I 9 1 10 I 11

l KH/04 - - - - 0.4 - 4.0 3.0 3.0 - 3.0 3.0 2.0 3.0

2 K2HP04 0.05 - o.s 0.4 - 3.0 - - 0.5

3 MgS04 . 7 H2o 0.5 1.0 0.5 0.4 0.1 0.5 0.5 - - 0.5 0.5 0.5 0.5

4 MgC1 2• 6 H2o - - - - - - 0.1 0.1 - - - - 0.1

5 NH4Cl - - - - - - - 0.1 0.1 3.0 - - 1.0 0.1

6 (NH4) 2so4 0.15 0.5 3.0 0.4 . 0.1 0.2 0.4 - - 0.4 0.2 .-7 NaCl - - - - 1.0

8 KCl 0.05 - 0.1 - - - - - - 0.1

9 CaCl 2" 2 1120 . - - - 0.03 0.25 0.25 0.1 0.1 - 0.25 0.25 0.1

10 Ca (NO.)? 0:01 - 0.01 - - - - - - 0.01 .) ~

11 MnS0_1• lll/l - - - 0.02

1'2 FeC]3. 6 H2o - - - - - - - - - - - 0.02

13 Al 2lSG4) 3. 12 Hz° - - - - 1.4

14 .FeS04 . 7 H2o 10 130 •44.2 33.3 10 - 0.01 - - - 0.01 0.01

15 Na 2s293. 5 H2o __ .· - - - - 5 5 - 5 - - 5

16 SULPHUR - - - - 10 - - 10 10 10 10

17 AGAR - _. - 4 - - 12.5 - - - - - - 12

18 pH 3.5 2.5 2.5 1.3 Nf\ 3.0 4.8 4.2 4.2 2.5 4.0 ND 4.5 4.2

19 ADJUST WITH - Cone. 1 ml 0. llN O .09ml ND ND ND ND ND ND ND ND ND

H2so4 10 N H2so4 Cone. Hz504 H2so4

20 SOLVENT ow DW DW DW ow ow DW DW ow ow DW ow ow DW

1- Leathen et al. 1956. 4- Tuovinen & Kelly, 1973. 7- Bacto thiobacillus agar. 10- Matin & Rittenberg, 1971. 2- Colmer et al. 1950. 5- Hutchinson et al. 1966. 8- Barton & Shively, 1968. 11- Parker & Prisk, 1953. 3- Silverman & Lundgren, 1959. 6- Waksman et al. 1923. 9- lfaksman & Starkey, 1923.

ND= Not described •. DW = Distilled water.

14

The purification of acidophilic members of the genus Thio"baciZZus

which are obligatelyautotrophic(ThiabaciZZus ferrooxida:ns, ThiobaciZZus

thiooxida:ns) on solid media is beset with problems. Agar cannot be

gelled properly because of its hydrolysis at the low pH required by the

organisms. A large number of investigators have tested and devised

modified agar media for the growth of Thio"baciZZus ferrooxidans and th~ir

reported L, inhibitory effects on the colony development (Bryner and

Jameson, 1958; Unz & Lundgren, 1961; Beck, 1967; McGoran et al., 1969;

Niemela & Tuovinen, 1972; Tuovinen et al., 1971-b). In attempts to

solve these problems workers (Leathen et al., 1951; Bryner & Jameson,

1958; Beck, 1960; Lapteva et al., 1971) have suggested the use of silica

gel for solidifying the medium. Gels prepared according to some pro­

cedures widely in use at present are either too soft or too cloudy to

be really useful. A method claimed to be satisfactory for the prepara­

tion of silica gel for growth of Nitrosomonas and Nitro"bacter species

has been described by Roslycky (1972). However, no attempts were made

to grow ThiobaciZZi on this medium. The absence of any suitable

method for colony development has made the conventional isolation of

low pH ThiobaciZZi from a single colony very difficult and has also

je.opardised performance of any classical genetic studies involving

these microorganisms. However, Tuovinen & Kelly (1973, 1974-c) have

reported a method for determining viable counts of Thio"baciZZus ferro-

oxida:ns using membrane filters placed on agar plates; specially

purified agar is used as the solidifying agent. According to these

workers, the purified agar does not inhibit the growth of Thio"baciZZus

ferrooxidans or ThiobaciZZus ne!X[JoZita,nus. That this technique suffers

from the drawback of lack of reproducibility is indicated from the

results of various workers (Unz & Lieberman,1973). This study showed

that the type of membrane filter as well as brand of agar used influ­

enced the colony development of ThiobaciZZi. A low-ferrous, phosphate

15

free, modified form of 9K medium for isolating acidophilic iron-

oxidisers from acid mine drainage has been reported to give well deve­

loped colonies on agar plates (Manning, 1975). No attempts to determ­

ine the percentage recovery of ThiobaaiZZus ferrooxidans with the new

methods were made. However, with some modification, this technique

provides the means for the development of acidophilic cultures from

a single colony. Pure suspensions of ThiobaaiZZus fewooxidans have

been standardised by using a coulter counter (Shuler & Tsuchiya, 1975)

and Gormely & Dwcan (1974) developed a method for estimating bacterial

populations in association with mineral particles by determining the

nitrogen contents of such mixtures.

The purification of non-acidophilic ThiobaaiZZi is not so

difficult as that of acidophilic ThiobaaiZZi, though it does involve

some peculiar problems, including inhibition of colony development

on agar media due to the toxicity of agar and other associated material.

They will grow on solid media prepared from pure grade agar using

some special techniques. On solid plates these bacteria (Thiobaai­

ZZus thioparus and others) need frequent transfer because of their

sensitivity to metabolically generated acid. They are best maintained

in liquid culture.

16

1.2.3 TAXONOMY OF THIOBACILLI

The taxonomy of ThiobaciZZi has been most confusing and

challenging since the beginning. As mentioned earlier, the basic

classification rests upon biochemical reactions, which in turn depend

upon various other factors. In the following, an endeavour has been

made to summarise the different traits of these microorganisms.

1.2.3.1 MORPHOLOGY

It has been generally accepted that all ThiobaciZZi are Gram­

negative, motile rods of diameter varying from 0.3 ~ 0.4 µm, and

about 1 ~ 3 µm long. They are easily stained with Gram stain and a

few (unconfirmed) reports exist which describe them as Gram-positive

rods (Starkey, 1935). The mode of motility of these bacteria indicates.

the presence of a single polar flagellum, which is very fragile and

frequently cannot be observed by ordinary optical microscopy employ-

ing conventional staining techniques. The flagellum is usually lost

during preparatory procedures. However, electron micrographs have

clearly shown the presence of one polar flagellum. The only non­

motile strain described is ThiobaciZZus noveZZus. The non-motility of

ThiobaciZZi depends upon a number of factors, viz. acidic environ­

ments or the exhaustion of substrate. The reason may be the lack of

energy under the 1.lllfavourable conditions, when cells are 1.lllable to

spend their energy for motility.

With the exception of a strain ThiobaciZZus thermophiZica

Imschenetskii nov. sp., isolated by Egorova & Deryugina (1963), all

ThiobaciZZi are non-sporulating. The spores were terminal and able

to withstand high temperatures. However, the position of this

microorganism in the genus ThiobaciZZus has been seriously questioned

by some workers (Hutchinson et al., 1967).

SPECIES

T. cur.dophil~a (II

T. delicatus 121

. T. dsnit"rificans

T. fenooridane

I. ntrJeZZ.ue

T. r.eapolitanua

.. "'. :nter1r,edi~UJ

*

.

,,. p€.rametat:olis

T. :rubellus <2>

t'r.WCxidans .

T. thioparu11 .

.

SHAPE

Short rods

Short rods

Short rods

Short. rods with rounded edges.

Short rods, coccoidal or ellipsoidal cells

Short rods

Thin short rods.

Thin short rods

Single rods.

Short rods

Thin

SIZE µm

0,5 - 0.8 X

1.0 - 1.5

0.45-0.50 X

0.7 -1.3

0.5 X

1.0 - 3.0

o.sx 1.0

0,4 - 1.0 X

0,6 - 4.0

0,5 • 1.0-1.5

0.5 l< LO· 2.0

0.5 X

1.0 - 2.0

1.0 - 1.6 x 1.7 - 3,0

0.5 X

1.0 - 2.0

0.5•1. 7

TABLE 1.5

MORPHOLOGICAL CHARACTERISTICS OF

THIOBACILLI

GRAM STAIN

Negative

Negative

Negative

Negative

Negative

Negative

NegativA

Negative

Negative

Negative

Negative

MOTILITY

Positive

Negative

ND

ND

Negative

Negative marine strain positive

ND

Positive

Positive

ND

Positive

FLAGELLUM

ND

ND

ND

Single (J)

polar.

Single polar

ND

Single, polar.

COLONY CHARAClERISTICS

Colonies on 9K glucose mediUll are discrete, small (1-2 mm in diame­ter), round, regular, convex, sl­ightly translucent and cream col­oured. Colonies on li yeast extract-0,5~ thiosulphate agar are 1 ncn ir. di­ameter with smoot..11 outer edge ,col­orless and transparent· turning whitish yellow. Colonies on thiosulphate agar th­in, clear, opalescent, deep agar colonies; star shaped glistening.

Colonies on thiosulphate agar are very thin & small with irregular margins, becoming whitish in the centre.

On nutrient agar slow growth, colonies colorless, moist, raised circular, 1 mm in diameter.Similar on thiosulphate agar but white.

Colonies on t~iosulphate agar are small {l-2rmn), circular ,convex, glistening whitish yellow, old colonies changing to pin~ in the centre.

On thiosulphate ngdr, small col­onies, (<l mm), yellow, opaque, with raised centres & flat c'.}.nd veil like fringes, on yeast extra-ct small, thin, clear and spreading colonies are product1d.

LIQUID CULTUi\E

9K gh,cose :::edium.

Cells are capable of growing in thiosulphace mineral :l\E:diw:i..

Uniform turbidity

Unifurm turbid.ity

Th iasulpha t:e brot..li. wii­f om.ly turbid, no f't,:.ll­icles. N;.itrier,t broth, slightly turbid ••i th gelati:1ous pellicle.

Uniform turbidity \\·i 't.h

pellicle~, containg pre cipit~ted sulphur.

)10

Colonies on lhiosulphate are barely Uniform turbidity wit..--i visible; on yeast extract, colcnies slow growth in yeast a"L"e 1·-3 mm in diameter, entire and extract. creamy.

Colonies on 0.1% yeast extract-0.5~ thiosulphate agar are smooth-outer edged, 1 mm in dL:unctt.."'rwiti1 reddish tinge, centre of colonies becoming brownish at'ter two weeks.

ND

Colonies on thiosulphatc agar, minu- Uniform turbidity. te, transparent or whitish yellow.

colonies on thiosulphate agar very thin& small(l-2 mm, diameter) ,whitish turnin~ to pink and brown.

Uniforn turbidic).

• Talen from Bergcy's Manual of Determinative Bacteriology, 8th Edition, 1974, Buchon~n & GibLons, 1974,

(1)- Cuay & Silvcr,1975. (2)- Mi:oguchi et ol.1976. (3)- London & Rittenberg, 1967. ND= Not determined.

I--' -..J

18

Cells occur singly or in pairs, and sometimes chains of cells

have been observed. Silverman & Rogoff (1961) reported alteration of

cellular morphology of ThiobaaiZZus ferrooxidans in response to exce­

ssively vigorous aeration. According to these authors, the normal

cellular rods were transformed into a coccoidal form with a diameter

of 1.0 µm, when saturated with air and strongly agitated. These cells

regained the normal morphology when grown en masse or with less vigo­

rous aeration. Cells grown in glucose have been reported to become

slightly rounder, with a tendency to form pairs (Lundgren et al., 1964).

ThiobaoiZZus ferrooxidans and ThiobaoiZZus thiooxidans were found

to possess two mechanisms of reproduction, by fission and by partition,

the former being the most common mode of cell division (Karavaiko &

Avakyan, 1970; 1971).

Table 1.5 summarises the morphological characteristics of the

genus ThiobaoiZZus as described in the latest edition of the Bergey's

Manual of Determinative Bacteriology (Buchanan & Gibbons,1974).

1.2.3.2 BIOCHEMICAL CHARACTF:R.ISTICS

Parker & Prisk (1953) carried out a comprehensive study of

ThiobaoiZZi whilst they were examining the utilisation of various red­

uced compounds of sulphur and classified them into different species.

They found that thiosulphate was oxidised by all ThiobaoiZZi and Thio­

baoiZZus thiooxidans, ThiobaoiZZus oonoPetivorus and ThiobaoiZZus X

converted it to tetrathionate and sulphite and then oxidised it

further to sulphate and free sulphuric acid. ThiobaoiZZus thiopaPUs

was found to convert thiosulphate to sulphite and sulphur followed

by partial oxidation of sulphur to sulphuric acid. Elementary sulphur

was found to be oxidised by ThiobaoiZZus thiooxidans, ThiobaoiZZus

oonoPetivorus, ThiobaoiZZus X, and ThiobaoiZZus thiopaPus; the rate of

oxidation was in that order. Hydrogen sulphide was oxidised by

THE GE~US THIOBACILLUS

~ OBLIGATELY AUTOTROPHIC FACULTATIVELY AUTOTROPHIC

AEROBIC

T. THIOPARUS

T. NEAPOLITANUS

T. FERROOXIDANS

T. THIOOXIDANS

FACULTATIVELY ANAEROBIC

T. DENITRIFICANS

T. ACIDOPHILUS

T. DELICATUS

T. NOVELLUS

FIG. 1.2 SUB-CLASSIFICATION OF THE GENUS THIOBACILLUS

MIXOTROPHIC

T. INTERMEDIUS

T. PEROMETABOLIS

T. RUBELLUS

...... I.O

20

Thiobacillus concretivorus (Thiobacillus thiooxidans) and Thiobacillus

X. Therefore, in the early studies the basic differentiating charact­

eristics serving to distinguish between the different forms of Thiobacilli

were considered to be the capacity of utilising certain specified

reduced compounds of sulphur as the substrate and the production of

certain metabolites. Both of these criteria have many disadvantages

and, indeed, have caused a good deal of confusion in the taxonomical

studies of the Thiobacilli. For example, in a number of recent inves­

tigations it has been amply demonstrated that the utilisation of

various sulphur compounds and the formation of products other than

sulphate are dependent on such conditions of growth as pH, oxygen

transfer, substrate level and so on. Therefore, these biochemical

characteristics can no longer be regarded as definitive diagnostic

tests for taxonomic purposes.

Earlier, microorganisms capable of oxidising or otherwise meta­

bolising thiosulphate were catalogued as Thiobacillus species; for

example, Thiobacillus coproliticus and Thiobacillus trau-tweinii

(Lipman & Mcless, 1940; Bergey et al., 1925). Those heterotrophs, now

known to possess the ability to oxidise thiosulphate without obtaining

energy from the process (Guittoneau, 1925; Starkey, 1935), are no

longer considered to be members of the genus Thiobacillus (Vainshtein,

1975). Those species which obtain energy from the oxidation of thios­

ulphate can be divided into three subgroups, namely obligately auto­

trophic, facultatively autotrophic and mixotrophic (Fig. 1.2). The

later subgroup includes heterotrophic microorganisms whichutilise compounds

sulphur and organiclsimultaneously (Rittenberg, 1969). The obligate

autotrophs are further divided into aerobic and facultatively anaero­

bic sub-sub-groups. The aerobic sub-sub-group is comprised of

(a) species which are capable of oxidising only sulphur compounds in

order to fulfil their energy requirements (Thiobacillus thiooxidans~

I • OPTH~: SPECIES I OPTIMUM pH-

TEMPERATURE I

T. acidophilua<ll 3.0 25 - 30 [LS - 6.0] .

T. denitrificana 7.0 30

I'. d.alwatus(Z) 5.0 - 7.0 30

* 6.0 - 7.0# T. i nt,ermediua 30

[1.9 - 7.0]

* T. f en'ac:::id.ans 2.5 - 5.8 28 - 35

[J.4 - 6.0]

. 1'. ri.eapo'Zit,-;r..us 6.2 - 7.0 28

[3.0 - 8.5)

. . T. nct;,eZZus 7.8 - 9.0 30

[5.0 - 9.2]

* T. perometabolis 2.6 - 6.8 30

'

'I'. roeZZus (2) s.o - 7.0 30

. T. thioo::.damJ 2.0 - 3.5 28 - 30

[0.5 - 6.0]

* T. th{..oparus 6.6 -7.2 28 - 30

[up to ID J

TABLE 1.6

BIOCHEMICAL CHARACTERISTICS OF

THIOBACILLUS

OXYGEN INORGANIC SUBSTRATES REQUIREMENT

Grows only on elemental Positive sulphur.

5203-, s•, s--. and Facultatively

polythionates. anerobic.

Thiosulphate·. Aerobic

Thiosulphate and other Aerobic

reduced compounds of sulphur.

Ferrous, thiosulpha,te ,ele- Aerobic

mental sulphur and other

compounds of sulphur.

Thiosvlphate, tetrathionate, Aerobic

elemental sulphur and hydrogen

sulphide .

Thiosulphate,no growth on Aerobic

elemental sulphur.

Sulphur, thiosulphate and tet- Aerobic

rathionatc are used only in

the presence of organic comp.

Thiosulphate in the presence of Aerobic·

organic compounds only .

Elemental sulphur, thiosulph- Aerobic

ate, polythionates.

Elemental sulphur, thiosulph- Aerobic

ate, tetra-and other polythio-

natcs; in some strains thio-

cyantc and hydrogen sulphide

are also oxidised.

C02 G + C FA.'<E ~RGi,'111 0\

FIXATION RATIO PROFILE ORGA.',lC Cu).(?.

ND 62.9-63.2 ND FACLILTATI\ELY AUTOTROPHIC

Positive ND II CBLIGATIL\

AITTOTROf'HiC

ND 67 + 3 ND FAGJLTATI\cl.Y

AUTOTROl'illC

Positive ND II MlXOTROrHI.::(J;

Positive 56 - 57 I OBLIGATELY

AlITOiROPl!lC

Positive 56 - 57 I OELlG.~Tii.Y (4~

AUTOTROi'lilC

Positive 62 - 68 II FACULTATI\1:LY

AUTOTROPH!C

ND ND ND OBLIGATEi.Y

MIXOTROPHIC

ND 65 + 3 ND OBLIGATHY

MIXOTROPHIC.

Positive so - 52 III OBLIGATELY

AlffOTROPHl C

Positive 62 - 68 II OBLIGATE LY

AlITOTROPHI C

• Ta:S:en from Bergey's Manual of Detel'minative Bacteriology, 8th Edition, 1974. (1}- Guay & SiZvez,, 1975. (2) Mizuguchi et at. 1966 .. (.li.'/:.tt.."r.for;;, 1Jc~ q W,]ql' values ir.::licate the :range of pH fo-r- gz,ovth. (4)- Aaaimil.ates organic compounds in the pNsence of sulp/;uz, =,;,l"..:n.is. fl Persumed vales. ND-Not dntennined. FAME- Fatty acid methyl estez,.

N I-"

22

ThiobaaiZZus thioparus etc.,) and (b) species which can oxidise either

ferrous ions or sulphur compounds ( ThiolxxciZZus ferrooxidans)

ThiobaciZZus denitrifieans is the only species known to oxidise

sulphur compounds with nitrate as the terminal electron acceptor in

place of oxygen, and it is consequently the only facultatively anaero­

bic species in the genus. According to a recent investigation it has

been claimed that both ThiobaciZZus feYTooxidans and ThiobaciZZus thio­

oxidans have the ability to grow anaerobically by oxidising elemental

sulphur in the presence of ferric ions, which actas achemical oxidant

(Brock & Gustafson, 1976).

The facultative autotrophs of the genus are differentiated on

the basis of the effect of organic compounds on the oxidation of in­

organic sulphur compounds. In one species, ThiobaciZZus noveZZus,

organic matter represses the oxidation of thiosulphate while in the

other species, such as ThiobaeiZZus intemzedius, ThiolxxciZZus rubeZZus,

ThiobaeiZZus deZicatus, this oxidation is not affected by organic

matter; in fact the latter two organisms oxidise thiosulphate and

organic substrates simultaneously and are classified as mixotrophic

(Mizoguchi et al., 1976).

This classification lays much emphasis upon the autotrophic

nature or otherwise of the microorganisms, a criterion which itself

is liable to fall prey to many drawbacks, the more important being the

lack of a precise definition of autotrophy (Kelly, 1971; Schlegel,

1975; Whittenbury & Kelly, 1977). It has been reported by many workers

that it is possible, with special techniques, to grow even the most

putatively obligate autotrophs on glucose (Shafia & Wilkinson, 1969);

the possibility of growing ThiobaciZZus thiooxidans on glucose as the

sole energy source has been discussed by Borichewski & Umbreit (1966).

The biochemical characteristics of the genus ThiobaciZZus are

tabulated in Table 1.6.

23

In order to resolve the controversy on whether the Thiobaaillus

genus consists of a spectrum of types (Baalsrud, 1954), or is a genus

containing distinct species (Vishniac & Santer, 1957; Parker & Prisk,

1953), Hutchinson et al. (1965, 1966, 1967) studied the taxonomy of

the genus exhaustively. They extended the method of numerical taxonomy

pioneered by Sneath (1957) to this group of microorganisms and conclu­

ded that the 'groups' and 'sub-groups' in the genus were well differ­

entiated and that they were unable to detect any intermediate organisms

in this genus. While considering these conclusions, the critical dis­

course on numerical taxonomy published by Pratt (1972) should be kept

in mind. According to this critique the fundamentals of numerical

taxonomy, namely' single character' and 'unit character' are inade­

quately and sketchily drawn assumptions.

Another technique which is helpful in determining the groups or

sub-groups within a genus is the base composition of DNA, a technique

now frequently applied to the taxonomy of a wide range of microorgani-

sms. The technique is useful in supplementing the results of other

taxonomical studies ( viz., numerical taxonomy, FAME profile) and has

helped in a number of cases to arrange the groups into sub-groups.

An example which can be cited is that of confirmation of the subdiv­

ision of the Staphyloaoaaus - Miaroaoaaus group into the two

distinct groups already indicated by the numerical taxonomical

results (Silvestri & Hill, 1965).

The DNA base composition of Thiobaailli was studied by

Jackson et al. (1968) and their results are summarised in Table 1.6.

Their analyses support to a certain extent the classification of

Thiobaailli into distinct species, as proposed by Hutchinson et al.

(1965-1967). A recent study, however, indicated some anomalies with

respect to DNA base composition of Thiobaaillus ferrooxida:ns when

grown on different substrates ( Guay et al., 1975; 1976).

24

Another criterion recently described for the classification of

Thiol>acilli is the fatty acid composition of the cells. These are

extracted, converted to methylesters, and their occurrence and relative

abundance determined by gas-liquid chromatography (Agate & Vishniac,

1973-a). It is possible to divide the Thiobacilli into three distinct

types, differing in their fatty acid content (FAME profiles). It has

been claimed by the authors that the method provides a rapid differen­

tiation between such closely related species as Thiobacillus neapoli­

tcmus and Thiobacillus thioparus. The three FAME-profiles are prese­

nted in Table 1.6, Table 1.7 summarises the composition of these FAME

profiles. The FAME-profile obtained by Levin(l971) from a Thiobacillus

thiooxidans strain contained a predominance of c19 cyclopropanic acid

and was different from the FAME-profiles obtained from other strains

of Thiol>acillus thiooxidans. There are profound changes in the

compositions of phospholipids during various phases of growth (Agate

& Vishniac, 1969; 1973) and therefore the accuracy of FAME-profiles

depends upon the conditions of growth and age of the cultures. Since

in a microbiological system it is difficult to reproduce similar cond­

itions the method should be used with some caution. Advantages and

disadvantages of chemotaxonomical methods employing FAME-profiles in

bacterial taxonomy have been recently discussed in an exhaustive

review by Lechevalier (1977).

The current status of the classification of Thiobacilli is

summarised in Table 1.8. As can be seen from this table, numerical

taxonomy recognizes eight independent species/groups existing distinc-

tly in the genus Thiobacillus. The results of chemotaxonomical

methods (viz., DNA base composition, FAME-profiles) tend to agree

closely with one another and in general with the numerical taxonomical

classification. The results obtained from the study of five Thiobaci­

Zli have indicated that they can be grouped into three distinct groups.

TABLE 1.7

TYPES OF FATTY ACID METHYL ESTER (FAME) PROFILES

FOUND IN GENUS THIOBACILLUS#

25

* TOTAL EXTRACTED FATTY ACIDS IN: FATTY ACID TYPE I TYPE II TYPE

% % %

c6 7

Cs 6 4 6

Cg 7

C10 6 3

Cu 2 10

C12 4 14

C13 7

C14: 1 30 2 16

C14 12 3 21

C15 2 32

C16:1 15 2

C16 9 7 4

C17: 1 15

C17 20

III

# Taken from Bergey's Manual of Determinative Bacteriology,

8th Edition, 1974.

* Number indicates the length of carbon chain; number to

right of colon indicates number of double bonds.

NO. I

1-

2-

3-

4-

5-

6-

7-

8-

9-

10-

11-

TABLE 1.8

GROUPS OR SPECIES OF THIOBACILLI RECOGNISED BY

THREE INDEPENDENT METHODS OF TAXONOMY

NUMERICAL TAXONOMY I NUCLEOTIDE COMPOSITION LIPID COMPOSITION

(GC-RATIO) (FAME-PROFILE)

T. thioparus T. thioparus }

T. thiopa.rus} Type

T. noveUus T. noveUus T. noveHus 11 }

ND T. acidophilus 62-68% ND }

ND T. :rubeUus NO }

ND T. del,icatus tl)

T. neapo li tanus T. neapo li tanus 6 % }5 -57

T. neapo 7,i tan.us }Type

T. ferTOo:xidans T. f erroozidans T. f eZTOcxcidans I

T. thioo:r:idans T. thioo:r:idans 50-52% T. thioo:z:idans Type 111

T. denit'l'ificans ND N)

T. intermedius ND N)

T. t'l'auweinii. N) JI)

ND= Not described.

N O'I

27

All the three methods agree in the fact that ThiobaeiZZus

thiopa:r'us, ThiobaciUus neapoZitanus, ThiobaciUus thiooxidans and

Thio'ba,ciZZus ferrooxidans (all of which are grouped together in Fig.

1.2) are distinctly different species. Thus, al though these biochemical

techniques are relatively new to taxonomy, they have helped already

towards the shaping of a useful basis. for the classification of the

ThiobaciUi.

1.2.3.3 SUBMICROSCOPIC ORGANIZATION OF THIOBACILLI

Thio'ba,ciZZi are capable of thriving on inorganic substances

such as elemental sulphur and ferrous ions. Some members of the genus

are also known to withstand very high hydrogen ion concentrations. On

account of their peculiar substrates and sharply defined environmental

requirements, there has been a wide interest in the study of their

ultrastructure.

Umbreit & Anderson (1942) were among the first workers to study

the structure of ThiobaciZZus thiooxidans with an electron micro­

scope. These workers could not confirm the 'dipolar' appearance of

the cell reported earlier by Umbreit et al. (1942) after examining

these cells mder optical microscope. They also could not recognize

a loose cell envelope, seen on some of their figures (viz., Figs. 4

& 6), which they regarded as either bacterial cell wall or an artifact

developed during the processing. Knaysi (1943) however, in an excell­

ent paper, was the first to point out the existence of slime-coat or

loose cell-envelope around the cells of ThiobaciZZus thiooxidans. He

also considered the 'halo structures' in the electron micrographs of

ThiobaciZZus thiooxidans reported by Umbreit & Anderson to be the cell

envelope.

The fine structure of ThiobaciZZus ferrooxidans was most

28

probably studied and reported for the first time by Lundgren and his

colleagues in 1964 (Lundgren et al., 1964). This group undertook the

investigations in the hope of demonstrating a correlation between

structure and function and of finding some unique and novel structural

components which would explain the organisms' special physiological and

biochemical properties. However, they could not find anything unusual

about the structure of ThiobaciZZus ferrooxidans and described its

cellular organization as being comparable to that of ordinary Gram­

negative bacteria, with a loose envelope which consisted of at least

two electron-dense areas, enclosing a non-electron-dense area in bet­

ween them. Inside the cell envelope a unit membrane structure contained 0

the cytoplasmic material. Many particles of 70 - 150 A in size,

which were believed to be polyribosomes, were also reported.

In 1965, a working model for the oxidation of ferrous to ferric

salts by ThiobaciZZus ferrooxidans was proposed by Dugan & Lundgren

(1965). The model was based on assumption that a complex is formed at

the cell surface of the bacterium. The concept of involvement of the

cell envelope in the oxidation process prompted Remsen & Lundgren (1966)

to undertake rigorous investigations of the cell envelope structure;

they employed both freeze-etching and chemical-fixation techniques

during their studies. They found that the structure of chemically­

fixed cells of FerrobaciZZi was similar to that of other Gram-negat­

ive bacteria ( e.g., Escherichia coZi, Pseudomonas sp. ) as well as

to that of ThiobaciZZus thiooxidans as described by Mahoney & Edwards

(1966). They also demonstrated that the cell envelope of chemically­

fixed cells comprised five separate layers, distinguishable by

their location and electron density. On the other hand, freeze-etched

cells revealed only three layers, measuring in thickness approximat­o

ely 100 A each, and identifiable as an outer lipoprotein-lipopoly­

saccharide layer, a middle layer (containing globular protein

29

attached to fibrillar mucopeptide) and an innermost layer, the cyto-0

plasmic membrane, which was covered with particles (100 - 120 A

diameter) considered to be enzyme particles.

The cytomembranes observed in ultrasections of a marine Thiobaai­

ZZus were less numerous than those found in other autotrophic micro-

organisms, like Nitrosomonas, Nitroaystis oaeanus and Nitrobaater

(Tilton et al., 1967-a). However, it was presumed that their presence

supported Murray's hypothesis, based on the ultrastructure of some

autotrophic bacteria, that a relationship exists between the degree of

energetic processes and the extent and complexitiesof cytomembranes

(Murray, 1963; Murray & Watson, 1965).

Recent studies demonstrate that the ultrastructure of a marine

ThiobaaiZZus, when examined by thin-sectioning and freeze-etching

techniques, is similar to that of terrestrial species of the Thiobaai­

ZZus genus (Murphy et al., 1974).

The ultrastructure of the facultatively autotrophic members of

the genus ThiobaaiZZus ( ThiobaaiZZus noveZZus) was first studied

in Czechoslovakia by Kocur et al. (1968), who were concerned with

finding any structural differences that might exist between the

facultative and obligate autotrophs and with explaining the diffe­

rences in terms of variations in their physiology. These workers

reported that ThiobaaiZZus noveZZus resembles the obligate auto-

troph, ThiobaaiZZus thiooxidans, in each detail of its structure, with

the exception of the presence of vacuoles in ThiobaaiZZus noveZZus.

However, they considered the vacuoles to be artifacts resulting from

plasmolysis of cells during preparations of the samples; this was

later confirmed by Van Caeseele & Lees (1969), who could find no

vacuoles in their samples. The autotrophically-grown cells of Thio­

baaiZZus noveZZus were folfild to be devoid of the electron-dense layer

present in the cell envelope of the heterotrophically-grown cells; the

30

latter contained large inclusions of polysaccharides as well (Van

Caeseele & Lees, 1969).

A comparative study of the ultrastructure of Thiobacilli was

made by Shively et al. (1970) in which they attempted to pin-point

the characteristic structural features peculiar to each species.

Although the structure of the cell envelope was similar to that found

in most Gram-negative bacteria, obvious differences were noted in the

middle layers of the cell envelopes of the seven species studied: in

Thiobacillus thiooxidans and Thiobacillus A2 it was very prominent;

Thiobacillus thiopaPus & Thiobacillus intermedius contained less

conspicuous middle layers; in Thiobacillus noveiius this structure

was either absent or very diffuse. These workers also reported the

presence of lamellar bodies, resembling those present in photosynthetic

bacteria, in a few cells of Thiobacillus thiopaPUs. However, this

finding remains unconfirmed. Paracrystalline bodies of unknown

function were demonstrated in a few cells of Thiobacillus intermedius.

Polyhedral inclusions were seen in four of the species, namely,

ThiobaciUus thiopaPUs, ThiobaciUus neapolitanus, ThiobaciUus

intermedius and Thiobacillus thiooxidans.

Thus it can be seen that the studies of the ultrastructures of

Thiobacilli so far made have revealed no specific organelle possessed

by them, not even the presence of the cytomembranes which were thought

to be possessed by all autotrophic bacteria. In fact, in structural

architecture, they most closely resemble the heterotrophic Gram-negative

bacteria.

THIOBACILLI REVISITED

The findings that Thiobacilli are closely related to Gram-negative

heterotrophic bacteria in their submicroscopic organization could not

be agreed upon by Russian workers and some other groups. Although

31

there was much evidence accumulated from previous studies to support

the above conclusion, the very llllique physiology of ThiobaciZZi such

as tolerance to very low pH and very high metal ion concentrations

caused research workers to presume that these microorganisms possess

an ultrastructure distinct from other Gram-negative bacteria. Among

these groups, the most prominent are the Russian biologists who have

studied the ultrastructures of ThiobaciZZi once again and published a

series of very interesting research papers since 1971. In the

following the ultrastructure of these ThiobaciZZi as seen by these

investigators is briefly discussed.

ThiobaciZZus ferrooxidans was the first bacterium to be

re-examined by Avakyan & Karavaiko (1970). The bacterial cells were

fixed chemically for electron microscopy. They noted no significant

difference in cell wall structure between their strains and those

previously described by Remsen & Lundgren (1966). However, they

compared the cell wall structures of a number of bacteria and concluded

in contrast with the conclusions of the other workers that, although

the structure of this organelle is similar in ThiobaciZZus thiooxidans

and ThiobaciZZus ferrooxidans, it is definitely different from those

of certain other Gram-negative bacteria but is closely related to the

cell wall of Escherichia coZi, Nitrosomonas spp & Nitrobacter spp.

An LU1confirrned observation by the authors was the presence of

intracellular membrane structures, which they considered similar to

the lamellae of photosynthetic bacteria or the membrane structure of

nitric bacteria. According to them, the data indicated that the

intracellular structure of ThiobaciZZus ferrooxidans is more

sophisticated than it is in the heterotrophic Gram-negative bacteria

and a number of its features, especially the presence of highly deve­

loped membrane structures, indicated that it more closely related to

chernotrophs like nitric bacteria.

32

The ultrastructure of ThiobaciZZus thiooxidans indicated that

the cell wall was similar to that of ThiobaciZZus feYTooxidans

(Karavaiko & Avakyan, 1971). The cells were fomd to contain intra­

cellular membranes of the mesosome type which occur as complex invagi­

nations of the cytoplasmic layers and which are covered with electron 0

dense particles of 40 - 70A diameter, possibly enzyme complexes. A

very important observation was that there were no lamella-like

membrane structures as detected in ThiobaciZZus thiooxidans previously

(Avakyan & Karavaiko, 1970).

Submicroscopic examination of ThiobaciZZus neapoZitanus revealed

that the cell wall was different from that of ThiobaciZZus thiooxidans

and ThiobaciZZus ferrooxidans (Karavaiko & Avakyan, 1971; Avakyan &

Karavaiko, 1970) as it lacked the middle dense layer and

was similar to that of many Gram-negative bacteria (Pivovarova &

Karavaiko, 1973). The cells were found to contain numerous membranous

structures representing loop-like invaginations of the cytoplasmic

membrane and were supposed to be concerned with the oxidation of

sulphur. The full mechanism of this oxidation is, however, still not

known. It is suggested by these workers that these intracellular

membranous structures are responsible for oxidation as well as

excretion of sulphur granules.

Unlike ThiobaciZZus neapoZitanus (Pivovarova & Karavaiko, 1973),

ThiobaciZZus thiocyanoxidans cell wall is complex and was found to

resemble in its organization that of ThiobaciZZus ferrooxidans and

ThiobaciZZus thiooxidans (Pivovarova & Karavaiko, 1973-a). The

ultrastructure of this ThiobaciZZus exhibited an abundance of

intracellular membranous structures, which differ in their shape and

structure from those of ThiobaciZZus neapoZitanus (Pivovarova &

Karavaiko, 1973). The presence of a middle layer in the cell wall and

the pecularity of the membrane structure are attributed to the sulphur

33

deposition. A general conclusion is that, in combination with the

membrane structures, the cell wall represents a unique mechanism for

the secretion of the colloidal sulphur produced within the cell.

However, it must be pointed out at this juncture that nature and

precise information about the mechanisms operating are still obscure.

Recently, the ultrastructure of ThiobaciZZus thiooxidans,

ThiobaciZZus thioparus, ThiobaciZZus interrnedius, ThiobaciZZus noveZ­

Zus and ThiobaciZZus neapoZitanus has been studied again, employing

both chemical fixation and freeze-etching techniques (Holt et al.,

1974) with the hope of obtaining some information about the

relationship between the structural organization and physiological

function of these microorganisms. These workers have confirmed that

the architecture of ThiobaciZZi is typical of Gram-negative bacteria.

They also observed certain characteristic features of individual

ThiobaciZZus species, like the paracrystalline inclusions in

ThiobaciZZus intermedius and the lamellar body in ThiobaciZZus

thioparus:, which were reported earlier by Shively et al. (1970).

Ultrastructural organization of freeze-etched cells revealed

the multilayered cell envelope as a closely juxtaposed structure, each

layer having a peculiar texture. An outer layer of the cell envelope

could bedemonstratedin only a few species, for example, ThiobaciZZus

neapoZitanus and ThiobaciZZus thioparus, but was not present in each

species. The middle layer contained particles of about 80 A in

size, except in ThiobaciZZus ferrooxidans (Remsen & Lundgren, 1966)

where this layer was reported to be smooth textured. One important

observation made by these investigators was the presence of 120 A

diameter, membrane-botmd particle studs on the plasma membrane; these

studs possessed some degree of surface differentiation, for example

'hole or channel' configurations. It has been suggested by this

group that these structures might be the connections between the outer

34

envelope and the inner cell by providing the channels or holes between

these bolllldaries.

This study, like many others, has confirmed the presence of 0

various cytoplasmic inclusions, ranging from 2150 A diameter granules 0

to 1300 A diameter membrane bolllld polyhederal inclusions which have

been recently recognized in Thiobacillus neapolitanus as the structures

containing the ribulose diphosphate carboxylase, and which have been

named carboxysomes (Shively et al., 1973). Each carboxysome is bolllld 0

by a monolayer membrane approximately 35 A thick with an array of 0

100 A particles of ribulose-diphosphate carboxylase with a central hole

or depression (Shively et al., 1973-a). Similar polyhedral bodies have

also been reported in Thiobacillus intermedius(Purohit et al., 1976-a).

Dense bodies are generally seen and reported in all Thiobacilli.

Their fllllction and nature still remain obscure. Recently, Shively,

in a review has summarised the occurrence and functions of these

inclusions in the Thiobacilli (Shively, 1974).

There is now sufficient evidence to conclude that the utilisat­

ion of inorganic sulphur compollllds by Thiobacilli is highly unlikely

to be related to the development of 'specialised' structures or

extensive membrane systems such as are known to occur in prokaryotic

phototrophs and ammonia- and methane-oxidising bacteria. As far as

the contradictory evidence for the presence or absence of cytomem-

branes in Thiobacilli is concerned this may be due to differences in

the strains which each of the workers has studied, or it may be the

result of the artifacts developed in the preparation of samples. However,

the idea of cytomembranes in Thiobacilli gets very little support from

the currently existing experimentar evidence and no evidence for stru­

ctural involvement is available regarding the oxidation of sulphur and

sulphur compollllds. The puzzles relating to how Thiobacilli survive at

low pH, while maintaining the internal pH at physiological level, the

35

mode of attack on solid substrate, the transportation mechanisms

involved, and many other characteristics of the genus are enough to

keep cytologists busy for another decade.

36

1.3 BIODEGRADATION OF MINERALS BY MICROORGANISMS

A world wide depletion of mineral resources within the coming

decades has been frequently forcast by reliable authorities and it

is generally believed that the supply of ores of copper, mercury, zinc

and of many other vitally important metals for present day technology

is becoming more and more uncertain. According to one estimate, the

known workable ore bodies of copper, cobalt, nickel, vanadium and

lead may be depleted by the end of the year 2000 (Brooks, 1972; Harts,

1973). In order to maintain the present rate of inflow of various

metals for technological developments, future technology for metal­

winning operations will possibly have to consider the following

sources or modifications in already existing operations:-

1- Development of efficient recycling metrods for metals

such as tin, lead, copper, zinc, etc.

2- Novel methods for recovering metals from the rocks in

which they exist at average abundance levels. Appli­

cations of PNE (Peaceful Nuclear Energy Explosion) for

crushing the secondary ores under ground and leaching

them in situ had been suggested (Nordyke, 1971), but

the study is in its initial stages.

3- Metal Extraction From Sea.

Sea water can be a potential source of metals. This

is apparent from the following estimates of the total

amounts of certain industrial metals which are conta­

ined in the oceans (Cloud, 1968).

( Calculations based on· total volume of

Sea= 3.3. 108 cubic miles.)

METALS MILLION TONS

Copper 5,000

Manganese 3,000

Mercury 50

Silver 500

Tin 5,000

Tungsten 150

Uranium 5,000

Zinc 15,000

Perhaps it is due to this huge magnitude of metals that

Warren (1973) has described the oceans as the " Mining

Frontiers of Future". The major difficulty in extract­

ing metals from sea water is their low concentrations ;

this means that the very large volumes required to be

treated to obtain sufficient amounts of metals would

affect the economics adversely. However, development &

isolation of microbial strains which are resistant to

and capable of accumulating heavy metals can be helpful

in concentrating these metals (Zajic & Chiu, 1972).

4- Development of substitutes for metals.

37

Microorganisms play an important part in the formation as well

as degradation of minerals in this planet (Trudinger, 1971). They

can be used for enhancing metal production from the various sources

and operations described under headings 1 to 3 above. So far, however,

they have not been employed in the synthesis of metallic substitutes.

Both heterotrophic and autotrophic microorganisms are known to

degrade minerals and rocks. In the following sections, the action of

micro bes in the degradation of minerals is described.

38

1.3.1 BIODEGRADATION OF MINERALS BY HETEROTROPHIC MICROORGANISMS

There is ample evidence that heterotrophic microorganisms take

a very active part in weathering and degradation of rocks to form soil

in nature. During this process mineral components of rocks are solu­

bilised. In a series of studies primarily concerned with the mechanism

of weathering, a number of workers have clearly demonstrated the abil­

ity of soil bacteria, actinomycetes and fungi to decompose a number

of natural and synthetic silicate minerals (Duff & Webley, 1959; Webley

et al., 1960, 1963; Duff et al., 1963; Henderson & Duff, 1963). The

most active bacterial isolate was found to produce 2-keto-gluconic

acid, whilst citric and oxalic acids were the products of the fungal

action. Fungi were fotmd to be most effective in degrading silicate

minerals; in some cases dissolution was nearly complete (calcium

silicate 94%, magnesium silicate 74%, zinc silicate 96%). Bacteria and

actinomycetes have been found to give an average dissolution of

calcium silicate within the limits of 83 - 87 %. There is sufficient

evidence to conclude that Pseudomonas cultures can destroy the

crystalline structure of silicates like olivine [(Mg.Fe) 2Si04 ] or

wollastonite [Ca 3 (Si0 3) 2 ]. The possible mechanism has been described

as the acidic action of 2-keto-gluconic acid.

Decomposition of tri-calcium phosphate and rock phosphate incre­

ase the fertility of agricultural land. Therefore, microorganisms

capable of solubilising rock phosphates are of special interest and

have been isolated from these rocks by a number of workers (Bardiya

& Gaur, 1974). Heterotrophic bacteria isolated from gold mines have

been found to dissolve gold (Pares~ 1964, 1964-a, b). Lyalikova and

Mokeicheva (1969) found that a culture isolated from the Zodsk gold

deposits, similar to Bacillus alvei, was able to dissolve 500 - 600

µg/t of gold in three weeks; consequently such microorganisms have

39

been presumed to play a vital role in the migration of gold deposits

in nature (Korobushkina et al., 1974).

Two mechanisms appear to be involved in the biodegradation of

minerals by these heterotrophic microflora:-

1- Acidification or alkalisation, and

2- Chelation of metals by organic metabolites.

Acidification or alkalisation.

Acidification may result either from secretion of acidic

metabolites or by a selective utilisation of alkaline components such

as ammonium ions; organic acids such as lactic, butyric, succinic and

gluconic are the usual end products of the carbohydrate metabolism in

these microorganisms. Alternatively, in certain cases the production

of ammonium ions by the utilisation of proteineous material increa-

ses the pH of the environment up to pH 10; at this pH certain minerals,

especially those of copper, are easily attacked and the process can be

compared to chemical leaching by ammonia (Le Roux, 1966).

Silverman & Munoz (1970) in a study involving Penicillum simpli­

cissimum conclusively showed that the solubilising agent produced

during the degradation of various minerals (basalt, granite, grano­

diorite, rhyotite, andesite, peridotites & quartzite) was citric acid.

Intracellular enzymes did not appear to be involved in the degradation,

since heated (80 °C, for 1 hour) and unheated spent acid media were

equally effective in solubilising basalt. That acid was involved in

this process was indicated by the change in the infrared spectra of

the rocks during the experiments; this was assumed to be due to the

disruption of the crystalline structure. The oxalic acid, produced by

metabolism of glucose by chemoorganotrophic microorganisms has been

considered responsible for the leaching of uranium ores in situ

(Magne et al., 1973). Agbim & Doxtader (1975) have shown that

40

solubilisation of synthetic zinc silicate and the minerals willemite

[Zn2(SiO4) ] and hemimorphite [Zn4Si2O7(OH)2 H2O] was facilitated

by soil inoculated glucose-mineral salt medium. Volatile and non­

volatile organic acid fractions of the culture liquids were folllld to

solubilise substantial amounts of these minerals. These studies are

in agreement with the previous findings of Silverman & Munoz (1970)

claiming that heterotrophic solubilisation is a non-enzymic phenomenon.

In another investigation, mineralysis of silica quartz has been

reported to be dependent upon growth rate of Bacillus caldolytics

(Lauwers & Heinen, 1974).

Chelation of metal ions by metabolites.

Certain organic metabolites of bacteria and fungi have the

ability to chelate important divalent metallic ions such as iron,

copper, zinc, magnesium, calcium and manganese. These metabolites may

function as metal-transporting agents in cellular metabolism. Amino

acids (glycine, histidine, glutamic acid), phenols (pyrocatechol,

salicylic acid), phosphates (ATP, ADP), organic acids (citric, 2-keto­

gluconic) and polyols (mannitol) are the usual bacterial metabolites

which have been found to have a very strong affinity for metabolically

important divalent metal ions (Bourne et al., 1959). During the disso­

lution of gold by Bacillus megaterium and Bacillus mesentericus niger,

Korobushkina et al. (1974) demonstrated the significant role played by

amino acids by employing various mutants of above strains.

Pyrocatechol, which isrelated to compoW1ds occurring in hurnic

substances, chelates iron to form the following complex and thus

accomplishes solubilisation of iron for transportation in soils.

41

FERRIOXAMINE B

ENTEROCHEL IN

Chemical structures of iron-transporting

substances produced by bacteria.

[ Doetsch & Cook, 1973]

42

Phenolic acids (2,3-dihydrobenzoic acid, 2,3-dihydroxybenzoyl

glycine, 2,3-dihydroxybenzoyl serine, salicylic acid, 6-methyl­

salicylic acid) are secreted by a number of bacteria and these acids

chelate iron in similar manner. These acids have been shown as

essential for growth of a mutant of Esaherichia coZi which was unable

to synthesise 2,3-dihydroxybenzoyl serine in the presence of iron.

A cyclic trimer of 2,3-dihydroxybenzoyl serine, known as"enterochelin",

produced by Escherichia coZi, has been regarded as the major factor

in iron transport in the coliform bacteria. Enterochelin chelates

iron as a hexadentate ligand through the phenolic hydroxyls.

Iron transport into bacterial cells can also be accomplished by

the so-called siderarnine groups of compounds which are complex hydro­

xamic acid derivatives with a high affinity for iron, and are

recognized as growth factors for these organisms. They include terre­

gene factors ( growth factors for Arthrobacter terregens ), coprogens

( growth factors for PhiZoboZus and also produced by Sarcina Zutea),

mycobactins ( growth factors for Mycobacterium pamtuber-euZosis) and

various ferrioxamines produced by Streptomyces species ( Doetsch &

Cook, 1973).

Arrieta & Grez (1971) isolated_ 10 soil fungi which solubilised

iron from minerals. They concluded that complex formation was the

most probable mechanism in this process, for even a very strong

cation-exchange resin was not able to extract the complexed iron from

43

the culture solution. Wenberg et al. (1969) have reported laboratory

scale leaching of copper minerals by PeniciUium species, under

neutral or slightly alkaline conditions. They too suggested a chelat­

ion mechanism for solubilisation. Certain marine microorganisms

have been known to solubilise large amounts of iron from limonite and

geothite. There are several indications that iron oxide in the minerals

is reduced enzymatically to its soluble form (De Castro & Ehrlich, 1970;

Ottow & Vonklopotek, 1969).

The understanding of mineral leaching by heterotrophs is still in

its infancy and vital information regarding kinetics, environmental

effects and mechanisms of dissolution is scarce.

1.3.2 BIODEGRADATION OF MINERALS BY THIOBACILLI

The first report concerning the microbial degradation of sulp-

hide minerals was published in the first quarter of the ninteenth

century by Rudolfs & Helbronner (1922), who put forward the idea of

employing bacterial cultures for recovering metals from low grade,

sulphide-bearing ores and wastes (Rudolfs, 1922; Rudolfs & Helbronner,

1922). The idea was not pursued again until 1947, when Colmer and

Hinkle succeeded in isolating and describing a chemoautotrophic,

acidophilic, iron-oxidising bacterium from acid mine drainage; these

workers demonstrated that these bacterial cultures were associated

with, and actually played a fundamental role in, sulphide degradation

(Colmer & Hinkle, 1947). Similar microorganisms were isolated by

Razzell & Trussell (1963-a) from Rio Tinto (Spain) leach water. These

authors confirmed the biological nature of the process responsible for

the natural leaching of chalcopyrites reported by Taylor & Whelan (1943).

Since the identification of ThiobaciZZus ferrooxidans by

Temple and Colmer in 1951, the organism has been found to be involved

44

in leaching operations in Utah (Bryner et al., 1954; Bryner & Anderson,

1957), British Columbia (Razzell & Trussell, 1963), Arizona (Razzell &

Trussell, 1963; Corrick & Sutton, 1961), England (Ashmead, 1955),

Russia (Ivanov et al., 1961) the Congo (De Cuyper, 1964), Australia

(Andersen & Allman, 1968; Agate, 1973), Japan (Imai, 1971) and in many

other countries of the world.

Vishniac & Santer (1957) summarised the physiological and bio­

chemical characteristics of the genus ThiobaciZZus as they were llllder­

stood at that date. Later, the role of microorganisms in formation

and degradation of minerals was reviewed by Silverman & Ehrlich in

1964. More recently, an introductory review pertaining to microbial

processes capable of attacking and degrading minerals was published

by Le Roux (1971), and Fletcher (1971) reviewed briefly the bacterial

leaching of low-grade ores. Since then Tuovinen & Kelly have published

a number of excellent review articles dealing with the biology and

mineral oxidative activities of the genus ThiolxiciZZus (Tuovinen, 1972;

Tuovinen & Kelly, 1972; 1974-d). These authors have painstakingly

collected and evaluated the huge amount of work published on the

subject. Exhaustive as they are, these reviews all lack a detailed

account of leaching mechanisms. This gap has been filled by a

detailed account of the known oxidative mechanisms involved in mineral

degradation by bacteria (Ralph, 1978).

The microbiological leaching of metal sulphides is now a common

practice throughout the world for recovering copper and uranium from

low grade ores. Torma (1977) has critically reviewed the biogenic

role of bacteria in hydrometallurgical operations.

45

1.3.3 FACTORS AFFECTING THE MICROBIAL LEACHING PROCESS

Microbial leaching of sulphide minerals by ThiobaciZZus species

can be considered as a particular case of the general interaction of

microorganisms with solid, insoluble substrates; extension of some

aspects of the general rationale of such transformations [ as developed

by Nickerson (1969) while studying the breakdown of natural organic

polymers] to mineral leaching studies might be advantageous. The two

important components involved in microbial leaching are the solid

substrates and the microbial agencies. The efficiency of the process

will be dependent upon the degree of associations, contacts or inter­

actions between these two components and will be influenced by the

nature of the substrate and microbial system present.

1.3.3.1 NATURE OF THE SUBSTRATE

Metal Distribution.

In an ore-body, sulphide minerals are generally associated with

other materials, such as quartz, silicates, carbonates and oxides.

The distribution pattern within the ore-body is one of the major

parameters determining procedures most suitable for extraction of the

metal from that ore-body. Generally, high grade ores are processed

by pyrometallurgical operations, whereas low grade ores are dumped at

a suitable place near the mine, and may subsequently be leached.

Metal leaching rates are enhanced by acidophilic members of the

ThiobaciZZus genus, namely, ThiobaciZZus ferrooxidans and ThiobaciZZus

thiooxidans, which proliferate actively in low pH conditions. When the

metal sulphide is associated with high proportions of alkaline matrix,

achievement of low pH becomes impossible due to'high consumption of

acid for neutralization of such alkaline materials. This may

46

effectively slow down or completely inhibit the leaching process.

Oxidative degradation of mineral sulphide depends upon the

access to it of water, various dissolved ions, oxygen and other rea­

gents; and the breakdown may be accelerated by the closer proximity of

bacteria. Access of these various components of the degradative sys­

tem will depend upon a number of factors including relative distri­

bution of mineral sulphides and matrix ( for example in veins, as

massive aggregates, finely disseminated etc.) and upon the diffusion

characteristics of the matrix in respect to the various reacting

components.

Properties of Component Minerals.

About two hundred and fifty metallic sulphides have physical and

chemical characteristics which allow them to exist as individual mineral

species in geological environments (Ralph, 1978). Each mineral sulp-

hide hasuniqµe characteristics which affect its behaviour as a

substrate in bacterial leaching systems. For example copper exists in

a number of minerals containing a sulphidic moiety (chalcocite Cu2S;

chalcopyrite CuFeS2 ; covellite CuS; enargite Cu3AsS4 ; bornite Cu5FeS4;

digenite Cu9S5). The degree of leachability of these minerals with

bacteria varies to a considerable extent and the mineralogy of copper

sulphide ores is well known to affect its ease of leaching, Razzell

& Trussell (1963-a) found chalcopyrite to be relatively resistant to

chemical leaching but readily oxidisable by bacterial cultures.

Covellite was auto-oxidised less readily than chalcocite, and conversely

was reported to be more readily pron~ to biological oxidation (Ehrlich

& Fox, 1967). Copper minerals containing iron in association ( i.e.,

bornite Cu 5FeS4 and chalcopyrite CuFeS2 ) are reported to be almost

completely oxidised by bacteria (Duncan and Trussell, 1964). In

contrast, enargite, which contains arsenic, has been found to be only

47

11 - 20 % oxidisable by these bacteria (Hao et al., 1972). The

difference in leaching behaviour can be attributed to the modification

of structure of these minerals brought about by different compositions.

Compositional variations may be accompanied by changes in the crysta­

lline form ( e. g. pyrrhotites) offering entirely different surfaces for

interaction with bacteria. However, these variations have been

little studied in relation to their possible effects on the rate of

biodegradation.

The difference in leaching behaviour exhibited by different

sulphide minerals is probably due to a complex of factors which include

the stoichiometry of the mineral, its crystallographic form, its degree

of substitution by other metallic ions, the basic chemistry of the

metal itself and perhaps more subtle characteristics of an electroche­

mical kind. The effect of such factors may well be overlaid by the

grosser effect of experimental parameters such as pH, Eh, temperature,

particle size and so on.

1.3.3.2 EFFECT OF PHYSICO-CHEMICAL FACTORS

Particle Size.

Particle size influences the leaching rate in a number of ways.

The smaller the size of the mineral particles the greater is the inter­

facial area between the solid and liquid phases and therefore the

higher the rate of transfer of materials and the greater the specific

area of interaction between microorganisms and substrate.-

Many investigators have reported an enhancement of bacterial

leaching rate on reduction of particle size of the mineral. For

example, Razzell & Trussell(l963) reported that doubling the surface

area of chalcopyrite (by reducing the particle size from a mean

diameter of 53µ to 44µ) doubled the rate of leaching. Duncan et al.

48

(1964), while demonstrating the effects of surfactants and shaking on

the leachability of chalcopyrite by Thiobacillus ferrooxidans, employed

mineral particles less than 44 µ in diameter; Ehrlich & Fox (1967)

observed an increase in the rate of oxidation of synthetic copper

sulphide when particles less than 62 µ instead of 177-125 µ were used.

MoS2 was fotmd to be oxidised bacteriologically at a faster rate in

the airlift column when particles smaller than 250 µ, rather than 600µ,

were employed. The highest rates of bacterial oxidation of pyrites

were achieved in airlift columns by using particles less than 44 µ

rather than larger particles (Malouf & Prater, 1961). About six times

more copper was microbiologically recovered from chalcopyrite using a

finely grotmd ore of less than 44 µ diameter (Razzell & Trussell, 1963-a).

A similar enhancement in the oxidation of pyrites by using particles

of less than 44 µ in a Warburg respirometer was reported by Silverman

et al. (1961). The effect of particle size on the extraction of copper

has also been reported by Bruynesteyn & Duncan (1971). Since reduction

of particle size essentially results in increasing the surface area

of substrate, Torma et al. (1970 & 1972) studied the effect of initial

specific surface area of zinc sulphide concentrates on the rate of

metal release by Thiobacillus ferrooxidans ( also see Torma & Subra­

manian, 1974). These authors were able to demonstrate that at 16%

pulp density zinc extraction rate was increased significantly by

increasing the specific surface area from 0.5 m2 /g to 1.37 m2/g,

a further increase in the specific surface area by using particles

smaller than 40 µ produced no further dramatic changes in the rate of

leaching (Torma & Legault, 1973; Torma et al., 1974; Torma & Guay,

1976). This rather surprising result, which is not matched in studies

on the chemical leaching of very finely divided chalcopyrite, may be

accotmted for by the formation of particle aggregates between the

finer particles and the microorganism (Torma & Gabra, 1976). Data

49

obtained by Torma et al. (1972) suggest that the rate of zinc extract­

ion at maximum surface area is limited by carbon dioxide.

A decrease in particle size, however, may not always result in

an enhancement of rate of metal release, especially in the case of

low-grade ores where the metallic compound (substrate) is sparsely

distributed in the host rock. Under such circumstances the properties

of the host rock itself may influence the leaching process and should

be considered carefully. Reduction of particle size in such cases may

not always expose new mineral surfaces. On the contrary, it can

increase host rock surfaces immensely with an adverse effect on the

leaching process. Attempts to simulate the natural gangue-mineral

situations, in laboratory conditions were made by 'diluting' pure

copper sulphide with quartz sand during airlift leaching experiments.

Results from such studies have indicated that the situation is a

complex one for the mineral-sand mixture did not behave as did the

low-grade ores (Bryner et al., 1954; Bryner & Anderson, 1957; Malouf

& Prater, 1961).

Interaction between size of particle and depth of column has been

successfully demonstrated by Audsley & Daborn (1963) while investiga­

ting leaching of uranium ores in columns. Their results showed that

the rate of uranium extraction depended upon the depth and particle

size of the ore-beds; air permeability of the column was concluded to

be a significant factor. The maximum rate of oxidation from Bica ore

was obtained in a 2 feet deep column, containing ore particles of mean

diameter 0.25 inches, which was irrigated at the rate of 25 ml per

kg of ore per week. In deep beds the rate of oxygen transfer rather

than the diffusion of solutes was shown to be rate limiting.

So far no data are available for the effect of microfined powders

of minerals on the rate of leaching by bacteria. It will be very

interesting to examine the rate of leaching of mineral particles when

50

they are reduced to size smaller than microorganism itself. It should

be noted that reduction of particle size is a very expensive process

on a large scale and is probably only of relevance for a bacterial

leaching process when such methods are used for the recovery of metals

from flotation residues and other bye-products of conventional meta­

llurgical techniques.

Surface Effects.

Bacterial activity in soil is reported to be enhanced by the

presence of solid surfaces (Stotsky & Rem, 1966) and an increase in the

oxidation rate of ferrous iron solution in the presence of an inert

solid surface was noticed by Malouf & Prater (1961). The explanation

put forward for this phenomenon was that nutrients accumulate in the

vicinity of bacteria due to surface adsorption. Recently Marshall

(1975) has reviewed various interactions and physico-chemical phenomena

observed during the contact of bacteria with solid particles.

The beneficial effects of surface active agents on the initial

rate of microbial leaching (Duncan et al., 1964) and isolation of

phosphatidylinositol, a surface active compound from the culture fluids

of ThiobaciZZus thiooxidans, by Schaeffer & Umbreit (1963) indicated

a possibility that wetting of surfaces by these compounds enabled the

bacteria to cover the mineral surfaces during the process of leaching.

However, data obtained in a recent investigation of leaching of chalco­

pyrite with ThiobaciZZus ferrooxidans in the presence of externally

added wetting agents (Tween 20, 40 & 60) are in disagreement with the

previous reports (Torma, 1976; Torma & Gabra, 1975, 1976). According

to these workers the wetting agent reduced the chalcopyrite oxidation

ability of ThiobaciZZus fer.rooxidans by reducing the surface tension

of the medium and thus affecting the oxygen mass transfer.

Earlier work presents some evidence of bacterial attachment to

51

crystals of elemental sulphur (Schaeffer et al., 1963). Adherence of

Thiobaaillus ferrooxidans to solid surfaces like pyrite and inert

surfaces like polyvinyl chloride has also been frequently reported

(Mehta & Le Roux,, 1974; Tributsch, 1976). Suggestion of the occurre­

nce of an organic complexing agent to bind the iron so that its redox

potential is decreased to a value enabling it to couple with the

electron-transport system of the cell during its oxidation (Blaylock

& Nason, 1963) implies that activity is concentrated on the solid

surface in a 'microenvironment' which is controlled by adsorption

properties as well as the capacity of the bacteria to modify their

microenvironment in such a way that energy transfer can occur most

efficiently.

Recent work has shown that the selective attachment of therrno­

philic microorganisrns of the genus Sulfolobus to molybdenite (MoS2 ),

pyrite (FeS2 ), or chalcopyrite (CuFeS2) is generally by adhesion

and not by pili (Berry & Murr, 1975; Murr & Berry, 1976). These

results emerged from the studies of a very important aspect of

mineral/bacterial interaction, i.e., the mode and place of attachment.

Further detailed investigation in this direction would no doubt add

much to our understanding of the fundamental mechanism involved in

bacterial leaching.

Temperature.

The effect of temperature on microbial leaching has been studied

extensively (Ehrlich & Fox, 1967; Leathen et al., 1956; Silverman &

Lundgren, 1959; Kinsel, 1960; Marchlewitz & Schwartz, 1961; Bryner et

al., 1966). Some of the early workers tried to differentiate between

the temperature optima for growth of the associated microorganisms and

for leaching processes. Leathen et al. (1956) had reported the

optimal temperature for growth of Thiobaaillus ferrooxidans as being

52

between 20 and 25°C, but according to Silverman & Lundgren (1959)

the optimal temperature for growth of ThiobaciZZus ferrooxidans was

28°C whilst the maximum rate of oxidation of ferrous ion was at 37°C.

However, more recently, workers have demonstrated that oxidative pro­

cesses carried out by ThiobaciZZi are growth-associated, having their

temperature limits between 30 and 40 °C with an optimum usually at 35 °C

(Landesman et al., 1966; MacDonald & Clark, 1970; Bryner et al., 1966;

Torma, 1971). Manometric experiments revealed somewhat higher tempe­

rature optima for mineral leaching by cell suspensions of ThiobaciZZus

ferrooxidans, and sulphur oxidation was found to be more sensitive than

iron oxidation to higher temperatures and was completely inhibited at

45°C (Beck, 1969). Torma et al. (1970) studied the effect of tempera­

ture on leaching of zinc sulphide concentrate by ThiobaciZZus ferro­

oxidans and found that microbial activity was limited at temperatures

greater than 45°C, and had an optimum at 35°C. Some recent studies

using fully monitored apparatus and continuous culture techniques

have demonstrated an optimum temperature for leaching by ThiobaciZZus

ferrooxidans between 30 and 35 °C (Gormely et al., 1975; Tomizuka

et al., 1976). (1972)

Tuovinen & Kelly are of the opinion that absolute limits of

temperatures cannot be precisely expressed for ThiobaciZZus ferrooxi­

dans, since, like other microorganisms, response . of these bacteria

to temperature depends upon their previous growth histories, Further,

in virtually all published work on the effect of physico-chemical

variables on leaching rates the assumption is made that there is no

interaction between the different variables. It might be deduced

from general considerations ( for example Brock's work on the environ­

mental limits) that at least some of the physico-chemical factors are

in fact highly interdependent and temperature and pH might well be two

of the most significant factors in this respect (Brock & Darland, 1970).

53

Under natural environments, active leaching dumps usually become

hot and temperatures upto 60-80°C have been reported, whereas in other

instances reports describing kindling the heap exist (Lyalikova, 1960).

High temperatures in the interior of the heap are mainly due to the

low thermal conductance of the rock mass and production of heat by

oxidative processes, both biological and abiological, whose sequence

is mainly unknown at present. Evidence that the initial steps in the

oxidative train are due to the participation of ThiobaciUus ferroox­

idans is presented by Lyalikova (1961) on the basis of direct observ­

ation of heating of the Kransogvardeisk deposits in the Middle Ural.

Effluent water temperatures well above the ambient temperature of 30 °C

from leaching heaps have been noted by Beck (1967) who showed that the

bacterial load of iron-oxidisers in the waters was highest ( 5.0 104

cells per ml) at 27°C and was dramatically reduced to 50 cells· per ml

in waters with a temperature of 39°C. This low population of bacteria

at higher temperature was also noticed during a microbial survey of

overburden heaps at Rum Jungle Mine (Khalid & Ralph, 1976); it indic­

ates abiological leaching at this temperature. Leaching studies by

Bryner et al. (1966) have shown a rapid rate of copper solubilisation ab­

ove. 45°C in a pure chemical system, but biological leaching was

maximum at 35°C with minimal activity at 45°C. Recently Corrans &

Scholtz (1976) have reported that chemical leaching of pentlandite is

highly temperature dependent.

Existence of thermophilic ThiobaciZZi has not so far been recor­

ded with certainty, although one unconfirmed and doubtful thermophile,

ThiobaciZZus thermophiZica Imchenestskii, has been reported by Egorova

& Deryugina (1963). Earlier attempts to isolate thermophilic Thiobaci­

ZZi from high temperature habitats were unsuccessful ( Marchlewitz &

Schwartz, 1961).

54

Isolation of therrnophilic ThiobaciZZus -like bacteria, capable of

oxidising sulphur and ferrous salts, has been carried out by Le Roux

et al. (1977) and Brierley & Lockwood (1977). These isolates have been

reported to oxidise metallic sulphides within a wide range of pH values

(Brierley & Le Roux, 1977). Caldwell et al. (1976) have reported the

isolation of a high-pH, facultatively anaerobic, facultatively autotr­

ophic therrnophile, Thermoth:rix thioparus, whose temperature optimum is

70 - 73°C. In recent years, therrnophilic sulphur oxidisers have been

isolated from hot spring waters (Brock et al., 1972; Brierley, 1966;

Brierley.&. Brierley, 1973; Williams & Hoare, 1972) and have been named

as SuZfoZobus species. Brierley & Murr (1973) have demonstrated their

capabilities for leaching sulphidic minerals and to facilitate the

solubilisation of molybdenum at 60°C (Brierley, 1974; 1977). It may

be possible that these species are associated with leaching operations

in the hot regions inside the heaps. However,there are no reports of

their isolation from a heap.

The lower limits of temperature for activity of ThiobaciZZi have

not been studied extensively. Their leaching capabilities appear to

be hindered drastically at lower temperatures; this is indicated by

unsuccessful heap leaching operations in the cold parts of the world,

for example in copper mines on the Kola Peninsula in the USSR which lie

north of the Arctic Circle with temperature as low as 4°C (Lyalikova,

1961); in low temperature Canadian mines higher recoveries of uranium

have been obtained by a process involving hot water.

Redox Potential a:nd pH.

Microorganisms in any environment are affected by the energy flow

processes mediated by electrons and hydrogen ions. Therefore, the

existence of microbial populations can be predicted in certain environ­

ments provided the oxidation-reduction characteristics of that

55

milieu are know. That occurrence of specific microflora is controlled

by the redox potential and the hydrogen ion concentration of the loca­

tion has been well doct.nnented (Baas-Becking et al., 1960).

Diagrams can be constructed, from the redox potentials (Eh) and

pH values, to represent the free energies of the chemical species

involved over a pH range; detailed methods for constructing such

diagrams are available (Garrels & Christ, 1965; Pourbaix, 1966). Eh-pH

diagrams, as these are referred to, indicate the precise conditions of

Eh and pH for effective energy transfer. These diagrams also contri­

bute significantly towards the development of hydrometallurgical

operations for metal sulphides. The metal sulphide, a common substrate

during microbial oxidation of sulphide minerals, forms one of the

components of the metal-sulphur-water system which is not a well under­

stood system thermodynamically. With the help of Eh-pH diagrams the

lack of thermodynamic data required for determining the mechanism of

oxidation of most of the sulphides has been overcome (Majima & Peters,

1969). Moss and Andersen (1968) extended the use of Eh-pH diagrams to

study the mechanism of heap-leaching at Rum Jungle.

Microbial activity itself is highly pH dependent. Although high

rates of leaching of metallic sulphides has been reported in aqueous

solutions at pH values between 2 and 4 with ThiobaciZZus ferrooxidans,

the maximum leaching rate of these sulphides was at pH 2.5 (Razzell,

1962; Razzell & Trussell, 1963-a). A large nt.nnber of investigations

tend to support the conclusion that the optimt.nn pH for microbial

leaching of sulphide minerals is 2.5, a value usually quoted as optimal

for their growth ( Gupta & Sant, 1970; Duncan & Teather, 1966; Moss &

Andersen, 1968).

There is evidence that ThiobaciZZus ferrooxidans and ThiobaciZZus

thiooxidans are capable of oxidising sulphur and fixing carbon dioxide

56

at pH values as high as 5.0 and the experiments of Rao and Berger

(1971) on ThiobaciZZus thiooxidans indicate the retention of oxidative

ability in respect of elemental sulphur and maintenance of via-

bility at pH as high as 7.0. This work noted that at these high

pH values there was an uncoupling of substrate oxidation and oxidative

phosphorylation and no growth. Although these microorganisms ( i.e.,

ThiobaciZZus ferrooxidans and ThiobaciZZus thiooxidans) were cap­

able of oxidising sulphur at pH values as high as 5.0, they seldom

oxidised metallic sulphides at pH values higher than 3.5 (Duncan et al.

1966; Rao & Berger, 1971). These studies have relied on measurement

of solubilised metal as measurement of metal degradation and at pH

values above 4.5 this would not be expected to occur for purely

chemical reasons, the oxidation of the sulphidic moiety to sulphate

can occur at relatively high pH without metal release (Mizoguchi

et al., 1976).

Some Russian work indicates that the pH optimum for covellite

and pyrite oxidation was 2.0 - 1.7, and that bacterial activity was

also affected by the concentration of metallic ions and the temperature.

According to these authors, if concentrations of iron, copper and zinc

were less than 5,000, 4,000 & 8,000 p.p.m. respectively at temperat­

ures below 50°C, the optimal pH value for microbial oxidation of the min­

eral was less than 1.5 (Ivanov, & Nagirnayak, 1962; Golomzik &

Ivanov, 1965). Watanabe (1968) has shown that the pH optimum for

ThiobaciZZus ferrooxidans towards high grade ores of copper sulphide

is 2.5 whereas pH 1.5 was found more satisfactory for low-grade

ores.

57

1. 3. 3. 3 NUTRITIONAL REQUIREMENTS OF THE MICROFWRA INVOLVED

IN LEACHING

In the previous section, factors relating to the substrate and

the microenvironment have been delineated and now some of the other

important parameters controlling microbial activities during mineral

degradation will be discussed. Besides the appropriate environmental

conditions of temperature, pH and Eh as described earlier, microorganisms

have specific requirements for nutrients in order to fulfil their

demands for the carbon, nitrogen, oxygen and phosphorous, etc., needed

for building cellular components. Trace elements are also required for

optimal biological functions.

Requirement for Carbon.

Chemolithotrophic bacteria are capable of acquiring carbon for

their biosynthetic activities from carbon dioxide. Carbon dioxide

fixation in these bacteria is achieved at the expense of free energy

released during oxidation of inorganic compounds and trapped in the

form of high energy compounds. ATP has been shown to be the major

energy trapping system in chemolithotrophs ( Aleem & Nason, 1960; Cole

& Aleem, 1973). Substrate level phosphorylation during sulphite oxida­

tion by APS-reductase has been well documented for ThiobaciZZus thiop­

arus ( Peck, 1960; Peck et al., 1965; Peck & Fisher, 1961). Schlegel

(1975) has recently summed up various mechanisms used by chemoautotr­

ophs to fulfil their carbon demands.

NADH is not produced by substrate oxidation in the chemolithotr­

ophic bacteria but it has been shown that it is possible to obtain

reduced NAD by means of a reversed electron transport system which,

like that of mitochondria, is ATP - dependent. Thus, although

58

oxidation of inorganic substrates by chemolithotrophs is linked directly

to an electron transport system, the ATP produced during these oxidat­

ive processes could be partially used for deriving a reverse electron

flow for producing the NADH required for Co2 fixation. The possession

of the enzyme necessary for such a complete respiratory chain by

Nitrosomonas, Nitrobacter & ThiobaciZZi was established by Aleem and

his coworkers (Aleem, 1966, 1966-a, b; 1969; 1970; Aleem et al., 1963;

Peeters & Aleem, 1970) in their demonstration of an energy dependent

NAO reduction by electron transport from reduced cytochromes. The

reports of cytochrome c reduction by NADH in ThiobaciZZus neapoZitanus

(Trudinger & Kelly, 1968) and by NADPH in ThiobaciZZus thiooxida,ns

(Tano & Imai, 1968) and of cytochromes c and a by NADH in extracts

of N. winogradski(Van Gool & Laudelou41967) have shown, however,

that normal electron flow pathways can also exist in obligate

lithotrophs.

There is a general agreement regarding the fixation of C02 by

chemolithotrophs via the reductive pentose phosphate pathway (Calvin

cycle) ( Elsden, 1962; Kelly, 1967, 1971). Evidence for operation of

this pathway consists of direct demon5tration of ribulose diphosphate

carboxylase ( carboxydismutase, EC 4.1.1.39) activity in extracts and

whole cells (by the fixation of 14co2 into 3-phosphoglyceric acid,

3PGA) and is available for ThiobaciZZus thiooxida,ns(Suzuki & Werkman,

1958, 1958-b; Iwatsuka et al., 1962; Iwatsuka, 1962), ThiobaciZZus

thioparus (Santer & Vishniac, 1955), ThiobaciZZus noveZZus (Aleem &

Haung, 1965), ThiobaciZZus neapoZitanus (MacElroy et al., 1968),

ThiobaciZZus ferrooxida,ns (Gale & Beck, 1967; Din et al., 1967) and

ThiobaciZZus denitrificans (Trudinger, 1955, 1956).

Three molecules of ATP and two molecules of reduced pyridine

nucleotide are required by the Calvin-Benson cycle for fixing one

molecule of carbon dioxide. The ATP is derived directly from the

59

oxidation of growth substrates, whereas pyridine nucleotides are

reduced by the reversal of the electron transport chain. That reduced

compounds of sulphur and ferrous ions can be used for this process has

been amply demonstrated by various workers (Aleem, 1977; Aleem et al.,

1963; Margalith et al., 1966; Silver, 1970; 1977; Silver & Torma, 1974).

An important feature of this system in the ThiobaciZZi is the

inhibition by AMP of ATP-dependent CO2 fixation via ribulose diphos­

phate carboxylase (Gale & Beck, 1966; Johnson, 1966). AMP inhibition

was suggested as being due to allosteric modification of phosphoribulo­

kinase (Johnson, 1966) and Gale & Beck (1966) fomd that in ThiobaciZZ­

us ferrooxidans CO2 fixation inhibition was in fact competitive with

AMP. Studies with phosphoribulokinase isolated from ThiobaciZZus

thioparu.s indicated that the enzyme was cooperatively affected by ATP

suggesting that inhibition of this enzyme by AMP may not be solely

competitive (MacElroy et al., 1968-a). In another study of RuDP

carboxylase from ThiobaciZZus noveZZus, high levels of inhibition (43%)

obtained with ADP seems to be indicative of a regulatory role for this

compomd also during Co2 fixation (McCarthy & Charles, 1973). This

inhibitory mechanism may be an effective control for preventing the

unnecessary wastage of ATP in Co2 fixation; in the case of depletion

of ATP, AMP produced from ATP would tend to shut off CO2 fixation by

inhibiting the ATP-consuming phosphoribulokinase.

In some of the chemolithotrophs, CO2 may also be fixed by carb­

oxylation of phosphoenolpyruvate (PEP) to produce oxaloacetate (Aubert

et al., 1957-a). PEP carboxylase (EC 4.1.1.31) activity has been

demonstrated in ThiobaciZZus thiooxida:ns (Suzuki & Werkman, 1957, 1958;

Howden et al., 1972), ThiobaciZZus thioparus (Hoban & Lyric, 1975),

ThiobaciZZus ferrooxida:ns (Din et al., 1967) and ThiobaciZZus noveZZus,

(McCarthy & Charles, 1974).

60

The significance of this pathway for C02 fixation during growth

of chemolithotrophs is not fully understood, for the Calvin-Benson

pathway appears to be the predominent pathway during Co2 fixation.

This is indicated by very heavy labelling of 3PGA ( >80%) compared

to only 8% labelling of aspartic acid during experiments performed

with cell extracts or whole cells. Furthermore, a close relationship

between the Calvin cycle and chemolithotrophy has been demonstrated by

repression of synthesis of ribulose diphosphate carboxylase during

growth of the facultative chemolithotroph Thiobacillus novellus on

organic substrates, whilst PEP carboxylase levels were relatively unaff­

ected (McCarthy & Charles, 1974), and by the fact that ribulose diphos­

phate carboxylase could not be detected in Thiobacillus intermedius

when grown heterotrophically on yeast extract (Purohit et al., 1976).

The presence of the cardinal enzyme of the Calvin cycle, ribu­

lose-1,5-diphosphate carboxylase, has been demonstrated in the

'carboxysomes' of ThiobaciUus neapolitanus (Shively et al., 1970,

1973-a) and recently Purohit and coworkers (1976-a) have presented

unequivocal evidence for the presence of polyhedral carboxysomes in

Thiobacillus intermedius during autotrophic growth.

According to the Bunsen Absorption Coefficient, Co2 solubility

in water is not affected drastically by sulphuric acid (Tuovinen &

Kelly, 1972). However, electrolytes have been found to retard C02

solubility in water due to a 'salting out' effect. This suggests that

the toxicity of high concentrations of metals may be due to their imp­

osition of a carbon dioxide limitation. The partial pressure of

carbon dioxide affects the solubility and more than 100% increase

in carbon dioxide fixation by resting cells of Thiobacillus ferroo­

xidans was noticed when the carbon dioxide partial pressure was

increased from 0.15 to 2.4% (v/v) with respect to oxygen in Warburg

flasks (Beck & Shafia, 1964).

61

Nitrogen SoUX'ae.

Thiobaailli have a preference for ammonium nitrogen. Nitrates

and nitrites, when present as the sole nitrogenous source, have been

reported to be unassimilated (Lundgren et al., 1964; Corrick & Sutton,

1965). Some amino acids ( alanine, lysine, arginine, histidine and

glutamic acid) have been found to substitute totally or partially for

ammonium ions during growth of Thiobaaillus ferrooxidans (Lundgren et

al., 1964; Remsen & Lundgren, 1963).

It has been demonstrated that Thiobaailli, in particular Thioba­

aillus ferrooxidans, have a very low nitrogen requirement (Tuovinen

et al., 1971) and this characteristic has been employed in designing

nitrogen-limiting medium for isolation of an iron-oxidising bacterium

(Beck & Elsden, 1958). This low requirement of nitrogen by acidophilic

iron-oxidisers has been explained as being due to the solubility of

atmospheric ammonia into acidic solutions (Tuovinen & Kelly, 1972).

This explanation is unconvincing when activities of microorganisms in

the natural milieu are compared with their nitrogen demand.

One of the possible means by which Thiobaailli can obtain nitro­

gen essential for their growth can be through fixation of atmospheric

nitrogen. An early report (Mackintosh, 1971) suggested that some

Thiobaailli possess nitrogen fixing capabilities. Further data on

this observation have recently been reported using tracer techniques

employing 15N2 -enriched gaseous nitrogen as the sole nitrogen source

for Thiobaaillus ferrooxidans (Mackintosh,1976). The results obtained

by this experiment showed unequivocal accumulation of the

isotope in the bacteria. In the natural milieu, there is the possibi­

lity that microorganisrns of different genera may exist together and

situationsin.which a nitrogen-fixing bacterium can coexist with

acidophilic Thiobaailli can be envisaged. In fact, recent studies

62

have shown that in a nitrogen-free medium ThiobaciZZus ferrooxidans is

capable of leaching nickel ores in the presence of the nitrogen

fixing Beijerinckia Zacticogenes, which apparently obtain its carbon

requirements from ThiobaciZZus ferrooxidans (Tsuchiya, 1977; Tsychiya

et al., 1974; Trividi & Tsuchiya, 1975). There is a great deal to be

learnt about suchmutualismeffects between the various species fowd

in a leaching heap.

Nitrogen-fixation could be one of the major agencies for provi­

ding ammonium nitrogen and in a natural leaching system a number of

reactions, microbiological as well as abiological, leading to this

end can be envisaged (Schrauzer & Guth, 1976).

Phosphate.

The bulk supply of phosphorus required for phospholipids,

nucleotides and their derivatives is derived from inorganic phosphate,

and its limitation can seriously affect Co 2-fixation, growth and assi­

milation of energy yielding substrates. Addition of phosphate to a

suspension of ThiobaciZZi enhanced substrate oxidation and Co2-fixat­

ion (Beck & Shafia, 1964; Beck, 1969). The possibility of a further

role for phosphate arises from the suggestion by Dugan & Lundgren

(1965) that phosphate complexes with ferrous ion during initiation of

its oxidation to ferric ion by ThiobaciZZi.

However, inhibitory effects of phosphate on mineral degradation

have also been noticed (Silverman & Lundgren, 1959-a; Corrick & Sutton,

1965; Razzell & Trussell, 1963-a; Beck, 1969). Torma et al. (1970)

from their study on the effect of K2 HP04 on the leaching of ZnS concen­

trate by ThiobaciZZus ferrooxidans concluded that concentrations of

K2HPO4higher than 0.5% did not increase the rate of leaching. One

63

of the important findings on the effect of phosphate on microbial

leaching is that the phosphate level influences the rate rather than

the total amomt of a metal released in a leaching process. Therefore,

in order to achieve a constantly fast rate in a leaching operation

constant addition of phosphate into the leaching system could be

profitable. However, the disadvantages of this replenishment in the

stream of nutrients during active leaching operations have been pointed

out by workers who observed that such additions in a system apparently

lowered the adsorption forces between the microorganisms and the ore

surfaces. They concluded that this effect could seriously affect

outputs ( Tuovinen et al., 1971; Ehrlich & Fox, 1967); but the results

of Le Roux et al. (1973) contradicted the earlier findings by showing

that contact between bacteria and minerals during leaching conditions

is very firm and could not possibly be interfered with under mild

conditions. The reason for the inhibitory effects of phosphate have

not be·en explained but it is conceivably due to the precipitation of

insoluble metal phosphates which render mineral surfaces passive to

bacterial action.

Sulphate.

Besides the biosynthesis of sulphur amino acids, sulphate has

been shown to play an important role in the growth of ThiobaciZZi and

in the activation of their enzyme systems (Lazaroff, 1963, 1975, 1977;

Beck, 1969; Tominaga & Mori, 1974). The demand for sulphate during

iron oxidation by ThiobaciZZus ferrooxidans has been confirmed by

investigations involving whole cells (Schnaitman et al., 1969; Tuovinen

et al., 1971) and cell envelopes (Barvinchak, 1975). Involvement of

sulphate as a complexing agent during the oxidation of ferrous iron

has been frequently demonstrated by a ntDnber of workers (Dugan &

Lundgren, 1965). Although certain other divalent ions, viz., HP04

64

HAs04 have been shown to be able to replace sulphate to some extent

(Schnaitman et al., 1969), latter studies suggest an obligatory requi­

rement for sulphate ions by ThiobaciZZus ferrooxidans growing on

ferrous ions as the sole energy source (Bodo & Lundgren, 1971). In a

recent study on ThiobaciZZus feYTooxidans an uptake of sulphate was

observed in cell suspensions incubated in the presence of ferrous ion

(Tuovinen et al., 1975). Barbie (1977) has shown that ThiobaciZZus

ferrooxidans can withstand high concentrations of sulphate. as

compared to chloride and nitrate.

When ThiobaciZZus ferrooxidans is grown in energy sources other

than iron, such as sulphur, thiosulphate or metallic suphides, the

requirement for sulphate is no longer obligatory. It is thought that

in such cases they most probably fufil their sulphate requirement ·

from the sulphate produced .from the oxidation of sulphur or other oxi­

disable sulphur compollllds.

No information regarding the sulphate demands of other ThiobaciZZi

is available in the literature.

Trace EZemen ts.

The need for trace elements by ThiobaciZZi has been demonstrated

in the case of non-acidophilic members of the genus. For acidophilic

members, such as ThiobaciZZus ferrooxidans & ThiobaciZZus thiooxidans,

it is a general belief that the trace elements needed can be derived

from the natural town-water supply or from the impurities of these

elements in the rocks which these microorganism usually degrade.

One trace element which has been investigated to some extent is magne­

sium. It acts as a co-factor for many enzymatic systems of ThiobaciZZus

ferrooxidans ( Gale & Beck, 1967; Howard & Lundgren, 1970). It is

required in such low levels [usually l,OOOth of the sulphate

65

requirement (Tuovinen et al., 1971) that Tonna et al. (1970) were

unsuccessful in their attempts to demonstrate an additional need for

magnesilllD in the presence of zinc sulphide concentrate; the impurities

in the mineral were thought to supply the necessary level.

Effect of Dissolved Ions on the Growth and

Leaching Behaviour of ThiobaciZZi.

High concentrations of ions usually inhibit the normal activities

of microorganisms by affecting the water activity, but inhibition by

specific metal ions is also common. Mechanisms for inhibition by metal

ions have been reviewed by Sadler & Trudinger (1967). Generally,

ThiobaciZZi are more tolerant to high concentrations of metallic ions

than are the heterotrophic microflora and it is conceivable that their

inhibition mechanisms differ from those of other microorganisms.

ThiobaciUus ferrooxidans can tolerate exceptionally high

concentrations of metallic ions and the experimental data indicate that

highly metal resistant strains can be selected by simply transferring

a parent strain from lower to higher metal ion levels.

The substrates on which they are cultivated seem to affect the

tolerance response of ThiobaciZZi to metal inhibition. Thus, whilst

ThiobaciZZus ferrooxidans grown on sulphur, sulphide or ferrous ion,

is relatively tolerant towards a diversity of metal ions, low concen­

trations of the same metal ions were found to be inhibitory when thio­

sulphate was used as energy source (Tuovinen et al., 1971-a). The

sensitivity of CO rfixation to metal ions is greater than is

ferrous oxidation, according to the manometric observations of

Tuovinen and Kelly (1972). Some works have indicated that the

phenomenon of metal toxicity is complex and that metal salt

toxicity should be considered separately (Barbie & Bacilovic, 1975).

CATIONS

Al.

Cd.

·co.

Cr.

Cu.

H. K. Mg.

Mn.

Na.

Ni.

U20. Zn.

TABLE 1.9

INHIBITORY LEVELS OF DIFFERENT METALLIC COMPOUNDS DURING IRON OXIDATION BY THIOMCILLUS FER.ROOXIDANS ..

.. -

SULPHATES CHLORIDES NITRATES -

CONCENTRATION CONCENTRATION OF CONCENlRATION CONCENTRATION OF CONCENTRATION CONCENTRATION OF

IN MOLES METALS(P,P,M,) IN MOLES METALS(P,P,M,) IN MOLES METAi S(P,P,M,) -· 0.18 - 0.20 9 710 - 10 790 0.10 - :J.12 2 700 - 3 240 0,02 - 0.05 540 - 135 0.05 - 0.10 5 620 - 11 240 0.05 - 0.06 5 620 - 6 740 0.01 - 0.02 1 240 - 2 250 0.48 - 0.52 28 290 - 30 650 0.03 - 0.06 1 770 - 3 540 0.01 - 0.03 590 - 1 770

0.02 - 0.04 1 040 - 2 080 0.02 - 0.03 1 040 - 1 560 0.26 - 0.28 16 520 - 17 790 0.08 - 0.10 5 080 - 6 350 0.04 - 0.06 2 540 - 381 0,22 - 0.23 0.12 - 0.13 0.035- 0.04 a.so - o.s5 39 100 - 43 010 0.25 - 0.30 9 770 -11 730 0.03 - 0.06 1170 - 2 350 0,50 - 0.55 12 160 - 13 370 0,07 - 0.10 1 700 - 2 430 0,02 - 0.04 480 - 970

< 0.70 < 38 5::JO 0.22 - 0.26 12 000 -lll 300 0.06 - 0.08 3 300 - 4 400 0.40 - 0.43 18 390 - 19 770 0.25 - 0.27 5 750 - 6 320 0.06 - 0.09 1 380 - 2 070

< 0,70 < 41100 0.06 - 0,09 3 520 - 5 280 0,03 - 0.05 1 760 - 2 930 0.002:-0.004 476 - 952 0.002- 0,004 476 - 952

< 0.70 < 45 760 0.10 - 0,14 6 540 - 9 150 0.04 - 0,06 2 610 - 3 920

• BARBIC, 1977.

en en

67

The inhibitory levels of some metal ions and cations on iron

oxidation by ThiobaciZZus ferrooxidans are tabulated in Table 1.9.

The increased concentrations of metal ions which develop during

a natural leaching situation may be responsible for upgrading the metal

tolerance of ThiobaciZZi (Fletcher, 1970, 1971-a) and tolerance towards

a metal previously toxic has been found to develop rapidly in Thio­

baciZZus ferrooxidans (Zinnnerley et al., 1958). Concentrations as

high as 72 g/t of zinc ion have been achieved in stirred continuous

leaching operations using ThiobaciZZus ferrooxidans, without any dele­

terious effects upon microbial acitivity (Torma at al., 1970; Gormely

et al., 1975).

The effects of heavy metal ions on the growth & iron-oxidising

activities of ThiobaciZZus ferrooxidans have recently been studied by

Imai et al. (1975) who have found that high levels ( 10- 2 -10- 3 M) of

cupric, zinc, cadmium and chromium ions had no significant toxic effects;

lead was found to delay the lag period but no inhibition of growth was

evident. Levels of mercurous, mercuric and silver ions (l0-3M) have

been found to be toxic and to produce 100% inhibition of growth and

iron oxidation by ThiobaciZZus ferrooxidans by these workers. In an

endeavour to study the mechanism of metal inhibition, Imai et al.

(1975) employed cell free extracts and found that the activity of

cytochrome oxidase ( cytochrome a 597) of the iron oxidising system

was specifically inhibited by 5 x 10- 4 M mercuric ions. A similar

finding was reported by Lundgren (1975) after investigation of AgN0 3

inhibition of the iron oxidising system of ThiobaciZZus ferrooxidans.

Silver ion_toxicity towards ThiobaciZZus ferrooxidans has been

recently studied by Hoffman & Hendrix (1976) who showed that silver

ions when supplied as silver sulphate, were extremely toxic; concen-

trations greater than 1 p.p.m. of silver inhibited completely the

68

growth and iron oxidising capabilities of this species.

Tuovinen & Kelly (1974, 1974-a, b) noticed severe plasmolysis

in the culture of Thiobacillus feYTooxidans exposed to uranyl ions.

However, relief of uranyl ion inhibition by divalent ions led

Tuovinen& Kelly to assume that uranyl ions inhibited the transport

system of cells by binding to specific surfaces in the cells, as

has been the case with yeast cells (Rothstein, 1954; Maxwell et al.1971).

Tolerance of Thiobacillus ferrooxidans towards uranium is low

but selective subculturing has resulted in the development of strains

which are capable of withstanding a significant amount of uranium in

solution ( Duncan et al., 1967; Duncan & Bruynesteyn, 1971). Toxicity

of uranium ions towards Thiobacillus ferrooxidans can be ameliorated

by monovalent & divalent cations ( Tuovinen & Kelly, 1974-a, b).

Ebner & Schwartz (1974) reported a high population of a uranium-tole­

rant strain of Thiobacillus ferrooxidans which was capable of proli­

ferating in a maximum concentration of 1 g uranium per litre ( as

U0 2S0 4 ). The most interesting finding of these workers was that the

Thiobacillus thiooxidans could tolerate significantly higher amol.lllts

of uranium ( 12.5 g uranium per litre). Previously it was commonly

believed that Thiobacillus thiooxidans species was comparatively less

resistant to metallic ions than was Thiobacillus ferrooxidans.

Schwartz's group also has demonstrated for the first time that

each heavy metal ion, when present together with other heavy metal ions,

affects the bacterial activities as if it were present alone; and

that the maximum tolerance value for each ion is independent, i.e., a

combined toxicity effect is not observed. These findings have been

confirmed by Barbie (1977).

Generally, anions are more toxic than cations. Ferrous oxidation

by Thiobacillus ferrooxidans has been found to be hampered by a number

69

of anions. Fluoride, molybdate, cyanide and azide ions are most

deleterious, even in small concentrations (less than 0.25 mM)

(Andersen & Lundgren, 1969; Tuovinen & Kelly, 1972). Barbie (1977)

has shown that Thiobacillus ferrooxidans is most tolerant of

sulphate, less of chloride and least of nitrate; the relative toxicity

of sulphate, chloride and nitrate is 1:3:10.

Concentrations of chlorides above 6,000 p.p.m. and nitrate more

than 2,000 p.p.m. retarded iron oxidation by iron-oxidising bacteria.

It is worth noting, however, that Le Roux (1977- unpublished informa­

tion made available to Professor B.J.Ralph) has shown that for other

strains of Thiobacillus ferrooxidans chloride inhibition is not shown

until levels of 6,000 p.p.m. chloride ions were reached and the bacte­

rial population remained viable at concentrations of chloride ions up

to 8,000 - 9,000 p.p.m. Further recent work in Department of Micro­

biology, University of Cardiff, have indicated that some Thiobacillus

ferrooxidans can be acclimatised to sea water media with reduced but

still substantial growth rates (Private communication to Professor

B. J. Ralph, 1977). Similar claims were made by Mayling (1966) in a

patent where iron-oxidising bacteria were grown on ferric chloride

solution. These findings also explain the anomalous behaviour of

Thiobacillus ferrooxidans strains during the covellite oxidation

observed by Vanselow (1976) and Golding et al. (1977) in a chloride­

rich media. The iron- oxidising bacteria which these workers emplo­

yed was isolated from a marine mud (Corrans, 1970).

Relatively little is known about anion toxicity in Thiobacilli

and the biochemical basis of both anion and cation inhibition in

Thiobacilli has not been studied extensively. Such studies should

be emphasised in the future, since the mechanism of metal toxicity

may open the door to our understanding of pollution control problems.

70

Effect of Organic Compounds.

Organic compounds normally capable of acting as nutrients for

heterotrophic microorganisms are known to inhibit growth and meta­

bolic activity of several species of chemolithotrophic nitrifying

bacteria and some species of the genus ThiobaciZZus. Smith & Hoare

(1977) have recently reviewed the influence of organic compounds on

growth of lithotrophs. The acidophilic ThiobaciZZi, i.e., ThiobaciZZus

ferrooxida:ns and ThiobaciZZus thiooxidans are adversely affected

by the presence of organic matter in the medium (Tuttle & Dugan, 1969).

Pyruvate or oxaloacetate exceeding a level of 2 - 4 x 10- 4 Min a

medium containing elemental sulphur prevented the growth of ThiobaciZZus 2

thiooxidans (Borichewski, 1967; Rao & Berger, 1970). 10- M ~-alanine

has also been shown to inhibit growth of this species (Butler &

Umbreit, 1966).

Organic compounds such carbohydrates, carboxylic acids, anionic

detergents, cationic surfactants, quaternary ammonium compounds, yeast

extracts, peptone and amino acids have frequently been reported to have

deleterious effects upon iron oxidation or growth,or both, by Thio­

baciZZus ferrooxidans (Dugan & Lundgren, 1964; Duncan et al., 1964;

Leathen et al., 1956; Silver et al., 1967; Silverman & Lundgren, 1959-a),

and inhibition of sulphur oxidation by primary alcohols, dicarboxylic

acids and protein hydrolysate has been reported by Kelly (1971) and

Landesman et al. (1966-a).

Conversely, there are reports that ThiobaciZZus thioparus and

ThiobaciZZus thiooxidans are not noticeably inhibited by organic matter.

In some instances, their growth is reported to be accelerated by the

addition of organic matter to their media (Butler & Umbreit, 1966;

Starkey, 1934; Matin & Rittenberg, 1971). Increased rates of oxidation

of sulphur by ThiobaciZZus thiooxida:ns in the presence of dicarboxylic

71

acids (Vogler et al., 1942) and an enhancement in the rate of oxida­

tion of thiosulphate by ThiobaciZZus thioparus when TCA cycle inter­

mediates, viz., malate, succinate and fumarate, were present in

concentrations ranging from 10- 3 - 10-1 M, have also been reported

(Vishniac & Santer, 1957).

Shafia & Wilkinson (1969) and Tabita & Lundgren (1970,1971) have

succeeded in cultivating ThiobaciZZus ferrooxidans in a glucose-ferrous

sulphate medium and they reported that glucose was dissimilated via

the Entner-Doudoroff pathway. The presence of enzymes of carbohydrate

metabolic pathways have also been detected and studied in other members

of the ThiobaciZZus genus, such as ThiobaciZZus perometaboZis, Thio­

baciZZus intermedius, and ThiobaciZZus ferrooxida,ns (Matin & Rittenberg,

1970; Tabita & Lundgren, 1971-a, b). In order to investigate the

mechanism of metabolic regulation in facultative ThiobaciZZi, hetero­

trophic growth of ThiobaciZZus A2 on labelled sugars and organic

acids has been studied; presence of enzymes essential to the operation

of three major catabolic pathways ( the Embden-Meyerhof glycolytic

pathway, the Entner-Doudoroff pathway and oxidative pentose phosphate

pathway) in ThiolxzciZZus A2 grown on glucose and other sugars has

been detected (Wood & Kelly, 1976; 1977; Wood et al. 1977).

Physiological and biochemical changes in the transition from

autotrophy to heterotrophy and vice versa may be explained in terms

of the induction,, repression and depression of enzymes demonstrated

with ThiobaciZZus noveZZus (McCarthy & Charles, 1974). Since the

identification of ThiobaciZZus feYTooxidans is invariably based on

its capability of oxidising ferrous-iron, the inability of heterotro­

phic ThiobaciZZus ferrooxida,ns to grow autotrophically on ferrous

iron (Shafia & Wilkinson, 1969) casts doubts over its taxonomic

position. Earlier works have considered the possibility of original

heterogeneous cultures(Tuovinen & Kelly, 1972; Zavarzin, 1972) which

72

has been recently demonstrated by the isolation of Thiobacillus

acidophilus from a culture of Thiobacillus ferrooxidans (Guay & Sil­

ver, 1975). A recent observation made by Tuovinen & Nicholas (1977)

suggests that the reputedly obligately heterotrophic Thiobacillus

ferrooxidans KG-4, cultured on glucose, contained a small proportion

of cells which grew autotrophically on ferrous iron. The current data

also suggest that some cultures areheterogeneous on the basis of their

DNA base composition depending on their growth substrate (Guay et

al. , 1975 & 1976). The close association of a mixed population indi­

cates a very close metabolic coupling and interdependence of acidophi­

lic Thiobacilli (Tuovinen & Kelly, 1978).

In practical leaching situations, organic levels are usually

very low and provide an ideal situation for the activities of autotro­

phic microbial populations. In certain cases, however, where sewage­

waters have been used for irrigating the heaps, leaching rates have

been found to decrease (Zajic & Ng, 1970), probably because of inhibi­

tory effects of organic residues present in the sewage.

At present there is a growing trend towards the recovery of metals

from the leach-liquor by solvent extraction processes (Itzkovitch et

al., 1974) during which organic reagents dissolved in organic solvents

are mixed with leach solutions and the phases then separated. The

aqueous phase from which most of the metals have been removed are

recycled. Recently, it has been shown that residual organic solvents.

in these spent liquors exert an inhibitory effect upon the bacterial

leaching of chalcopyrite and should be removed by treatment with

activated charcoal prior to recycle (Itzkovitch & Torma, 1976; Torma

& Itzkovitch, 1976). These findings have been confirmed (Dr. John F.

Madgwick- personal communication).

So far there is no single common explanation for anomalies in

reports on the effects of organic compounds on acidophilic

73

ThiobaciZZi. However, it is suggested that a major factor contributing

to the inhibitory effects on iron oxidation may be the relative

electronegativity of the organic molecules (Tuttle & Dugan, 1976) and

that organic compotmds may inhibit iron oxidation in the following

manner:

1- Directly affecting the iron oxidising systems.

2- Reacting chemically with iron compounds.

3- Reacting with phosphate and sulphate and thus influencing

their roles in iron oxidation.

4- Interfering with the transport systems of cells by

disrupting cell membranes or envelopes; leakage of

cellular material from ThiobaciZZus ferrooxidans by

cell envelope disruption in the presence of organic acids

supports this hypothesis (Tuttle et al., 1977).

74

@

three.fold

@

Fig. 1.3 STRUCTURE AND AT(I.UC ARRANGEMENT OF ZnS.

(A)- SPHALERITE. (B)- WURTZITE.

(Berry & Mason, 1959)

75

1.4 BIOGEOCHEMISTRY OF ZINC SULPHIDE

Zinc sulphide (ZnS) minerals occur in nature in two polytypic

forms cubic (sphalerite) and hexagonal (wurtzite). Zinc, like cadmium

and mercury, always forms divalent sulphides. Zinc disulphide, ZnS2 ,

which is formed at very high pressures, also retains this divalent

nature of the cation (Bither, 1967; Bither et al., 1968).

The most common form in which zinc sulphide occurs in nature is

sphalerite,which has face-centered cubic stacking of the zinc sulphide

tetrahedral layers. Many specimens are mixtures of the two varieties

with predominance of the cubic type. Sphalerite is the stable form at

ordinary temperatures and, on heating to 1020°C, undergoes molecular

rearrangement resulting in the hexagonal stacking of ZnS layers to

form wurtzite:

1020 °C Sphalerite Wurtzite

The sphalerite - wurtzite inversion has been folllld to be pressure

and temperature dependent (Majumdar & Roy, 1965). The two types differ

only in the stacking pattern so it is possible to change from one to

the other by introducing a stacking fault. Several stacking sequences

longer than ABAB (hexagonal) or ABCABC (cubic) have been noted by

Smith (1955).

The main characteristics of these zinc sulphide forms are

described in the Table 1.10.

The structure of sphalerite (Fig. 1.3a) is analogous to that of

diamond, with four zinc atoms at the points of a face-centred cubic

lattice (comparable to four of the carbon atoms in diamond)and four

atoms of sulphur (comparable in position to the other four atoms of

76

carbon of diamond) at the points of a face-centred cubic lattice

displaced one quarter along the body diagonal of the first cube. In

sphalerite, each zinc atom is coordinated with four sulphur atoms and

each sulphur with four zinc. These tetrahedra of SZn4 are all oriented

in the same way, with a triangular face of the tetrahedra parallel to

the 111 face, and the ZnS4 tetrahedra are all oriented in the opposite 0

way. The atomic distance between adjacent cations, Zn2 +, is 5.406 A

Several minerals such as chalcopyrite, CuFeS2 , are structural

analogues of sphalerite.

TABLE 1.10

CHARACTERISTICS OF MAIN CRYSTALLOGRAPHIC FORMS

OF ZINC SULPHIDE#

PROPERTY

Crystal System

Space Group *

Crystal Class

Axial Elements

Lattice

Cell Dimensions (Pure ZnS)

Cell Content

Density

ZnS per cc.*

Zn - S *

s - s *

SPHALERITE

Cubic

216; 1 4 3 m

F 0

a= 5.406 A

Z = 4

4.096

2.52. 1022 0

2.34 A 0

3.82 A

WURTZITE

Hexagonal

186; c4 5V

6 mm

a:c = 1:1.638

p 0

a = 3 .82 A 0

C = 6 .26 A

Z = 2

4.086

2.52 • 1022 0

2.34 A 0

3.82 A

# Data taken from Berry & Mason, 1959, with the exception of*

* Data taken from Shuey, 1975.

Fig 1.3b shows the structure of wurtzite; the distance between

the adjacent zinc atoms in this crystallographic form of zinc sulphide

• 0

1s 3.82 A along a and b axes. In the hexagonal type the zinc and

sulphur atoms are on a similar lattice but zinc atoms are displaced

77

along the c axis. Along the c axis the distance between the adjacent 0

cations increases to 6.26 A.

In a purely ionic model, ZnS should consist of Zn2 + with a full

3d shell and s2 - with a full 3p shell. Direct covalence between

S - S atoms can be neglected due to the S - S distance being larger 0 0

than the ionic diameter (3.68 A). The Zn - S distance (2.34 A) is 0

distinctly less than the sum of ionic radii (2.58 A), and covalence

may occur by combination of Zn 4sp3 and S 3sp3 tetrahedral orbitals

reducing the effective charge on the atoms.

Due to the similarity in the ionic radii of zinc and iron atoms,

iron can occasionally replace zinc in the lattice of zinc sulphide

crystals resulting in the iron-containing sphalerite called' marmatite '

This substitution of iron in sphalerite has been found to be tempe­

rature dependent and the iron contents in sphalerites are often used

as a geological thermometer, indicating the temperature of formation

of rocks (Korostelev & Chainikova, 1966; Kullerud & Neumann, 1953).

However, some discrepancies in the data relating percentage of iron in

sphalerite and temperature of formation have been pointed out, chall­

enging the validity of the relationship (Sanzonov, 1961;

Cleveland, 1964).

Applications of ZnS in photoelectronic devices stimulated the

comprehensive study of atomic defects in ZnS. The results of these

investigations have been reviewed by Aven and Prener (1967), Ray (1969)

and Shuey (1975). The major effect of isoelectronic substitution is

a shift of the energy bonds. Thus when Cd replaces Zn, the conduction

band is lowered, whereas substitution of Se for S raises the valence

band. Copper, silver, gold, sodium and potassium in the lattice act

as acceptor defects. Transitional metal ions substituted for zinc

can in general act as donors or acceptors. Nickel, cobalt and iron

78

are known as 'killers' of the luminescence in ZnS commercial phosphors,

due to their similar behaviour when substituted in ZnS. Manganese is

apparently different and the paramagnetic resonance of Mn2+ is easily

observed and no evidence of another charged state of Mn in ZnS is ava­

ilable . Cadmium and manganese are noticed to have no effect on elect­

rical conductivity of ZnS whereas iron can produce free carriers.

The various proportions of these cations, especially iron, affect

the surface characteristics of the minerals and markedly influence the

flotation properties of sphalerites. It has been reported that marma­

tite can be floated more easily than pure zinc sulphides (Chainikova,

1962). Hardness, reflectance, density and lattice parameters of

sphalerite crystals have been found to be influenced by chemical

composition of the minerals. Substitution by Fe, Mn, and Cd affects

the crystal dimensions as well as physical properties of the sphalerite.

It has been found that the one-unit cell edge size of synthetic sphale­

rite bearing Fe, Mn and Cd is a linear function of the composition

expressed in mole% (Skinner, 1960; Hall, 1961; Takimoto et al.,1960).

Definite mathematical correlations between these properties and

mole percentages of Fe, Mn and Cd in these minerals have been proposed.

(Pudovkina et al., 1968). The lattice parameter 'a' of a substituted

crystal is given by the relationship: 0

a= ( 5.3985 + 0.0006 X + 0.00147 y + 0.00232 z) A

where x, y, and z are percentage mole fractions of Fe, Mn and Cd resp­

ectively present in the sphalerite specimen. Similarly the hardness

(H) of sphalerite has been found to be decreased by substitution of

the elements in the lattice. The following relationship has been

found to hold for the hardness of sphalerite specimens:

H = ( 3 Zn - 2.5 Fe - 58 Cd) kg/nnn2

The presence of these cations in otherwise pure sulphide samples has

79

been fomd to affect the chemical leaching behaviour of the minerals

(Scott & Dyson, 1968).

1.4.1 MICROBIOLOGICAL OXIDATION OF ZINC SULPHIDE

Unlike copper, iron and manganese, zinc does not exhibit variable

valency states in its common compomds, e.g., sphalerite, wurtzite and

zincite, and is unable to provide energy to oxidative mcroorganisms.

Most probably, the divalent zinc ions found in its common minerals are

not attacked per se by these microorganisms. The sulphidic moiety

present in zinc sulphides ( sphalerites, wurtzites and marmatites),

however, is easily oxidisable by microorganisms to sulphate and at

low enough pH sulphate ions are released into solution.

Scott & Dyson (1968) reported that chemical oxidation of zinc

sulphides at high pressures ( ~1700 kPa) and at elevated temperatures

(113 °C) takes place according to the following equations:

ZnS

ZnS

Reaction [2] can take place in the absence of acid while an acidic

environment is necessary for reaction [l]. Both reactions proceed

extremely slowly with pure zinc sulphides.

[l]

[2]

An electrochemical model has been proposed by Pawlek (1969) in

which zinc sulphide reacts anodically according to the reaction:

ZnS -+ Zn2 + + S0 + 2 e [3]

The cathodic reduction of chemisorbed oxygen, the presence of which

may vary the number of charge carriers within ZnS owing to its semi­

conduction properties, occurs according to the reaction:

[4]

80

The overall kinetics could not be explained on the basis of electronic

conduction alone, indicating that other rate controlling processes were

also involved. One of the factors involved was found to be H2S, which

is formed during the leaching of ZnS and its continual removal was

found to increase the leaching rate approximately 4-fold , while its

addition at a pressure of 2 atmospheres completely inhibits the leach­

ing reaction. This is due to the equilibrium in the reaction:

[5]

The removal of H2 S from a system may proceed by one of the following

reactions; of which reaction [6] takes place in highly acidic solutions,

+ 2 S 0

+ so~-

[6]

[7]

[8]

and reaction [8] apparently occurs at higher temperatures. The middle

reaction [7] is very slow; this reaction can be catalyzed by activated

charcoal, thereby increasing the leaching rate of zinc sulphide by a

factor of 5 (Pawlek, 1969).

Sphalerite (ZnS) and marmatite are the only zinc sulphides of

any commercial significance and their dissolution behaviour in acidic

ferric solutions and in acidic bacterial solutions containing iron has

been widely investigated. Sphalerite is found to be leached at signi­

ficant rates by acidic ferric sulphate solutions at temperatures

ranging from 80 - 100°C. The general equation for this dissolution is:

ZnS [9]

The reaction at high temperatures is rapid and the elemental sulphur

produced during oxidation does not impede the rates of dissolution.

The rate controlling step in ferric leaching appears to be the

availability of trivalent ferric ions since the dissolution rate of

81

sulphides is directly proportional to the amount of ferric ions pres­

ent. Initial concentrations of zinc sulphate ( 0 - 80 g/i) and

ferrous sulphate ( 0 - 60 g/i) have no significant effect on the

leaching. However, acid concentrations are critical to the extent of

+ leaching and an increment in the acidic content from 2 to 20 g of H

per litre depressed the amount of zinc dissolved from 75 to 50 %

(Dutrizac & MacDonald, 1974).

When ferric ion is used for leaching zinc sulphide, ferrous

sulphate and elemental sulphur are produced in addition to ZnS0 4 , as

shown in equation [9]. Under acid conditions, oxygen can convert the

reduced ferrous species to ferric state where it can be reused in the

system:

[10]

Zinc sulphide was probably the firstsulphidic mineral to be

oxidised microbiologically in the laboratory for the recovery of zinc

metal (Rudolf & Helbronner, 1922). Since that time many workers have

studied the bacterial oxidation of sphalerite ores. Zimmerley et al.

(1958) claimed, in a patent issued to them, that with ThiobaciZZus

ferrooxidans zinc sulphide was solubilised to give a concentration of

17 g/t . Zinc concentrations up to 25 - 50 g/t have been obtained by

various workers (Marchlewitz & Schwartz, 1961; Silverman & Ehrlich,

1964; Moss & Andersen, 1968) and more recently semi-industrial studies

performed at the University of British Columbia with zinc sulphide

concentrate resulted in a very fast zinc extraction of about

1300 mg/(i.h) (Gormely et al., 1975) and a dissolved zinc concentration

as high as 120 g/i (Torma et al. 1972).

In the presence of the iron oxidising bacterium ThiobaciZZus

ferrooxidans, oxidation of ferrous iron at acidic pH is catalyzed and

proceeds at a much faster rate, consequently accelerating reaction [10]

82

in the forward direction. The elemental sulphur produced during

reaction [9] is also oxidised by these bacteria to produce sulphuric

acid:

so [11]

Since ferric sulphate contributes so significantly to the leach­

ing of metallic sulphides it is possible that some of the early results

on bacterial leaching included an indirect chemical ferric leaching

component. Indeed a number of investigators, including Ivanov (1962,

1962-a) and Zirnrnerley et al. (1958), stated that the only part played

by the bacteria in the leaching process is the regeneration of ferric

iron from the ferrous iron produced during reaction [9].

It has been recently established that microorganisms are capable of

attacking zinc sulphide minerals directly according to the following

reaction:

ZnS Bacteria [12]

and significant rates of zinc leaching have been obtained by optimizing

the conditions of growth of bacterial populations (Duncan et al., 1966,

1967, 1967-a; Duncan & McGoran, 1971). Enhancement of leaching rates

by optimizing the microbial growth indicates that direct bacterial

attack on ZnS is significant. Duncan et al. (1967-b) also suggested

that attack on the sulphur moiety is the primary mechanism during

bacterial degradation. This view is also supported by Beck & Brown

(1968). Torma (1971), who leached pure synthetic zinc sulphide with

Thiobacillus ferrooxidans, reported reasonably high leaching rates

of 1150 mg/(t.h). The initial increase in the pH of the medium, noticed

by Torma during the bacterial leaching of the pure synthetic zinc

sulphide, was attributed to the consumption of acid as predicted from

the proposed oxidative mechanism on ZnS indicated in reaction [l].

Ferric iron, as stated earlier, is a good leaching agent, at

83

80-100°C, for sulphide minerals but its efficiency has been found to

be reduced in conditions which are necessary for rapid growth of

bacteria. It has been shown that addition of ferric ion at 35°C to

leaching experiments had no effect on the rate of release of copper

from covellite or zinc from marmatite (Duncan & Walden, 1972). The

data presented by these workers show that, during the microbial leach-

ing of chalcopyrite, ferric iron inhibited copper release. During

their investigations they noticed that iron precipitated out in spite

of the fact that the pH was low enough to prevent precipitation of

ferric hydroxide according to the equation [13].

+ -+ 2 Fe(OH) 3 + [13]

The iron was found to precipitate in the form of basic ferric

sulphates, frequently found in the form of jarosite-minerals (Jennings

et al., 1972; Torma & Legault, 1973; Rossi, 1974):

3 Fe (SO) + 12 H 0 2 4 3 2

-+ 2 HFe3(SO4) 2(OH) 6+5 H2so4 [14]

Jarosite

Hydronium ion in the jarosite can be replaced by the cations sodium,

potassium and ammonium etc.

After 60% of the metal in the leaching reactor has been leached,

copious amounts of these precipitates covered the mineral surfaces

thereby inhibiting the reaction. In certain cases these precipitates

were found to completely preclude the further leaching of the mineral

by forming a barrier between the mineral surface and bacteria ( March­

lenwitz & Schwartz, 1961; Guay et al., 1975). Beck (1967) also reported

that if iron-rich solutions are used for dump leaching there exists the

danger that jarosite will precipitate locally and plug the dump ,thereby

greatly reducing the leaching efficiency. Exposure of new surfaces by

regrinding the leach-residues has frequently been suggested as an

84

effective remedy for the inhibitory effects of jarosite precipitation

in laboratory experiments (Torma, 1972; 1976). Methods involving one­

stage(Torrna, 1975) and two-stage regrinding process have been described

(Duncan & Bruynesteyn, 1971).

Since the 9K basal salt medium of Silverman and Lundgren (1959)

contains only potassium and ammonium cations it is most probable that

jarosite formation can strip these from the medium. It has been

demonstrated that, during jarosite formation in the presence of

ThiobaciZZus ferrooxidans, both potassium and ammonium cations were

made insoluble (Duncan & Walden, 1972). These experiments indicated

that the ammonium concentration in solution was decreased from 800

mg/t to 100 mg/t, suggesting that medium may have become nitrogen

limiting for bacterial growth.

Inhibition of leaching by iron precipitation can be avoided by

using sulphur oxidising bacteria instead of the commonly employed

iron-oxidisers. The sulphur oxidising bacterium ThiobaciZZus thio­

oxidans can oxidise the sulphidic moiety of zinc sulphide, producing

sulphuric acid. Since this bacterium is incapable of oxidising

ferrous iron this remains in solution; at pH values below about 4.5

the chemical oxidation of the ferrous iron is comparatively slow.

The only reason for not using this bacterium in commercial leaching

operations is its incapability of tolerating high concentrations of

metallic ions. However, recent work has indicated that ThiobaciZZus

thiooxidans can tolerate high amounts of zinc ions in solution

(20,000 - 30,000 mg/t)

At the present time, information on the leaching of zinc sulphide

concentrates by ThiobaciZZus ferrooxidans is available. Much of the

information describes the optimum cultural conditions and other related

parameters, such as particle size, surface area, nutrient level etc.,

likely to influence the extraction of zinc. This information has

been reviewed previously in section 1.3.3.

1.4.2 EFFECT OF CRYSTAL AND IATTICE STRUCTURE ON RATES

OF LEACHING OF ZINC SULPHIDE

85

It has been reported (Trussell et al., 1964) that marmatite

was completely and rapidly leached (100%) by ThiobaciZZus ferrooxidans

whereas sphalerite was relatively recalcitrant and leached only to the

extent of 7 - 11% There appears to be no systematic study reported

in the literature on the effects of crystallographic forms of zinc

sulphide upon leaching rate. How substitution of ions of different

species in the crystal lattice of ZnS affects its leaching behaviour

is also not known with certainty. The behaviour of the marmatite

forms of sphalerite differ markedly in their chemical leaching

behaviour and in flotation characteristics from pure zinc sulphide

mineral and it might be anticipated that similar effects would be

observed during bacterial leaching, as suported by an observation

by Trussell et al. (1964).

Scott & Dyson (1968), while investigating the effect of various

catalysts on the pressure leaching behaviour of zinc sulphide, found

that several metals in solution catalyzed the release of zinc, increa­

sing in catalytic activity in the order Fe > Mo> Ru> Bi > Cu.

Copper and ruthenium were found to have appreciable catalytic activity

in the absence of acid. Although many other ions in addition to those

listed have the correct ionic radii to exchange with zinc in the zinc

sulphide lattice (e.g., Ag, Hg, Pb, Sn) these did not influence the

kinetics of dissolution. The effect of the catalysts was attributed to

the change in the electrical conductivity caused by penetration of

86

these ions into the lattice, thus pennitting electrochemical dissolu­

tion to occur according to equation [3]. The specific catalytic

activity of Fe, Mo, Ru, Bi, and Cu was attributed to the effect of

the catalyst on the reduction of oxygen at active surface sites.

There is some limited evidence to suggest that the crystalline form of

minerals does significantly influence rates of oxidative breakdown

under purely chemical conditions. This fact is clearly shown with the

a and the Sforms of chalcopyrite (Ferreira & Burkin, 1975); microbiol­

ogical analogies to this behaviour can be cited from the studies of

Silverman & Ehrlich (1964) on the behaviour of the two crystalline

forms of FeS2 (pyrite and marcasite).

Some of the work described in this thesis throws further light

on the influence of crystal structure and lattice substitution upon

leaching rates in the case of various zinc sulphide minerals with

different species of ThiobaciZZi.

87

1.5 AIMS OF THE CURRENT INVESTIGATION

It is well established that the acidophilic iron oxidiser,

ThiobaaiZZus feYTooxidans, can oxidise commercial and pure zinc

sulphides and there can be little doubt regarding the signific­

ance of this ThiobaaiZZus species in bacterial leaching systems

at low pH. One of the aims of the current investigation was to

determine whether other ThiobaaiZZus species ( e.g., ThiobaaiZ­

Zus thioparus and ThiobaaiZZus thiooxidans) can oxidise zinc

sulphides significantly and to establish a comparison, under

similar conditions of surface area, cell-density and temperatu­

re, between the behaviour of such species. In order to study

these effects it was essential that the purity of species empl-

oyed be ascertained with utmost precision. Therefore the

three isolates were purified and characterised.

Nature of crystal structure and types of bonds existing

between different atoms in the crystals of minerals are princi­

pal factors in controlling the properties and leaching behavio­

ur of these minerals. Occurrence of zinc sulphide in two well

defined crystalline structures, i.e., wurtzite and sphalerite,

prompted investigation of leachability of these zinc sulphide

minerals with the three species of ThiobaaiZZus.

..

. I

I r.

..c • @·

r

.. !-,,

·, ~,

;-'

' , , ~ .

t~

)' .

I

~>

• .

. ... . ~.

"· : I~;

.......

• >\:

.. it,~

.,

•

;

. ' I\,

..

"

; '

' .. ' .

1 ~

•it : r

" f.,';

... -~ .

..~ ' ·~ • t • . ,

,._...:.wt, . . .•

'i

«, . !I

~

.. ... ,,,r.

.;; >.

' ....

!;_;> ..... ·r 1..

. ... . .,. j

' .. 1

,,

"' \

~:

' ,. .. ,¥' .. ~..,..·.

~ '

',

'f'

" I

·, J

. .. I·~~-' · .. , .;A;N.D

•• f',

"""

. .,. .. .....

' . -• r

-1,

. '~·

>.

88

2.1 PREPARATION OF SULPHUR SUBSTRATES

A number of sulphur compounds, viz. polythionates and sulphides

etc., were required in these investigations and most of them are not

commercially available. They were prepared as required.

2.1.1 POLYTHIO NATES

The polythionates were prepared as the potassium salts because

of ease of crystallisation and purification.

Potassium trithionates and pentathionates were prepared by the

method of Stamm & Goehring (1942) and Goehring & Feldman (1948)

respectively as modified by Roy & Trudinger (1970). Potassium tetra-

thionate was synthesised by the method of Trudinger (1961), using

iodine and sodium thiosulphate.

2.1.2 SYNTHETIC ZINC SULPHIDE

Attempts were made to prepare synthetic zinc sulphide by

direct combination of metallic zinc with a small stoichiometeric

excess of elemental sulphur. The reaction was carried out in an

evacuated tube at 800 °C but in a number of experiments completion of

the reaction was never achieved. It is now known that this method

requires the application of pressure for complete reaction and anneal­

ing (Personal discussion with Mr W. M. B. Roberts, Bureau of Mineral

Resources, Canberra;and Roberts, 1965.)

Preparation of synthetic zinc sulphide was effected by precipi­

tation, with gaseous hydrogen sulphide, from M/2 zinc sulphate

solution, which had been saturated with ammonium sulphate and made

alkaline with ammonia. The precipitate was thoroughly washed with

hot water and dried at 105° C overnight. The reaction was very slow

89

and gave extremely poor yields (10-15%). Zinc sulphide produced by

this method exerted enormous pressures and exploded the sealed tubes

during attempted annealing at 800° C.

In a view of these drawbacks, a synthetic zinc sulphide which was

prepared by Ajax & Co. was used instead. This zinc sulphide assayed

(AAS) at Zn 66.2% (theoretical 67.1%), and contained small amounts of

iron (0.01%), copper (0.005%) and nickel (0.005%) [See Table 3.3.2

section 3.3.2].

2.1.3 NATURAL ZINC SULPHIDE MINERALS

Two sphalerites (from Oklahoma and Spain) and a triboluminescent

wurtzite with some sphalerite (from Beaver County, Utah) were obtained

from Wards Natural Science Establishment Inc., and a marrnatite from

Zinc Corporation Ltd., Broken Hill, New South Wales.

2.1.4 PREPARATION OF ZINC SULPHIDE SUBSTRATES

The zinc sulphide mineral samples (moisture content 0.10%)

were ground in an agate mortar to pass 400 mesh screen. They were

stored in air-tight, screw-top jars until required.

The specific surface area of each sample was determined by the

B.E.T. method (Brunauer, Emmett & Teller, 1938) with the assistance of

Mr G. Roach of School of Metallurgy, University of New South Wales.

Krypton was used as absorbent gas and specific area was calculated by

a computer programme, supplied by Mr Roach, which analysed the experi­

mental data using a least squares method. Specific surface area of

zinc sulphide minerals is presented in Table 3.3.3., section 3.3.5.

Each mineral sample was analysed for principal elements by

atomic absorption spectroscopy (see section 2.2.5) and for minor

components by semiquantitative emission spectroscopy (see section 2.2.4).

90

2.2 ASSAY PROCEDURES

2.2.1 THIOSULPHATE

The residual sodium thiosulphate in fermentation broths was

estimated either iodometrically or by the spectrophotometeric method

of Sorbo (1957).

Iodometeria Estimation of ThiosuZphate (Roy & Trudinger, 1970).

This well known method involves iodometric titration in 5% (v/v)

acetic acid, with thyodene (Purkis, Williams Ltd., 60 Brewery Road,

London, N. 7) as the indicator.

The sample was centrifuged immediately at 10,000. gin a

Sorval RC-2B centrifuge for 10 - 15 minutes. The cell-free superna­

tant (1-2 ml) was pipetted into a 50 ml conical flask and 4% (w/v)

formaldehyde solution (0.5 ml) added to eliminate interference from

sulphite, if any. 10% acetic acid (1.0 ml) and thyodene (0.5-1.0 g)

were added and the mixture titrated with N/100 iodine solution,

previously standardised as described by Vogel (1962). Usually a

titration was carried out at least in triplicate. The following

relationship was used to calculate the amount of thiosulphate present

in the sample:

1 ml of N/100 Iodine 10 Micranoles of Thiosulphate.

Colorimetric Estirrution of ThiosuZphate (Sorbo, 1957).

The method involves the measurement of thiocyanate formed by the

interaction of thiosulphate with cyanide in the presence of copper ions,

which act as a catalyst. The procedure used was as follows:-

Centrifuged supernatant (1 ml) was added to a 25 ml volumetric

91

flask and the pH adjusted to 10 with 5% (w/v) sodium hydroxide solution

with thymolphthalein as indicator. The volume was adjusted to 25 ml

with distilled water. Aliquots of this solution (4.2 ml) were placed

in test tubes and 0.1 M potassium cyanide (0.5 ml) and 0.1 M cuprous

chloride (0.3 ml) were added while agitating the tubes to ensure rapid

mixing. Ferric nitrate reagent [ 100 g Fe(N0 3 ) 3 • 9 H2o+ 200 ml nitric

acid (65%, v/v), made up to 1000 ml with distilled water] (0.5 ml) was

added to dissolve the precipitate. A blank was also prepared, by add­

ing ferric nitrate reagent and cuprous chloride solution to the samples

followed by the potassium cyanide reagent. The absorbance was measured

at 460 nm using a Hitachi Perkin-Elmer 139 UV-VIS Spectrophotometer.

Since phosphate is reported to interfere with the method, and

the growth medium contained phosphate, the method was standardised

with and without medium present. The similarity of the standard

curves (Fig. 2.1) indicated negligible interference from medium

components of Vishniac & Santer's medium (1957).

Distilled water (.) 0.4 Growth medium (o)

[Vishniac & Santer, 1957. ]

0.3 I= i::::

0

'° s::t"

0.2 FIG. 2.1 E-o

<i:: STANDARD CURVES FOR 1-Ll

u ESTIMATING THIOSUL- z

<i::

PHATE SPECTROPHOTO- ~ 0.1 0 Cl)

METRICALLY. a:i <i::

0

,/ /

,, I v,o'

.,, //

,P 'Y

k{'

i/''Y 0 40 80 120

Sodium thiosulphate

• /0

/

160 ( µg )

92

2.2.2 POLYTHIONATES

In order to obtain a general picture of by-products of thiosul­

phate oxidation by the organism under study, polythionates were studi­

ed and roughly estimated by the method suggested by Starkey (1934-a),

in which polythionates are hydrolysed, in the presence of potassium

hydroxide, to sulphite and thiosulphate both of which can reduce

iodine. The presence of polythionates was detected by the difference

in iodine titrations before and after alkali treatment of culture

broth.

Culture broth (5 ml), centrifuged at 10,000.g for 15 minutes

and filtered free of any suspended sulphur particles, was titrated

with N/100 iodine solution, as described previously under section

2.2.1., to obtain a measure of the amount of residual thiosulphate

present. To another aliquot (5.0 ml) 10% (w/v) potassitDil hydroxide

solution (5.0 ml) was added and thoroughly mixed while heating in a

boiling water bath for five minutes. After cooling, 10% (v/v) acetic

acid (10 ml) was added and the solution was titrated with N/100 iodine

solution, as described under section 2.2.1. An increase in the titra­

tion over that initally performed for residual thiosulphate indicated

the presence of polythionate.

No attempts were made to identify or estimate individual polythio­

nates produced in fermentation broths. The difference in iodine titres

before and after potassitDil hydroxide treatment was measured as such, as

a semi-quantitative measure of 'polythionates'

2.2.3 FERROUS AND FERRIC IONS

Ferrous iron was measured spectrophotometrically as described

by Welcher (1962).

Ferrous ions form a stable, orange-coloured complex with

o-phenanthroline, the absorption of which bears a reasonable

93

proportionality to the concentration of ferrous ions when measured at

520 nm. The reaction is so sensitive that ferrous concentrations bet­

ween 1 and 7 p.p.m. can be accurately measured. The sample, if

turbid, was centrifuged at 5,000. g for 15 minutes, and diluted with

dilute sulphuric acid (pH 2.5) so that the concentration of ferrous

ions was within the limits of 1 - 7 p.p.m. An aliquot (10 ml) of

this solution was pipetted into a 25 ml volumetric flask and 0.25% (w/v)

o-phenanthroline solution (2.0 ml) was added. The volume was made up

to 25 ml with dilute sulphuric acid (pH 2.5). Absorbance was measured

at 520 nm in a 1 cm glass cell against a blank containing o-phenanthr­

oline in dilute sulphuric acid (pH 2.5). A calibration curve was

constructed from standard ferrous solutions and the concentration of

samples read from this curve.

Although it was not necessary to reconstruct the standard curve

each time analyses were carried out, two concentrations of standard

ferrous solution ( 2 & 5 p.p.m.) were always included as a check on

the standard curve. In all these cases the deviations from the

standard curve were very small, i.e., within the limits of 2-5%.

The method was found to be specific for ferrous ions, but interference

from ferric ions can occur at very high ferric concentrations. Impor­

tant factors for the reproducibility of results were found to be the

pH of the reaction mixture and the use of sulphuric acid (pH 2.5) for

dilution instead of water.

Ferric ions were estimated by a difference method. Total soluble

iron was determined by atomic absorption spectroscopy (see section

2.2.5 for procedure) and the amount of soluble ferric ion was then

determined by substracting the amowt of ferrous ions, estimated by the

Welcher (1962) method, from the total iron concentration.

94

2.2.4 EMISSION SPECTROSCOPIC ANALYSIS OF MINERALS

The semi-quantitative composition of minerals was determined

by emission spectroscopic analysis. With the assistance of Mr Finlay­

son of the Department of Analytical Chemistry, the emission spectra of

minerals were recorded by using a BAIRD ATOMIC-3M grating spectrogr­

aph, Model GX.

The zinc sulphide substrates were prepared as described tmder

section 2.1.4. An appropriate amotmt (9-11 mg) of the substrate was

mixed with an equal amount of spectroscopically pure carbon in a

sample bottle. The mixture was packed into the cavity (approximately

1/8 "diameter and 3/16" deep) of a pure carbon electrode and burned

in an electric arc.

Two 10" x 4" photographic plates [Ilford, No. 30] sensitive to 0 0

wavelenths of 2,300 A - 5,200 A were used to record the spectral lines

from these minerals. Using the iron spectrum as standard, spectral

lines for various elements were identified. Concentrations of elements

were determined from the intensities of their spectral lines when

compared with various standard concentrations.

Results of spectral analyses are shown in Table 3.3.1.

2.2.5 MINERAL ANALYSIS BY ATOMIC ABSORPTION SPECTROSCOPY

Atomic absorption spectroscopy was used to estimate quantitatively

the amounts of metallic ions leached by bacterial action on minerals

and also the composition of minerals used during these investigations.

Mineral Analysis.

An appropriate amount of finely divided mineral, prepared as

described under section 2.1.4 (0.5 - 0. 75 g), was weighed accurate,ly

in triplicate into clean dry test tubes. Concentrated hydrochloric

95

acid (10 ml) was added to each tube and the tubes were covered with

watch glasses. The acid was swirled around the test tubes so that the

sample was thoroughly wetted. The tubes were heated in an aluminium

block for 15-20 minutes. After digestion, the tubes were removed from

the aluminium block and concentrated nitric acid (2.5 ml) was poured

down the sides of the test tubes and the glass cover was replaced as

quickly as possible. When the reaction had lost its vigor, the tubes

were digested as before, by placing them in the heated aluminium block

for 15 minutes, until all the reaction had ceased, as indicated by the

losss of vigorous boiling. The residue at this stage should be light

brown coloured or, if the sample contained organic compounds, black.

If the residue was still black, a further addition of concentrated

nitric acid was followed by 16-20 minutes digestion and then the

glass cover was removed and the contents were allowed to evaporate to

a volume of approximately 5.0 ml. Care was taken that the samples were

not evaporated to dryness.

Concentrated hydrochloric acid (20 ml) was added to a 100 ml

volumetric flask and the contents of the tube were transferred to the

flask with distilled water. These samples were allowed to settle

for 3-4 hours at room temperature before flaming in the atomic absor­

ption spectrophotometer.

Liquid Sa.mple Analysis.

Soluble metal contents of liquid samples obtained from fermenter

or shake-flask cultures were determined by spraying them directly into

atomic absorption spectrophotometer after suspending materials had

been removed from these liquid broths. In order to remove bacterial

and mineral suspensions, the solutions were either centrifuged at

10,000. g for 10 minutes and the supernatant collected or alter­

natively they were filtered through a membrane filter removing

96

bacterial populations of the broth as well [ Sartorius membrane filter,

SM 11307].

It is important that the acid concentrations of standards and

samples are the same. Therefore, all dilutions were made with 3N

hydrochloric acid. Standard solutions were made from the stock solut­

ion of 1000 p.p.m., purchased from B.D.H. The atomic absorption spectro-

photometer used was a Varian Techtron, Type AA3, fitted with digital

printer and digital indicator, built by Techtron Pty., Ltd., Melbourne,

Australia.

The spectral lines, wavelenths, gas mixtures and lamps used

for various elements during these analyses are shown in appendix 6.5.

2.2.6 X-RAY DIFFRACTION MEASUREMENTS

Representativesamples of minerals were finely grotmd in an

agate mortar. Mineral residues from shake flask experiments were

thoroughly washed with distilled water tmtil free from soluble sulphate

and dried overnight at 105°C.

A capillary tube loaded with sample was fixed in the centre

of a 6" camera loaded with Kodak X-ray film, 154, 8870. A copper

X-ray tube with nickel filter, operating at 30 kv and 25 mA, was used

to bombard the sample for 45-60 minutes. For marmatite, which

contained a higher percentage of iron, a chromium X-ray tube was used

and film was exposed for 90 minutes.

With the assistance of Mr F. Scott of the School of Metallurgy,

the repeat distances of the specimens were calculated and compared with

the standard values described in the Index Cards for sphalerite,

wurtzite and marmatite.

97

9

0 hr 8

I: I I I I I• I

I I I 'I ' I

7

6

~ 5 2

----+--+----+--ll-----'r C D

1

FIG. 2.2. WATER SAMPLER.

1- Lead sinker. 2- Sample bottle.

3- Rubber stopper. 4- Stainless steel tube for

5- Stainless steel tube inlet of sample.

for outlet of air. 6- Plastic tubing.

7- Steel wire with depth 8- Stop cock

markings. 9- Grip

98

2.3 ISOLATION OF THIOBACILLI

2.3.1 ISOLATION OF NON-ACIDOPHILIC THIOBACILLI

The isolation of Thiobacilli which are tolerant to pH values

between 4 & 9 (referred to herein as 'non-acidophilic Thiobacilli')

was carried out by employing enrichment techniques in both open and

closed systems.

Source Samples.

were:

The source materials used for the isolation of these bacteria

(i) Soil Samples.

About 300 soil samples from numerous areas of New South Wales.

(ii) Sewage Water Samples.

Water samples from various sewage treatment plants were collected

in pre-sterilised sample bottles and brought to the laboratory the same

day.

In order to get water samples from.various depths of oxidation

ponds, a special water sampler, designed and fabricated in the labo-

ratory with the assistance of Mr Thomas Babij, was used. It consisted

of a sample bottle (serum-bottle) fixed horizontally to a 'lead sinker'

with metal clamps. The wateFtight rubber stopper of the bottle was

fitted with two bores, one for inlet of the water sample and the other,

to which a sufficient length of plastic tubing was attached, for the

escape of air. The other end of the plastic tubing was connected to

a simple stop cock. This whole assembly was attached to a strong cord

marked for depth. For sampling at different depths, the stop cock was

closed and the sampler lowered to the desired depth. The air inside

99

the bottle was then released by opening the stop-cock, allowing the

water sample to rush in. When the bottle was filled, the stop-cock

was closed and sampler was removed from the water.

The water sample thus collected was poured into a sterile,glass

stoppered sample bottle and stored in a cold room ( 4° C ). The samp­

ler bottle was washed with sodium hypochlorite ( 5% w/v) and then

thoroughly rinsed with sterile water before next sample collection.

Fig. 2.2 illustrates the sampler.

Media.

The following two media were used for isolating the non-acidophi-

lie Thiobaci Ui.

COMPONENTS

(NH4) 2so4

NH4Cl

K2HPo4

KH2Po4

MgS04.7H20

MgC12.6 H2o

CaC12

FeC13.6 H2o

MnS04 .4 H2o

NaHC0 3

Na2s2o3 .5 H2o

pH

Distilled Water

1- Starkey (1934-a).

2- Beijerinck (1904).

MEDIUM A1 MEDIUM B2

0.10 g

0.10 g

4.00 II 0.20 II

4.00 II II

0.10 II

2.50 II

0.10 II

0.02 II

0.02 II

1.00 II

10.00 II 5.00 II

6.6 9.1

1,000 ml 1~ 000 ml

100

Medium A.

The basal salts (i.e., all components except thiosulphate)

were dissolved in 900 ml of distilled water and the solution dispensed

into test tubes (4 ml per tube) and sterilised by autoclaving at 15

psi for 15 minutes. The sodium thiosulphate was dissolved separately

in 100 ml of distilled water and sterilised by filtration through a

membrane filter (0.2µ pore size, SM 11307 , Sartorius ). 0.5 ml of

sterilised sodium thiosulphate solution was added aseptically to

each tube of sterile basal salts medium.

Medium B.

The basal salts were dissolved in 900 ml of distilled water and

4-5 ml portions dispensed into test tubes. The tubes were then auto­

claved at 15 psi for 15 minutes. The sodium thiosulphate and sodium

bicarbonate solutions were sterilised separately by filtration using

the same type of filter as described for medium A. 0.5 ml of this

solution was added aseptically to each test tube containing medium B

basal salt solution.

Solid media were prepared by adding 1% (w/v) 'Ionagar-2' to the

broths described above.

Isolation Procedu:res.

0.5 - 1.0 ml of each sewage water sample or 0.5 - 1.0 g of

each soil sample, withdrawn after thorough mixing, was aseptically

introduced into test tubes of medium A and B. The tubes were incubated

at 30°C. After 2 and 7 days the tubes were examined for evidence of

growth, decrease of pH and presence of residual sodium thiosulphate.

The pH was determined with narrow range pH papers (Whatman-BDH).

The samples showing any sign of growth were re-inoculated into

500 ml Erlenmeyer flasks containing the corresponding medium and

101

incubated on a reciprocal shaker at 30°C (5 cm stroke, 90 + 2 strokes

per minute). These flasks were also monitored for their residual

concentration of sodium thiosulphate and for pH.

Purification of the enriched cultures thus obtained was achieved

on solid medium plates, using conventional procedures.

2.3.2 ISOLATION OF ACIDOPHILIC THIOBACILLI

The isolation of Thiobacillus species tolerant to pH values

between 1.0 and 4.0 (referred to herein as 'acidophilic Thiobacilli'),

was achieved after enrichment in shake flasks.

Sou:t'ce Samples.

(i) Soil Samples.

Soil samples were taken from various mineral heaps, sulphur

stores and from the vicinity of eroding concrete structures.

(ii) Mineral Samples.

Mineral or rock samples obtained from various places.

(iii)Mine Drainage.

Acidic drainage solution from Mol.IDt Lyell Mine was fol.IDd to

be excellent source of these bacteria.

Mediwn.

The basal medium used for the isolation of acidophilic Thio­

bacilli was the 9K medium devised by Silverman & Lundgren (1959); its

composition is shown in Table 1.4B. The basal salts were dissolved

in 900 ml of distilled water. 90 ml portions were then dispensed into

500 ml Erlenmeyer flasks and sterilised by autoclaving at 15 psi for

15 minutes.

102

Isolation ProcedUl'es.

For the isolation of ThiobaciZZus ferrooxida:ns, ferrous sulphate

solution (40%, w/v) was sterilised separately by filtration; (pH was

previously adjusted to 2.5 with dilute sulphuric acid to avoid its

rapid oxidation). 10 ml of this sterilised solution was added

to each flask aseptically. The flasks were then inoculated with a soil,

mineral or mine drainage water sample and incubated at 30° Con a

reciprocating shaker ( 5 cm stroke; 90 ~ 2 strokes per minute). Evid­

ence of growth was obtained by monitoring the oxidation of ferrous ion.

Isolates were obtained by serial dilution of the enriched culture.

For the isolation of ThiobaciZZus thiooxida:ns, the 9K basal

medium was supplemented with 2% (w/v) elemental sulphur which had

previously been sterilised by Tyndallization over three consecutive days.

A decrease in pH, due to the production of sulphuric acid by the

oxidation of elemental sulphur, was taken as evidence for the presence

of ThiobaciZius thiooxidans. The purification was achieved on

thiosulphate agar, pH 4.0.

2.3.3 MAINTENANCE OF CULTURES

The preservation of ThiobaciZZi is still problematical. Therefore,

the cultures of 'non-acidophilic ThiobaciZZi' isolated during these

investigations were maintained on solid as well as liquid cultures.

The petri dishes with colonies were stored at 4°C, and they were

regularly transferred into fresh plates at fortnightly intervals. It

was found that, if there were delays of more than two weeks in the

transfer of these cultures onto fresh medium, the cultures lost

viability, most probably because of overproduction of acid. The same

was true of the shake flask cultures.

103

Preservation of the more acidophilic members of the genus was

carried out by the same method, with the exception that only liquid

cultures were preserved at 4°C. It was noticed that ThiobaciZZus

ferrooxidans cultures could be maintained viable for long periods

(6 - 8 weeks) by suspending them in dilute sulphuric acid ( pH 2.5)

instead of growth medium. ThiobaciZZus thiooxidans suspensions in

dilute sulphuric acid (pH 2.5) were also viable for 8-10 weeks.

The purity of each culture was checked by routine microscopic

examination of Gram-stained slides.

104

2.4 TAXONOMICAL INVESTIGATIONS

Taxonomical investigations were carried out according to

conventional microbiological procedures. However, due to the difficulty

in obtaining colonies on agar medium in the case of some species, some

tests had to be modified. The various tests performed to identify

these microorganisms are described in the following:

2. 4.1 MICROSCOPIC EXAMINATIONS

These microscopic tests were carried out according to the

general procedures described by Skennan, 1967.

1- Gram staining.

Actively growing cells were stained by Gram-stain and examined

under a microscope.

2- FZageZZurn staining.

The cells for this test were obtained from actively growing

liquid cultures. The culture was centrifuged and the cell pellet

suspended in deionised sterile water; the staining procedure of

Leifson was then applied ( Skerrnan, 1967).

3- MotiZi ty test.

For motility tests cells from the logarithmic growth phase of

liquid cultures were examined microscopically by phase contrast.

4- MorphoZogiaaZ s-t;udies.

The morphology of BJR-451 (ThiobaaiZZus thioparus) and BJR-KOl

(ThiobaaiZlus thiooxida:ns) was studied after growing them on thiosul­

phate agar plates. For BJR-451, the solid medium of Vishniac & Santer

(1957) was used whereas BJR-KOl was grown on thiosulphate agar medium

105

made by adding 1% (w/v) sodium thiosulphate to the 9K medium of

Silverman & Lundgren (1959). ThiobaciZZus ferrooxidans (BJR-Kl)

was grown in the 9K medium with ferrous ion as the oxidisable energy

source.

Unfixed cells were examined microscopically under phase contrast.

This examination provided more reliable information regarding the shape

and size of the cells than would have obtained from fixed cells.

2.4.2 BIOCHEMICAL TESTS

The following biochemical tests were performed.

1- Growth on nutrient agar.

Cultures were streaked on the nutrient agar plates and were

incubated at 30°C for one week, after which they were examined for

development of colonies. The test was carried out to determine the

autotrophic nature of the isolates.

2- Growth on different sulphur substrates.

The ability of these isolates to oxidise various reduced sulphur

compollllds was tested by growing them in the presence of these substrates

as the sole energy source.

The sulphur salts were substituted for the usual substrates

(sodium thiosulphate for BJR-451, and elemental sulphur for BJR-KOl)

in liquid medium. Growth, indicated by the presence of turbidity or

increase in the acidity, or augmentation of the cell numbers measured

by direct enumeration under a microscope, was considered a positive

sign of the substrate having been utilised.

3- Oxidation of ferrous ion.

Distinction between ThiobaciZZus thiooxidans and Thiobaci­

ZZus ferrooxidans was made by examining an isolate's capability of

oxidising ferrous salt. Each isolate was inoculated into the broth

106

containing ferrous sulphate ( 2%, w/v) and incubated at 30°C. Cultu­

res showing oxidation of ferrous ion as indicated by the disappearance

of ferrous species from the medium and development of a brownish

coloration were classified as Thiobacillus ferrooxidans.

4- Anaerobic growth.

In order to examine the capabilities of the isolates to utilise

nitrate as a terminal electron acceptor in the absence of molecular

oxygen, they were grown anaerobically in the presence of nitrate and

a suitable energy substrate; (in most cases sodium thiosulphate was

used as energy source). The cultures were examined for growth after

2, 7 and 14 days' incubation at 30°C.

5- Nitrate reduction.

The reduction of nitrate in the presence of oxygen was tested standard bacteriological

by the L procedure. Tests for the accumulation of nitrite were

carried out as recommended by the standard procedure described by

Skerman, 1967.

6- Detemination of DNA base composition.

The DNA base composition of Thiooocillus thioparus was

determined by following the method described by Hill (1968).

Thiobacillus thioparus (BJR-451) cells were collected from the

outstream of a continuous flow fermenter and were centrifuged in

sterile centrifuge tubes in a Sorvall centrifuge operating at 12,000 .g

for 10-15 minutes. The cells were washed twice with the basal

salt medium in order to remove extracellular materials including

sulphur.

The nucleic acid was extracted from the washed cells by the

method described by Marmur (1961). Purified nucleic acids were

analysed by recording their UV-absorption spectrum, which is the

summation of the individual spectra of four bases; ( a highly purified

107

DNA isneeded, as traces of proteins with UV-absorption maxima at 280

nm interfere). Due to its relative simplicity and reliability, the

method described by Hirschman & Felsenfeld (1966) was used for spectrum

analysis of nucleic acids. The absorption of DNA solutions in phosph­

ate buffer, pH 7.0, containing 0.01 M sodium chloride was read at 5 nm

intervals from 235 nm to 290 nm with a Hitachi-Perkin Elmer 139 UV-VIS

spectrophotometer.

The absorbance values for three different concentrations were

obtained and are shown in appendix 6.4a. The method of calculating

the [A+ T] ratio from the data is shown in appendix 6.4.

7- Determination of fatty acid composition.

The determination of fatty acid composition of the cells of

BJR-451 was carried out by the following method described by Babij

et al. (1969). Bacteria were grown in a chemostat at optimum growth

conditions with respect to pH and temperature and cells were harvested

by centrifuging the outflow at 10,000.g for 15 minutes. As recommended

by Agate and Vishniac (1973-b), the cell pellet was used as such

without washing.

Bacterial lipids were extracted by the technique used by Bloor

(1928), Floch et al. (1951) and Mcfarlane (1942). The cell pellet was

transferred to a sample tube and frozen to -20°C. The frozen cell

mass was transferred quantitatively into two volumes of ethanol:

diethyl ether [3:1 (v/v], and allowed to thaw at room temperature in

a nitrogen atmosphere. The solvent was decanted and the cells re­

extracted with a ethanol:chloroform [2:1 (v/v] mixture for two hours.

All extractions were carried out under a nitrogen atmosphere in order

to avoid oxidation.

The combined solvent extracts, after filtration through

Whatrnan No. 1 filter paper, were evaporated at 50°C under vacuum. The

residue was refluxed for 30 minutes with 10 N methanolic potassium

108

hydroxide to saponify the lipids. The fatty acids were separated by

acidifying the contents to pH 2.0 with hydrochloric acid and were

methylated by refluxing for 30 minutes with 5% methanolic hydrochloric

acid. The methyl esters were concentrated by pouring the refluxed

mixture into cold saturated sodium chloride solution. They were

collected from the top of the sodium chloride solution and

at -20°C.

stored

The methylated esters of the fatty acids were analysed iso­

thermally at 200°C in a Beckman GC-4 gas chromatograph fitted with a

column packed with 10% polyethylene glycol succinate on acid washed

Cromosorb W. The carrier gas pressure was 30 psi.

109

2.5 CULTIVATION OF THIOBACILLI

The ThiobaciZZi were cultivated in batch and continuous flow

systems. The degree of control of various parameters differed from

one system to another. In this section the apparatus, culture vessels

and control systems used during cultivation of Thio'baciZZi employed in

these studies are described.

2.5.1 CULTURE VESSELS

Erlenmeyer flasks of one litre capacity containing 200 ml medium

were frequently employed for growth of inoculum and during leaching

experiments. These flasks were shaken on a reciprocating shaker oper­

ating at 5 cm stroke and 90: 2 strokes per minute. It was housed in

a temperature controlled room (30 ± 1 °C). One of the serious draw­

backs of shake flasks is the lack of proper pH monitoring facilities

for the system. Therefore, the pH of the medium during these experi­

ments was controlled manually. pH was adjusted with appropriate vol­

umes of sterilised sodium hydroxide or sulphuric acid.

In order to achieve a better control of pH, temperature and

dissolved oxygen, a fermenter which could be operated both as a batch

reactor and as a continuous culture vessel was employed. The fermenter

with its control system is shown in Fig. 2.3.

2.5.2 PREPARATMNOFMEDM

Batch cuZtUPe.

Unless otherwise stated, the non-acidophilic ThiobaciZZus thiop­

arus BJR-451 was cultivated in Vishniac & Santer medium (1957). Basal

salts of Vishniac & Santer medium was dissolved in distilled water.

The basal salt solution was dispensed in Erlenmeyer flasks of one litre

capacity (180 ml). The flasks were sterilised by autoclaving at

110

FIG. 2.3. A DIAGRAMATIC REPRESENTAION OF THE CULTURc

VESSEL AND THE AUXILLIARY EQUIPMENT.

1- Medium reservoir. 2- Mackley Air Filter, Microflow Ltd. 3- Medium pump (peristaltic finger pump). 4- Air filter packed with glass-wool. 5- Precision air pressure regulator valve,

(Negretti & Zambra, London). 6- Fine needle valve. 7- Air flow meter. 8- Air filter packed with glass-wool. 9- Drip feed.

10- Vibromix (El, Chemap, AG, Alte Landstr, 415, 8708 Mannedrof, ZH, Schweiz.).

11- Air outlet moisture trap. 12- Mackley Air Filter, Microflow Ltd. 13- pH-controller, (Dynaco, 21 A, England). 14- DOT meter (Scrool of Biological Technology). 15- Temperature controller (Ether, "MINI", Type 19-90 I, Pye). 16-17-} Acid and Alkali reservoirs.

18-} Acid and Alkali pumps (Delta Watson & Marlow Ltd.). 19-20- Infra red heating lamp (Osran, England). 21- Fermenter. (Volume 2 litres) 22- pH-electrode, combined, sterilisable. 2 3- Condenser. 24- Oxygen electrode (Borkowski & Johnson probe). 25- Hallow sparger of Vibromix. 26- Sterile neoprene seal. 27- Overflow tube. 28- Thermister. 29- Steam sterilisable outlet. 30- Mackley Air Filter, Microflow Ltd. 31- Culture collecting cylinder. 32- Steam sterilisable sample collector. 33- Mackley Air Filter, Microflow Ltd. 34- Sterilised culture collecting vessel.

'· 4

t AIR IN

7

FIG. 2.3.

8

34

A DIAGRAMATIC REPRESENTATION OF THE CULTURE

VESSEL AND IBE AUXILLIARY EQUIPMENT

111

112

14 psi for 15 minutes. Sodium thiosulphate (10 g) was dissolved in

distilled water (100 ml) and sterilised by passing through a membrane

filter (0.2 µ pore size, SM 11307, Sartorius:). An appropriate

aliquot of this solution (20 ml) was added aseptically to each flask.

9K medium (Silverman & Lundgren, 1959) was used for culturing

acidophilic Thiobacilli. Thiobacillus ferrooxidans was grown in the

presence of ferrous ion as the sole energy source. The medium has been

described in section 2.3.2 which also details the growth medium for

Thiobacillus thiooxidans.

Continuous cultu.re.

The media for continuous culture investigations were the same

composition as those for batch culture and were prepared in bulk by

filtration through a membrane filter using the specially designed

apparatus described by Babij et al. (1971). The components were mixed

together by stirring in a stainless steel container and were then

passed through a pre-filter to remove insolubles. After prefiltration

the medium was pumped through a sterilised membrane filter into

sterilised storage bottles. Approximately 60 litres medium was pre­

pared in one operation.

2.5.3 MEASUREMENT OF DI~SOLVED OXYGEN

The dissolved oxygen tension (DOT) in the culture broth

was measured by using a modified form of the oxygen electrode descri­

bed by Johnson et al. (1964). The anode was a pure lead sheet

cylinder instead of the spiral as recommended by the above authors and

the silver anode was sealed in a glass capillary tube to protect it

from the corrosive action of the electrolyte. The output from the

113

probe was fed into a recorder.

The media used for cultivating ThiobaaiZZi in the fermenter

frequently contained thiosulphate and other sulphur compounds which

reacted chemically with the silver anode of the oxygen electrode and

lowered its sensitivity. The reaction between the silver anode and

sulphur compomds was prevented to some extent by covering the anode

with double layer of teflon membrane,which also prolonged the stabi­

lity of the electrode.

2. 5. 4 MEASUREMENT OF CO 2 IN THE EFFLUENT GAS STREAM

The utilisation of co2 by ThiobaciZZi was determined by

measuring the residual co2 in the exit stream of gases from the

fermenter. A known amount of co2 was pumped into the fermenter at a

known rate, measured by the flowmeter and the co2 present in the out­

let stream was determined by an infrared co2 analyzer (Hartman and

Braun, Germany).

The output from the co2 analyzer was transferred to a recorder

(Rikadenki-Multipen Recorder •. Model B-2021, Kogyo Co., Japan). The

instrument was previously calibrated with an accurate and pure mixture

of 0.49% co2 in pure nitrogen (CIG-pure mixture). The zero on the

recorder was calibrated with pure nitrogen (CIG). The amount of co2

utilised by the culture was detennined by difference.

2.5.5 DETERMINATION OF TOTAL BACTERIAL POPUIATION

The total number of bacteria in the fermentation broths was

determined by counting them in a bacterial counting chamber under a

microscope. Since the young cultures were actively motile, they were

killed with 5% (w/v) phenolic solution in water ( usually 2 ml

cell suspension was mixed with 1 ml of phenolic solution). The

114

mixture was thoroughly mixed on a vortex mixer. The counting chamber

was then filled with the bacterial suspension and the number of micro­

organisms seen by phase-contrast microscopy corn1ted. The bacteria

present in at least 20 squares were corn1ted and their average was

calculated.

3 RESULTS & DISCUSSION

115

3.1 ISOLATION AND CHARACTERISATION OF THIOBACILLI

About 300 soil and water samples from sewage works and mines

were screeened for non-acidophilic sulphur oxidising bacteria (appendix

6.1). Endeavours were also made to isolate iron-oxidisers and acido­

philic sulphur bacteria from soil, sewage works and mine water samples

(appendices 6.2 & 6.3).

3.1.1 ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING BACTF:R.IA

Appropriate amounts of soil or water samples (soil sample 1-2 g

or water sample 1 ml) were incubated in test tubes containing either

medium A or medium B. As already mentioned under section 2.3.1 medium

A was as devised by Starkey (1934-a), adjusted to pH 6.6, whereas

medium B was Beijerinck's medium used for isolating microorganisms

with pH optima near or above neutrality and was adjusted to an initial

pH of 9.1 (Beijerinck, 1904). Sodium thiosulphate was the sole source

of energy provided in these media. After 2 and 7 days' incubation at

30°C, pH and thiosulphate levles were monitored with narrow range pH

papers and a standard iodine solution. Production of any turbidity,

sediments or pellicles was also recorded (appendix 6.1). Those samples

showing any sign of growth were reinoculated into fresh media; only

one sample (451) gave good growth and complete utilisation of sodium

thiosulphate. The strain isolated from this sample was designated

BJR-451, and was selected for further study.

The fact that chemical changes occurring during growth of a mixed

population in a closed system are very complex and highly uncontrollable

makes the reproducibility of the classical enrichment techniques very

difficult. A new type of growth selection for studying the mutation

116

rates in microorganisms was discussed and practised with the introduc­

tion of continuous cultivation techniques (Novick & Szilard, 1950-a).

In contrast to batch culture, continuous culture provided the facility

of selecting microorganisms in a controlled environment on the basis

of their different growth rates at different substrate concentrations.

Surprisingly, continuous culture has been used very sparingly for the

selection and enrichment of microorganisms. Veldkamp (1970) discussed

the use of continuous culture methods for a possible enrichment

technique for isolating prokaryotes and Harder et al. (1977) recently

published an excellent appraisal of the various methods employed for

microbiological selection by using continuous culture.

Starkey's medium ( Starkey, 1934-a) (2 litre) was dispensed in

the fermenter described in section 2.5.1 and was inoculated with 10-15

ml growth suspension obtained by incubating soil sample 451 as descr­

ibed earlier. Temperature and pH were controlled at 30°C and 6.6

respectively. When growth occurred in the fermenter (after 3-4 days,

indicated by production of white suspension and depletion of sodium

thiosulphate from the culture broth) fresh medium was pumped in at a

definite flow rate lllltil a steady state was reached. The flow rate

was progressivley increased to washout. The culture from the outlet

was collected and plated out on Starkey agar plates and incubated at

30°C. The strain derived from the highest dilution rate (0.12 h-l)

was designated BJR-451 and was reinoculated into a shake flask and

further studied.

3.1.2 ISOLATION OF ACIDOPHILIC IRON OXIDISING BACTERIA

The isolation of iron oxidising bacteria was carried out by

inoculating samples into test tubes containing 5.0 ml of 9K medium

of Silverman and Lundgren (1959) with 4% (w/v) ferrous sulphate as the

sole energy source. The tubes were incubated at 30°C and examined for

117

oxidation of ferrous ion after 2,7 and 14 days.

Only one water sample (from Mount Lyell) oxidised the ferrous

ion to a significant extent (appendix 6.2). Further transfers were

carried out in liquid medium and finally the culture was purified

from a single colony developed on a silica gel plate. The culture

was maintained in dilute sulphuric acid (pH 2.5) and was designated by

the code number BJR-Kl.

3.1.3 ISOLATION OF ACIDOPHILIC SULPHUR OXIDISING BACTERIA

For isolating acidophilic sulphur oxidising bacteria, samples

were inoculated into test tubes containing 5.0 ml of 9K medium of

Silverman & Lundgren (1959) containing elemental sulphur as the

energy source.

According to growth measurement, only one sample was able to

oxidise elemental sulphur at a significant rate at the low pH of 2.5.

It presumably contained acidophilic sulphur oxidisers; the soil

sample was obtained from sulphur storage pad at Sulphide Corporation

Ltd., at Boolaroo, N.S.W. (appendix 6.3) . The enriched sample

was reinoculated into a flask and finally purified on a thiosulphate

agar plate (pH 4.0). The organism isolated was designated by the

code number BJR-KOl.

3.1.4 TAXONOMY OF THE ISOLATES

The taxonomical characteristics of the three isolates described

under sections 3.1.1, 3.1.2 & 3.1.3 are summarised in Table 3.1.

BJR-451

BJR- 451 is short Gram-negative, non-sporulating rod, 0.6-1.6 µ

long and 0.5-0.75 µ wide. It was actively motile when in contact

with substrate but motility was lost after exhaustion of sodium

118

0

c;

PLATE 1

ELECTRON MICROGRAPH OF BJR-451 GROWN IN VISHNIAC AND SANTER'S

MEDIUM .

0

0

119

thiosulphate from the medium. All attempts to stain the flagellum

were unsuccessful, apparently because of its extremely fragile nature.

However, electron micrographs of a young culture, treated with phospho-

tungstic acid, reveales a single polar flagellum (Plate 1). The

microbial cells appeared to be embedded in a thick slime.

Growth in thiosulphate broth was rapid, the medium becoming

turbid within 24 hours. As growth proceeded, white pellicles appeared

on the surface of the broth. Complete decomposition of thiosulphate

occurred within 30-40 hours, with decrease of pH from 6.5 to 4.0.

When grown on thiosulphate agar plates, small colonies, about

1.0 - 1.5 mm in diameter, appeared within 24 - 36 hours (Plate 2).

These colonies were white and circular with entire edges. Fresh

colonies were transparent to translucent but developed a whitish

hue, due to the precipitation of sulphur, with the passage of time.

One week old colonies were usually opaque due to precipitation of

elemental sulphur whereas 2-3 weeks old colonies acquired a brownish

tinge with ageing.

Since in pure culture it does not grow on nutrient agar, this

organism may be an obligate autotroph. Sometimes contaminants grew

on nutrient agar; however, when these were streaked back onto

thiosulphate agar no growth was noticeable, indicating the absence of

any facultative autotrophic bacteria in the culture.

When inoculated into liquid broth containing elemental sulphur

instead of sodium thiosulphate, the organism oxidised the sulphur

slowly as compared to thiosulphate. Uniform turbidity was obtained

after three weeks and pH was dropped to 4.5.

BJR-451 was also capable of oxidising a number of other reduced

sulphur compounds, for example, sodium sulphide, tetrathionate, tri­

thionate and pentathionate. Sodium sulphite and sodium thiocyanate were

not oxidised.

PLATE 2 COLONIES OF BJR-451 ON VISHNIAC

AND SANTER'S MEDIUM.

120

121

TABLE 3 .1

CHARACTERISTICS OF BJR-451, BJR-Kl & BJR-KOl.

CHARACTERISTICS

MORPHOLOGY:

CELL SHAPE &

SIZE.

MOTILITY

BJR-451

Short rods.

0.6-1.6 µ long, 0.5-

0.75µ wide, single

cells, occasionally

occurring in pairs.

Young cultures were

actively motile but

tended to lose their

motility with age.

FLAGELLUM Single, polar.

GRAM-STAINING Gram-negative,

easily stained.

SPORE FORMATIO! Non-sporulating.

COLONY

CHARACTERISTIC/

LICUID

BROTH.

Colonies on thiosul­

phate agar at pH 6.0

were small, circular

with complete edges;

1-1.5 mm in diameter.

Initially colonies

were whitish yellow

turning brown after

prolonged incubation

for 2-3 weeks.

Liquid medium with

thiosulphate became

uniformly turbid,

BJR-Kl

Short rods.

1-2 µ long, 0.5-0.75p

wide, single cells.

Non-motile.

Gram-negative,

easily stained.

Non-sporulating.

Colonies could not

be produced on agar

plates. probably due

to the toxicity of

agar towards this

BJR-KOl

Rods.

1.0-2.5 11 long, 0.5 -

1.0µ wide, single

cells.

Young cultures were

motile but motility

appeared to be lost

as the pH decreased

with growth.

Single, polar.

Gram-negative,

easily stained.

Non-sporulating.

Colonies on thiosu­

lphate agar (PH 4.0)

were small, circular,

varying in size from

0.5-1.0 mm in diam-

bacterium. On silica eter. They were

gel-ferrous sulphate

plates tiny colonies

of brownish red

whitish yellow in

colour and sometimes

the edges of colonies

colour were obtained. were clear due to

The colonies were

circular, sometimes

developing serrated

edges.

No growth was

noticeable in thio­

sulphate broth. 9K

oxidation of precip­

itated sulphur.

Uniform turbidity

i•n sulphur broth was

obtained and medium

CHARACTERISTICS

PHYSIOLOGY:

GROWTH ON

NUTRIENT AGAR

PLATES.

UTILISATION OF

SULPHUR COMP-­

OUNDS:

Thiosulphate.

Trithionate.

Tetrathionate.

Elemental

Sulphur.

Sodium

Sulphide.

TABLE 3.1 (Contd.)

CHARACTERISTICS OF BJR-451, BJR-Kl & BJR-KOl.

BJR-451

with pellicles and

occasionally contained

precipitated sulphur.

The pH dropped from

6.5 to 4.0.

BJR-Kl

medium with ferrous

sulphate supported

growth and produced

uniform turbidity.

After 2 days' incub-

122

BJR-KOl

became extremely acid

(pH 0.7). Growth in

thiosulphate broth

was sparse and produ­

ced a uniform turbi-

ation ferrous ion dity.

No growth.

Uniform turbidity.

Uniform tubidity.

Uniform turbidity

was obtained after

one week'sincubation.

Uniform turbidity

was obtained after 3

three weeks, pH

dropped to 4.5.

Uniform turbidity

was obtained.

was oxidised to ... i.

ferric state turning

the broth from

bluish green to red-

dish brown.

No growth.

No growth.

No growth.

No Growth.

Initially slow

growth but after two

weeks adaptation

strain oxidised sul­

phur at rates compa -

rable to that given

by B.JR-K0l.

Not determined.

No growth.

oxidises very slow­

ly.

Very slow oxidation

occurred after prolo­

nged period of 3 weeks

incubation.

Uniform turbidity

was obtained after

two weeks'incubation.

Uniform turbidity

was obtained after

two days,dropping the

pH to 0.5.

Not determined.

CHARACTERISTICS

Thiocyanate.

Ferrous

Sulphate.

Oxygen requi­

rement.

DNA Base

Composition.

FANE Profile.

pH Range for

growth.

TABLE 3.1 (Contd.)

CHARACTERISTICS OF

BJR-451

No growth.

No growth.

0bligately aerobic.

G+c = 64.8%

Cl2, Cl6 & Cl8:l

were predominant fatty

acids.

7.0 to 4.0.

BJR-451, BJR-Kl f, BJk-t:01.

BJR-Kl

Not Determined.

oxidised ferrous

iron to ferric.

0bligately aerobic.

Not determined.

Not determined.

4.0 to 1.0

123

BJR-KOl

Not determined.

No growth or oxi­

dation of ferrous iron

was observed.

0bligately aerobic.

Not determined.

Not determined.

4.0 to 0.5

124

TABLE 3.2

FATIY ACID METHYL ESTER PROFILE

FOR BJR-451

* FATTY ACID CHAIN LENGTH

LAURIC C12

MYRISTIC C14

PALMITIC C16

PAIMITOLEIC C16:1

OLEIC C1a:1

TOTAL EXTRACTED

FATTY ACIDS (%)

24.1

1. 7

25.9

6.9

41.38

* Nl.llilber to right of colon indicates nl.llilber of

double bonds.

125

BJR-451 is aerobic and does not grow anaerobically in the

presence of sodium nitrate as an electron acceptor. It was also

unable to oxidise ferrous sulphate.

The DNA base composition for GC was found to be 64.8%. The

complete calculations have been described in appendix 6.4. Fig.3.1

is FAME (fatty acid methy ester) profile of BJR-451. The strain

was cultivated at its optimum pH and temperature (pH 5.5, temperature

30°C) in a chemostat and lipids were extracted with ethanol and

diethyl ether [3:1 (v/v)]with a final extraction by methanol chloroform

mixture [2:1 (v/v)] as described by Babij et al. (1969). Table 3.2

presents the percentage composition of the fatty acids detected and

identified from Fig. 3.1. Oleic acid was in predominance (41.38%),

followed by !auric and palmitic acids in almost equal proportions

(24.1 and 25.9% respectively) and myristic and palmitoleic acid were

present in smaller proportions ( 1.7 & 6.9 % respectively).

Since BJR-451 is a strict autotroph capable of oxidising

reduced sulphur compounds and its cells are motile rods without sulphur

granules, it presumbly belongs to the genus ThiobaciZZus, the known

species of which are ThiobaciZZus thioparus, ThiobaciZZus neapoZitanus,

ThiobaciZZus denitrificans, ThiobaciZZus noveZZus, ThiobaciZZus inter­

medius, ThiobaciZZus perometaboZis, ThiobaciZZus thiooxidans and

ThiobaciZZus ferrooxida:ns (Buchanan & Gibbons, 1974). Recently some

workers have claimed the isolation of some new species of the genus

ThiobaciZZus, viz., ThiobaciZZus rubeZZus, ThiobaciZZus deZicatus

(Mizoguchi et al., 1976), and ThiobaciZZus acidophiZus (Guay &

Silver, 1975).

BJR-451 is a non-acidophilic sulphur oxidising organism as

acidic conditions (pH less than 4.0) were highly unfavourable for its

growth, therefore, the possibility of its being ThiobaciZZus

126

100-----------------r--------,-------,

80 I----+-----------·--·--------------

60 ---+--------~----~- - -t-------1

! I I

'

40 1-1--~------1----i

... . . >-- . 1---------i

20 ---- ---+-------·-------·-------··--·---- -- - -- - ·-----·---·------- -

Flg.3.1 FAME Profile Of BJR- 451.

127

thiooxida:ns, ThiobaciZZus ferrooxidans and ThiobaciZZus acidophiZus,

which are the acidophilic members of the genus ThiobaciZZus, is

eliminated .. ThiobaciZZus ferrooxidans is also an iron oxidiser,

obtaining its energy requirements from the oxidation of inorganic

ferrous ion to ferric ion; since BJR-451 could not oxidise ferrous

salts, it cannot resemble this species.

ThiobaciZZus denitrificans oxidises sodium thiosulphate anaero­

bically in the presence of nitrate as hydrogen acceptor, producing

nitrogen and the incapability of BJR-451 to grow anaerobically in

the presence of nitrate distinguishes it from ThiobaciZZus denitri­

ficans. BJR-451 is obli~atelv autotrophic which identifies it as facul tatively

different from the L autotrophic and mixotrophic species of

the genus ThiobaciZZus (i.e.,ThiobaciZZus deZicatus, ThiobaciZZus

rubeZZus, ThiobaciZZus perometaboZis, ThiobaciZZus intermedius and

ThiobaciZZus noveZZus). The only possibilities left are ThiobaciZZus

thiopa:rus and ThiobaciZZus neapoZitanus . The GC fraction of BJR-451 's

DNA was 64.8%, a value markedly different from that of ThiobaciZZus

neapoZitanus (56-57%), but comparable to ThiobaciZZus thioparus

(62-68%). Thus BJR-451 bears a close resemblance to this species in

its characteristics.

The fatty acid methyl ester (FAME) profile of ThiobaciZZus

thioparus contains a major fraction of higher fatty acids (Type II,

Table 1.2.6 & 1.2.7). The fatty acid methyl ester profile of BJR-451

as described in Table 3.2 differs from the standard profile for

ThiobaciZZus thiopa:rus described in Bergey's Manual of Determina-

tive Bacteriology (Buchanan & Gibbons, 1974); the major fatty acid

component was oleic C1 a: 1 instead of saturated c15 component.

Anomalies in the fatty acid methyl ester profile have previously

been reported for the FAME profile of a ThiobaciZZus thiooxidans

strain which was found to vary considerably from the standard

128

description (Levin, 1971). It is well established that variations in

the cultural conditions of many organisms are often reflected by qual­

itative changes in the fatty acids produced by them ( O'Leary, 1962 &

1970). Fatty acid profiles of ThiobaciZZi have been found not to

be affected by changes in cultural parameters such as phosphate

concentration, temperature, agitation (Levin, 1972; Agate & Vishniac,

1973-a & b). The effect of changes of pH, oxygen tension or other

nutrients on the fatty acid composition of ThiobaciZZi have recently

been studied by Dunn et al. (1977).

Heterotrophically grown cells of ThiobaciZZus noveZZus have

been reported to contain more unsaturated ( C1a:1; C19 ) fatty eye

acids, the percentage of saturated fatty acids ( c18 & c19) was

considerably increased when the cells were grown autotrophically

(Levin, 1972). Although the fatty acid profile of BJR-451, bears

a resemblance to the pattern of fatty acids found in ThiobaciZZus

noveZZus, described by Levin (1972), in certain other characteristics,

especially its obligately autotrophic nature, it resembles more

closely ThiobaciZZus thioparus.

BJR-Kl

This a short, Gram-negative rod, about 1-2 µ in length and 0.5

to 0.75 µ wide. Cells occur singly and are non-motile. It

oxidises ferrous to ferric ion in liquid cultures and produces a brow­

nish precipitation when cultivated at pH 2.5. When cultivated at pH 2.0

or below, the brownish precipitate does not appear and a culture with

129

uniform turbidity is produced.

All attempts to grow the culture on ferrous sulphate agar as

described by Tuovinen & Kelly (1973) failed, indicating the extreme

toxicity of organic matter. Small, circular, brownish colonies were

obtained on silica gel plates containing ferrous sulphate. These

colonies were initially brown and in the latter stages of growth, salts

precipitated around them.

When suspended in 9K medium of Silverman & Lundgren (1959) contain­

ing sodium thiosulphate, at pH 4.0, the cells did not grow even if·

incubated for periods as long as three to five weeks. Elemental

sulphur could support growth of BJR-Kl; however, a lag of one week

occurs after the first transfer from mediwn with ferrous sulphate

to the medium containing elemental sulphur as the sole energy for~.

The organism was unable to grow on nutrient agar.

It is classified as Thiobaciiius ferrooxidans. Such classification

is decided upon after consideration of the following arguments which

have been put forward previously in the literature.

In 1947, Colmer & Hinkle reported an organism capable of partici­

pating in the aerobic oxidation of ferrous ion in mine waters. The

organism was able to oxidise sulphur and thiosulphate and was mor­

phologically very similar to the previously known sulphur oxidising

bacteria, Thiobaciiius thiooxidans. _

Further physiological studies of the organism showed it to be an auto­

trophic, aerobic organism, capable of proliferation at low pH values

C Colmer et al., 1950; Temple & Colmer, 1951; Lyalikova,1959, 1960).

According to its morphological similarities to other ThiobaciZZus species

and its ability to grow on inorganic media containing ferrous ion as

the only oxidisable source of energy, it was classified as ThiobaciZZus

ferrooxidans (Colmer et al., 1950).

A similar organism was isolated from analogous environments by the

130

Leathen group in 1949 (Leathen & Madison, 1949; Leathen & Braley,

1954; Leathen et al., 1956). The organism was found to be capable of

oxidising ferrous ions only and not sulphur and its other reduced

compounds. The name Fer>robaaiUus ferrooxidans was suggested for it

by Leathen & Braley (1954).

Later in 1960, Kinsel isolated a new species Ferrobaaillus

sulfooxidans, which was very similar to the Thiobaaillus ferrooxidans

and Ferrobaaillus ferrooxidans species already mentioned. Kinsel's

organism was similar to the other two in that it could utilise ferrous

alone as the sole source of energy but differed from them in its ability

to use elemental sulphur as the sole source of energy i.n the presence of

ferrous ions. Thus, there appeared three descriptions of closely related

bacterial types, all capable of autotrophic growth on media containing

ferrous sulphate as the energy source but differing in their ability or

inability to utilise elemental sulphur or thiosulphate as a sole source

of energy. Lyalikova (1960) consequently raised doubts as to the

independent e.~istence of ThiobaaiUus ferrooxidans & FerrobaaiUus

ferrooxidans as separate species. Works of other investigators

describe these three. closely related, autotrophic microorganisms

(Beck & Elsden, 1958; Bryner & Jameson, 1958; Beck, 1960). Ivanov &

Lyalikova (1962) discussed the various characteristics of these three

strains and pointed out the following:-

1- The inability of Leathen's strain to utilise thiosulphate

was probably. due to the fact that it was an old culture which

had been repeatedly grown on ferrous ion, which causes a loss of

thiosulphate oxidising ability in Thiobaaillus ferrooxidans,

(Starkey,1935; Vishniac & Santer, 1957; Lyalikova, 1959).

2- Insufficient proof for inability of Fer>robaaillus ferrooxidans

to oxidise elemental sulphur was obtained by Leathen et al. ( 1956 ) .

3- Production of elemental sulphur by the spontaneous decompo-

131

sition of thiosulphate in sterile acid media, places in doubt the

statement about the inability of FerTobaciZZus ferrooxida:ns

to utilise elemental sulphur as the sole source of energy.

With these points in mind these three strains in question could

well be identical and Ivanov & Lyalikova (1962) suggested that the name

ThiobaciZZus ferrooxidans should be retained for all three. Recently,

Kelly & Tuovinen (1972) also suggested that the names FerrobaciZZus

ferrooxida:ns and FerrobaciZZus suZfooxida:ns should be regarded only

as subjective synonyms of ThiobaciZZus ferrooxidans, since the so

called differences in their abilities to oxidise sulphur or thiosulphate

are invalid in as much as all three can grow. on either sulphur or

thiosulphate. Consequently, BJR-Kl was named ThiobaciZZus ferrooxidans.

The pure strain of BJR-Kl was found to be capable of oxidising both iron

ferrous and elemental sulphur when present as the sole energy source.

It resembled the strain described by Colmer & Hinkle (1947) in its

inability to oxidise thiosulphate and its ability to oxidise sulphur

only slowly during its first transfer to sulphur medium. After the

second transfer the ability to oxidise sulphur was recovered markedly

and there was no prolonged lag period.

The strain was strictly autotrophic and could not be adapted to

grow on glucose; organic matter in traces was toxic and growth was

inhibited. Transition from autotrophic strains to organotrophic types

has been studied by Shafia & Wilkinson (1969) who reported that two

out of eight strains of ThiobaciZZus ferrooxidans were capable of

growing on sucrose. They also dernonstrate'd that putative facultatively autotro-

phic strain (KG-4) of ThiobaciUus ferrooxida:ns which was capable

of growing on glucose lost its ability to oxidise ferrous iron.

Oxidation of ferrous ion to ferric being the only real criterion for

ThiobaciZZus ferrooxidans, the taxonomic position of such organotrophic

132

strains becomes highly dubious. In .fact, various workers have suggested that

so called standard strains of ThiobaciZZus ferrooxidans may be a

heterogenous mixtures, associated with some other heterotrophic satellite

(Tuovinen & Kelly, 1972; Zavarzin, 1972). This idea gets further

support from a recent report describing the isolation of a facultative

autotroph, ThiobaciZZus acidophiZus from a standard culture of

ThiobaciZZus ferrooxidans (Guay & Silver, 1975). The strongly inter-

dependent nature of the various components of such heterogenous asso­

ciations is further indicated by the report that an apparentorganotrophic

strains of ThiobaciZZus ferrooxidans (KG-4) also possess small iron­

oxidising population (Tuovinen & Nicholas, 1977).

Attempts to isolate some heterotrophic satellites from a suspen­

sion of BJR-Kl, by method used by Guay & Silver (1975) for isolating

ThiobaciZZus acidophiZus

of such heterotrophic form.

were unsuccessful, indicating the absence

Although this culture of BJR-Kl was developed

from a single colony, the purity of culture containing one strain

cannot be ascertained since the culture developed only from one cell can

be called as a pure culture.

133

BJR-K0l

This strain is a Gram-negative rod, approximately 1-2.5 µ in

length and 0.5-1.0 µ in diameter. It is motile and non-sporula-

ting. The flagellum could not be detected by ordinary flagella stains

but detached flagella were observed in the electron micrographs of young

cultures which had been negatively stained with phosphotungstic acid.

The cells were examined by electron microscopy at different growth

phases but in no case were flagella attached to the cells detected.

This suggests an extremely fragile nature.

The colonies on thiosulphate agar (pH 4.0) were very tiny varying

from 0.5 - 1.0 mm in diameter. Initially, colonies were whitish yellow

in colour due to the precipitation of elemental sulphur. As they became

old their edges were cleared due to the oxidation of precipitated sulphur.

Growth in thiosulphate broth with 9K medium of Silverman & Lundgren (1959)

was very slow and produced uniform turbidity. When inoculated into

9K basal salt solution containing elemental sulphur at pH 2.5, copious

growth was obtained. The pH of the broth was decreased to 0.5. When

inoculated into 9K basal salt solution at higher pH value ( 6.5),

in the presence of elemental sulphur, no growth could be detected.

Both nitrate and ammonium nitrogen were utilised as nitrogen source,

with preference towards the latter. Nutrient agar did not support

growth and the microorganism did not exhibit any ability to oxidise ferr­

ous iron. Strain BJR-K0l is an acidophilic sulphur oxidising

bacterium which can grow profusely at low pH values, oxidising elemental

sulphur for its energy requirements. It was unable to oxidise ferrous

salts and in this and other respects resembles ThiobaeiZZus thiooxidans

description.

134

3.2 OPTIMISATION OF ISOLATES

The pure isolates obtained as described earlier (section 3.1)

were studied for their optimum growth conditions and nutrient requi­

rements.

Erlenmeyer flasks of one litre capacity containing 200 ml medium

were frequently employed for growth studies of the isolates as well as

for growing inoculum. For batch studies, 190 ml of Vishniac & Santer

medium (1957) was distributed into Erlenmeyer flasks of one litre

capacity and sterilised at 15 psi for 15 minutes. Sodium thiosulp­

hate (40%, w/v) solution was sterilised separately by filtration

through a membrane filter. 5 ml sterilised thiosulphate solution were

dispensed aseptically into the flask containing Vishniac & Santer med­

ium. 5 ml inoculum was used and flasks were shaken on a reciprocal

shaker ( 5 cm stroke; 90 ± 2 strokes per minute) at an appropriate

temperature.

A fermenter, in which temperature and pH were controlled and

oxygen tension was measured continuously, was employed for determining

the optimum pH and temperature for BJR-451. At regular intervals

5 ml samples were withdrawn aseptically from the flasks/ fermenter and

total cell count was determined immediately as described in ,section

2.5.5. The sample was centrifuged at 10,000 . g for 15 minutes, and

residual sodium thiosulphate was determined in the supernatants by

the two procedures already described in section 2.2.1.

3.2.1 SELECTION OF AN APPROPRIATE GROWTH MEDIUM FOR

NON-ACIDOPHILIC BJR-451

135

The non-acidophilic sulphur oxidiser, BJR-451, had been isolated

on media_ designed by Beijerinck (1904) and Starkey (1934-a). As

mentioned earlier (section 3.1) relatively poor rates of thiosulphate

oxidation after 4-5 transfers on the same medium indicated that the

culture had lost its capacity for vigorous growth on this medium.

None of the trace elements required as enzyme-cofactors, which are

essential for growth (Pfenning, 1961), are added to Starkey's medium.

The medium of Vishniac & Santer (1957), which contains a wide spectrum

of trace elements, was therefore used in a comparative study of the

growth of BJR-451 in each medium concurrently.

Erlenmeyer flasks of one litre capacity containing 180 ml of

basal salts solution of Vishniac & Santer or Starkey medium,

were sterilised by autoclaving at 15 psi for 15 minutes. 20 ml of

sodium thiosulphate (10 %, w/v) previously sterilsed by filtration

through a membrane filter were added to each flask. The flasks were

then each inoculated with 10 ml of a freshly prepared inoculum of

BJR-451 and incubated at 30 ± 0.5°C on a reciprocating shaker ( 5 cm

stroke; 90 t 2 strokes per minute). Thiosulphate oxidation and changes

in pH were measured at regular intervals of time.

In the Vishniac & Santer medium 100% oxidation of thiosulphate

was achieved in 48 hours, as compared to 184 hours in the Starkey-

medium (Fig. 3.2.1). The stimulation was particularly apparent in

the fonner medium's ability to decrease the lag period before

thiosulphate consumption and acid production.( Maintenanace of the

pH at 5.0 for a relatively long duration in the Starkey medium

reflects the buffering capacity of this medium at pH 5.0. This pH

value was not achieved however, witil 18 hours after all the

50

40

10

0

136

-------- - - ... -- --------···---·----------- --- ------------ --..... ~10

.... .... ......

...... ...... .....

0---0-· -o ----o \

---- -----1------,- .... -------1----·-··--- 8

0

------ ~-- - -· - ·-\

\

\ I­I

()

' ' ' '

___________ _., __ -\

\

\

\

\

' o_

\ • -•--

o, ---~- - ---- ---- -------- --------· ---- ·- 6

' '

.... ' ' '

'c., - •'"-,---- .... _

.... ...... -- - ~ '9 ·,

\ ' ' '

\ \ I

I -·- ·----- -----------'---'.:....._----------- 4

\ ' a \ \ I \ I I

\ \

' ' •

-------··---- ----. - - ------- - . -- --------',· ------~

..

\ 40

------- -----80

TIME (h)

120

FIG. 3.2.1. lITILISATION OF THIOSULPHATE BY BJR-451 IN

STARKEY MEDIUM AND VISHNIAC & SANTER MEDIUM.

(6)- pH changes in Vishniac & Santer medium

C•)- pH changes in Starkey medium.

\ \

\

' \ \

\ \

\

160

( o)- Residual concentration of thiosulphate in Starkey medium.

' \ \

\

' 200

2

0

( •) -Residual concentration of thiosulphate in Vishniac and Santer medium.

137

thiosulphate of the Vishniac & Santer medium had disappeared.)

The results clearly indicated the beneficial effects of the

trace elements in the Vishniac and Santer mediwn, which was therefore

adopted as the medium in which further growth optimising studies

were conducted.

3.2.2 BATCH GROWTH OF BJR-451 IN FLASK

pH, thiosulphate and cell number were monitored during growth,

under conditions identical with those described in section 3.2.1.

According to the pH curve, oxidation of thiosulphate involved

three stages (Fig. 3.2.2). During the first stage, which lasted over

the first 12 hours, the pH decreased gradually from 6.5 to 5.6, 18%

of the thiosulphate was oxidised and the bacterial count increased

slowly. These changes are typical of the lag-phase of a microbial

process.

In the next stage, which lasted over the next six hours, the

remaining 80% of the sodium thiosulphate was oxidised, and the cell

number increased whilst the pH remained stable at 5.5.

The third stage, which was the stationary phase according to

cell counts, was characterised by active production of hydrogen ions

(pH decreased from 5.5 to 3.7). During this phas~ the cultural broth

became turbid, due to precipitation of free elemental sulphur. If

allowed to continue for 60-80 hours, the culture became sterile,

preswnably as result of sensitivity to low pH.

3.2.3 BATCH GROWTH OF BJR-451 IN FERMENTER

Further information regarding the growth parameters of BJR-451

was obtained by cultivating it at 30°C in a fermenter. pH, dissolved

oxygen, cell number and residual thiosulphate were monitored over the

course of 300 hours. Fig. 3.2.3 reveals that the patterns of

8.0

7.9

7.8 ,-J :E: 0:: 7.7 w 0..

0:: w 7.6 ~ :::i z

7.5 r.J) ,-J ,-J w u 7.4 c.:, 0 ,-J

7.3

7.2

7.1

138

45 ----------------------9.0

40

_o Oo

30

i w E--<20 ::c: 0.. ~- • ,-J :::i r.J) 0 ..... ::c: E-

10 .\ •

0

0 10 20

TIME (h)

30 45

FIG. 3.2.2. OXIDATION OF THIOSULPHATE BY BJR-451

(Shake Flask Data)

( • )- Thiosulphate

( e )- pH

( 0 )- Cell concentration

7.0

5.0

3.0

1.0

::c: 0..

139

utilisation of thiosulphate, changes in pH, and cell growth were very

similar to those recorded previously in Fig. 3.2.2. However the

experiment also revealed that the thiosulphate was initially converted

to polythionates(rnainly tetrathionate) and that during this phase,

which included phases 1 and 2 of the previous experiment, growth

occurred and· the pH changed little. Thiosulphate was completely

exhausted by 38 hour, after which the oxygen consumptjon rate

decreased markedly (as indicated by a sudden increase in the dissolved

oxygen tensiom from 57% to 93% saturation). During the next six hours

the dissolved oxygen tension decreased to 65% saturation as 30% of

the polythionate was consumed and acid was produced. By the time the

pH had decreased to 4.8, polythionate consumption had ceased and the

oxygen tension had begun to increase again. It continued to increase

to a final value of 98% saturation, whilst more acid was produced.

Cell growth continued, at a decelerated rate, during the period of

polythionate consumption, and then growth and polythionate consumtion

terminated concurrently. In the very late stages of culture (50-300

hour) cell lysis was accompanied by oxygen uptake (inidicated by a

decrease in oxygen tension) and continued acid production whilst the

polythionate concentration remained unaltered.

Since ThiobaciZZus thioparus is not acid tolerant, it was

presumed that cessation of biological activity was enforced by the

acidic environment produced by thiosulphate oxidation. Previous

reports also have indicated the lethal effects of low pH environments

on the growth and viability of ThiobaciZZus thioparus(Parker & Prisk,

1953). In order to confirm the deleterious effect of pH, ThiobaciZZ­

us thioparus was grown in the fermenter tmder conditions which were

identical to those described above, except that, when the thiosulphate

had been totally depleted, the pH was automatically controlled at 5.5

by the addition of sterilised sodium hydroxide solution (2N). The

0 "::T ..-i

...:I ::E

0:: i:.u c. 0:: i:.u Q:l ~ ::) z Cl.) ...:I ...:I i:.u u C.!)

3

0 0)

00

1./) r--

00

1./)

-.:I"

00

1./) .-I . 00

1./) 00

r--

FIG. 3.2.3. OXIDATION OF THIOSULPHATE BY BJR-451 IN FERMENTER

(0)- D. 0. T.

( o ) - Thiosulphate

( .t. )- Cell concentration

( • ) - Polythionates ( • )- pH

~ /~

~ ~ ~ .--:I--:~ ~~ \, Ao ' E- ri • i <t: '\ &

5 "'° 0 / ) ~ 'o~o .)l.-• ;• o ·-•-~

>- ~: I 00) ~ o - A ®-"-AD / T -2..~ \ '.!;": Q 00

& -----~-~ /~~-~~ :2 ;- \ • c. • \ \ So ~ · ~ .-I / ...

\ "--•-t ....

C 0 .-I

,-.. z

00 00 .....

0

'°

0 -.:I"

E­<t: 0:: ::)

~ Cl.)

0\0

. E-

0 . 0

~ 7·-.-----· 0 .'---.

[I

o-----'--------L-------IL...---.....l..--'-----... , _ __._ _ __,_ __ o

0 10 20 30 40 50 150 250 350 N

TIME (h)

r--

'° 1./)

~

t'-J·

:I: 0..

FIG. 3.2.4. OXIDATION OF THIOSULPHATE BY BJR-451 IN FERMENTER

( o )- Thiosulphate ( • ) - Polythionates ..--i

o:i- ( e) - D. 0. T. ( 6) - pH ..--i I i----------------------0 01"" O 100 ~\ ~

\ _.,.,__ r·· ..,,..:.,...-1:,._ q --0 "7.A~ e

~ I

o \ /~ i '-. IO ; r') ~{ '\. t • , 00 , l,C)

~ · r;-,___ . ___ I \. Z O • 'v" •-l - • \ 0 A,;, 1 ,-, ~ I -- -:-_ - I • ~

>- ~ " ~ I E-~ ...:I • / \ •• 0 0 • ~ \ C. N 'J / Cl \. • o;::,, ~ ut; \ \ ~ • .; '°~

...:J \ ·-· J C/)1 ::c: Ul I ~ a\ / \. f o,\'> 0-, E- ' . ~ E- 9.. . ......,,

! \K .. (\\ / 8 ~\,\ I ~ Cl) • r ::c:

~ ~ /• \ f 0.. \ j O o, N E- \ .. I, I~

/ lr

.,, . r! \ \ I

~ i ~ l 0 0 ___ ...._ __ __._ ___ ..__..;i..-&... __ __., ___ .._~ N - 0

0 20 40 60 80 90 100 110 120 130 140

TIME (h)

142

results,• shown in Fig. 3.2.4, revealed that oxygen consumption was

restored (as indicated by a decrease in oxygen tension) as soon as

the pH was restored to 5.5 and continued until the polythionates

were completely utilised. This confirms the observation of many

workers, including Parker & Prisk (1953) that tetrathionate was

completely oxidised by ThiobaciZZus X, when the pH of the cultural

broth was controlled manually.

Again, as in the test recorded in Fig. 3.2.3, cell growth

occurred during tetrathionate consumption but at a slower rate than

when sodium thiosulphate was present as the energy source.

The experiment confirmed that pH control is vitally important

to this organism and that pH values below 4.8 drastically reduce its

activity and viability.

3.2.4 DETERMINATION OF THE OPTIMUM pH FOR GROWTH OF

BJR-451

The pH optima for growth and thiosulphate utilisation were

determined by cultivating this bacterium at 30 ± 0.5°C, in a

fermenter, at various pH values. The effect of pH over the complete

growth cycle was monitored by analysis of 5 ml samples collected

aseptically at regular intervals from the fermenter.

pH 5.5 proved to be optimal for both growth and thiosulphate

utilisation. Figs. 3.2.5 - 3.2.8 demonstrate the greater sensitivity

of growth and thiosulphate utilisation to decrease, as compared to

increase in pH. The growth pH optimum was so well defined in this

respect that growth rate decrease to 1/l0th when the pH was decreased

from 5.5 to 5.0. Due to extremely slow growth rate, growth curve

for pH 5.0 in Fig. 3.2.7 could not be shown.

45

30

10

0

0 20 40 60 70 TIME (h)

FIG. 3.2.5. EFFECT OF pH ON SUBSTRATE UTILISATION

BY BJR-451.

143

(6) - pH 6. 5 (e) - pH 6. 0 ( o ) - pH 5. 5 (o) - pH 5. 0

3.0 ,-.. ..c: o.l .._, -i IJ.l E-

~ 0. ,-J ~ 2.0 t/) 0 1-t :c E-u. 0

z 0 1-t E-< t/) 1-t ,-J 1-t 1.0 5 u. 0

5

0

144

~-- ...

I ----·-- -----~ ,

------- -----

4.5 5.0 5.5 6.0 6.5 7.0

pH

FIG. 3.2.6. EFFECT OF pH ON THE RATE OF UTILISATION

OF THIOSULPHATE BY BJR-451

DURING LOG PHASE

145

9.2

....:i 8 . 6 1------+----I-J~-~--~---+----+-----+-----t ~

0:: µ_J 0..

0:: ~ 8 . 4 --------+--H--+--+----1-----+-----+-----t-------,

~ z

7_4._ _______ ..._ ___ .,_ ___ .,_ __ __,,,.,_ __ ...,ii.... __ _,

0 10 20 30

TIME (h)

40 50

FIG. 3.2.7 EFFECT OF pH ON THE GROWTH OF BJR-451

( • )- pH 5 .5 ( 0 )- pH 6 .0 ( e) - pH 6 .5

60 70

,....... U) riJ

Ei z 1-1 ~ '--,j

1500

~ 1400

~ (3 300 I.I-, 0

~ 250

§ z 1-1

100

4.5 5.0

•

5.5 pH

6.0 6.5

FIG. 3.2.8. EFFECT OF pH ON GENERATION TIME OF

BJR-451

146

7.0

147

3.2.5 DETERMINATION OF THE OPTIMUM TEMPERATURE FOR

GROWTH OF BJR-451

The effects of temperature variation on growth and sodium thios­

ulphate utilisation by BJR-451 were studied over a temperature range

of 25-40°C in a fermenter. The results are plotted in Figs. 3.2.9 -

3.2.12.

Figure 3.2.9 reveals that the utilisation of thiosulphate was

facilitated by rise of temperature from 25°C to 30°C, above which the

utilisation rate of thiosulphate decreased. Up to 35°C, the duration

of lag phase was not significantly affected, but at 40°C, the lag phase

was prolonged.

The effect of temperature on the rates of growth and of sodium

thiosulphate utilisation during the logarithmic growth phase are shown

in Figs.3.2.10 and 3.2.11, respectively. The shapes of the curves are

typical of the temperature dependence of biological reation rates.

The maximum growth ( generation time 150 minutes) and sodium

thiosulphate utilisation rates were obtained when BJR-451 was cult­

ivated at 30°C.

The optimum of 30°C recorded for both parameters is in

agreement with values reported by other workers who quote a range

from 28°- 35° C for growth and thiosulphate utilisation (Vishniac &

Santer, 1957). At 40°C thiosulphate utilisation rate had decreased

to less than half that recorded at the optimum temperature, 30°C

(see Table 3.2.1).

Using the experimental data in Fig. 3.2.11,values for the

temperature coefficient [ Q10 ], as defined by the expression below,

were calculated and tabulated in Table 3.2.1.

= K 1

10 [ ---=-------,=- ]

; - T1 (Miller-& Litsky, 1976).

50

40

30

~

~ ~ 0.. ...l 20 :::> V) 0 1-4

~

10

0

0 25

•

so TIME (h)

'·

75 100

FIG. 3.2.9. EFFECT OF TEMPERATURE ON THIOSULPHATE

UTILISATION BY BJR-451.

148

(•)- 40°C (0)- 35°C (•)- 30°C (o)-27°C (•)- 25°C

,-.. Cl) i:.u

~ z ~

~ ~

~ :3:: 0 ex: c.:,

'-1-. 0

i:.u Cl)

~ 0..

c.:, 0 ...J

z I-{

~ ~

E--

z 0 I-{

~ 1,.1.l z l'.-U c.:,

149

400

300

200

100

0 40 36 32

TEMPERATURE °C

•

28 24

FIG. 3.2.10. EFFECT OF TEMPERATURE ON GENERATION TIME OF

BJR-451 DURING LOG PHASE

150

-------,-------r-------,-------,4.0

•

3.0

•

• 2.0

• 1.0

L..------L-----L--------'---------0 24 28 32

TEMPERATURE °C 36

FIG. 3.2.11. EFFECT OF TEMPERATURE ON RATE OF UTILISATION OF

THIOSULPHATE BY B,JR-451

DURING LOG PHASE

40

,-., ...c:: ~ .._, -~ z 0 ..... E-<i: en ..... ....J ..... ~ i:.u E-

~ 0.. ....J :::, en 0 ..... :I: E-

ii. 0

i:.u

~

151

TABLE 3.2.1

VALUES OF Q10 FOR THIOSULPHATE OXIDATION

BY BJR-451.

TEMPERATURE oc

25

27

30

35

40

RATE OF OXIDATION

[ mM/(i,h)]

1.20 }

2.40

3.48}

2.08}

1.44}

10

4.8

- 1.20

- 1. 38

152

For chemical reaction Q10 values range from 2 to 4. Since

numerous chemical reactions are involved during the growth of bacteria,

this law is usually not upheld during bacterial growth except in the

middle of the active temperature range (Litchfield, 1976). Values

of Q10 obtained for utilisation of thiosulphate by ThiobaciZZus

thioparus are typical of bacterial behaviour, the Q10 value of 10

between 25 and 27° C decreasing to 4.8 as the optimum temperature

(30°C) was approached. Negative values were of course obtained

as the temperature was increased above its optimum value.

153

:3.2.6 GROWTH OF I3JR-451 IN A CHEMOSTAT

Organisms can be cultured continuously at specific growth rates

less thanµ in a chemostat, as first described by Novick & Szilard, max

1950. The maximum specific growth rate of the organism can be

calculated by growing it at a series of dilution rates. The exact

relationship between dilution rate (D) and the growth rate (µ)

of organisms in a chemostat can be calculated from the following:

Increase in bacterial concentration= Growth - Output

i.e., dx Dx dt = µx -

i.e., dx (µ D) [I] where x concentration of dt = X - = cells.

It follows from equation [I] that if dx µ > D, dt will be positive and

the concentration of organisms in the culture will increase with time.

On the other hand, if dx µ < D, then dt will be negative and the cell

concentration will decrease with time until the culture eventually

'washes out' of the fermenter. During steady state, the concentration

of microorganisms in the fermenter remain constant; therefore, dx dt is

zero, andµ will be equal to the dilution rate, D. The continuous

culture of organisms in a chemostat therefore depends on provid-

ing conditions in which the specific growth rate(µ) and the dilution

rate (D) are equal and invariant with time. Since the growth rate is

controlled by the dilution rate, it can be adjusted, up to an upper

limit, to any value desired. The upper limit is the critical dilution

rate (Dc), which approaches µrnax If the dilution rate is set to

a value greater than Dc, it will exceed the maximum growth rate (µmax)

and the culture will be progressivley washed out from the fermenter.

Determination of D is thus a measure of C

Thiobacillus thioparus, BJR-451, was grown continuously in the

9.0

8.5

~ ...J u.. 5 0

z H

...J ~

8.0 i:i::: UJ Q..

i:i::: UJ ,:x:i

$ z ...J ...J UJ u C.:J 0 7.5 ...J

7.0

0 ......

L/'l . r--

,-.. ~ -._,

0

L/'l

UJ

~ ::r:: Q.. ...J :::> Cf) 0 1-1

::r:: E-

L/'l

N

0

0

154

2000

... --~ , ... ~

1500

,-... ..c:: -bi)

s ._,

UJ E-

:ii • • 1000 ~

:::> I Cf)

0 I H

I '---~, ::r:: E-

I u.. 0

\ z • 0 I H

E-<

\ Cf)

500 H ...J

I H

~1 5 • :1 ';j'I

•

I g I rt

0 0 .1 0.2 0.3

DILUTION RATE h-1

FIG. 3.2.12. GROWTH OF BJR-451 IN A CHEMOSTAT

( ... ) - Cell concentration (.) ThiosuJphate in outflow

( . ) - Utilisation of thiosulphate.

155

chemostat illustrated in Fig. 2.3 and described in section 2.5, with

sodiwn thiosulphate as the growth-limiting substrate. The pH was

controlled at 5.5, and the temperature at 30 ± 0.5°c, throughout the

cultivation. 1% (w/v) sodiwn thiosulphate was used in Vishniac &

Santer mediwn (1957) in the inflow (S ), and dissolved oxygen tension r

was maintained at approximately 50-60% saturation by passing sterilised

atmospheric air through the fermenter.

The cell number and substrate concentration in the outflow

during steady state at various dilution rates are shown in Fig. 3.2.12.

A maximum growth rate (dilution rate) of 0.24 h- 1 was recorded. A

steady state could not be obtained at a dilution rate greater than

o.24 h- 1 ; at a dilution rate of 0.25 h- 1 wash-out occurred. At low

dilution rates ( < 0.05 h- 1) there was a complete utilisation of

sodium thiosulphate. A conspicuous wall-film of elemental sulphur

containing microbial cells was developed at these dilution rates.

BJR-451, behaved· normally in the chemostat giving standard shapes of

its cultivation curves. During steady states, the cell population

remained constant, but as'wash-out' was approached the cell population

decreased with time.

ThiobaciZZus thiopa:rus and other ThiobaciZZi have been

frequently cultivated in continuous culture. For example, Kuenen &

Veldkamp (1973) cultivated a marine strain of Thio"baciZZus thiopa:rus

in a chemostat with thiosulphate as the growth-limiting substrate and

recorded aµ of more than 0.3. max

156

._,,

.........

~

4.0

3.0 U-l

~ 0.. ...J :::, U) 0 ..... :c: r' Lt. 0 :z 2. 0 0 ..... ~ U) ..... ...J ..... 5 Lt. 0

~ ~ 1.0

0

/' ~-

••

I

0 400 800 1200

CARBON DIOXIDE(MICRO MOLES PER HOUR)

FIG. 3.2.13. EFFECT OF CARBON DIOXIDE SUPPLEMENTATION

ON TIIIOSULPHATE UTILISATION BY BJR-451

IN A CHEMOSTAT

157

3.2.7 EFFECT OF CARBON DIOXIDE ON GROWTH OF BJR-451

ThiobaciZZi, like other autotrophic microorganisms, fulfil their

carbon requirements by fixing Co2 from the atmosphere. In this experi­

ment the effect of carbon dioxide on the growth of BJR-451, while

growing in a chemostat, was studied.

Co2 present in atmospheric air was removed by passing it through

10% (w/v) solution of sodium hydroxide and scrubbing it with sterilised

water. C02 -free atmospheric air was mixed with known volumes of a

standard C0 2 mixture. BJR-451 was cultivated in a chemostat, with

thiosulphate as the growth-limiting substrate, at a dilution rate of

0.1 h- 1 (approximately 1/3 rd of itsµ ). max

At steady state, known amounts of C02 were fed into the fermenter,

and residual co2 in the gas-outflow was analysed by passing it

through an infrared gas analyser, as described in section 2.5.4.

The concentration of thiosulphate in the outflow was measured till a

new steady state was indicated by constant value.

Increased C02 in the chemostat up to 770 µmoles/hour resulted

in enhancement of thiosulphate utilisation, indicating that the system

was limited with respect to carbon source (Fig. 3. 213 ) . By increasing

the input of Co2 from 100 to 402 µmoles/hour, thiosulphate utilisa­

tion rate was improved 2.0 to 2.5 rnM/(i.h), indicating that more

carbon was being fixed. Effect of carbon dioxide limitation during

leaching of zinc sulphide by acidophilic ThiobaciZZus ferrooxidans

have been studied by Torma et al. (1972), who showed that

with increased concentration of carbon dioxide, bacteria leached more

concentrate. However, it is generally suggested that carbon dioxide

contents in the air of a modern city are sufficient for supplying the

carbon dioxide demands of these bacteria.

3.2.8

158

TOXICITY OF ZINC IONS TOWARDS THIOBACILLUS THIOPARUS,

THIOBACILLUS FERROOXIDANS AND THIOBACILLUS THIOOXIDANS

Effect of zinc ion concentration on growth and metabolic activity

of three strains of ThiobaciZZi was tested in shake flasks. The

following procedure was used. For acidophilic members, the basal

salt solution of 9K medium (pH 2.5) of Silverman & Lundgren (1959)

was used. 180 ml of this basal salt solution were contained in

Erlenmeyer flasks of one litre capacity and sterilised by autoclaving

at 15 psi for 15 minutes. Ferrous sulphate solution (40%, w/v) was

sterilised separately by filtration. For ThiobaciZZus ferrooxida:ns,

10 ml of sterilised ferrous sulphate solution was added to the flasks

aseptically; and for ThiobaciZZus thiooxidans, 2% (w/v) sterilised

flowers of sulphur were added to the flasks aseptically. For Thiobac­

iZZus thioparus, Vishniac & Santer medium (1957), (pH 5.5), (180 ml)

dispensed in to Erlenmeyer flasks of one litre capacity and the flasks

sterilised at 15 psi for 15 minutes. 10 ml sodium thiosulphate (20%,

w/v) solution previously sterilised by filtration, were added

aseptically. Sterilised zinc sulphate to provide different zinc ion

concentrations were added to these flasks which were then inoculated

with the respective species. The flasks were incubated at 30° Con

a reciprocating shaker ( 5 cm stroke; 90 ± 2 strokes per minute),

After regular intervals 5 ml samples were aseptically withdrawn from

the flasks and total bacterial cells were counted immediately. Resid­

dual concentrations of the respective substrates were also analysed.

pH was manually adjusted during the experiment. (ThiobaciZZus thiooxi­

dans and ThiobaciZZus ferrooxidans at pH 2.5;ThiobaciZZus thioparus

at pH 5.5).

ThiobaciZZus ferrooxidans was found to be tolerant to zinc ions

upto 20,000 p.p.m. The rate of oxidation of ferrous iron in the

presence of 20,000 p.p.m. zinc ion was not markedly different from that

,--.. 0 0 0 ..... ><

e

0..

159

(0)- 20, 000 p.p.m. Zn (+)- 15, 000 p.p.m. Zn ( e)-10,000 p.p.m. Zn

( • ) - 8, 000 p. p. m. Zn ( 1::..) - 4, 000 p. p. m. Zn ( • ) - 2,000 p. p. m. Zn

( o )- 1, 000; 500 p.p.m. Zn and Control.

'-' 6 1-------~r--i--1

z 0 1-4

~ ~ Ul u z 0 u

:z:4 ~

2

0

0 40 80 120 TIME (h)

FIG. 3. 2. 14. EFFECT OF ZINC ION CONCENTRATION ON IRON

OXIDATION BY THIOBACILLUS FERROOXIDANS.

160

,-J

~

160

( 0 )- 20,000 p.p.Jll. Zn ( ·)- 15,000 p.p.m. Zn

( . ) - 10,000 p.p.m. Zn ( . ) - 8,000 p.p.m. Zn

( .6 ) - 4,000 p.p.m. Zn ( 0 ) - 2,000 '

1000; SOO p.p.m. Zn

and control.

9.0------,.-----.,...,-------,------,-------,

,.u - - _l) - -~::-:--_ --- - ---:.:~

/I>

8.6 D

- - -- ----··-- ~- --=---= -..- --=----- ---0_ ---

•

a: 8.2 ----------1.J,J

0..

0:: LU co ~ :::> z

5 7 .8 ,-J

LU u c.? 0 ,-J

7.4

7.0

• .1· I

0

----1--- - --------- -----

6.8 '--------J~-------L-------'------------------0 40 80

TIME (h)

120

FIG. 3.2.15 EFFECT OF VARIOUS CONCENTRATIONS OF ZINC ON

GROWTH OF THI()BACILLUS FERROOXIDANS

160 200

...:I ~

0::

161

~ 8. 01--------+-+,~.._----+----,r-+-----=-t'----+---------f 0:: UJ i:o

!5 z ...:I ...:I UJ u I:.:)

0 ...:I

0 50 100

TIME (h)

(·)- 20,000p.p.m. Zn (@)-15,000 p.p.m. Zn

(0)- 8,000 p.p.m. Zn (0)-_ 4,000 p.p.m. Zn

(8)- 1,000 p.p.m. Zn (a)- SOO p.p.m. Zn

150 200

(tl)- 10,000 p.p.m. Zn

C•)- 2,000 p.p.m. Zn

(e)-Control.

FIG. 3.2. 16. EFFECT OF ZINC ON GROWTH OF THIOBACILLUS

THIOOXIDANS

162

in the control. The effect of increased concentrations of zinc ion

on growth was to increase the duration of the lag phase and decrease

the growth rate, without significantly affecting the final cell mass

(Figs. 3.2.14 & 3.2.15). Similar effects were recorded in the case of

Thiobacillus thiooxidans (Fig. 3.2.16) which was comparatively less

tolerant to increased concentration of zinc ion; the maximum tolerable

concentration of zinc ion for this strain was 15,000 p.p.m. 20,000

p.p.m. severely retarded growthbutwas not lethal.

Zinc sulphate solution, when added to Vishniac & Santer medium

(pH 5.5), suddenly decreased the pH to 3.5. Neutralisation of this

solution produced copious amollllts of zinc hydroxide precipitate, which

associated with the dissociation products of sodium thiosulphate, pro­

duced by acidification. Under these circumstances it was very diffic­

ult to determine the toxic levels of zinc for Thio/Jacillus thiopa:r>us.

Generally, acidophilic members of the genus Thiobacilli are

tolerant to high metallic concentrations. Bacteria tolerant to very

high concentrations of zinc (120 g/i) have been reported (Gormely

et al., 1975) but molybdenum and silver are reported to be very toxic

to these bacteria (Imai et al., 1975; Hoffman & Hendrix, 1976).

3.3 LEACHING OF ZINC SULPHIDE BY THIOBACILLUS FERROXIDANS,

THIOBACILLUS THIOOXIDANS AND THIOBACILLUS THIOPARUS

163

Preliminary (sections 3.3.1, 3.3.2, 3.3.3 and 3.3.4) and

definitive (section 3.3.5) experiments were conducted with the aim

of comparing the abilities of three Thiobacillus species to leach

various zinc sulphides, and to explore the effects on leaching rates

of crystal structure, lattice substitution and other parameters, Two

sets of experiments were designed; in one set (section 3.3.2, 3.3.3,

& 3.3.4) leaching rates were studied in the presence of equal pulp

density of minerals whereas the other set (section 3.3.5) employed

equal surface area of minerals for comparison of leaching rates.

Unless otherwise stated, the following standard experimental

procedure was followed. For ThiobaciZZus thiooxidans and ThiobaciZZus

ferrooxidans the growth medium was the 9K basal salts medium of

Silverman & Lundgren(l959) adjusted to pH 2.5 with sulphuric acid

(2 N), lacking a soluble energy source; for ThiobaciZZus thioparus

the growth medium was identical to that of Vishniac and Santer (1957)

except that it lacked thiosulphate, and was adjusted to pH 5.5. 190

ml of the appropriate medium, contained in Erlenmeyer flasks of one

litre capacity, were sterilised by autoclaving at 15 psi for 15 minutes.

2 g of zinc sulphide, previously ground to pass through a 400 mesh

(37 micron) screen and sterilised by propylene oxide, were transferred

aseptically to each flask. 10 ml of inoculum (prepared as described

in individual test) were added to each flask .to give an initial

concentration of approximately 107 cells per ml. An uninoculated

flask, with otherwise identical contents to that of the test flask, was

prepared with each test and was incubated under identical conditions.

164

The flasks were shaken at 30 ± 0.5°C on a reciprocating shaker: ( 5 cm

stroke; 90 + 2 strokes per minute).

The pH was controlled manually at pH 2.5 or 5.5,as appropriate,

with sterile 2N sulphuric acid or 2N sodium hydroxide throughout the

course of each test. At appropriate intervals the flasks were removed

from the shaker and allowed to stand for 30 minutes to permit settling

of the zinc sulphide; 5 ml of culture were then withdrawn aseptically

from each flask and transferred to centrifuge tubes; the 5 ml were

replaced with 5 ml of fresh, sterile medium before returning the flasks

to the shaker. The clear supernatants, obtained by centrifugation at

10,000 g for 15 minutes, were analysed for zinc, ferrous and ferric

ions and their pH measured. Cell counts were determined on tennination

of each experiment.

3.3.1 LEACHING CAPABILITIES OF THIOBACILLUS THIOPARUS (BJR-451)

This preliminary experiment was designed to test the potential

of BJR-451 for leaching sulphide minerals of zinc, copper and nickel.

The three sulphides were tested concurrently by suspending each in a

shake flask containing a medium which was identical to that of Vishniac

and Santer (1957) except that it lacked thiosulphate. The initial pH

of the medium was adjusted to 5.5, the optimum pH for growth of

BJR-451. pH was not subsequently controlled. Controls containing the

medium and mineral but no microbial cells, were treated similarly

and incubated concurrently. The flasks were shaken on a reciprocating

shaker at 90 ± 2 strokes per minute with 5 cm stroke, at 30°C.

Every 3-4 days over a period of three weeks the flasks were removed

from the shaker, the mineral allowed to settle and 5 ml of culture

withdrawn aseptically and an equal volume of fresh sterile medium

added. The samples were centrifuged at 10,000 . g for 15 minutes and

the clear supernatant analysed for copper, zinc or nickel.

165

6.0

::r: i:i.. 5.0

4.0

• . ZnS

El 3.0 . p.. . p..

z 70 Zn 0 H

~ 60 •

~~ (J.J

r-iu oz

0 •..-tU 40 z

Cl) .,z

::so UH

u H ...l 20 ...l <I'. E-

~ 0

0 • • • 0 1 2 3

TIME (WEEKS)

FIG. 3.3.1. LEACHING CAPABILITIES OF BJR-451

166

The results of the metal releasing ability of BJR-451, illustra­

ted in Fig. 3.3.1, revealed that in the presence of the bacterium,

zinc ions were released into solution. The copper and nickel ion

concentrations in the control and inoculated flasks were almost

identical and no significant changes in pH were discernable. It

was concluded that BJR-451 was incapable of degrading copper and nickel

sulphides under these conditions, but was capable of degrading zinc

sulphide. The pH of the medium containing zinc sulphide decreased

from 6.0 to 3.5 as the mineral was solubilised. During the first 14

days the zinc ion concentration increased progressively from 15 p.p.m.

to 65 p.p.m. Thereafter, there was no change in zinc ion concentration

and the pH remained constant at its minimum value of 3.5. Since the

bacterium had been shown to be inhibited at pH values lower than 4.5

(see section 3.2) it seems reasonable to assume that cessation of

activity was due to production of acid. Most probably the complex

reaction involving the oxidation of the sulphidic moiety of zinc

sulphide is represented stoichiometrically by:

s (Torma, 1972)

Because of its ability to leach zinc sulphide, ThiobaciZZus

thioparus was included in the comparative study of the leaching of

several zinc sulphide samples.

TABLE 3.3.1

SEMI-QUANT ITATlVE ANALYSIS OF ZINC SULl'IIIIJE Ml NERALS

BY EMISSION SPECTROSCOPY ( 0o)

167

Element Zinc Marmatite Sphalerite Sphalerite Wurtz1te Sulphide (N. S .W.) (Okla.) (Spain) (Utah)

Ag <0.0005 0.0010 0.0005 <0.0005 0.0030

Al 0.0200 <0.0050 <0.0050 <0.0050 0.2000

As ,0.1000 <0.1000 •0.1000 <0.1000 <0.1000

B 0.0005 0.0005 0.0005 0.0005 0.0005

Ba <0.1000 ,0.1000 ·0.1000 ·0.1000 <0.1000

Be <0.0002 <0.0002 ·0.0002 ,0.0002 <0.0002

Bi •0.0010 <0.0010 •0.0010 •0.0010 <0.0010

Ca 0.0200 0.0200 0.0200 0.0200 0.0200

Cd 0,0010 0.2000 0.6000 0.0700 0.2000

Co 0.0600 0.0050 ,0.0010 ·0.0010 ·0.0010

Cr 0.0005 ·0.0005 ·0.0005 ·0.0005 ·0.0005

Cu 0.0010 2.0000 0.1000 0.0100 0.0600

Fe 0.0600 3.0000 0.0800 0.0300 0.0600

K <0.1000 ·0.1000 <0.1000 <0.1000 <0.1000

Li <0.0050 <0.0050 <0.0050 <0.0050 <0.0050

Mg 0.0200 <0.0030 <0.0030 <0.0030 ,0.0030

Mo <0.0010 0.0020 <0.0010 <0.0010 0.0010

Mn <0.0010 0.1600 <0.0010 <0.0010 0.0100

Ni <0.0010 <0.0010 <0.0010 <0.0010 <0.0010 p <0.1000 ,0.1000 <0.1000 <0.1000 <0.1000

Pb <0.0050 0.6000 0.0050 0.0050 1.2000

Sb ,0.0050 <0.0050 ,0.0050 <0.00~0 0.6000

Si 0.2000 0.2000 o. 3000 0.1000 4.0000

Sn <0.0007 0.0007 0.0007 0.0007 0.0007

Ti <0.0010 •0.0010 ,0.0010 <0.0010 0.0050

V •'.0.0010 •0.0010 ,0.0010 ,0.0010 •0.0010

Zr <0.0030 <0.0030 ·0.0030 <0.0030 <0.0030

Zn The maJor component ls Zinc.

168

3.3.2 LEACHING OF ZINC SULPHIDES BY THIOBACILLUS FERROOXIDANS

ThiobaaiZZus ferrooxidans is well-known for its ability to

degrade zinc sulphide (Zimmerley et al., 1958; Torma et al., 1972;

Gormely et al., 1975). The aim of this experiment was to examine the

the leachability of different zinc sulphides by a strain of ThiobaciZ­

Zus ferrooxidans isolated from a mine drainage at Mount Lyell. The

elemental compositions,, of the five zinc sulphides tested are given

in Tables 3.3.1 and 3.3.2. Semiquantitative analysis of these

minerals revealed that arsenic, cadmium, lead, iron and silicon were

present as impurities in them.

Quantitative analyses of these five minerals for

principal metals like zinc, iron, lead, copper and nickel as shown

in Table 3.3.2, which reveals that synthetic zinc sulphide, and

museum grade specimens of sphalerites from Spain and Oklahoma, are

almost identical in their mineral compositions, except that the latter

contained a higher iron content. Mannatite from Broken Hill, N.S.W.,

contained 12.2% iron and had a high percentage (1.35%) of lead

associated with it. The lowest zinc content was that of wurtzite from

Utah (46.9%). This sample also had a high percentage of lead (5.6%)

associated with it.

The experiment was performed in one litre flasks containing 190

ml of the 9K basal salt medium of Silverman & Lundgren (1959). The

experimental procedure as described earlier was followed (see section

3. 3 ) .· ThiobaciUus ferrooxidans grown on ferrous sulphate as the

sole source of energy was used as inoculum. Leaching results are

shown in Fig. 3.3.2. The curves in Fig. 3.3.2 were obtained from the

net value of microbial leaching, i.e., the differences between the zinc

concentrations in the inoculated flask and in the control flasks.

169

TABLE 3.3.2

CONTENT OF PRINCIPAL METALS

ZINC IRON LEAD COPPER NICKEL

ZINC SULPHIDE 66.2 0.01 0.05 0.005 0.05 (Synthetic)

MARMATITE 50.4 12.2 1.35 0.48 0.05 (Broken Hill,

N.S.W.)

SPHALERITE. 67.7 0.3 0.05 0.03 0.05 (Okla.)

SPHALERITE 65. 7 0.2 0.05 0.005 0.05 (Spain)

WURTZITE 46.9 0.7 5.6 0.12 0.05 (Utah)

Percentage composition (w/w) by atomic absorption

spectroscopy.

Theoretical zinc content of ZnS = 67.1 %

170

Leaahing of Marmatite.

Leaching of this mixed sulphide by Thiobacillus ferrooxidans was

revealed by a progressive increase in the concentration of solubilised

zinc ions in the inoculated flasks. A maximum concentration of 1,920

p.p.m. was recovered with 145 p.p.m. in control. The lag period was

approximately 2 - 2.5 days; zinc release then occurred up to the 12th

day of experiment, after which its rate of release decelerated. By

the 7th day of the experiment, precipitation had occurred and the

degree of precipitation was such that brown particles of marmatite

were hardly visible; they were embedded in the hard reddish brown

precipitate.

During these experiments endeavours were made to monitor the

soluble ferrous and ferric ions in the flasks. It was folllld that,

whilst the soluble ferrous content was negligible at all stages, the

soluble ferric contents progressively increased up to the 7th day

(see Fig. 3.3.2). By the 8th day it had begllll a progressive decrease

which was accompanied by the formation of the heavy precipitate

referred to above. The maximum concentration of ferric ions at 7th day

of experimentation was 80 p.p.m. (average of three experiments).

Insolubilisation of virtually all the ferric ion in the form of

precipitate, commonly reported as jarosite by other authors (Dllllcan

& Walden, 1972), was coincident with the cessation of zinc ion

solubilisation.

Leaahing of Synthetia Zina Sulphide.

Synthetic zinc sulphide was also leached by Thiobacillus ferroo­

xidans grown on ferrous sulphate as energy source. The shape of the

curve was very similar to the marmatite leaching curve. The lag was

also 2-2.5 days and after the 12th day of the experiment the zinc solu­

bilisation decelerated. During this leaching period a maximum

2000

1500

. ~ 1000 p..

p..

u z H N

SOO

0

171

----------------,,.--------,--------,100

I al I

' I

b

I

I I

I

~ I

I I

/,Q. / \

~ \ / \

D

'

\

I I • ~r;;,_,...

\ \ \

\

\ \

'o \

\

\ R: <1 --I> 0 <1- -l>-<1 - ....- .. - .. I>- \ .,._ -_..,..-~ \

\ 0

/ j ~ .,,.,,< ,b /• ------ ... \ ___ _....._ .- •--...

75

so

25

/ ~ ··----= i- • ·- -· -·--?9~ -•-•- 0-..- --o--O a::.;;--;.;;.--;.;;.,;:,..__ __ _;,•• ____ • _ _,1., _______ _._ _______ ..i,.. _______ ,.. 0

0 5 10

TIME (Days)

15

FIG. 3.3.2. LEACHING OF ZINC SULPHIDE MINERALS BY

THIOBACILLUS FERROOXIDANS

(. )- Marrnatite ( 0) - Synthetic zinc sulphide

c• )- Sphalerite (Spain) ( 6) - Sphalerite (Oklahoma)

(. )- Wurtzite ( D ) - Ferric iron (Total iron) (From rnarrnatite)

20

. E

0.. . 0..

z 0 0::: H

172

concentration of 2,000 p.p.m. zinc ion was recovered with 210 p.p.m.

in the control.

Leaching of Museum Grade Zinc SuZphid,e Minero.ls.

Comparatively pure museum grade specimens of zinc sulphide mine­

rals in the form of sphalerite were also leached by ThiobaciZZus ferro­

oxidans. Their leaching rate was slower than that of marmatite or the

relatively pure synthetic zinc sulphide. Wurtzite, a hexagonal form of

zinc sulphide was, however, very recalcitrant to leaching by the organism.

Maximum concentrations of 467 p.p.m. and 405 p.p.m. of zinc were obta­

ined from sphalerite samples of Oklahoma and Spanish origins, respect­

ively. The concentrations of solubilised zinc ions in the controls

were 30 and 32 p.p.m. The maximum concentration of solubilised zinc

from the wurtzite sample was 125 p.p.m. with 15 p.p.m. in the control.

Discussion.

As previously stated, zinc sulphide is among the earliest mineral

sulphides examined for microbial leachability. Rudolf & Helbronner

(1922) were the first to apply bacterial leaching to the extraction of

zinc from zinc blende. Subsequently, numerous studies on the microbial

leaching of zinc sulphide by ThiobaciZZus ferrooxida:ns have been made,

including those of Ivanov et al.(1961), Torma et al.(1970), Gormely et

al.(1975). It is now well documented that zinc sulphide concentrates

are amenable to microbial degradation. Most of the information has

been obtained from studies with marmatite as the zinc mineral, which

contains appreciable amotmts of iron in it. The so called 'indirect

mechanism' (which limits the role of the iron oxidising bacteria to

ferrous oxidation, with the ferric ions produced acting as the leachi­

ng agents) had been considered as the main leaching mechanism during

earlier investigations. Generally all natural sulphides contain iron

as one of the impurities, and according to the indirect mechanism

173

of microbial leaching this iron can be constantly recycled.

The stationary phases reached after leaching of marmatite and

synthetic zinc sulphide in the current investigation preswnably resul­

ted from either some nutrient or other cultural requirement becoming

limiting. The main energy sources for the bacteria in the case of

marmatite were the sulphide and ferrous components. At stationary phase

ample amounts of these components were still present in the medium and

available to the bacteria. It is conceivable that oxygen and carbon

dioxide, which are important nutrients for bacteria, may have become

limiting. However, in any consideration of limiting factors, it must

be born in mind that one of the most important factors in any solid­

bacterial interaction is the availability of surfaces to the bacteria;

such access is improved by contact between the solid substrate and the

bacterial cells. Whether such contact changes the bacterial response

towards the solid substrate is still unknown but a number of reports

exist in which the importance of contact between solid surface and

bacteria has been suggested by the relationship between the rate of

oxidation of solid substrate (viz., sulphur) and shaking speed.

It has been observed, for example, that an increased shaking rate

sometimes decreases the oxidation rate of elemental sulphur by Thio­

bacillus thiooxidans ( Starkey et al., 1956). Reaction rate can also

be limited by the precipitation of materials on surfaces, which inact­

ivate the contact points between the bacteria and solid substrate.

The situation would simulate substrate limitation.

The formation of a complex ferric precipitate (jarosite) during

microbial leaching has been observed previously by many investigators

(Duncan & Walden, 1972; Torma & Legault, 1973; Rossi, 1974). The

current results indicate that, after the 7th day, the concentration

of soluble ferric ions in the medium starts decreasing and at the 14th

174

day almost all the ferric ions are precipitated out. This coincides

with the cessation of zinc release from marmatite. Thus it is most

likely that when marmatite is the zinc source the formation of ferric

precipitates could inhibit the rate of leaching by blocking the contact

between the bacteria and the mineral particles. Similar findings have

been reported by a number of workers, who have shown that by regrinding

the mineral particles more of the mineral can be extracted (Torma,

1977). Dtmcan & Walden (1972) demonstrated that during bacterial

leaching of marmatitic zinc sulphide, iron concentration was increased

for the first 100 hours of the process, after which iron was precipi-

tated out from the medium with a concomitant depletion of ammonium ion

from the medium.

The ability of Thiobacillus ferTooxidans to degrade pure, synth­

tic minerals has been demonstrated by many workers including Torma

(1971). In this study the aim was to compare, under identical condit­

ions ( e.g. bacterial concentrations, pulp density, temperature etc.)

the leaching pattern of relatively pure, synthetic zinc sulphide with

that of other natural zinc sulphide minerals. The leaching pattern

was surprisingly similar to that of marmatite considering that the

synthetic zinc sulphide had a low iron contentbut (presumably in

compensation) was very finely divided and hence presented a greater

surface area (w/w) to the bacterium than did the marmatite.

One of the most striking findings of this test was the relatively

slow leaching rates of the two natural museum grade sphalerite minerals

and the almost zero response of the organism to the wurtzite. An

early report by Trussell et al. (1964) also recorded the low leaching

rates of sphalerites by Thiobacillus ferrooxidans. They found that

almost 100% of the marmatite but only 4-9% of the sphalerite was

leached. The current experiment confirms that different sulphides of

the same metal do not respond identically to bacterial leaching.

175

That structural differences in the sulphides affect their leachability

is indicated by the fact that wurtzite, which belongs to a crystal

system which is different from that of sphalerite, is virtually resis-

tant to these bacteria. No previous reports on

ble for comparison with the current findings.

wurtzite are availa-

Iron contents of these minerals seemed to be overlapped by the

structural influence in determining the leaching behaviour. Thus,

although wurtzite contains a significant amotmt of iron in its lattice

(0.7%), it was not leached readily. The higher content of lead (5.6%)

present may be another inhibitory factor affecting the leachability

of wurtzite by these bacteria.

176

3.3.3 LEACHING OF ZINC SULPHIDES BY THIOBACILLVS THIOOXIDANS

As previously mentioned, ThiobaciZZus thiooxidans is generally

not considered a suitable leaching bacterium; consequently little work

has been reported previously on the leaching behaviour of this Thio­

baciZZus species. In the current experiment, the inoculum was

prepared as follows:

ThiobaciZZus thiooxidans (BJR-KOl) was grown with elemental

sulphur as the sole energy source, at 30°C, pH 2.5, in a shake flask

culture; the residual sulphur was removed from the suspension by fil­

tration through Whatman No. 1 filter paper and the filtrate centrifu­

ged at 10,000. g for 15 minutes; the cell pellet, suspended in 9K

basal salt solution of Silvennan & Llllldgren (1959) was used to inocu­

late the growth flasks at a level of approximately 107cells per ml.

The amollllts of zinc released from the various zinc sulphides are

illustrated in Fig. 3.3.3.

Leaching of Marmatite.

ThiobaciZZus thiooxidans released a total of 1,250 p.p.m. of

zinc from the marmatite during 18 days of incubation. Ferrous ion

concentration increased as the dissolution of marmatite proceeded.

The maximum concentration of ferrous ion was 395 p.p.m. at the maximum

total soluble iron concentration of 410 p.p.m.; the difference, which

was presumably a measure of ferric ion, is negligible and therefore

indicates the relatively pure state of the ThiobaciZZus thiooxidans in

terms of its freedom from iron oxidising bacteria. In contrast to the

tests with ThiobaciZZus ferrooxidans, no precipitati0n occurred in the

flasks, and the mineral particles were at all times clearly visible.

20

15

. e . p..

p..

0 0 10 .... ><

u z H N

5

17 7.

--------r--------,--------,.-------1100

o--°--0 1---------+--------+------....,....~~,..----------175

0 5 10

TME (Days)

. •

----0

V V

~ J-•---Y+-• 1--V--- .. - - . ------

15

FIG. 3.3.3. LEACHING OF ZINC SULPHIDE MINERALS BY

( • ) - Marrnati te

THIOBACILLUS THIOOXIDANS

( o) - Synthetic zinc sulphide

( 6.) - Sphaleri te (Oklahoma)

e . p.. . p..

50 ~ ><

z 0 IX H

U) :::i 0 IX IX tJ.l

25 i:.r..

20

(~) - Sphaleri te (Spain)

( • )- Wurtzite (0)- Ferrous iron.(From marmatite)

178

Leaching of Synthetic Zinc Sulphide.

The leaching curve for the relatively pure zinc sulphide was

steeper than that for marmatite and the maximum concentration of

leached zinc, 1,550 p.p.m. was greater than that obtained from marmat­

ite (1,250 p.p.m.). The leaching rate had decelerated by the 15th

day and the leaching had ceased by the 18th day.

Leaching of Museum Grade Zinc Sulphide Minerals.

The museum grade specimens of sphalerite were not as effectively

leached as were the marmatite and synthetic zinc sulphide. The maximum

concentrations of zinc ion released from the Spanish and Oklahoma

sphalerites were 180 and 230 p.p.m. respectively. Wurtzite was

particularly recalcitrant to the action of Thiobacillus thiooxidans &

only 100 p.p.m. of zinc were solubilised.

The viable bacterial population at the end of experiments with

the two sphalerites and the wurtzite was 2 . 10 8 organisms per ml,

which was about one tenth of the population obtained after growth on

the marmatite and zinc sulphide samples.

Discussion.

The results indicate that strain BJR-KOl is capable of degrading

sulphide minerals. It released 1,250 p.p.m. zinc from marmatite into

solution in 18 days, compared with the 1,920 p.p.m. released by

Thiobacillus ferrooxidans under similar conditions. Therefore, the

marmatite leaching activity of Thiobacillus thiooxidans is about

65% of that of Thiobacillus ferrooxidans for leaching marmatite.

Since sulphur oxidisers can oxidise only the sulphur moiety in

the crystal lattice, it can be assumed that all metal released from

sulphides by Thiobacillus thiooxidans is due to direct bacterial

activity. In the case of iron oxidisers, such as Thiobacillus

179

ferrooxidans, which oxidise ferrous ions preferentially over sulphide

ions, the leaching of mannatite cannot be considered as being entirely

due to direct bacterial oxidation, since the ferric product of the

ferrous oxidation can serve as a good leaching agent and thus contrib­

ute a degree of chemical leaching to the process. If we were to

consider that the sulphur oxidising capacities of ThiobaaiZZus

thiooxidans and ThiobaaiZZus ferrooxidans were equal, then the diff­

rence between the maximum concentrations of zinc ions released by

ThiobaaiZZus thiooxidans (1,250 p.p.m.) and ThiobaaiZZus ferrooxidans

(1,920 p.p.m.) would be due to ferrous/ferric oxidative activity,

i.e., due to chemical leaching. However, such a simplistic concept

cannot be proposed when it is known that ThiobaaiZZus ferrooxidans

oxidises ferrous ions preferentially over sulphide ions. Account

must also be taken of the fact that the former reaction involves a

one electron transfer and the second (for complete oxidation to

suphate) an eight electron transfer._ Moreover, the relative availab­

ilities and . rates of oxidation of ferrous and sulphide ions would

be extremely significant factors in the relative contributions of the

direct and indirect mechanism to leaching by ThiobaaiZZus ferrooxidans.

Vanselow (1976) has studied the relative contributions of the two

processes in detail by comparing rates of leaching of covellite

(CuS) by one strain of ThiobaaiZZus ferrooxidans in the presence and

absence of ferrous ions. A similar observation was made in the

current study (see section 3.3.6). Nevertheless it is worthy of n-0te

that ThiobaaiZZus feYTooxidans leached 35% more zinc from marmatite

than did ThiobaaiZZus thiooxidans and that the increase could perhaps

relate to its iron oxidation ability.

The iron (mainly in the ferrous form) released from mannatite

remained in solution and reached a maximum concentration of 415 p.p.m

(7.4 rnM) compared with a maximum soluble zinc concentration of

180

1,250 p.p.m. (19.12 mM), i.e., soluble iron: soluble zinc= 1:2.6.

The iron content of the original marmatite was 12.2 % and zinc content

50.4% (Table 3.3.2) which would give marmatite an empirical formula

of zn3 • 5Fe s5 • Therefore the molar ratio of zinc and iron is 4.1 : 1.

If metal release of iron and zinc had been proportional, they would

have maintained the same stoichiometric ratio in the solution, i.e.,

1:4.1 rather than the 1:2.6 observed.

The difference between the theoretical and observed stoichiometry

represents 37% deviation from proportional leaching of iron and zinc

at the concentration in which they occurred in the marmatite sample.

Conclusions regarding this deviation are difficult to draw. However,

a situation whereby the iron atoms are solubilised and extracted to

a greater proportional extent than are the zinc ions would leave

'holes' inside the crystal lattice and this could facilitate more

leaching. If this were so then marmatite should leach more readily

than synthetic zinc sulphide. The fact that it did not could however

relate to differences in the specific surface areas of the two

substrates. This variable was removed in the definitive experiments

in section 3.3.5.

The activity of Thioba.ciZZus thiooxidans towards marmatite had

virtually ceased by the 15th day. Metal toxicity and/or oxygen

limitations are among the possible causes of cessation of activity;

further work would be required before conclusions regarding termination

of the leaching process could be made.

A maximum concentration of 1,550 p.p.m. zinc was released from

synthetic zinc sulphide which was 19.4% greater than marmatite. A

high microbial population ( 5 • 109 bacteria per ml) after the end of

the experiment with synthetic zinc sulphide indicated the acceptiblity

of synthetic zinc sulphide as a suitable substrate for ThiobaciZZus

thiooxida:ns. A microbial population of 3.5 . 109 cells per ml was

181

recovered in the culture growing with mannatite.

In common with the ThiobaciZZus ferrooxidans tested, ThiobaciZZus

thiooxidans was unable to leach sphalerites or wurtzite to any

significant extent. The reason for their low leachability by these

bacteria may be due to a very stable crystal structure, or to a

stability induced by the natural fonnation of minerals; the data

collected are however an insufficient basis for more than very

tentative speculation.

182

3.3.4 LEACHING OF ZINC SULPHIDE BY THIOBACILLUS THIOPARUS

The study with ThiobaciZZus thioparus (BJR-451) was carried out

at pH 5.5 in shake flasks ( one litre Erlenmeyer flasks) containing

190 ml of the basal salt mediun of Vishniac & Santer (1957). After

sterilising the medium at 15 psi for 15 minutes, 2 g of zinc sulphide

minerals previously sterilised with propylene oxide gas for 12-18

hours, were aseptically added. The bacterial inoculum was prepared

as follows:

ThiobaciZZus thioparus (BJR-451) was cultivated in a chemostat

with thiosulphate as the growth limiting substrate, at a dilution rate

of 0.1- 1 h; the cells were collected after centrifugation at 10,000 g

for 15 minutes, washed with and resuspended in sterilised Vishniac

& Santer medium (1957). 10 ml of this inoculum was introduced into

each shake flask, so that the final concentration was approximately

107 cells per ml. The flasks were shaken in a reciprocal shaker (at

5 cm stroke and 90 ± 2 strokes per minute) and samples withdrawn and

analysed as described in section 3.3.2 and 3.3.3. The results are

illustrated in Fig. 3.3.4.

Leaching of Marmatite.

ThiolxiciZZus thioparus leached marmatite at a considerably

slower rate than did the acidophilic bacteria ThiolxiciZZus ferrooxi­

dans and ThiolxiciZZus thiooxidans. The maximum soluble zinc concen­

tration recorded was 90 p.p.m. after 19 days' incubation. The

bacterial cell collllt at the end of of experiment was 3 . 108 bacteria

per ml of which 90% viable.

The ferrous ion concentration was less than 0.1 p.p.m. and the

total soluble iron was 3 p.p.m.

20

15

. 10 e 0..

0..

0 .-I

><

u z 1-1 N 5

0

0 5 10

TIME (Days)

15

183

_. ..

FIG. 3.3.4. LEACHING OF ZINC SULPHIDE MINERALS BY

THIOBACILLUS THIOPARUS

( • )- Marmatite

(A)- Sphaleri te (Spain)

( • )- Wurtzite

( o )- Synthetic zinc sulphide

( 6 )- Sphalerite (Oklahoma)

(+)- Control

20

184

Leaching of Synthetic Zinc sulphide.

The leachability of synthetic zinc sulphide was greater than

that of marmatite, the maximum recorded concentration of zinc ion in

the medium being 167 p .p .m. The lag phase was shorter and the leaching

curve was steeper than in the case of marmatite. The viable population

at the end of the experiment was between 3 and 4 . 108cells per ml,

a comparable figure to that recorded in the marmatite culture.

Leaching of Natural Zinc SUlphid,e Minerals.

The action of Thiobacillus thioparus on natural sphalerite was

not so significant. The leaching of two samples of sphalerite was

relatively slow, and the maximum recorded concentration of zinc ion

was 60 and 43 p.p.m. for Spanish and Oklahoma variety respectively.

No significant degree of leaching over that recorded in the control was

observed when the zinc sulphide was in the form of wurtzite, the

maximum recorded concentration of soluble zinc being 25 p.p.m.

Discussion.

The leaching capabilities of non-acidophilic members of the

genus Thio'bacillus have not been examined previously to any signifi­

cant extent. The results obtained from current experiments suggest

that the non-acidophilic Thiobacillus thioparus is relatively slow

in its leaching activity towards the various zinc sulphide minerals

studied. However, the pattern of leaching which has emerged from the

current study is very similar to the one observed on the study of the

leaching of these minerals by Thiobacillus thiooxidans (see

185

section 3.3.3.). Synthetic zinc sulphide was leached at the fastest

rate followed by marmatite, and the natural sphalerites were partially

recalcitrant, whilst wurtzite, was almost completely recalcitrant.

Whenever marmatite is leached, its iron component would be expected

also to be leached out, as it was when marmatite was leached by the

acidophilic ThiobaciZZi. Negligible amounts of soluble iron were

recorded however when marmatite was leached by ThiobaciZZus thio­

parus. The maximum being less than 3 p.p.m.It is believed that the

ferrous iron released at pH 5.5 was converted to insoluble ferric

complexes, such reactions being favoured at that pH. This phenomenon

may also have contributed to the relatively low leachability of marma­

tite (compared with the leachability of synthetic zinc sulphide)

by passivating the surfaces of the zinc sulphide particles with

ferric hydroxide precipitates. However, a valid comparison can only

be made when samples of equal specific surface area are presented to the

organisms ( see section 3.3.5). The increase in viable counts of

ThiobaciZZus thioparus on synthetic zinc sulphide and marmatite from

107 to 3-4.108 bacteria per ml indicates a reasonable growth activity

of this member of the ThiobaciZZi on sulphide minerals. Whilst the

relatively low concentrations of solubilised zinc in the cultures

indicate that this species is not a good leaching agent, it must be

kept in mind that at pH 5.5 the solubility of metals is drastically

affected; equlibriurn shifts towards hydroxide formation can occur and

render the solubilized metal once more insoluble; in other words,

a conversion of metal sulphide to metal hydroxide could occur. By

analogy, when galena (PbS) is oxidised microbiologically, the concen­

tration of lead in solution does not change significantly, since the

final product of bacterial oxidation, PbS04,is also practically

insoluble (Tomizuka, 1976). It is not unreasonable to assume

186

therefore that, under relatively high-pH conditions, a correct measure

of leaching of any metal sulphide cannot be obtained by determining

the soluble metal concentration alone. In order to have a reasonable

assessmentof mineral leaching under such conditions, other products

of bacterial oxidation, such as sulphate, should also be measured

and the presence of any new insoluble mineral in the medium should

be checked by X-ray crystallographic methods.

187

TABLE 3.3.3

SPECIFIC SURFACE AREA OF * ZINC SULPHIDE MINERALS

MINERAL SURFACE AREA (m2/g)

ZINC SULPHIDE 30.00 (Synthetic)

MARMATITE 9.14 (Broken Hill, N.S.W.)

SPHALERITE (Okla.) 7.60

SPHALERITE (Spain) 5.70

WURTZITE (Utah) 6.40

* Determined by B.E.T. Method.

188

3.3.5 INFLUENCE OF STRUCTURE OF ZINC SULPHIDE ON MICROBIAL

LEACHING BY THIOBACILLI

From the studies described earlier in this section it was evident

that our three strains of Thiobacillus species each leached the

different zinc sulphide minerals at different rates and to different

extents. It was however realized that (a) not only were two different

crystallographic forms, i.e., sphalerite (cubic) and wurtzite (hexa­

gonal), represented in the zinc sulphide samples but (b) these samples

probably had different specific surface areas. In the current

experiment the second variable was removed in order to investigate

the effect of the first more definitely.

In the preliminary experiments equal pulp densities were used,

but since in solid-substrate microbiology it is the surface area which

effectively determines the substrate concentration, a comparison was

made of the surface area of these zinc sulphide samples. The speci­

fic surface areas of the ground minerals used in the previous experi­

ment were determined by the B.E.T. method (Brunauer, Eramet & Teller,

1938), and are recorded in Table 3.3.3. The synthetic zinc sulphide

was relatively fine, with a specific area of [ 30 m2/g], 3 to 5 times

that of the other minerals. Mineral weights were adjusted to provide

equal surface area of 100 m2/litre in the present experiment.

Thiobaaillus ferrooxidans and Thiobacillus thiooxidans cultures

were first acclimatised to the individual mineral substrates in 9K

basal salt solution (Silverman & Lundgren, 1959). In the case of

wurtzite, where growth rate was poor, these thiobacilli were grown

with the mineral plus elemental sulphur as the substrate.

Because of its relatively poor leaching capabilities, the non­

acidophilic, Thiobacillus thioparus, bacterium was acclimatised only

to synthetic zinc sulphide. Acclimatisation in all cases was conducted

189

in shake flasks at 30°C for 4 - 5 weeks, and the cells harvested by

centrifugation of the filtrate after filtration through Whatman No.I

filter paper. ThiobaciZZus ferrooxidans and ThiobaciZZus thiooxidans

were then suspended in the 9K basal salt medium (pH 2.5) of Silverman

and Lundgren (1959), and ThiobaciZZus thioparus suspended in the basal

salt medium of Vishniac and Santer (1957). The cell suspensions

served as inocula for the leaching experiments.

For the leaching experiments, an appropriate weight of mineral,

providing a surface area of 20 m2 and previously sterilised by

propylene oxide, and 190 ml of sterile basal salt medium were added

aseptically to sterile Erlenmeyer flasks of one litre capacity. The

basal salt media used were those described above for suspension of

inocula. 10 ml of inoculum was added to each flask to give an initial

level of 107 bacteria per ml.

The flasks were incubated at 30°C on a reciprocating shaker

(5 cm stroke; 90 + 2 strokes per minute). The pH was manually adjusted

to its initial level ( 2.5 for ThiobaciZZus thiooxidans, ThiobaciZZus

fePPooxidans; 5.5 for ThiobaciZZus thioparus) at time intervals of

12 hours. At appropriate times (after every 24 hours) the flasks were

removed from the shaker and allowed to stand for 20-30 minutes at 30°C.

5 ml of mineral-free culture were withdrawn aseptically from each

flask, and replaced with appropriate sterile medium. The samples

withdrawn were centrifuged at 10,000 g for 10 minutes and the

supernatant was analysed for solubilised zinc, total iron and ferrous

iron. Results are illustrated in Figs. 3.3.5 - 3.3.9 and summarised

in Table 3.3.4.

Comparative Leaching of MaPmatite.

Fig. 3.3.5 represents the leaching of marmatite by ThiobaciZZus

ferrooxidans and ThiobaciZZus thiooxidans species. Under identical

20

15

e a: a: 0 0 ,-)( 10

(J C: ·-~

5

0

I • • 1/ -----· • • /i / :

190

100

·--- . I----------+--------•- --- __ , - -· - - - ------- ------ ·- ·- _ -· -- _ ------ 7 5

0

•

•

---o----o) __ _,.. _,--i----o-o .,,

(b i/if 7f-- ------ ---- -----------1 so a

0 ,

/ : ~,.__.__. __ . 0 '

C: c,,

' . •/t ~ /' ~

{_-/-- I t--------~ 25 .;..• 0 / -- - t---------- ---- -- ~

• I

•0 • I • ..'--+-_ .. :...----._ ....... ---~~t .__.. ~-+-..- • •

I 5 10

Days

15

FIG. 3.3.5. MICROBIOLOGICAL LEACHING OF MARMATITE

Zinc by: ( •) - Thiobaci l lus f er>r>ooxidans

( O) - Thiobaci l lus thiooxidans

(+)- Control

( .&)- Ferrous iron. (From T. thiooxidans leaching).

20

0

e q. a.

0 .....

60

45

)( 30

CJ C: ·-N

15

0 0

191

•

5 10

Days

15

.--­.~

··--·-20

FIG. 3.3.6. MICROBIOLOGICAL LEACHING OF SYNTHETIC ZINC SULPHIDE

Zinc by: ( •) -Thiobaci Uus ferrooxidans

( 0 ) -Thiobaci llus thiooxidans

( +) - Control.

192

conditions of pH, temperature and initial specific surface area,

marmatite leaching behaviour was very similar to that observed before.

Marmatite was more rapidly leached by ThiobaciZZus ferrooxidans than

by ThiobaciZZus thiooxidans . The ThiobaciZZus ferrooxidans'lag

period was comparatively shorter and the maximum concentration of zinc

ions found in solution was 1,875 p.p.m. after 11 days' incubation;

the soluble .Fe2 + & Fe 3+concentrations were too low to be measured but

ferric salt precipitation was observed. The viable ThiobaciZZus

ferrooxidans at the termination of the experiment was found within

the range of 3 - 4. 109 cells per ml and its maximum leaching rate

of zinc was 230 mg/(i.day).

ThiobaciZZus thiooxidans leached marmatite at a maximum rate of

130 mg/(i.day) and a maximum concentration of 1,270 p.p.m. zinc

was obtained at the end of the experiment; the iron released was

substantially ferrous and its maximum concentration at the end of the

experiment was 425 p.p.m.

The leachability of marmatite at the higher pH value of 5.5 by

ThiobaciZZus thioparus was comparatively slow and the maximum concen­

tration of leached zinc was 80 p.p.m. The rate of leaching was

about 4.9 mg/(i.day). Negligible amounts of soluble iron were found

in the medium. The viable counts of ThiobaciZZus thioparus at the

end of the experiment were in the range of 2 - 3. 108 cells per ml.

Comparative Leaching of Synthetic Zinc Sulphide.

Leaching of synthetic zinc sulphide by ThiobaciZZus ferrooxidans

and ThiobaciZZus thiooxidans is shown in Fig. 3.3.6. In contrast to

marmatite, ThiobaciZZus thiooxidans leached zinc sulphide at a

faster rate than did ThiobaciZZus ferrooxidans. The maximum concen­

trations of solubilised zinc were 530 p.p.m. and 385 p.p.m. in the

ThiobaciZZus thiooxidans & ThiobaciZZus ferrooxidans cultures

20

15

I 0 10

5

0 0

193

• ---· •

--+-•------=.~--..

5 10

Days

15

FIG. 3.3.7. MICROBIOLOGICAL LEACHING OF SPl~LERITE (SPAIN).

Zinc by: ( •) - Thiobaci Uus f errooxidans

( 0 ) - Thiobaci Z Zus thiooxidans

(+)- Control.

20

194

respectively and the solubilisation rates were 40 and 24 mg/(i.day)

respectively. The soluble Fe2 +& Fe 3+ iron concentrations were too

low to be measured. The viable bacterial populations of Thiolxicillus

ferrooxidans and Thiobacillus thiooxidans at the end of the experiment

were 2. 109 and 3-4 . 10 9 cells per ml respectively. Total zinc ions

solubilised by Thiolxicillus thioparus were 59.5 p.p.m. and only

traces of iron ions could be detected. The maximum rate of zinc

solubilisation with this species was only 6.9 mg/(i.day) and at the

end of the experiment only 2-3 . 10 8 cells per ml were recorded.

Comparative Leaching of Natural Zinc Sulphide Minerals.

Leaching curves for sphalerite are shown in Figs. 3.3.7 and

3.3.8. Both sphalerites responded similarly towards the acidophiles,

Thiolxicillus ferrooxidans and Thiobacillus thiooxidans in that the

maximum concentration of zinc ions liberated by each organism was

less than that released from marmatite or synthetic zinc sulphide.

The higher purity of the Spanish .sphalerite, compared with the

Oklahoma sphalerite, was presumed to be the reason why it was less

degradable by either acidophilic Thiobacillus species than was the

latter variety.

The maximum solubilisation rate of zinc from the Oklahoma and

Spanish varieties by Thiobacillus ferrooxidans, was 22 and 15 mg/(i.day),

respectively (Table 3.3.4). On the other hand, Thiolxicillus thio­

oxidans leached both sphalerites at an almost equal rate of 12 - 13

mg/(i.day).

Leaching with Thiobacillus thioparus produced the relatively

meagre soluble zinc ion concentration of 27-31 p.p.m. after a maximum

rate of solubilisation of 3.1 - 3.9 mg/(i.day). With such a small .

amotmt of zinc ions in solution, it was difficult to ascertain which,

if either, sphalerite variety was more amenable to degradation by

4

3

e· 0'. a: 0 0 ... 2 )(

CJ C: ·-"'

1

0 0

195

5 10

Days

/. /.

•

/ /0

JI·--· •

15

FIG. 3.3.8. MICROBIOLOGICAL LEACHING OF SPHALERITE (OKLAHOMA)

Zinc by: ( •) - Thiobaci Z Zus feY'Y'Ooxidczns

( 0 ) - Thiobaci Z Zus thiooxidans

C•)- Control

20

196

Thiobacillus thioparu.s. In the presence of either sphalerite, the

viable counts of Thiobacillus thioparus were 1.5 . 108 cells per ml

at the end of the experiment.

Comparative Leaching of Natural Wurtzite.

The result of the comparative study of wurtzite leaching by

three Thiobacilli are shown in Fig. 3.3.9 and Table 3.3.4. The maxi­

mum concentration of zinc in the Thiobacillus thiooxidans and Thio­

!Jacillus ferrooxidans flasks were 85 and 102 p.p.m. respectively and

the maximum leaching rates were 6.5 and 9.0 mg/(t.day) respectively.

The wurtzite sample containing 0.7% (w/w) iron as an impurity

(Table 3.3.2); a total of 25 p.p.m. of this was released as ferrous

iron into the solution by Thiobacillus .thiooxida:ns but only a total

of 2.5 p.p.m. iron could be found in the soluble form in the iron

oxidising Thiobacillus ferrooxida:ns culture. The viable count of

bacteria at the end of the experiment was within the range of 6. 107

cells per ml.

Negligible amounts of zinc ions were solublised from wurtzite

by the non-acidophilic species, Thio!Jacillus thioparus, at the end

of the experiment only slightly more than 3. 10 7 cells per ml were

found.

Discussion.

The results of this experiment suggest that the leaching

capability of Thiobacilli varies from species to species and that

both the structure and the composition of substrates may be factors

governing the leachability of the minerals.

The results demonstrate that the non-acidophile, Thio!Jacillus

thioparus (BJR-451), is the least active of the three species studied

in leaching zinc sulphides. None of the minerals studied, except

197

...

50l--------+-------+-------+-------t

e ~100--------+--~ u C: .... ...

~-0--+--0--0

50 1----------+

o o

·----•--ir--•--•---· .,----

5

.---10

Dav• 15

FIG. 3.3.9. MICROBIOLOGICAL LEACHING OF WURTZITE

Zinc by: ( • ) - Thiobaci Z Zus ferrooxidans

( o ) - Thiobaci Z Zus thiooxidans

(+)- Control

20

198

TABLE 3.3.4

LEACHING OF ZINC SULPHIDE MINERALS BY THIOBACILLI

Zinc Marmatite Sphalerite Sphalerite Wurtzite Sulphide (NSW) (Okla.) (Spain) (Utah)

Thiobaai l lus ferrooxidans # FeT (ppm) <1.0 3.5 1.1 <1.0 2.5

Fe 2+ (ppm)* 0.1 <0.1 <0.1 <1.0 <0.1

zn 2+solubil-isation Rate 24.0 230.0 22.0 15.0 9.0 (mg/Jl,/day)

Max. Cone.

Zn 2+ (ppm) 385 1800 296 178 102

Thiobaai l lus thiooxidans

f'eT (ppm) , 1. 0 425 8.0 '1. 0 15.5

Fe 2 + • (ppm) < 0. 1 415 5.0 < 0. 1 14.0

zn 2+solubil-isation Rate 40.0 130.0 13. 0 12.0 6.5 (mg/ Jl,/day)

Max. con.

Zn 2+ (ppm) 530 1270 204.0 155 85.0

Thiobaci Uus thioparus

FeT (ppm) < 0. 1 3.0 <0.1 <0.1 <0.1

Fe 2+ (ppm)* <0.1 <0.1 <0.1 <0.1 <0.1

zn 2+Solubil-isation Rate 6.9 4.9 3 .1 3.9 Ng (mg/R,/day)

Max. cone.

Zn 2+ (ppm) 59.5 80 27.5 31. 5 <6

* At conclusion of experiment Ng= Negligible

# Significant amounts of ferric compounds precipitated during course of experiment. FeT = Total Iron

NOTE: Zn2+ solubilisation rates were determined during log phase.

199

synthetic zinc sulphide, was degraded appreciably. However, the

strain was able to maintain its viability and some growth did occur

when these minerals were supplied as the only source of energy. The

degree of leachability of zinc sulphide minerals by ThiobaciZZus

thioparus varied between O and 2.69% (Table 3.3.5). Leachability of

wurtzite was negligible whereas 2.69% of synthetic zinc sulphide was

leached. Of all five minerals tested, synthetic zinc sulphide was

maximally leached with, ThiobaciZZus thioparus as well as with

ThiobaciZZus thiooxidans. Since synthetic zinc sulphide was almost

iron-free, the only possible mechanism of degradation can be the

direct oxidation of the sulphidic moiety of the minerals. The leaching

behaviour towards synthetic zinc sulphide of the two sulphur

oxidisers (ThiobaciZZus thioparus and ThiobaciZZus thiooxidans) was

very similar, except that a higher percentage of leachability was

obtained by the former (24 %); this can be attributed to the low pH

of the medium.

One of the obvious reasons for the low soluble metal content of

ThiobaciZZus thioparus cultures may be the relatively high pH (5.5)

of the leaching medium. At this pH the solubility of zinc ions might

have been drastically reduced because of formation of a new (insoluble)

complex. Consideration was given to clarifying the results by

determining the sulphate released into the medium as a product of

oxidation of the sulphide moiety. Attempts, however, to obtain a

meaningful determination of sulphate ion in this medium were seriously

affected by the presence of the large quantities of phosphate present

in the Vishniac & Santer medium (1957). It has been reported that

the, leaching capabilities of the newly isolated ThiobaciZZus

rubeZZus and ThiobaciZZus deZicatus species were also very small

compared with those of the acidophilic members of the

No.

1-

2-

3-

4-

5-

TABLE 3.3.5

PERCENTAGE LEACHABILI1Y OF ZINC SULPHIDE MINERALS

BY THIOBACILLI

200

MINERAL SPECIES PERCENTAGE LEACHABILI1Y

T. thiooxidans Zn = 23.00 Fe= 32

MARMATITE T. f errooxidans Zn = 33 Fe= 0.3

T. thioparus Zn = 1.45

T. thiooxidans Zn = 24.04

ZINC SULPHIDE T. ferrooxidans Zn = 17

(Synthetic) T. thioparus Zn = 2.69

T. thiooxidans Zn = 1. 34

SPHALERITE T. ferrooxidans Zn = 1.55

(Spain) T. thioparus Zn = 0.27

T. thiooxidans Zn = 2.33

SPHALERITE T. f errooxidans Zn = 3.31

(Oklahoma) T. thioparus Zn = o. 31

T. thiooxidans Zn = 1.12

WURTZITE T. ferrooxidans Zn = 1. 36

T. thioparus Negligible

201

genus Thiobacillus (Mizoguchi et al., 1976). Bacterial leaching of

various ores with an alkaline medium using Thiobacillus thioparus and

Thiobacillus thiooxida:ns has been studied by Mayling (1969), who has

suggested the use of a Thiobacillus thioparu.s strain at pH 8.4 for

leaching copper and zinc ores. But further study in this direction has

not yet been pursued, presumably because of the extremely slow

reactions and low solubilisation rates obtained.

In comparison with Thiobacillus thioparu.s both Thio"bacillus

thiooxidans and Thiobacillus ferrooxidans were relatively good

leaching agents. In the case of the natural minerals, Thiobacillus

ferrooxidans was more efficient in leaching zinc than was the sulphur

oxidiser, Thiobacillus thiooxidans. However, the relatively pure,

synthetic zinc sulphide was more effectively leached by

Thiobacillus thiooxidans than by Thiobacillus ferrooxidans. 41%more

zinc was released by the sulphur oxidising bacterium than by Thio­

"bacillus ferrooxidans from the synthetic zinc sulphide, whilst the

zinc concentration released from marmatite by Thiobacillus ferroo­

xidans was 43% greater than that released by Thio"bacillus thiooxid­

ans. The most plausible reason for the increased activity of

Thio"bacillus ferrooxidans is that metal release by Thiobacillus

thiooxidans is mostly due to the direct attack of bacteria, whereas

both direct and indirect mechanisms are likely to operate in the

case of Thio"bacillus ferrooxidans. (It is notable in this context that

the marmatite samples contain 12.2% iron while the synthetic zinc

sulphide contained only 0.06%.) This was further studied in the next

experiment, where the role of iron on leaching was investigated.

Leachability of zinc sulphide minerals by different Thio"bacilli

is tabulated in Table 3.3.S. It is evident that marmatite was leached

to a maximum degree of 33% by Thiobacillus ferrooxidans, which leached

,......,_ >. c,:I

"C . ~ .._,

..........

s . 0-, . 0-,

UJ CJ) <!'. UJ ...:I UJ 0::

u z ..... N

u.. 0

UJ E--t

~

202

400-------~-------,---------,--------,

300

200

100

0 0 5 10

TIME (Days)

15

FIG. 3.3.10. RATES OF ZINC RELEASE IN SOLUTION DURING

LEACHING OF MARMATITE

( • ) -Thiobaci l lus f errooxidans

( o) -Thiobaci l lus thiooxidans

20

203

17% of synthetic zinc sulphide and 1.55 and 3.31% of museum grade

sphalerites. Wurtzite leachability was a minimum (1.36%). It is

interesting to note that even with a small increase in the iron content

of sphalerite from Oklahoma (Table 3.3.2), its leachability was increa­

sed 114% when compared with the other sphalerite sample, from Spain.

Thiobaaillus thiooxidans was capable of releasing 23% zinc from

marmatite and its maximum leaching (24.04%) was observed in the case

of synthetic zinc sulphide. Wurtzite, again was attacked least (1.12%

leachability). Thiolxiaillus,thioparus was the least effective leac­

hing agent. It is interesting to note that leachability of zinc from

synthetic zinc sulphide, by both acidophilic species (Thiobaaillus

thiooxidans and Thiolxiaillus ferrooxidans), differed signifi-

cantly (Table 3.3.5). However, leachability of marmatite zinc was

43% greater with Thiobacillus ferrooxidans than with the sulphur oxi­

dising bacterium, whilst the leachability of marmatite iron was 100-fold

greater with the latter than with the former.

The rates of release of zinc during leaching of the different

zinc sulphides by the three Thiolxicilli are presented in Figs. 3.3.10-

3.3.14. These curves share some similarity in their pattern of rate

changes during the release of zinc ion into solution. During marmati­

te leaching the rate of release of zinc by Thiolxicillus ferrooxidans

increased steeply until 20.3 % zinc was released, then the rate

decreased rapidly (Fig. 3.3.10). A similar pattern was observed

during leaching of marmatite with Thiobaaillus thiooxidans. The rate

curve (which was non-linear) reached a maximum by eight ~and started

decreasing after remaining constant for two days. Marmatite was

leached at a much higher rate by Thiobacillus ferrooxidans than by

Thiobacillus thiooxidans. Rates of zinc released during leaching of

synthetic zinc sulphide by Thiobacillus thiooxidans and Thiobacillus

ferrooxidans are shown in Fig. 3.3.11. Synthetic zinc sulphide was

204

80 -------,-.------,---------,-------it

s 0..

0..

60

i:.u 40 r:f) c:i:: i:.u ,._J

~ u z ..... N

t.L. 0

~20 ~

0 0 5

I • r

10

TIME (Days)

15

FIG. 3.3.11. RATES OF ZINC RELEASE IN SOLlITION DURING

LEACHING OF SYNTHETIC ZINC SULPHIDE

( • ) - Thiobaci Uus f errooxidans

( o ) - Thiobaci Z Zus thiooxidans

20

205"

leached rapidly by ThiobaciZZus thiooxida:ns until 14% zinc was

released into solution after which it decreased rapidly. However,

pattern of the rate curve for zinc release by ThiobaciZZus ferrooxi­

dans is different from that for marmatite. In this case the rate of

zinc release increased non-linearly over the first eight days of the

experiment, remained constant for the next six days and then decreased.

The total metal leached ( 17% ) in this case was less than that

leached by ThiobaciZZus thiooxida:ns (24%).

Rates of release of zinc into solution

during leaching of sphalerite samples from Spain and Oklahoma are

depicted in Figs. 3.3.12 & 3.3.13. The rates of leaching the

sphalerite sample containing a higher percentage of iron (Oklahoma)

was (like marmatite, but in contrast to synthetic ZnS) higher with

ThiobaciUus ferrooxida:ns than with ThiobaciUus thiooxidans.

Leaching rates of zinc from wurtzite and spanish sphalerite also

increased during the early stages of the experiment and then decrea­

sed (Figs. 3.3.12 &' 3.3.14). There was little difference between

the rates of the two organisms . From all these rate curves no

unifying concept emerges which distinguishes the rate pattern of

ThiobaciUus ferrooxida:ns from that of ThiobaciUus thiooxidans.

With some minerals the curves were steeper for one organism but with

other minerals the reverse was the case.

It is apparent from these Figs (3.3.10 - 3.3.14) that the rate

of zinc release increased upto certain maximum and then declined.

The increases and the decreases in rates as well as their maximum

values varied with the mineral and with the organism. What caused the

rate to decline is not known. It cannot have been due to substrate

limitation per se, as maximum leachability achieved during these

experiments was 33% zinc from marmatite . With marmatite,

,--, >-. ('j

-0 . ~ '-' -E . p.. . p..

(.J..l rf) <i: (.J..l ~ (.J..l 0:::

u z ..... N

r.i.. 0 (.J..l

~

20

15

10

5

0 0

206

5 10

TIME (Days)

0

15

FIG. 3.3.12. RATE OF ZINC RELEASE IN SOLUTION DURING

LEACHING OF SPHALERITE (SPAIN)

( •) - Thiobaci "l "lus f err>ooxidans

( O ) - Thiobaci 7., "lus thiooxidans

20

,-.. >.. ro

"O . ~ ~ -. s . 0..

0..

U.l U)

c:i:: U.l ..J U.l 0::

u z ..... N

LI-,

0

U.l E--o

~

207

40

30

20

10

o _______ __._ _______________________ ...,.

0 5 10 TIME (Days)

15

FIG. 3.3.13. RATES OF ZINC RELEASE IN SOLUTION DURING

LEACHING OF SPHALERITE (OKLAHOMA)

( • )-Thiobaci Uus ferrooxidans

( o ) -Thiobaci l lus thiooxidans

20

208

20

,...._ >. ~ 15 . ~ .._, -s p.. . p..

c.u U)

-ex: c.u ...J c.u 0:::

u z H N

i:.i.. 0

c.u E-o

~

10

I 5

0 '-, ______ _._ ______ ....,j, _______ ...... ______ __

0 5 10

TIME (Days)

15

FIG. 3.3.14. RATES OF ZINC RELEASE IN SOLUTION DURING

LEACHING OF WURTZlTE

( • ) - Thiobaci l lus f errooxidans

( o ) - Thiobaci l lus thiooxidans

20

209

fonnation of an insoluble precipitate may have been responsible for

deceleration in the rate of leaching by ThiobaciZZus ferrooxidans.

However, this explanation is not valid for ThiobaciZZus thiooxidans;

perhaps the ferrous concentrations released into solution, which have

been shown as being inhibitory to this microorganism (see section 3.3.6),

contributed to the slowing down of the leaching rate. The nature of

the limiting factors in the case of synthetic zinc sulphide remains

unresolved. There are apparently no other toxic metals or other comp­

onents present in this mineral, and the maximum zinc contents released

into solution are definitely not injurious to these bacteria. The

inhibition might be due to the non-accessibility of oxygen, which is

one of the important nutrients for these bacteria or due to the forma­

tion of new surface characteristics. The changes brought about at

the surfaces of the minerals could contribute to inhibition of mineral

leaching because of their associated changes in the surface-bacteria

relationship. The postulate of Corrans et al. (1972), re:garding the

formation of a passive sulphur layer over covellite and other copper

sulphide minerals during chemical leaching could conceivably be

extended to zinc sulphide system, where perhaps rate of formation of

a passive sulphur layer over the mineral exceeds the rate of its

removal by bacteria. It is obvious that further work is needed to

detennine the exact nature of the factors limiting microbial leaching

of zinc sulphides.

As previously stated (see section 3.3.3), zinc and iron leaching

rates are different when they are present together in mannatite.

Leachability of the iron in marmatite was 39% more than that of zinc in

the case of T.thiooxidans(Table 3.3.5) but was only 1% of the leachability

of zinc in the case of T.ferrooxidans. In order to study the comp-

arative leachability of iron and zinc from marmatite, the molar ratio

5

4

,....., Q)

LI. ......... -,....., i::: N .........

LI. 0

0 3 H E-

~ 0::: <: ~ 0 :E:

2

1 0

210

----------- --------- ------------

5

Ini ial value of [Zn /[Fe] in

marmatite.

_..,,--.---• -- ---,,--,,.--.

10 15 20

TIME (Days) FIG. 3.3.15. MOLAR RATIO OF ZINC TO IRON DURING MICROBIOLOGICAL

LEACHING OF MARMATITE

ThiobaciZZus ferrooxidans

( e-• ) - Molar ratio in mineral

( •--• )- Molar ratio in solution

Thiobacillus thiooxidans

( 0-0 )-Molar ratio in mineral

( 6-6 )-Molar ratio in solution

211

of [Zn]/[Fe] in solution was plotted in Fig. 3.3.15. These results

indicate that during leaching of marmatite by either of the acidophilic

species of Thiobacillus this ratio decreases abruptly from an initial

value of 2.75 to 2.0, and therefore suggest that in the initial stages

of leaching more iron molecules are released than zinc. The ratio

remained constant between the third and sixth days of the

experiment, the time which coincided with the active period of

bacterial leaching by Thiobacillus ferrooxidans (Fig. 3.3.10). The

ratio then increased and remained constant at a value similar to that

in the original solution. An interesting extrapolation of this observ­

ation was to calculate the molar composition of the residual mineral.

The molar ratio of [Zn]/[Fe] in the marmatite (initially 3.52) incre­

ased during bacterial leaching (Fig. 3.3.15), the increase being

slower and of lower maximum value when Thiobacillus thiooxidans repla­

ced Thiobacillus ferrooxidans as the leaching catalyst. This is

consistent with the fact that Thiobacillus ferrooxidans oxidises

iron much more rapidly than does Thiobacillus thiooxidans. At the

end of the experiment the zinc to iron ratio in the marmatite subjected

to Thiobacillus ferrooxidans was 4.55 whilst that subjected to the

sulphur oxidising bacterium (Thiobacillus thiooxidans) was 3.98. The

third component, sulphur was not estimated and it was not possible to

determine the change in [Zn]/[S] ratio, a value helpful in-predicting

the mechanism of leaching.

These results have therefore indicated that the basic leaching

capacity· of Thiobacilli in decreasing order is Thiobacillus

ferrooxidans > Thiobacillus thiooxidans > Thiobacillus thioparus

in the case of natural minerals and Thiobacillus thiooxidans >

Thiobacillus ferrooxidans > Thiobacillus thioparus in the case of

synthetic, relatively pure zinc sulphides.

212

The results also provide a basis of comparison of the leaching

of the various zinc sulphides by each individual Thiobacillus. It was

obvious that wurtzite was the least amenable to attack by any of the

three Thio/Jacillus species employed. The hexagonal crystallographic

structure in which the component ions of wurtzite are arranged contr­

asts with the cubical forms of sphalerite and marmatite, and this may

contribute to its difference in reactivity. However, it must be kept

in mind that results of the kind described can only be considered def­

initive if obtained with properly annealed, high purity, synthetic

minerals. Such facilities were not available at the time of experi­

mentation, and therefore natural minerals of known composition were

used instead.

A limited arnomt of published information suggests significant

variations in the behaviour of different crystalline forms towards

microbial attack and that further effects can arise from substitutions

in the lattice. A high degree of resistance to oxidation by museum

grade pyrite in the presence of acidophilic, iron oxidising bacteria

was noted by Leathen et al.(1953), who reported the relative ease of

degradation of high grade museum specimen of marcasite by the same

bacterial species. In more detailed investigations of these two iron

sulphide minerals, Silverman et al.(1961) confirmed the resistance of

coarsely crystalline pyrite to oxidative attack by Thiobacillus thioo­

xidans and Thiobacillus ferrooxidans and observed that both these

species readily degraded marcasite. Leachability of sphalerite and

marmatite was investigated by Trussell et al.(1964), who reported that

the marmatitic form of zinc sulphide was rapidly and completely leached

by Thiobacillus ferrooxidans but that, mder similar conditions, iron­

free sphaleriteshowed only a minor release of zinc ions. These findi­

ngs are thus compatible with the results of thecurrent investigation

which indicates that leachability of minerals is dependent upon the

213

structure and composition of the mineral.

Further evidence that the composition of minerals has a major

effect on their leachability is seen in the work of Scott & Dyson

(1968). Zinc sulphide containing copper and iron were shown to be

more readily leached chemically than was the pure mineral. The

catalytic effect of some ions on leaching is so pronotmced that

even 0.05% copper in zinc sulphide is sufficient to affect the

leaching rate significantly (Scott & Dyson, 1968). The catalytic

effect of these ions has been attributed to the possibility of

activating the metal in its lattice and at the same time promoting

the reduction of dissolved oxygen at the cathodic sites on the zinc

sulphide mineral. Sufficient detailed work is not however available

for formulating the precise mechanism operating under such conditions.

-e a: a:

4

3

0 0 02 ->c -u C:

N

1

0

0

214

FIG. 3.3.16. EFFECT OF FERROUS CONCENTRATION ON THE

LEACHING OF SYNTI-IETIC ZINC SULPHIDE BY THIOBACILLUS

FERROOXIDANS

fil

---------------+----- ----------1

---------1----------------- - ------------l

e.--1-----i "---· 0 •

100 200 Hours

eNo ferrous; " 5 p.p.m. ferrous; o100p.p.m ferrous; .soop,p.m. ferrous:

02000 p.p.m ferrous:+4000 p.p.m. ferrous;

300 400

o 10 p.p.m. ferrous: .. 1000 p.p.m. ferrous

r-1 Marmat ite.

215

3.3.6 EFFECT OF FERROUS IONS ON LE:ACHING OF SYNTHETIC ZINC

SULPHIDE BY THIOBACILLUS FERROOXIDANS & THIOBACILLUS

THIOOXIDANS

The effect of ferrous iron concentration on the leaching rate of

synthetic zinc sulphide was detennined on the presence of Thiobacillus

ferrooxida,ns and ThiobaciUus thiooxiclans. These experiments were

carried out according to the procedure described in section 3.3.S,

except that the pulp density was doubled.

Addition of low concentrations (5-10 p.p.m.) of ferrous iron had

no significant effect upon the leaching curve of the sulphide by Thio­

baciZZus ferrooxidans (Fig. 3.3.16) but increasing ferrous concentra­

tions between 100 and 2,000 p.p.m. caused a progressive increase in

leaching rate. The total amount of zinc ions leached was increased from

600 p.p.m. at 10 p.p.m. ferrous to 2,575 p.p.m. in the presence of

2,000 p.p.m. ferrous. Marmatite, under similar conditions but without

the addition of ferrous ions, was leached more rapidly than was synth­

etic zinc sulphide in the presence of saturating concentrations of

ferrous ion.

The maximum. rates of leaching, and the total amount of zinc

leached in the presence of various concentrations of ferrous ion are

tabulated in the Table 3.3.6. These values indicate that the rate of

zinc release was proportional to the amount of iron present, but

that the system was saturated with respect to iron at concentrations

of 2,000 or greater p.p.m. Fig. 3.3.17 represents the Lineweaver and

Burk plot of values of ferrous ion versus rate of zinc leaching. The

straight line obtained indicates that the addition of ferrous ion to

synthetic zinc sulphide increases zinc leaching in accordance with the

Michaelis-Menton equation. The value of K determined by this plot m

was 555.S p.p.m. (9.92 mM Fe) which closely agreed with the value

obtained by plotting the Eadie-Hofstee plot (586 p.p.m.), (White,

,......_ ..c: -. s . p., . p., '-'

> -...-t

216

0.25----------------------------

0.201-------~---------<~------------1-------t

0.15

0.1

0 0.002 0.004 0.006 0.008

1/ [S] p.p.m.

FIG. 3.3.17. Lineweaver and Burk plot for effect of

initial concentration of ferrous on initial rate of

leaching of synthetic zinc sulphide by Thiobacillus

fer>r>ooxidans.

0.01

217

TABLE 3. 3. 6

EFFECT OF FERROUS IRON CONCENTRATION ON THE LEACHING

OF IRON-FREE ZINC SULPHIDE BY THIOBACILLUS FERROOXIDANS

AND THIOBACILLUS THIOOXIDANS

Ferrous Iron T. ferrooxidans T. thiooxidans Concentration 2+ 2+ 2+ 2+

(ppm) Rate of Zn Tot-al Zn Rate of Zn Total Zn Solution Concn. Solution Concn. (ppm/hr) (ppm) (ppm/hr) (ppm)

0 1. 50 550 2.4 730

5 1. 65 570 2.45 790

10 1. 50 600 2.60 820

100 5.2 1475 2.00 670

500 15.8 2150 1.80 610

1000 20.8 2500 1.8 570

2000 29.0 2575 1. 75 550

4000 28.0 2700

Marmatite 31. 0 3575

218

Handler & Smith, 1973; Lehninger, 1970).

Low concentrations of ferrous ion stimulated leaching by Thio­

baciZZus thiooxidans, but concentrations of 100 p.p.m. or more were

inhibitory (Fig. 3.3.18 and Table 3.3.6). For example there was 8%

increase in the leaching rate in the presence of 10 p.p.m. ferrous but

500 p.p.m. ferrous inhibited the rate of zinc release by 25%. Maximum

inhibition (27%) was observed in the presence of 2,000 p.p.m. ferrous

ions.

Discussion.

The environment in which leaching occurs normally contains

dissolved iron in either the ferric or ferrous form. The prominent

role played by iron species during chemical, as well as microbiological,

leaching of sulphide ores has been well documented previously (Scott

& Dyson, 1968; Dutrizac & MacDonald, 1974; Tuovinen & Kelly, 1974;

Forward, 1960; Duncan & Walden, 1972). The results reported in this

and the previous section revealed that, in the absence of additional

iron, the synthetic zinc sulphide was leached slightly more rapidly

by ThiobaciZZus thiooxuia:ns than by ThiobaciZZus ferrooxidans.

With a relatively pure zinc sulphide such as this, virtually the only

oxidisable source for the ThiobaciZZi is the sulphidic moiety on the

solid surface of the substrate and consequently all zinc release is

presumably due to the 'direct action' of the bacterial oxidation of

the sulphur atoms in the crystal lattice, with perhaps some contri­

bution from the sulphuric acid produced in accordance with the

following equaion:

+ so + l½ 0 Bacteria

ThiobaciZZus ferrooxidans has been reported to obtain its energy by

e Q. Q.

6 0 0 .... )(

(,)

C: ·-N

4

0

219

FIG. 3.3.18. EFFECT OF VARIOUS CONCENTRATIONS OF FERROUS ION

ON LEACHING OF SYNTHETIC ZINC SULPHIDE BY THIOBACILLUS

THIOOXIDANS

100

Ferrous iron concentrations:

200 Hours

300 400

(0)- 5 p.p.m., (e)- 10 p.p.m., (.6)- 100 p.p.m., (D) SOO p.p.m.,

( .a)- 1000 p.p.m., C• )- 2000 p.p.m., ( •)- Control.

220

oxidation of metallic ions per se occurring in the lattice,

especially in the case of copper minerals (Nielsen & Beck, 1972;

Golding et al., 1974). Since zinc does not exhibit variable oxidation

states in its minerals, there is very little probability of a direct

oxidation of the zinc species in the crystal lattice (Zajic, 1969).

Addition of ferrous ion to a Thio"baaiZZus ferrooxida:ns culture

provides another energy source for this organism. In the

current investigation its addition stimulated the leaching rate of

zinc from synthetic zinc sulphide by ThiobaaiZZus ferrooxida:ns to

values which exceeded those achieved by ThiobaaiZZus thiooxidans.

The question remaining is whether it did so directly or indirectly.

In any attempt to assess the possible mechanistic roles of ferrous

iron in this situation the following points require consideration:

(a) ferric ions produced by Thio"baaiZZus ferrooxidans' oxidation of

ferrous iron,are an efficientlixiviant and can oxidise sulphides

chemically to sulphates, regenerating ferrous ion again for recycling;

(b) since growth of ThiobaaiZZus ferrooxida:ns on ferrous ions is

luxurious as compared to that on insoluble substrates such as zinc

sulphide, larger population densities become available for direct

leaching of the sulphides. It is hard to differentiate between (a)

and (b), but the following do provide some guidance towards a logical

conclusion. It is a common observation. that the leaching rates of

sterile sulphides by ferric ion at temperatures (30-40°C) favourable

for bacterial growth are slow compared with rates in the presence of

ThiobaaiZZi. Such an observation provides a strong basis for the

assumption that the direct bacterial oxidation of sulphide in the

current investigation contributes more than did the chemical oxidation.

Even stronger evidence for this assumption lies in the fact that addi­

tion of ferric sulphate to a leaching medium in the presence of Thio­

baaiZZus ferrooxida:ns has been shown to exert no influence on the

leaching of commercial zinc sulphide

1972). Further evidence against

221

concentrates (Duncan & Walden,

a significant contribution

from direct chemical oxidation of the sulphide by ferric iron was

provided by the demonstration in the current results that only a small

concentration of soluble ferric ions were detectable in the culture

media. Once formed, most of the ferric form precipitated, presumably

as jarosite salts of ammonium. Although the formation of jarosite

would decrease the availability of ammonium ions, the only suitable

nitrogen source for the bacteria, the ammonium ion concentration

evidently remained sufficiently high to permit increased growth in

the presence of the iron supplement.

This was the first study to reveal inhibition of zinc leaching

by ThiolJaeiZZus thiooxidans in the presence of high ferrous ion

concentrations. Such inhibition, which could explain, at least

partly, why a comparatively low population of sulphur oxidising bacte­

ria is found in natural milieu, could be due to the one or both of

the following possibilities; (a) adsorption of ferrous ions into the

surface of the zinc sulphide particles, which would thereby become

resistant to ThiolJaeiZZus thiooxidans' action, (b) inhibition of the

activities of ThiolJaeiZZus thiooxidans per se, by perhaps, affecting

the cell surfaces and transport mechanisms.

The zinc from marmatite (unsupplemented with additional iron)

was released at a faster rate than from synthetic zinc sulphide

by both species of ThiolJaeiZZi; this indicates the possibility of

some contribution from the interstitial positioning of the iron ions

to the leachability of minerals. The amount of iron released during

marmatite leaching by ThiobaeiZZus ferrooxidans is difficult to

assess, since most of it was precipitated as jarosite salts. However an

assessment can be made from the leaching behaviour of marmatite

with the ThiobaeiZZus thiooxidans, which does not oxidise ferrous

222

ions. In such experiments it was folllld that ferrous and zinc ions

were released in the approximate ratio of 1:2.99 (section 3.3.3) from

marmatite by ThiobaciZZus thiooxidans. If it is assumed that the

same proportion of zinc and ferrous ions would be released from

marmatite by ThiooociZZus ferrooxidans, then for the final zinc conc­

entration of 3,575 p.p.m. recorded, the ferrous concentration would

be 1196 p.p.m.

The rate of release of zinc ions from synthetic zinc sulphide by

ThiobaciZZus ferrooxidans in the presence of 1196 p.p.m. is 22.2

mg/(i.h) compared to 31 mg/(i.h) from mannatite . 39% increase in

the rate of release of zinc might have been related to the fact that

the iron atoms were present in the solid marmatite, rather than in

solution. In this form they were released slowly into the solution

without adversly affecting the bacterial growth as the excess ferric

ion has been reported to be inhibitory to both ferrous ion and sulphide

oxidation(Landesman et al., 1966; Razzell & Trussell, 1963a; Dllllcan &

Walden, 1972; Wong et al., 1974). In the solid form they should

affect the electrochemical behaviour of zinc sulphide crystal lattice

or even perhaps the excitation state of the iron/oxygen complex at the

mineral interface. Another possible explanation could be that ferrous

iron leaching from the lattice proceeds that of zinc or sulphide and

leaves 'holes' in the crystal for further activation and penetration

of bacterial and chemical agents. Section 3.3.3 reports that in the

presence of ThiobaciZZus thiooxidans, at least, iron leaches out in

a greater proportion than that expected from its proportion in the

marmatite. This observation needs further investigation before a

more precise theory regarding the mechanism of leaching of minerals of

different composition can be proposed.

4 CONCLUSIONS

223

4. CONCLUSIONS

A comparison of the leaching action of three principal species

of ThiobaciZZi on different zinc sulphide minerals has been studied

with the intention of providing some information regarding leachability

of zinc sulphide within a pH spectrum of 2.0 - S.S. Two principal

species, isolated indigenously, were ThiobaciZZus thiooxidans and

ThiobaciZZus ferrooxidans, representing acidophiles, while ThiobaciZZ­

us thioparus was selected to represent non-acidophilic members of the

genus. It was ascertained that isolated species are closely related

to the above mentioned principal species. Taxonomical studies revealed

no strikingly different characteristics of these species other than

those described in Bergey's Manual of Determinative Bacteriology,

(Buchanan & Gibbons, 1974). The FAME (fatty acid methyl ester) profile

of BJR-451 was foillld to be different from that of described by Agate

and Vishniac (1973-a); the F.AM:E profile of BJR-451, however, was later

studied by Dillln et al.(1977), employing more sophisticated and elabor­

ate techniques, and was found to be identical to ThiobaciZZus thio­

parus. These workers also confirmed, by FAME profile examination,

the identity of two other strains, which were identified as Thiobaci­

ZZus thiooxidans and ThiobaciZZus ferrooxidans (section 3.1.4).

The ability of ThiobaciZZus ferrooxidans to utilise elemental

sulphur besides ferrous ion, and its possession of a pH optimum for

growth very similar to that of ThiobaciZZus thiooxidans, enhance the

possibility of its existence in acidophilic cultures of ThiobaciZZus

thiooxidans. Isolation of the new associate, ThiobaciZZus acidophiZus,

from a standard culture of ThiobaciZZus ferrooxidans indicates the

possibjle usage of 'mixed' cultures in the past (Guay & Silver, 1975)

and casts doubt on the validity of certain comparative. studies made.

224

Lack of availability of specific media for isolation of each disti-

nct ThiobaeiZZus species can lead to the isolation of mixed species

and a need to devise such media is very urgent for ecological surveys

of mine fields and exhausted dumps. However, in the current investig­

ation, particular attention has been paid to purification of each

strain studied. Although micro-manipulation for single cell iso­

lation was not employed, the isolated suspensions behaved homogeneou­

sly and yielded reproducible results. Absence of high ferric ion

concentration in solution when mannatite was leached by ThiobaeiZZus

thiooxidans, even in the presence of ferrous ion,, indicated that the

sulphur-oxidiser was free from ThiobaeiZZus ferrooxidans.

BJR-451 was studied in detail and its pH and temperature optima

determined. Its pH curve was sharp and optimal at S.S. (Acidic

environments (pH below 4.5) were bacteriostatic, whereas lower pH

values (4.0 - 3.5) were bacteriocidal.

Tolerance of the three ThiobaeiZZi to zinc ion was determined,

and it was found that ThiolxieiZZus ferrooxidans could withstand

concentrations of zinc as high as 20,000 p.p.m. However, ThiobaeiZZus

thiooxidans was capable of tolerating zinc concentrations only up to

15,000 p.p.m. without deleterious effects on its growth being observed.

ThiobaeiUus thioparus' maximum zinc tolerance level could not be

determined, because of the rapid changes in pH resulting from the

addition of zinc sulphate solution. A most notable observation was

that high zinc concentrations influenced growth only by prolonging the

lag period which was followed by growth at a rate similar to that of

the control without zinc). This suggests a complete adaptation by

these bacteria to zinc. The low metal tolerance observed in the case

of ThiobaeiZZus thiooxidans might have been due to its growth on

sulphur; this would accord with the suggestion that ThiobaeiZZus

ferrooxida:ns grown on thiosulphate as the sole energy source is less

225

tolerant to metals than cells harvested from medium containing ferrous

ions (Tuovinen et al., 1971). Changes in metal tolerance constitute

another very important field in which further research should be

carried out before definitive statements regarding species differen­

ces can be made. Metal resistant plasmids have been reported in

some bacteria (Summers et al., 1975) and, with the advent of modern

genetic engineering technology, a suitable metal tolerant plasmid

should provide a desirable ThiobaaiZZi for each particular assign­

ment when introduced into the appropriate bacterium.

The observation made by Mayling (1969) for extracting alkaline

ores of zinc and copper could not be confirmed by using ThiobaciZ Zus

thioparus. The leaching capabilities of this species was found to

be minimwn when compared with the two acidophilic species (i.e.,

ThiobaciUus thiooxidans and ThiobaciUus fe:rTooxidans). Low metal

solubilisation by ThiobaciZZus thioparus might have been due to one or

more of the following possibilities: viz., low rate of oxidation of

sulphide; metal toxicity; the pH of the environment; the high concent­

ration of phosphates in growth medium. At pH 5.5 zinc phosphates and

hydroxides might have been released into solution and have precipitated.

However, no direct evidence in support of this postulate is forwarded.

ThiobaciZZus thiooxidans leached synthetic zinc sulphide more

effectively than did ThiobaciZZus ferrooxidans.

An early observation made by Trussell et al.(1964), regarding

the i higher leachability by ThiobaciUus ferrooridans of marrnatite

compared with sphalerite, was confirmed. The work was extended to a

study of the behaviour of various zinc sulphides, differing in

their composition and structure. This was the first investigation

into the effects of structures and compositions of minerals on their

226

relative leachability. It was fotmd that, in addition to the physico­

chemical parameters (which have been already discussed by many workers

and are summarised in section 1.3.3.), two other factors influence the

leaching rates: (a) the bacterial species involved; and (b) the

mineral used. It has been observed that, when leaching natural

museum-grade mineral specimens, the capabilities of Thio"baciZZi to

leach them are, (in decreasing order) Thio"baciZZus ferrooxida:ns >

ThiobaciZZus thiooxida:ns > ThiobaciZZus thioparus and for

synthetic mineral the capabilities are,(in decreasing order)

Thio"baciZZus thiooxidans > Thio"baciZZus ferrooxida:ns > Thiobac­

iZZus thioparus. It would be most informative to determine the rela­

tive behaviour of every species of the genus Thio"baciZZus in its

ability to leach other zinc ( and other metal) sulphides.

It was shown that the hexagonal species of ZnS (wurtzite) is

very inert towards leaching by all species of ThiobaciZZi; natural

sphalerite specimens were leached by these bacteria, but. not to a

significant degree. Maximum leachability, however, was found in

the case of synthetic zinc sulphide and marmatite. Present data

are insufficient for postulating a reason for wurtzite's inertness.

It may be due to the difference in the arrangement of sulphur tetra­

hedra in the crystal of wurtzite. Further investigation involving

electrochemical and electron-probe studies of the wurtzite before and

after leaching are needed before any firm conclusion can drawn.

Marmatite ( Zn 3 • 5Fe S5) was released by both acidophilic species

to give a soluble zinc to iron ratio which progressively decreased in

the early stages of leaching as a result of a iron being released

at a rate which was faster than the rate of zinc release,consequently,

the ratio of zinc to iron in the residual solid mineral increased.

The general nature of atomic point defects in ZnS indicates that

Fe is inserted in 'holes' rather than in valency bands, and an

227

explanation for the higher leachability of marmatite can be based on

the reasoning that bacteria extract ' ferrous' from these holes,

and that subsequently zinc is leached as a result of the collapse of

unit cells. However, a rigorous study should be made involving the use of

electron probe and thin section techniques in an attempt to obtain

definitive evidence for pitting and changes in the relative concen­

trations of the component metal atoms.

Zinc sulphide crystals can accommodate many different atomic

species in their 'holes' (Shuey, 1975), viz., noble elements (Cu, Ag,

Au) and alkali metals of group I (Li, Na) which produce acceptor

defects, as well as Br, Cl and I (as substituents for sulphur), which

produce donor effects. It is known that the presence of Cu, Cd, or Mn

in traces catalyzes the pressure leaching of zinc (Scott & Dyson,

1968). However no systematic study involving the effects of substi­

tuents in the zinc sulphide lattice on ZnS leaching by bacteria is

known; such information could provide very useful guidelines for deter­

mining the relationship between mineral structure and leaching.

It was shown that the presence of ferrous ions in the leaching

medium were inhibitory to the sulphur oxidising bacteria. (Previously,

no information regarding the effects of iron on the activities of these

bacteria was available.) On the other hand, enhanced rate of leaching

of zinc from synthetic zinc sulphide by ThiobaciUus ferrooxidans

was observed on the addition of ferrous ions to the system. The

effects of iron present in the marmatite crystals and that added exte­

rnally to synthetic zinc sulphide were not comparable; the iron

present in the marmatite was shown to be more efficient than was added

iron.

A study employing marmatite of various ferrous contents would

be extremly useful in determiningthe definitive role of iron in

leaching. Such studies would provide a significant contribution to

228

the newly emerging field of biohydrometallurgy.

229

5. R E F E R E N C E S

AGATE, A. D., 1973. Survey of ThiobaciZZus ferrooxidans in solubil­

isation of copper sulphide ores. NML Technical Journal 15: 25-27.

AGATE, A. D. and VISHNIAC,W.,1969. Changes in the lipid composition

of ThiobaciZZus neapolitanus. Bacteriological Proceedings P29.

AGATE, A. D. and VISHNIAC, W., 1973. Changes in phospholipids compo­

sition of ThiobaciZZus neapolitanus during growth. Archives

fur Mikrobiologie 89: 247-256.

AGATE, A. D. and VISHNIAC, W., 1973-a. Characterization of Thiobaci­

ZZus species by gas-liquid chromatography of cellular fatty

acids. Archives fur Mikrobiologie 89: 257-267.

AGBIM, N. N. and DOXTADER, K. G., 1975. Microbial degradation of

zinc silicates. Soil Biology and Biochemistry 7: 275-280.

ALEEM, M. I. H., 1966. Generation of reducing power in chemosynthesis.

II-Energy linked reduction of pyridine nucleotides in the chemo­

autotroph Nitrosomonas europae. Biochimica et Biophysica Acta

113: 216-224.

ALEEM, M. I. H., 1966-a. Generation of reducing power in chemosyn­

thesis. III- Energy linked reduction of pyridine nucleotides in

ThiobaciZZus noveZZus. Journal of Bacteriology 91: 729-736.

ALEEM, M. I. H., 1966-b. Generation of reducing power in chemosyn­

thesis. IV- Energy linked reduction of pyridine nucleotides by

succinate in ThiobaciZZus noveZZus. Biochimica et Biophysica

Acta 128: 1-12.

ALEEM, M. I. H., 1969. Generation of reducing power in chemosynthesis.

VI- Energy linked reduction in the chemoautotroph ThiobaciZZus

230

VI- Energy linked reduction in the chemoauto troph ThiobaciZZus

neapoZitanus. Antonie van Leeuwenhoek, Journal of Microbiology

and Serology 35: 379-391.

ALEEM, M. I.H., 1970. Oxidation of inorganic nitrogen compounds.

Annual Review of Plant Physiology 21: 67-90.

ALEEM, M. I. H., 1975. Biochemical reaction mechanisms in sulfur

oxidation by chemosynthetic bacteria. Plant and Soil 43: 587-607.

ALEEM, M. I. H., 1977. Coupling of energy with electron transfer

reactions in chemolithotrophic bacteria. Symposia- Society for

General Microbiology 27: 351-381.

ALEEM, M. I. H. and HUANG,E., 1965. Carbon dioxide fixation and

carboxydismutase in ThiobaciZZus noveZZus. Biochemical and

Biophysical Research Communications 20: 515-520.

ALEEM, M. I. H. and NASON, A., 1960. Phosphorylation coupled to

nitrite oxidation by particles from the chemoautotroph Nitrobacter

agiZis. Proceedings of the National Academy of Sciences of the

United States of America 46: 763-769.

ALEEM, M. I. H., LEES, H. and NICHOLAS, D. J. D., 1963. ATP-dependent

reductase of nicotinamide adenine dinucleotide by ferro-cytochrome

c in chemoautotrophic bacteria. Nature 200: 759-761.

ANDERSEN, J.E. and ALLMAN, M. B., 1968. Some operational aspects of

heap leaching at Rum Jungle. Proceedings of the Australian

Institute of Mining and Metallurgy 225: 27-31.

ANDERSEN, K. J. and LUNDGREN, D.G., 1969. Enzymatic studies of the

iron-oxidizing bacterium FerrobaciZZus ferrooxidans : Evidence

for a glycolytic pathway and Krebs cycle. Canadian Journal of

Microbiology 15: 73-79.

231

ARRIETA, L. and GREZ, R., 1971. Solubilisation of iron-containing

minerals by soil microorganisms. Applied Microbiology 22: 487-490.

ASHMEAD, D., 1955. The influence of bacteria in the formation of acid

mine water. Colliery Guardian 190: 694-698.

AUBERT, J. P., MILHAUD, G. and MILLET, J., 1957. Carbon metabolism

in chemosynthetic autotrophism. Carbon dioxide fixation on

phosphoenol pyruvic acid. Comptes Rendus Des Travaux du Labora­

toire Carlsberg 244: 398-401.

AUBERT, J. P., MILHAUD, G. and MILLET, J., 1957-a. The path of

carbon dioxide assimilation in chemoautotrophic bacteria.

Annales de l'Institut Pasteur 92: 515-524.

AUDSLEY, A. and DABORN, G. R., 1963. Natural leaching of uranium ores.

II- A study of experimental variable$. Transactions, Institute

of Mining and Metallurgy 72: 235-246.

AVAKYAN, A. A. and KARAVAIKO, G. I., 1970. Submicroscopic organization

of Thiobacillus ferrooxidans. Mikrobiologiya 39: 855-861.

AVEN, M. and PRENER, J. S., 1967. Physics and chemistry of II-VI

compounds. North Holland, Amsterdam, 844 pp.

BAALSRUD, K. 1954. Physiology of Thiobacilli. Symposia- Society

for General Microbiology 4 : 54- 67 •

BMLSRUD, K. and BAALSURD, K. S., 1954. Studies on Thiobacillus

denitrificans. Archives fur Mikrobiologie 20: 34-62.

BAAS-BECKING, L.G. M., KAPLAN, I. R. and MOORE, D., 1960. Limits

of the natural environment in terms of pH and oxidation-reduction

potentials. The Journal of Geology 68: 243-284.

232

BABIJ, T., DOBLE, R. and RALPH, B. J., 1971. Mobile equipment for

the sterilisation of fermentation media by filtration.

Chemistry and Industry pp. 1045-1046.

BABIJ, T., MOSS, F. J. and RALPH, B. J., 1969. Effects of oxygen

and glucose levels on lipid composition of yeast Candida utilis

grown in continuous culture. Biotechnology and Bioengineering

11: 593-603.

BARBIC, F. and BRACILOVIC, D., 1975. Effect of mineral acids on

iron bacteria. Zeitschrift fur Allgemeine Mikrobiologie 15:

307-313.

BARBIC, F. F., 1977 . Effects of different compounds of metals and

their mixtures on the growth and survival of Thiobacillus

ferrooxidans. Zeitschrift fur Allgemeine Mikrobiologie 17:

277-281.

BARDIYA, M. C. and GAUR, A. C., 1974. Isolation and screening of

microorganisms dissolving low grade rock phosphate.

Folia Microbiologica 19: 386-389.

BARTON, L. L. and SHIVELY, J.M., 1968. Thiosulfate utilization by

Thiobacillus thiooxidans ATCC 8085. Journal of Bacteriology

95: 720.

BARVENIK, F. W. and JONES, G. E., 1969. Chemosynthetic productivity

in a coastal pond. Bacteriological Proceedings G38, p. 124.

BARVINCHAK, G., 1975. Investigation of the mechanism of iron

oxidation by cells and cell envelopes of Thiobacillus ferrooxi­

dans. Ph.D. Thesis, Syracuse University, U.S.A. [cited from

233

Lazaroff, 1977].

BECK, J. V., 1960. A ferrous iron oxidizing bacterium. I- Isolation

and some general physiological characteristics. Journal of

Bacteriology 79: 502-509.

BECK, J. V., 1967. The role of bacteria in copper mining operations.

Biotechnology and Bioengineering 9: 487-497.

BECK, J. V., 1969. Thiobacillus ferrooxidans and its relation to the

solubilization of ores of copper and iron. In: Perlman, D., Ed.,

Fermentation Advances pp. 747-771. Academic Press, London.

BECK, J. V. and BROWN, D.G., 1968. Direct sulfide oxidation in the

solubilization of sulfide ores by Thiobacillus ferrooxidans.

Journal of Bacteriology 96: 1433-1434.

BECK, J. V. and ELSDEN, S. R., 1958. Isolation and some characteris­

tics of an iron oxidizing bacterium. Journal of General

Microbiology 19: i.

BECK, J. V. and SHAFIA, F. M., 1964. Effect of phosphate ion and

2:4-Dinitrophenol on the activity of intact cells of ThiobaciZZ­

us ferrooxidans. Journal of Bacteriology 88: 850-857.

BEIJERINCK, M. W., 1904. Uber die bakterienwelche sich in dunkeln

mit kohleusaure als kohleusloffquelle ernahren konneu.Zentralbl­

att fur Bakteriologie, Parasitenkunde. II Abt. 11: 593-599.

~ited from Roy & Trudinger, 1970, p.210.]

BERGEY, D. H., HARRISON, F. C., BREED, R. S., HAMMER, B. W. and

HUNTOON, F. H., 1925. Bergey's Manual of Determinative Bacteriology,

2nd. Edition. pp. 1-462. The Williams & Wilkins Co., Baltimore.

BERRY, L. G. and MASON, B., 1959. Mineralogy - pp. 308-317. W. H.

Freeman and Company, San Francisco & London.

234

BERRY, V. K. and MURR, L. E., 1975. Bacterial attachment to molybde­

nite: An electron microscope study. Metallurgical Transactions B,

6B: 488-490.

BITHER, T. A., 1967. Second International Conference on Solid

Compounds. Trans. Elem. Enschede. p.26.

[ Cited from: JELLINEK, F., 1968].

BITHER, T. A., BOUCHARD, R. J., CLOUD, W. H., DONOHUE, P. C. and

SEIMONS, W. J., 1968. Transition metal pyrite dichalcogenides.

High-pressure synthesis and correlation of properties.

Inorganic Chemistry 7: 2208-2220.

BLAYLOCK, B. A.and NASON, A., 1963. Electron transport system of

the chemoautotroph Ferrobacillus ferrooxidans. Journal of

Biological Chemistry 238: 3453-3462.

BLOOR, W. R., 1928.

The determination of small amounts of lipids in blood plasma.

Journal of Biological Chemistry 77: 53-73.

BODO, C. A. and LUNDGREN, D. G., 1971. Uptake and oxidation of iron

Thiobacillus ferrooxidans. Bacteriological Proceedings P44. p.131.

BORICHEWSKI, R. G., 1967. Keto acids as growth limiting factors in

autotrophic growth of Thiobacillus thiooxidans. Journal of

Bacteriology 93: 597-599.

BORICHEWSKI, R. G. and UMBREIT, W.W., 1966. Growth of Thiobacillus

thiooxidans on glucose. Archives of Biochemistry and Biophysics

235

116: 97-102.

BOURNE, E. J., NERY, R. and WEIGEL, H., 1959. Metal chelates of

polyhydroxy compounds. Chemistry and Industry, August 1,

pp. 998-999.

BRIERLEY, J. A., 1966. Contribution of chemoautotrophic bacteria

to the acid thermal waters of the Geyser Spring Group in

Yellowstone National Park. Ph. D. 'l'hesis, Montana State Unive­

rsity, Boseman, Montana, U.S.A.

BRIERLEY, C.L., 1974. Molybdenite leaching: use of a high-temperature

microbe. Journal of Less-Common Metals 36: 237-242.

BRIERLEY, J. A., 1977. Thermophilic microorganisms in extracion

of metals from ores. Developments in Industrial Microbiology

18: 273-284.

BRIERLEY, C. L. and BRIERLEY, J. A., 1973. A chemoautotrophic and

thermophilic microorganism isolated from an acid hot spring.

Canadian Journal of Microbiology 19: 183-188.

BRIERLEY, C. L. and MURR, L. E., 1973. Leaching: Use of a thermophi­

lic and chemoautotrophic microbe. Science 179: 488-490.

BRIERLEY, J. A. and Le ROUX, N. W., 1977. A facultative thermophilic

Thiobacillus like bacterium: Oxidation of iron and pyrite.

GBF Monograph Series, No. 4 (August, 1977), 55-66, Conference

Bacterial Leaching 1977.

BRIERLEY, J. A. and LOCKWOOD, S. J., 1977. The occurrence of

thermophilic iron-oxidizing bacteria in a copper leaching system.

236

FEMS Microbiology Letters 2: 163-165.

BROCK, T. D. and DARLAND, G. K., 1970. Limits of microbial exiatence:

Temperature and pH. Science 169: 1316-1318.

BROCK, T. D. and GUSTAFSON, J., 1976. Ferric iron reduction by

sulfur and iron-oxidizing bacteria. Applied and Environmental

Microbiology 32: 567-571.

BROCK, T. D., BROCK, K. M., BELLY, R. T. and WEISS, R. L., 1972.

Sulfolobus: A new genus of sulfur-oxidizing bacteria living at

low pH and high temperature. Archives fur Mikrobiologie 84:

54-68.

BROOKS, H., 1972. Materials in a steady state world.

Metallurgical Transactions 3: 759-768.

BRUNAUER, S., EMMETT, P. H. and TELLER, E. J., 1938. Adsorption of

gases in multimolecular layers.

Society 60: 309-319.

Journal of American Chemical

BRUYNESTEYN, A, and DUNCAN, D. W., 1971. Microbiological leaching of

sulphide concentrates. Canadian Metallurgical Quarterly 10:57-63.

BRYNER, L. C. and ANDERSON, R., 1957. Microorganisms in leaching

sulfide minerals. Industrial and Engineering Chemistry 46:

1721-1724.

BRYNER, L. C. and JAMESON, A. K., 1958. Microorganisms in leaching

sulphide minerals. Applied Microbiology 6: 681-687.

237

BRYNER, L. C., BECK, J. V., DAVIS, D. B., and WILSON, D. G., 1954.

Microorganisms in leaching sulfide minerals. Indastrial and

Engineering Chemistry 46: 2587-2592.

BRYNER, L. C., WALKER, R. B. and PALMER, R., 1967. Some factors

influencing the biological and non-biological oxidation of

sulfide minerals. Transactions of Society of Mining Engineering

238: 56-61.

BUCHANAN, R. E. and GIBBONS, N. E. (ed.), 1974. Bergey's Manual of

Determinative Bacteriology, 8th Edition, pp. 456-461. The

Williams and Wilkins Company, Baltimore, U.S.A.

BITTLER, R. G., 1975. Pyruvate inhibition of the carbon dioxide

fixation of the strict chemolithotroph Thiobacillus thiooxidans.

Canadian Journal of Microbiology 21: 2089-2093.

BITTLER, R. G. and UMBREIT, W.W., 1966. Absorption and utilization of

organic matter by the strict autotroph Thiobacillus thiooxidans

with special reference to aspartic acid. Journal of Bacteriology

91: 661-666.

CALDWELL, D. E., CALDWELL, S. J. and LAYCOCK, J. P., 1976. ThePmo­

thrix thioparus gen. et sp. nov. a facultatively anaerobic

facultative chemolithotroph living at neutral pH and high

temperature. Canadian Journal of Microbiology 22: 1509-1517.

CHAINIKOVA, N. A., 1962. Flotation properties of sphalerite varieties

having various iron contents (Maritime Territory Deposits).

Soobschch. Dal'nevost. Filiala. Sibirsk. Otd. Akad. Nauk. SSSR

238

No. 15, 47-51.[cited from Chemical Abstracts, 7425b : 62: 1965].

CLEVELAND, J. H., 1964. Variation of iron sulfide content in

sphalerites of Mississippi Valley Deposits. Proceedings of

Indiana Academy of Sciences 74: 271-277.

CLOUD, P., 1968. Mineral resources from the sea. In Resources and

Man, p. 135. W. H. Freeman & Co., San Francisco.

COCCUZZA, A. and NICOLETTI, G., 1961. Metabolic characteristics of

certain chemosynthesizing species of autotrophic Thiobacteria

isolate. Atti. Soc. Peloritana Sci. Fis. Mat. Nat., 7: 769-775.

COLES, J. S.III, and ALEEM, M. I. H., 1973. Electron transport linked

with proton induced ATP generation in Thiobacillus novellus.

Proceedings of National Academy of Sciences of the United

States of America 70: 3571-3575.

COLMER, A. R. and HINKLE, M. E., 1947. The role of microorganisms

in acid mine drainage- A preliminary report. Science 106: 253-256.

COLMER, A. R., TEMPLE, K. L. and HINKLE, M. E., 1950. An iron

oxidizing bacterium from the acid drainage of some bituminous

coal mines. Journal of Bacteriology 59: 317-328.

CORRANS, I. J., 1970. Studies on the bacterial leaching of natural

and synthetic iron copper sulfide minerals. M.Sc. Thesis,

University of New South Wales.

CORRANS, I. J., HARRIS, B. and RALPH, B. J., 1972. Bacterial

leaching: An introduction to its application and theory and a

study of its mechanism of operation. Journal of the South African

Institute of Mining and Metallurgy 72: 221-230.

CORRANS, I. J. and SCHOLTZ, M. T., 1976. A kinetic study of the

leaching of pentlandite in acidic ferric sulphate solutions.

Journal of the South African Institute of Mining and Metallurgy,

239

76: 403-411.

CORRICK, J. D. and SUTTON, J. A., 1961. Three chemosynthetic autotro­

phic bacteria important to leaching operations at Arizona Copper

Mines. United States Bureau of Mines Investigation Report 5719.

CORRICK, J. D. and SUTTON, J. A., 1965. Copper extraction from a low

grade ore by Ferrobaaillus ferrooxidans. Effects of environmnetal

factors. United State Bureau of Mines Investigation Report 6717.

De CASTRO, A. F. and EHRLICH, H. L., 1970. Reduction of iron oxide

minerals by a marine Bacillus . Antonie van Leeuwenhoek Journal

of Microbiology and Serology 36: 317-327.

De CUYPER, J. A., 1964. Bacterial leaching of low-grade copper and

cobalt ores. In: Wadesworth, M. E. & Davis, F. T. (Eds.), Unit

Processes in Hydrometallurgy 24: 126-142.

De KRUYFF, C. D., Van DER WALT, J. P. and SCHWARTA, H. M., 1957.

The utilization of thiocyanate and nitrate by Thiobaailli. Antonie

van Leeuwenhoek Journal of Microbiology and Serology 23: 305-316.

DIN, G. A., SUZUKI, I. and LEES, H., 1967. Carbon dioxide fixation

and phosphoenol pyruvate carboxylase in Ferrobaaillus ferrooxidans.

Canadian Journal of Microbiology 13: 1413-1419.

DOESTSCH, R. N. and COOK, T. M., 1973. Bacterial attack on

minerals. In: Introduction to Bacteria and their Ecobiology

pp.342-247. University Park Press, Baltimore, Maryland, U.S.A.

DUFF, R. B. and WEBLEY, D. M., 1959. 2-ketogluconic acid as a natural

chelator produced by soil bacteria. Chenistry and Industry,

pp. 1376-1377.

DUFF, R. B., WEBLEY, D. M. and SCOTT, R. 0., 1963. The solubilisation

of minerals and related materials by 2-ketogluconic acid

producing bacteria. Soil Science 95: 105-114.

DUGAN, P.R. and LUNDGREN D. G., 1964. Acid production by Ferro­

bacillus ferrooxidans and its relation to water pollution.

Developments in Industrial Microbiology 5: 250-257.

240

DUGAN, P. R. and LUNDGREN, D. G., 1965. Energy supply for the chemo­

autotroph Ferrobacillus ferrooxidans. Journal of Bacteriology

89: 825-834.

DUNCAN, D. W. and BRUYNESTEYN, A., 1971. Enhancing bacterial activity

in an uranium mine. Canadian Mining and Metallurgical Bulletin

64: 32-36.

DUNCAN, D. W. and MCGORAN, C. J.M., 1971.Rapid bacteriological extr-

action from materials containing metallic sulphides,

u. s. Patent 3,607,235.

DUNCAN, D. W. and TEATI-IER, C. J., 1966. Method. of extracting metals

from sulphide ores using bacteria and an accelerating agent.

u. s. Patent 3,266,889.

DUNCAN, D. W. and TRUSSELL, P. C., 1964. Advances in microbiological

leaching of sulfide ores. Canadian Metallurgical Quarterly 3:

43-55.

DUNCAN, D. W. and WALDEN, C.C., 1972. Microbiological leaching in

the presence of ferric iron. Developments in Industrial Microb­

iology 13: 66-75.

DUNCAN, D. W., TRUSSELL, P. C. and WALDEN, C.C., 1964. Leaching of

chalcopyrite with Thiobacillus ferrooxidans-Effects of surfacta­

nts and shaking. Applied Microbiology 12: 122-126.

DUNCAN, D. W., WALDEN, C.C. and TRUSSELL, P. C., 1966. Biological

leaching of mill products. Canadian Mining and Metallurgical

241

Bulletin 59: 1075-1079.

DUNCAN, D. W., WALDEN, C. C. and TRUSSELL, P. C., 1967. Accelerated

microbiological ore extraction process. u. s. Patent 3,305,353.

DUNCAN, D. W., WALDEN, C. C., TRUSSELL, I'. C. and LOWE, E. A., 1967-a.

Recent advances in the microbiological leaching of sulfides.

Transactions of Society for Mining Engineers (AIME), 238: 122-128.

DUNCAN, D. W., TRUSSELL, P. C. and WALDEN, C. C., 1967-b. Role of

Thiobacillus ferrooxidans in the oxidation of sulfide minerals.

Canadian Journal of Microbiology 13: 397-403.

DUNN, N. W., RALPH, B. J. and RHODES, S. H., 1977. Further observa­

tions on the FAME profiles of Thiobacillus species. GIAM v

Symposium, Bangkok, November, 1977. (To be published).

DITTRIZAC, J.E. and MacDONALD, R. J. C., 1974. Ferric ion as a

leaching medium. Minerals Science and Engineering 6: 59-100.

EBNER, H. G. and SCHWARTZ, W., 1974. Geomikrobiologische untersuch­

ungen. XII- Verhalten von mikroorganismen auf uranhaltigen

gesteinen. Zeitschrift fur Allgemeine Mikrobiologie 14: 93-102.

EGOROVA, A. A. and DERYUGINA, A. P., 1963. The spore-forming thermo­

philic thiobacterium, Thiobacillus thermophilic Imschenetskii.

Mikrobiologiya 32: 439-446.

EHRLICH, H. L. and FOX, S. I., 1967. Environmental effects on

242

bacterial copper extraction from low grade copper sulphide ores.

Biotechnology and Bioengineering 9: 471-485.

ELSDEN, S. R., 1962. Photosynthesis and lithotrophic carbon dioxide

fixation. In: Cunsalua, I. C. & Stanier, R. Y. (Eds.), The

Bacteria pp. 1-40. Academic Press, London.

FERREIRA, R. C. H. and BURKIN, A. R., 1975. Acid leaching of

chalcopyrite. In: Burkin, A. R. (Ed.), Leaching and Reduction

in Hydrometallurgy pp. 54-66. The Institute of Mining and Meta­

llurgy, London.

FINDLEY, J., APPLEMAN, M. D. and YEN, T. F., 1974. Degradation of

oil-shale by sulfur-oxidizing bacteria. Applied Microbiology

28: 460-464.

FLETCHER, A. W., 1970. Metal winning from low grade ores by

bacterial leaching. Transactions of the Institute of Mining

and Metallurgy 79: C247-C252.

FLETCHER, A. W., 1971. Copper recovery from low grade ores by

bacterial leaching. In: Miller, J. D. A. (Ed.), Microbial

Aspects of Metallurgy pp. 183-194. MTP, Chiltern House

Aylesbury, England.

FLETCHER, A. W., 1971-a. Metal winning from low grade ores by

bacterial leaching. Discussion & contributions. Transactions

of the Institute of Mining and Metallurgy, BO: Cll4-Cll6.

FLOCH, J., ASCOL!, I., LEES, M., MEATH, J. A., and Le BARON, F. N.,

1951. Preparation of lipids extracts from brain tissue.

Journal of Biological Chemistry 191: 833-841.

FORRESTER, J. A., 1959. Destruction of concrete caused by sulfur

243

bacteria in purification plant. Surveyor 118: 881-884.

GALE, N. L. and BECK, J. V., 1966. Competitive inhibition of phospho-

ribulokinase by AMP. Biochemical and Biophysical Research

Communications 24:792-796.

GALE, N. L. and BECK, J. V., 1967. Evidence for the Calvin cycle and

hexose monophosphate pathw~y in ThiobaciZZus ferrooxidans. I

Journal of Bacteriology 94: 1052-1059.

GARRELS, R. S. M. and CHRIST, c.: L., 1965.Solutions, Minerals and

Equilibria. Harper and Ro~e. New York, Evanston and London.

GILCHRIST, F. M. C., 1953. Mic~obiological studies of the corrosion

'

of concrete sewers by sulfuric acid producing bacteria. South

African Industrial Chemistr:y 7: 214-215.

GOEHRING, M. and FELDMANN, U., 1!948. ,Nue Verfahren zur Darstellung van

Kaliumpentathionat und van Kaliumhexathionat. Zeitschrift fur

anorganische Chemie. 257: 223-226.

GOLDING, R. M., RAE, A. D., RAL~H, B. J. and SULLIGOI, L., 1974.

A new series of polynuclear copper Dithiocarbamate copper halide

polymers. Inorganic Chemis~ry 13: 2499-2504.

GOLDING, R. M., HARRIS, B., RAU~H, B. J., RICKARD, P.A. D. and

VANSELOW, D. G., 1977. The nature of the passivation film on

covellite exposed to oxygen. GBF Monograph Series No. 4,

191-200. Conference Bacterlial Leaching, 1977.

GOLOMIZK, A. I. and IVANOV, V. l., 1965. Izv. Vyssh. Ucheb. Zaned.

244

8: 33 [cited from Gupta & Sant, 1970].

GORMELY, L. S. and DUNCAN, D. W., 1974. Estimation of ThiobaciZZus

ferrooxidans concentrations. Canadian Journal of Microbiology

20: 1453-1455.

GORMELY, L. S., DUNCAN, D. W., BRANION, R. M. R. and PINDER, K. L.,

1975. Continuous culture of Thiobacillus ferrooxidans on a

zinc sulphide concentrate. Biotechnology and Bioengineering

17: 31-49.

GUAY, R. and SILVER, M., 1975. ThiobaciZZus acidophiZus sp nov.:

Isolation and some physiological chacteristics. Canadian

Journal of Microbiology 21: 281-288.

GUAY, R., SILVER, M. and TORMA, A. E. , 1975. DNA base composition

of some bacteria oxidizing ferrous iron and metal sulphides.

IRCS Medical Science: Biochemistry, Microbiology, Parasitology

and Infectious Diseases .3: 417.

GUAY, R., TORMA. A. E. and SILVER, M., 1975-a. Oxydation de l'ion

ferreux et mise en solution de !'uranium d'um rninerai par

ThiobaciZZus ferrooxidans. Annales Microbiologie (Institute

Pasteur) 126B: 209-216.

GUAY, R. SILVER, M. and TORMA, A. E., 1976. Base somposition of DNA

isolated from ThiobaciZZus ferrooxidans grown on different sub­

strates. Revue Canadienne de Biologie 35: 61-67.

245

GUITTONNEAU, G., 1925. Sur la formation d'hyposulfites aux depens du

soufre par les microorganisms du sol. Comptes Rendus Hebbomadaires

des seances De L'Academie de Sciences. Paris. 180: 1142-1144.

GUPTA, R. C. and SANT, B. R.,1970. Beneficiationof low grade ores by

microbial leaching. Journal of Scientific and Industrial

Research (India) 29: 372-377.

HALL, W. E., 1961. Unit cell edges of cobalt and cobalt-iron bearing

sphalerites. u. s. Geological Survey Profess. Papers. No. 424-B

271-3. [ Cited from: Chemical Abstracts, 9541 i, 1962.]

HAPPOLD, F. C., JOHNSTONE, K. I., ROGERS, H.J. AND YOUATT, J.B.,

1954. The isolation and characteristics of an organism oxidizing

thiocyanate. Journal of General Microbiology 10: 261-266.

HAPPOLD. F. C. JONES, G. L. and PRATT, D. B., 1958. Utilization of

thiocyanate by ThiobaciZZus thiopa.rus and ThiobaciZZus thio­

cyanoxidans. Nature 182: 266-267.

HAO. P. L. C., CHANG, W. S. and WANG, N. W., 1972. Microbiological

leaching of copper from low grade enargite. Proceedings of IV

International Fermentation Symposium, Fermentation Technology

Today 509-512.

HARDER, W. KUENEN, J. G. and MATIN, A., 1977. A review: Microbial

selection in continuous culture. Journal of Applied Bacteriology

43: 1-24.

HARTS, L. H., 1973. Mineral science and the future of metals.

Mining Engineering 25: (April issue) pp. 57-61.

HENDERSON, M. E. K. and DUFF, R. B., 1963. The release of metallic and

silicate ions from minerals, rocks and soils by fungal activity.

Journal of Soil Science 14: 236-246.

HILL, L. R., 1968. The determination of deoxyribunucleic acid base

composition and its application to bacterial taxonomy. In: Gibbs,

B. M. and Shamton, G. (Eds.), Identification Methods for

246

Microbiologists: Part B, pp. 177-186. Academic Press, New York.

HIRSCHMAN, S. Z., and FELSENFELD, D. G., 1966. Determination of DNA

composition and concentration by spectral analysis. Journal of

Molecular Biology 16: 347-358.

HOBAN, D. J. and LYRICS, R. M., 1975. Phosphoenolpyruvate carboxylase

of Thiobaeillus thioparus. I- General properties. Canadian

Journal of Biochemistry 53: 875-880.

HOFFMAN, L. E.and HENDRIX, J. L., 1976. Inhibition of Thiobacillus

ferrooxida:ns by soluble silver. Biotechnology and Bioengineering,

18: 1161-1167.

HOLT, S. C., SHIVELY, J.M. and GREENAWALT, J. W., 1974. Fine struct­

ure of selected species of the genus Thiobacillus as revealed by

chemical fixation and freeze-etching. Canadian Journal of

Microbiology 20: 1347-1351.

HOWARD, A. and LUNDGREN, D. G., 1970. Inorganic phosphatase from

Ferrobacillus ferrooxidans. Canadian Journal of Microbiology

7: 381-396.

HOWDEN, R. L., LEES, H. and SUZUKI, I., 1972. Phosphoenolpyruvate

carboxylase of Thiobacillus thiooxida:ns. Kinetics and metabolic

control properties. Canadian Journal of Biochemistry 50: 158-165.

HUTCHINSON, M., JOHNSTONE, K. I. and WHITE, D., 1965. The taxonomy

of certain Thiobaeilli. Journal of General Microbiology, 41:

357-366.

HUTCHINSON, M., JOHNSTONE, K. I. and WHITE, D., 1966. Taxonomy of

acidophilic Thiobacilli. Journal of General Microbiology

44: 373-381.

HUTCHINSON, M., JOHNSTONE, K. I. and WHITE, D., 1967. Taxonomy of

anaerobic Thiobacilli. Journal of General Microbiology 47:17-23.

247

IMAI, K., 1971. Bacterial leaching in the mining industry. In:

Sakaguchi, K., Uemura, T. & Kinoshita, S.(Eds.), Biochemical and

Industrial Aspects of Fermentation- Recent Contributions From

Japa.n, pp. 329-336. Kodaush- Tokyo.

IMAI, K., SUGIO, T., TSUCHIDA, T. ans TANO, T., 1975. Effect of

heavy metal ions on the growth and iron-oxidizing activity of

ThiobaciZZus ferrooxidans. Agricultural and Biological Chem­

istry 39: 1349-1354.

IIDA, T., KOZU, H. and USAMI, S., 1975. Rust removal by chemoautotr­

ophic bacteria. Journal of Fermentation Technology 53: 347-353.

ILYALETDINOV, A. N. and KANATCHINOVA, M. K., 1964. Microbiological

transformation of sulfur compounds in periodically flooded soils

of Kzyl-Orda region. Mikrobiologiya 33: 118-125.

ITZKOVITCH, I. J. and TORMA, A. E., 1976. Influence of organic copper

extractants in activity of ThiobaciZZus ferrooxidans. IRCS Medi­

cal Sciences: Biochemistry, Environmental Biology, Medical Micro­

biology, Parasitology and Infectious Diseases 4: 155.

ITZKOVITCH, I. J., ETTEL, V. A. and GEUDRON, A. S., 1974. Copper

removal from Thompson nickel anolyte by solvent extraction.

CIM Bulletin 67: 92-96.

IVANOV, M. V., 1957. Role of microorganisms in depositing sulfur in

hydrogen sulphide springs of Sergleo Mineral Waters.

Mikrobiologiya 26: 338-345.

IVANOV, V. I., 1962. Role of sulphur bacteria in the leaching of

sulphide ores. Dokl. Akad. Nauk. SSr., 146: 447-449.

[Cited from Dutrizac & MacDonald, 1974.]

IVANOV, V. I., 1962-a. Iron oxidation by ThiobaciZZus ferrooxidans.

Mikrobiologiya 31: 795-799.

248

IVANOV, V. I., 1962b. Effect of some factors on iron oxidation by

cultures of Thiobacillus ferrooxidans. Mikrobiologiya 31: 645-648.

IVANOV, V. I. and LIALIKOVA, N. N., 1962. On the taxonomy of iron­

oxidizing Thiobacilli. Mikrobiologiya 31: 468-469.

IVANOV, V. I. and NAGIRNYAK, F. I., 1962.Tsvet. Metally. Mosk. 35: 30.

[Cited from Gupta and Sant, 1970].

IVANOV, V. I., NAGIRNOYAK, F. I. and STEPANOV, B. A., 1961., Bacterial

oxidation of sulphide ore. I- The role of Thiobacillus ferrooxi­

dans in the oxidation ofchalcopyrite and sphalerite. Mikrobiolo­

giya, 30: 688-692.

IWATSUKA, H., 1962. Studies on the metabolism of a sulfur oxidising

bacterium. III- Postoxidative fixation of carbon dioxide.

Plant and Cell Physiology. 3: 167-173.

IWATSUKA, H., KUNO, M. & MARUYAMA, M., 1962. Studies in the metabol­

ism of a sulfur oxidizing Bacterium. II- The system of carbon

dioxide fixation.in, T-hiobacillus. thiooxidans. Plant and

Cell Physiology 3; 157-166.

JACKSON, J. F., MORIARTY, D. J. W. and NICHOLAS, D. J. D., 1968.,

Deoxyribonucleic acid base composition and taxonomy of Thiobacilli

and some mitrifying bacteria. Journal of General Microbiology,

53: 53-60.

JELLINEK, F., 1968.,Sulphides. In: Nickless, G., (Ed.)Inorganic Sulp­

hur Chemistry pp. 669-747. Elsevier Publishing Company,

Amsterdam, London, New York.

249

JENNINGS, P.H., TARASSOFF, P. and FORD, R. E., 1972. Heap tests on

Gaspe copper oxide ore. Paper presented at 101st Annual

Meeting of the American Institute of Mining Engineering,

San Francisco, 1972.

JOHNSON, E. J., 1966. Occurrence of the adenosine monophosphate

inhibition of carbon dioxide fixation in photosynthtic and chem­

osynthetic autotrophs. Archives of Biochemistry and Biophysics

114: 178-183.

JOHNSON, M. J., BORKOWSKI, J. and ENGBLOM, C., 1964. Steam sterli­

sable probes for dissolved oxygen measurements. Biotechnology

and Bioengineering 6: 575-467.

JONES, G. L. and CARRINGTON, E, G,, 1972. Growth of pure and mixed

cultures of microorganisms concerned in the treatment of

carbonization waste liquors. Journal of Applied Bacteriology

35: 395-404.

KAPLAN, L.B., 1956. Evidence of microbiological activity in some

of the geothermal regions of New Zealand.

nal of Science & Technology 37B: 639-662.

New Zealand Jour-

KARAVAIKO, G. I., 1959. Biological influence in oxidation of sulfur

compounds of Rozdolski Deposits. Mikrobiologiya 28: 846-849.

KARAVAIKO, G. I. and AVAKYAN, A. A., 1970. The mechanism of Thiobaci­

llus ferrooxidans multiplication. Mikrobiologiya 39: 950-952.

KARAVAIKO, G. I. and AVAKYAN, A. A., 1971. Submicroscopic organization

of Thiobacillus thiooxidans. Mikrobiologiya 40: 278-283.

KARAVAIKO, G. I., IVANOV, .M. V. and SREBRODOLSKII, B. I., 1962.

Oxidation of stored sulfur ores. Soviet Geology 5: 133-139.

250

KARAVAIKO, G. I. and PIVOVAROA, T. A., 1973. Oxidation of elementary

sulfur by ThiobaeiZZus thiooxidans. Mikrobiologiya 42: 345-350.

KELLY, D. P., 1967. Incorporation of acetate by the chemoautotroph

ThiobaeiZZus neapolitanus strain C. Archives fur Mikrobiologie

58: 99-101.

KELLY, D. P., 1971. Autotrophy: Concepts of lithotrophic bacteria and

thie organic metabolis. Annual Review of Microbiology 25:177-210.

KELLY, D. P. and TUOVINEN, 0. H., 1972. Recommendation that the

mames FerrobaeiZZus ferrooxidans, Leathen & Braley and Ferroba­

eiUus sulfooxidans, Kinsel, be recognized as synonyms of Thio­

baeiZZus ferrooxidans, Temple and Colmer. International Journal of

Systematic Bacteriology 22: 170-172.

KHALID, A. M. and RALPH, B. J., 1976.Unpublished information on the

microbial ecology of the Rum Jungle Mine area, Northern Territory,

Australia. (Australian Atomic Energy Commission Research Contract

74/F/40).

KHALID, A. M. and RALPH, B. J., 1977. The leaching behaviour of

various zinc sulphide minerals with three ThiobaeiZZus species.

GBF Monograph Series No. 4 ( August, 1977). Conference Bacterial

Leaching pp. 165-173.

KINSEL, N. A., 1960. New sulphur oxidizing iron bacterium, Ferro­

baei Z Zus suZ f ooxidans Sp . Nov. Journal of Bacteriology 80: ::- ·,'

628-632.

KNAYSI, G., 1943. A cytobiological and microbiological study of

ThiobaeiZZus thiooxidans. Journal of Bacteriology 46: 451-461.

KOCUR, M., MARTINEC, T. and MAZANCE, K., 1968. Fine structure of

ThiobaeiZZus noveZZus. Journal of General Microbiology 52:

343-345.

251

KOROBUSHKINA, E. D., CHERNIAK, A. S. and MINEEV, G. G., 1974. Dissolut­

ion of gold by microorganisms and their metabolism. Mikrobiologiya

43: 49-54.

KOROSTELEV, P. G. and CHAINIKOVA, N. A., 1966. Possible use of

FeS-ZnS systems as geological thermometers. Vop. Geol. Sev.

Zap. Sekt., Tikhookean, poyasa, Valadivostok. 185-187.

[Cited from: Chemical Abstracts 67: 34748 y, 1967.]

KUENEN, J. G. and VELDKAMP, H., 1973. Effects of organic compounds on

growth of chemostat cultures of Thiomicrospira pelophila,

Thiobacillus thioparus and Thiobacillus neapolitanus.

Archives fur Mikrobiologie 94: 173-190.

KULLERUD, G. and NEUMANN, H., 1953. The temperature of granitization

in the Rendalsvik area, northern Norway. Norsk. Geol. Tidsskr.

32: 148-155. [Cited from: Chemical Abstracts 4377d, 1954.]

LANDESMAN, J., DUNCAN, D. W. and WALDEN, C.C., 1966. Iron oxidation by

washed cell suspensions of the chemoautotroph Thiobacillus ferro­

oxidans. Canadian Journal of Microbiology 12: 25-33.

LANDESMAN, J., DUNCAN, D. W. and WALDEN, C. C., 1966-a. Oxidation of

inorganic sulphur compounds by washed cell suspensions of Thio­

bacillus ferrooxidans. Canadian Journal of Microbiology 12:957-964.

LAPTEVA, A. M., KRIUCHKOV, V. A. and GOLOMZIK, A. I., 1971. Application

of gel plates impregnated with the medium 9K for quantitative

control and isolation of Thiobacillus ferrooxidans. Mikrobiologiya

40: 572-574.

LAUWERS, A. M. and HEINEN, W., 1974. Bio-degradation and utilization of

silica quartz. Archives fur Mikrobiologie 95: 67-78.

252

LAZAROFF, N., 1963. Sulfate requirement for iron oxidation by Thio­

baaiZZus ferrooxida:ns. Journal of Bacteriology 85: 78-83.

LAZAROFF, N., 1975. Specificity of the anionic requirement for iron

oxidation by cells of ThiobaaiZZus ferrooxidans. Abstracts Annual

Meeting of American Society for Microbiology, I 119.

LAZAROFF, N., 1977. Specificity of anionic requirement for iron

oxidation by ThiobaaiZZus ferrooxida:ns. Journal of General

Microbiology 101: 85-91.

LEATHEN, W.W. and BRALEY, S. A., 1954. A new iron-oxidizing bacterium:

·FerrobaaiZZus ferrooxida:ns. Bacteriological Proceedings

G64, p 44.

LEATHEN, W.W., BRALEY, S. A. and MCINTYRE, L. D., 1953. The role of

bacteria in the formation of acid from certain sulfuric constitu­

ents associated with bituminous coal. II- Ferrous irom oxidizing

bacteria. Applied Microbiology 1: 65-68.

LEATHEN, W.W. and MADISON, K. M., 1949. The oxidation of ferrous

iron by bacteria found in acid mine water.. Bactei:iological

Proceedings. p.64.

LEATHEN, W. W, KINSEL, N. A. and BRALEY, S. A. 1956. FerrobaaiZZus

ferrooxidans:A chemosynthetic, autotrophic bacterium. Journal of

Bacteriology 72: 700-704.

LEATHEN, W.W., MCINTYRE, L.D.and BRALEY, S. A., 1951. A medium for the

study of the bacterial oxidation of ferrous iron. Science 114:

280-281.

LECHEVALIER, M. P., 1977. Lipids in bacterial taxonomy- A taxonomist's

view. CRC Critical Reviews in Microbiology, 5: 109-210.

LEHNINGER, A. L., 1970., Biochemistry. Worth publishers, Inc.,

253

70 Fifth Avenues, New York, pp. 832.

Le ROUX, N. W., 1966.

Annual Report, Division of Plant Industry, Commonwealth Scientific

and Industrial Research Organization, Australia. pp.147-148.

Le ROUX, N. W., Mineral attack by microbiological processes.

In: Miller, J. D. A. (Ed.), Microbial Aspects of Metallurgy

pp. 173-182. MTP, Chiltern House, Aylesbyry, England.

Le ROUX, N. W., NORTI-1, A. A. and WILSON, J.C., 1973. Bacterial

oxidation of pyrite. Proceedings of the 10th International

Mineral Processing Congress, London, 2-14 April, Paper No. 10.

Institute of Mining and Metallurgy, London.

Le ROUX, N. W., WAKERLEY, D.S. and HUNT, S. D., 1977. Thermophilic

Thiobacillus-type bacteria from Iceland thermal areas.

Journal of General Microbiology 100: 197-201.

LEVIN, R. A., 1971. Fatty acids of Thiobacillus thiooxidans.

Journal of Bacteriology 108: 992-995.

LEVIN, R. A., 1972. Effect of cultural conditions on the fatty acid

composition of Thiobacillus novellus.

Journal of Bacteriology 12: 903-909.

LIESKE, R., 1912. Untersichungen uber die physiologie denitrifiziere­

nder Schwefel bacterien. Ber. deut. bot. Gesell, 30: 12-22.

[ Cited from: Baalsrud & Baalsrud, 1954.]

LIPMAN, C. B. and McLESS, E., 1940.

Soil Science 40: 429.

[Cited from Roy & Trudinger, 1970. ]

LITCHFIELD, J. H., 1976. Food Microbiology. In: Miller, B.M.

and Litsky, W. (Eds.), Industrial Microbiology. pp. 257-308.

254

McGraw-Hill Book Company, 1976.

LONDON, J., 1963. ThiobaciZZus noveZZus nov. sp - A novel type of

facultative autotroph. Archives fur Mikrobiologie 46: 329-337.

LONDON, J. and RITTENBERG, S. C., 1966. Effects of organic matter on

the growth of ThiobaciZZus intermedius. Journal of Bacteriology

91: 1062-1069.

LONDON, J. and RITTENBERG, S. C., 1967. Thiobacillus perometabolis,

nov.sp. a non-autotrophic ThiobaciZZus. Archives fur Mikro-

biologie 59: 218-225.

LUNDGREN, D. G., 1975. Microbiological problems in strip mine

areas: Relationship to the metabolism of ThiobaciZZus ferro­

oxidans. Ohio Journal of Science 75: 280-287.

LUNDGREN, D. G., ANDERSEN, K. J., REMSEN, C. C. and MAHONEY, R. P.,

1964. Cutlture. structure and physiology of the chemoautotroph

FerrobaciZZus ferrooxidans. Developments in Industrial

Microbiology 6: 250-259.

LYALIKOVA, N. N., 1959. Fiziologiya i Ekologiya ThiobaciZZus

ferrooxidans v Svyazi s Yego Rol'yu v Okinlenii Sul'fidnykh

Rud. [ The physiology and ecology of ThiobaciZZus ferrooxidans

in relation to its role in the oxidation of sulfide ores].

Diss. Bibl-ka Otd. Biol. Nauk AN SSSR.

[ Cited from: Kuznetsov, S. I., Ivanov, M. V., and Lyalikova,

N. N. (Eds.), Introduction to Geological Microbiology, pp. 252.

McGraw-Hill Book Company, 1963.]

LYALIKOVA, N. N., 1960. The part played by ThiobaciZZus ferrooxidans

in oxidizing the sulfide ores in the copper pyrite deposits of

the Middle Urals. Mikrobiologiya 29: 773.

255

LYALIKOVA, N. N., 1961. The role of bacteria in the oxidation of the

sulphide ores in the copper-nickel deposits of the Kola Pensisula.

Mikrobiologiya 30: 135-139.

LYALIKOVA, N. N. and MOKEICHEVA, L. Ya., 1969. The role of bacteria in

gold migration in deposits. Mikrobiologiya 38: 805-810.

MacDONALD, D. G., and CLARK, R.H., 1970.The oxidation of aqueous ferr­

ous sulpate by Thiobaaillus ferrooxidan. Canadian Journal of

Chemical Engineering 48: 669-676.

MacELROY, R. D., JOHNSON, E. J. and JOHNSON, M. K., 1968. Characteri­

zation of ribulose diphosphate carboxylase and phosphoribulo­

kinase from Thiobaaillus thioparus & Thiobaaillus neapolitanus.

Archives of Biochemistry and Biophysics 127: 310-316.

MlcELROY, R. D., JOHNSON, E. J. and JOHNSON, M.K., 1968-a. Allosteric

regulation of phosphoribulokinase activity. Biochemical and

Biophysical Research Communications 30: 678-682.

MACKINSTOSH, M. E., 1971. Nitrogen fixation by Thiobaaillus ferroo­

xidans. Journal of General Microbiology 66: i-ii.

MACKINTOSH, M. E., 1976. Fixation of 15N2 by Thiobaaillus

ferrooxidans. Proceedings of the Society of General Microbiology

4 (Part 1) 23.

MAGNEM, de MM. R., BERTHELIN, J. and DOMMERGIJES, Y., 1973. Solubilis­

ation de l'uranium dans les roches, par des bacteries n'appatenant

pas au genne Thiobaaillus. Comptes Rendus Hebdomadaires des Seances

de l'Academie des Sciences de Paris t 276 (9 mai) series D-

2625-2628.

256

MAHONEY, R. R. and EDWARDS, M. R., 1966. Fine structure of Thiobaeillus

thiooxidans. Journal of Bacteriology 42: 487-495.

MAJIMA, H.and PETERS, E., 1968. Electrochemistry of sulfide dissolution

in hydrometallurgical systems. VIII International Mineral Process­

ing Congress, Leningrad.

MAJUMDAR, A. J., and ROY, R., 1965. Pressure-temperature dependence of

the sphalerite and wurtzite inversion in ZnS. American Mineralo­

gist 50: 1121-1125.

MALOUF, E. E. and PRATER, J. D., 1961. The role of bacteria in the

alteration of sulphide minerals. Journal of Metals 13: 353-356.

MANNING, H. L., 1975. New medium for isolating iron-oxidizing and

heterotrophic acidophilic bacteria from acid mine drainage.

Applied Microbiology 30: 1010-1016.

MARCHLEWITZ, B. and SCHWARTZ, W., 1961. Investigations of microbial

association of acid mine waters. Zeitschrift fur Allgemein

Mikrobiologie 1: 100-114.

MARGALITH. P., SILVER, M. and LUNDGREN, D. G., 1966.Sulfur oxidation

by the iron-bacterium Ferrobaeillus ferrooxida:ns. Journal of

Bacteriology 92: 1706-1709.

MARMUR, J., 1961. A procedure for the isolation of deoxyribonucleic

acid from microorganisms. Journal of Molecular Biology 3:

208-218.

MARSHALL, K. C., 1975. Clay mineralogy in relation to survival of

soil bacteria. Annual Review of Phytopathology 13: 357-373.

MATIN, A. and RITTENBERG, S. C., 1970. Utilization of glucose in

heterotrophic media by Thiobacillus intermedius. Journal of

Bacteriology 104: 234-238.

257

MATIN, A. RITTENBERG, S. C., 1971. Enzymes of carbohydrate metabolism

in ThiobaciZZus species. Journal of Bacteriology 107: 179-186.

MAXWELL, W. A., METZLER, R. and SPOERL, E., 1971. Uranyl nitrate

inhibition in Saccharomyces cerevisiae. Journal of Bacteriology

105: 1205-1206.

MAYEUX, J. V. and JOHNSON, E.J., 1967. Effect of AMP, ADP and

reduced NAO on ATP-dependent carbon dioxide fixation in the

the autotroph ThiobaciZZus neapoZitanus. Journal of Bacteriol­

ogy 94: 409-414.

MAYLING, A. A., 1966. Cyclic~ leaching process employing iron

oxidizing bacteria. Canadian Patent 744, 701.

MAYLING, A. A., 1969. Bacterial leaching of basic metal sulfide

ores. u.s.Patent 3,455,679.

McCARTHY, J. T. and CHARLES, A. M., 1973. Purification and purine nuc­

leotide regulation of ribulose-1,5, diphosphate carboxylase from

ThiobaciZZus noveZZus. FEBS Letters 37: 329-332.

McCARTHY, J. T., and CHARLES, A. M., 1974. Co2 fixation by the

facultative autotroph ThiobaciZZus noveZZus during autotrophy­

heterotrophy interconversions. Canadian Journal of Microbiology

20: 1577-1587.

McFARLANE, A. S., 1942. Behaviour of lipoids in human serum.

Nature 149: p. 439.

McGORAN, C. J.M., DUNCAN, D. W. and WALDEN,C.C.,1969. Growth of Thio­

baciZZus ferrooxidans on various substrates. Canadian Journal

of Microbiology 15: 135-138.

MEHTA, K. B. and Le ROUX, N. W., 1974. Effect of wall growth on conti­

nuous biological oxidation of ferrous iron. Biotechnology and

258

Bioengineering 16: 559-563.

MEYER, C. W. and YEN, T. F., 1976. Enhanced dissolution of oil shale

by bioleaching with Thiobacilli. Applied and Environmental

Microbiology 32: 610-616.

MILLER, B. M. and LITSKY, W. (Eds.). 1976. Industrial Microbiology

MgGraw-Hill Book Company, Sydney. 465 pp.

MIZOGUCHI, T., SATO, T. and OKABE, T., 1976. New sulfur-oxidizing

bacteria capable of growing heterotrophically, Thiobacillus

rubellus nov sp & Thiobacillus delicatus nov. sp. Journal

of Fermentation Technology 54: 181-191.

MOSS, F. J. and ANDERSEN, J.E., 1968. The effect of environment on

bacterial leaching rates. Proceedings of the Australian Institute

of Mining and Metallurgy 225: 15-25.

MURPHY, J. R., GIRARD, A. E. and TILTON, R. C., 1974. Ultrastructure

of a marine Thiobacillus. Journal of General Microbiology 85:

130-138.

MURR, L. E. and BERRY, V. K., 1976. Direct observations of selective

attachment of bacteria on low-grade sulfide ores and other mineral

surfaces. Hydrometallurgy 2: 11-24.

MURRAY, R. G. E., 1963. The organelles of bacteria. In: Mazia, D. &

Tyler, A, (Eds.), General Physiology of Cell Specialization pp 28-

52. MgGraw-Hill Book Company, Inc. N.Y.

MURRAY, R. G. E. and WATSON, S. W., 1965. Structure of Nitrocystis

ocea:nus and comparison with Nitrosomonas & Nitrobacter.

Journal of Bacteriology 89: 1594-1609.

MUTZE, B. and ENGEL, H. 1960. Bacterial oxidation of sulfur in the

river Elbe. Archives fur Mikrobiologie 35: 303:309.

259

NATHANSOHN, A., 1902. Uber eine neu gruppe von schwefelbakterien und

ihren stoffwechsel. Mitt. z.ool. stn. Neapol. 15: 655-680.

NICKERSON, W. J., 1969. Microbial transformations of naturally

occurring polymers. In: Perlman, D. (Ed.), Fermentation Advances,

631-634.

NIELSEN, A. M. and BECK, J. V., 1972., Chalcocite oxidation and

coupled carbon dioxide fixation by ThiobaciZZus ferrooxida:ns.

Science 175: 1124-1126.

NIEMELA, S. I. and TUOVINEN, O.H., 1972. Acidophillic ThiobaciZZi in

river Sirppujoki. Journal of General Microbiology 73: 23-28.

NORDYKE, M. D., 1971. Other unique applications of PNE(Peaceful

Nuclear Explosions). In: Peaceful Nuclear Explosions II- The

Practical Applications pp 223-232. International Atomic

Energy Agency, Viena.

NOVICK, A., and SZILARD, L., 1950. Description of the chemostat.

Science 112: 715-716.

NOVICK, A., and SZILARD, L., 1950-a. Experiments with the chemostat on

spontaneous mutation of bacteria. Proceedings of the National

Academy of Sciences (Washington) 36: 708-719.

O'LEARY, W. M., 1962. The fatty acids of bacteria. Bacteriological

Reviews 26: 421-447.

O'LEARY, W. M., 1970. Bacterial lipid metabolism. In:Florkins, M. &

Stotz, E. H. (Eds.), Comprehensive Biochemistry 18: 229-264.

Elsevier Publishing Co. Amsterdam.

OTTOW, J.C. and VON KLOPOTEK, A., 1969. Enzymatic reduction of

iron oxide by fungi. Applied Microbiology 18: 41-43.

260

PARES, Y., 1964. Intervention des bacteries dans la solubilisation

du cuivre. Annales de L'Institut Pasteur 107: 132-135.

PARES, Y., 1964-a. Action de Serr'atia macescens dans le cycle biologi­

que des m'etaux. Annales de L'Institut Pasteur 107: 136-141.

PARES, Y. 1964-b. Action d'Agrobacteriwn twnefaciens dans la mise

en solution de l'or. Annales de L'Institut Pasteur 107:141-143.

PARKER, C. D., 1945. The corrosion of concrete. I- The isolation of a

species of bacterium associated with the corrosion of concrete

exposed to atmospheres containg H2S. Australian Journal of

Experimental Biology and Medical Science 23: 81-90.

PARKER, C. D. and PRISK, J., 1953. The oxidation of inorganic compounds

of sulphur by various sulphur bacteria. Journal of General

Microbiology 8: 344-364.

PAWLEK, F. E., 1969. Research in pressure leaching. Journal of

_South African Institute of Mining and Me.tallurgy 69: 632-:-654.

PECK, H. D. Jr., 1960. Adenosine-5'-phosposulfate as an intermediate

in the oxidation of thiosulphate by Thiobacillus thioparus.

Proceedings of the National Academy of Sciences, U.S.A., 46:

1053-1057.

PECK, H. D. Jr., DEACON, T. E. and DAVIDSON, J. T., 1965. Studies on

APS-reductase from Desulphovibrio desulphuricans& Thiobacillus

thioparus. I- The assay and purification. Biochimica et Biophysica

Acta 96: 429-446.

PECK, H. D. Jr. and FISHER, E.Jr. 1961. The oxidation of thiosulfate

and phosphorylation in extracts of Thiobacillus thioparus.

Journal of Biological Chemistry 237: 190-197.

PEETERS, T. & ALEEM, M. I. H., 1970. Oxidation of sulfur compounds

261

and electron transport in ThiobaciZZus denitrificans. Archives

fur Mikrobiologie 71: 319-330.

PFENNIG, N., 1961. Eine vollsynthetische nahrlosung zur sele ktiven

a neicher ung einiger schwefelpur purbaketrien. Die Natur­

wissenschaften 48: 136.

PIVORAROVA, T. A., and KARAVAIKO, G. I., 1973. Submicroscopic organi­

zation and function of some structures in ThiobaciZZus neapoZitanus.

Mikrobiologiya 42: 672-676.

PIVORAROVA, T. A. and KARAVAIKO, G. I., 1973-a. Submicroscopic organi­

zation of ThiobaciZZus thiocyanoxidans. Mikrobiologiya

42: 1078 - 1080.

POSTGATE, J. R., 1966. Media for sulphur bacteria.

Laboratory Practice 15: 1239-1244.

POURBAIX, M. J. N., 1966. (Ed.) Atlas of Electrochemical Equlibria

in Aqueous Solutions. Pergammon Press, Oxford.

PRATT, V., 1972. Numerical Taxonomy- A critique. Journal of Theoretical

Biology 36: 581-592.

PUDOVKINA, I. A., RYABEVA, E. G., AKSENOVA, E. K. and DUBAKINA, L.S.,

1968. Effect of chemical composition on some physical properties

of sphalerite ores. Miner. Syr'e. No. 18, 13-28.

[Cited from: Chemical Abstracts 134999 c: 72: 1970]

PUROHIT, K., McFADEN, B. A. and COHEN, A. L., 1976. Purification,

quaternary structure, composition and properties of D-ribulose-

1-5- bisphosphate carboxylase fron ThiobaciZZus intermedius.

Journal of Bacteriology 127: 505-515.

PUROHIT, K., M:FADEN, B. A. and SHAYKH, M. M., 1976-a. D-ribulose

262

1,5-bisphosphate carboxylase and polyhederal inclusions bodies

in Thiobacillus intePmedius. Journal of Bacteriology 127: 516-522.

RALPH, B. J., 1978. Oxidative processes. In: Biogeochemical Cycling

of Mineral-forming Elements. Elsevier Press. (In press).

RAO, G.. •. -~. _ 2nd BERGER, L. R., 1970. Basis of pyruvate inhibition

in Thiobacillus thiooxidans. Journal of Bacteriology 102: 462-466.

RAO, G. S. and BERGER, L. R., 1971. The requirement of low pH for growth

of Thiobacillus thiooxidans. Archive fur Mikrobiologie 79:338-344.

RAY, B., 1969. II-IV Compounds. Pergarnon Press, Oxford, 268 pp.

RAZZELL, W. E., 1962. Bacterial leaching of metallic sulphides.

Canadian Mining and Metallurgical Bulletin 55: 190-191.

RAZZELL, W. E. and TRUSSELL, P. C., 1963. Isolation and properties of

and iron-oxidizing Thiobacillus. Journal of Bacteriology 85:

595-603.

RAZZELL, W. E. and TRUSSELL, P. C., 1963-a. Microbiological leaching of

metallic sulphides. Applied Microbiology 11: 105-110.

REMSEN, C. C. and LUNDGREN, D. G., 1963. The heterotrophic growth of

the chemoautotroph Ferrobacillus ferrooxidans. Bacteriological

Proceedings 33.

REMSEN, C. C. and LUNDGREN, D. G., 1966. Electron microscopy of the

cell envelope of Ferrobacillus ferrooxidans prepared by freeze­

etching and chemical fixation techniques. Journal of Bacteriology

92: 1765-1771.

RITTENBERG, S. C., 1969. The role of exogenous organic matter in the

physiology of chemolithtrophic bacteria. Advances in Microbial

Physiology 3: 159-196.

ROBERTS, W. M. B., 1965. Recrystallization and mobilization of

263

sulfides at 2,000 atmospheres and in the temperature range

50° - 145°. Economic Geology 60: 168-180.

ROGOFF, M. H., SILVERMAN, M. P. and WENDER, I., 1960. Elimination o,f, '.

sulfur from cpal by microbiological action. American Chemical

Society, Division of Gas & Fuel Chemistry 2: 25-36.

ROGOVSKAYA, T. I. and LAZAREVA, M. F., 1961.Intensification of bio­

chemical purification of industrial sewage. III- Microbiologi­

cal characteristics of the biofilm purifying H2S containing

sewage. Mikrobiologiya 30: 699-702.

ROSLYCKY, E. B., 1972. Reliable procedure for silica gel preparation.

Applied Microbiology 24: 844-845.

ROSSI, G., 1974., Microbial leaching of ores: Progress and prospects.

Bollettino della Associazione Mineraria subalpina. 11: 1-40.

ROTHSTEIN, A., 1954. Enzyme systems of the cell surface involved

in the uptake of sugars by yeasts. Symposium of Society for

Experimental Biology 8: 165-201.

ROY, A. B. and TRUDINGER, P.A., 1970. The Biochemistry of Inorganic

Compounds of Sulphur. Cambridge University Press, Cambridge. pp.400.

RUDOLFS, W., 1922. Oxidation of pyrite by microorganisms and their

use for making mineral phosphate available. Soil Science

14: 135-147.

RUDOLFS, W. and HELBRONNER, A., 1922. Oxidation of zinc sulphide

by microorganisms. Soil Science 14: 459-465.

264

SADLER, W.R. and TRUDINGER, P.A., 1967. The inhibition of microo­

rganisms by heavy metals. Mineralium Deposita 2: 158-168.

SANTER, M. and VISHNIAC, W., 1955. co2 incorporation by extracts of

ThiobaaiZZus thioparus. Biochimica et Biophysica 18: 157-158.

SANTER, M., BOYER, J. and SANTER, U., 1959. ThiobaaiZZus noveZZus .

I- Growth on inorganic and organic media. Journal of Bacteriology

78: 197-202.

SANZONOV, V. D., 1961. Determination of temperature of sphalerite

from its iron content. Tr. Inst. Geol. Akad. Nauk. Tadzh. ssr.

4: 215-233. [ Cited from: Chemical Abstracts 1241h, 1963]

SARGEANT, K., BUCK, P. W., FORD, J. W. S. and YEO, R. G., 1966.

Anaerobic production of ThiobaaiZZus denitPifiaans for the

enzyme rhodanese. Applied Microbiology 14: 998-1003.

SCHAEFFER, W. I. and UMBREIT, W.W., 1963. Phosphotidylinositol as a

wetting agent in sulfur oxidation by ThiobaaiZZus thiooxidans.

Journal of Bacteriology 85: 492-493.

SCHAEFFER, W. I., HOLBERT, P. E. and UMBREIT, W.W., 1963. Attachment

of Thiobacillus thiooxidans to sulfur crystals. Journal of

Bacteriology 85: 137-140.

SCHLEGEL, H. G., 1975. Mechanism of chemo-autotrophy. In: Kinne, 0.

(Ed.), Marine Ecology, Volume II, Part I, pp 9-60. John Wiley

& Sons, New York.

SCHNAITMAN, C. A., KORCZYNYSKI, M. S. and LUNDGREN, 1969. Kinetic

studies of iron oxidation by whole cells of FePPobaaiZZus

fePPooxidans. Journal of Bacteriology 99: 552-557.

SCHRAUZER, G. N. and GUTH, T. D., 1976. Hydrogen evolving systems.

I- The formation of hydrogen from aqueous suspensions of Fe(OH) 2

265

and reactions with reducible substrates, including molecular

nitrogen. Journal of the American Chemical Society 98: 3508-3513.

SCHWARTZ, A. and SCHWARTZ, W., 1965. Geomikrobiologische Untersuchungen.

VII- Uber das Vorkommen von Mikroorganismen in Solfataren und

heiben Quellen. Zeitschrift fur Allgemeine Mikrobiologie 5:395-405.

SCOTT, T. R. and DYSON, N. F., 1968. The catalyzed oxidation of zinc

sulphide under acid pressure leaching conditions. Transactions of

the Metallurgical Society of AIME 242: 1815-1821.

SHAFIA, F. and WILKINSON, R. F., 1969. Growth of Ferrobacillus ferro­

oxidans on organic matter. Journal of Bacteriology 97: 256-260.

SHIVELY, J.M., 1974. Inclusion bodies of prokaryotes. Annual Review

of Microbiology 28: 167-187.

SHIVELY, J.M., DECKER, G. L. and GREENWALT, J. W., 1970. Comparative

ultrastructure of Thiobacilli. Journal of Bacteriology 101:618-627.

SHIVELY, J.M., BALL, F. L., BROWN, D. H. and SAUNDERS, R. F.,1973.

Functional organelles in prokaryotes: Polyhederal inclusions

(carboxysomes) of Thiobacillus neapolitanus. Science 182:584-586.

SHIVELY, J.M., BALL, F. L. and KLINE, B. W., 1973-a. Electron

microscopy of carboxysomes (polyhederal bodies) of Thiobacillus

neapolitanus. Journal of Bacteriology 116:1405-1411.

SHUEY, R. T., 1975. Sphalerite-ZnS. In: Semiconducting Ore Minerals

319-327. Elsevier Scientific Publishing Company. Amsterdam,Oxford.

SHULER, M. L. and TSUCHIYA, H. M., 1975. Determination of cell numbers

and size for Thiobacillus ferrooxidans by electrical resistance

method. Biotechnology and Bioengineering 17: 621-624.

SILVER, M., 1970. Oxidation of elemental sulfur and sulfur compounds

and co2 fixation by Ferrobacillus ferrooxidans. Canadian Journal

of Microbiology 16: 845-849.

266

SILVER, M. 1977. Metabolic mechanisms of iron-oxidizing Thiobacilli.

In: Murr, L. E., Torma, A. E. & Brierley, J. A. (Eds.) Metallurgi­

cal Applications of Bacterial Leaching and Related Microbiological

Phenonena. The Academic Press, N. Y. (In press).

SILVER, M. and TORMA, A. E., 1974. Oxidation of metal sulphides by

Thiobacillus ferrooxidans grown on different substrates.

Canadian Journal of Microbiology 20: 141-147.

SILVER, M. , MARGALITH, P. and LUNDGREN D. G., 1967. Effects of gluco­

se on carbondioxide assimilation and substrate oxidation by

Ferrobacillus ferrooxidans. Journal of Bacteriology 93: 1765-1769.

SILVERMAN, M. P. and EHRLICH, H. L., 1964. Microbial formation and

degradation of minerals. Advances in Applied Microbiology 6:

153-206.

SILVERMAN, M. P. and LUNDGREN, D. G., 1959. Studies on the chemoauto­

trophic bacterium Ferrobacillus ferrooxidans. I- An improved

medium and a harvesting procedure for screening high cell yields.

Journal of Bacteriology 77: 642-647.

SILVERMAN, M. P. and LUNDGREN, D. G., 1959-a. Studies on chemoautotro­

phic iron bacterium Ferrobacillus ferrooxidans. II- Manometric

studies. Journal of Bacteriology 78: 326-331.

SILVERMAN, M. P. and MUNOZ, E. F., 1970. Fungal attack on rocks:

Solubilisation and altered infrared spectra. Science 169: 985-987.

SILVERMAN, M. P. and ROGOFF, M. H., 1961. Morphological variations in

Ferrobacillus ferrooxidans related to iron oxidation.

Nature 191: 1221-1222.

SILVERMAN, M. P., ROGOFF, M. H. and WENDER, I., 1961. Bacterial

oxidation of pyritic materials in coal. Applied Microbiology 9:

491-496.

267

SILVESTRI, L. G., HILL, L.R., 1965. Agreement between deoxyribonucleic

acid base composition and taxonometeric classification of Gram­

positkve Cocci. Journal of Bacteriology 90: 136- 140.

SKERMAN, V. B. D., 1967. A Guide to the Identification of the

Genera of Bacteria. 2nd. Edition. The Williams & Wilkins Company,

Baltimore.

SKINNER, B. J., 1960. Unit-cell edges of natural and synthetic

sphalerites. American Mineralogist 46: 1399-1411.

SMITH, A. J. and HOARE, D.S., 1977. Specialist phototrophs, lithotro­

phs and methylotrophs: a unity among a diversity of procaryotes?

Bacteriological Reviews 41: 419-448.

SMITH, F. G., 1955. Structure of zinc sulphide minerals. American

Mineralogist 40: 658-675.

SNEATH, P.H. A., 1957. The application of computer to taxonomy. Jour­

nal of General Microbiology 17: 201-226.

SOKOLOVA, G. A., 1960. Microbiological production of sulfur from

sulfide water strata. Mikrobiologiya 29: 888- 893.

SOKOLOVA, G. A. and KARAVAIKO, G. I., 1962. Biogenic oxidation of

sulfur of the Rozdol ore under laboratory conditions.

Mikrobiologiya 31: 984-989.

SOKOLOVA, G. A. and KARAVAIKO, G. I., 1963. Biogenic oxidation of

sulfur in Rozdol ore in laboratory conditions.

Mikrobiologiya 31: 797-800.

SORBO, B., 1957. A colorimetric method for the determination of

thiosulphate. Biochimica et Biophysica Acta 23: 412-416.

STAMM, H. and GOEHRING, M., 1942. Zur kenntnis der polythionsauren und

ihren bildung. Neu verfahren zur darstellung von kaliumtrithionat

und von kallumtetrathionat. Zeitschrift fur anorganische und

268

allegmeine chemie 250: 226-228.

STARKEY, R. L., 1934. Cultivation of organisms concerned in the

oxidation of thiosulfate. Journal of Bacteriology 28: 365-386.

STARKEY, R. L., 1934-a. The production of polythionates fron thiosulfate

by microorganisms. Journal of Bacteriology 28: 387-400.

STARKEY, R. L., 1935. Isolation of some bacteria which oxidize

thiosufate. Soil Science 39: 197-219.

STARKEY, R. L., JONES, G. E. and FREDERICK, L. R., 1956. Effects of

medium agitation and wetting agents on oxidation of sulfur by

ThiobaciZZus thiooxidans. Journal of General Microbiology

15: 329-334.

STOTSKY, G. and REM, L.T., 1966. Influence of clay minerals on

microorganisms. I- Montmorillonite and kaolinite on bacteria.

Canadian Journal of Microbiology 12: 547-563.

SUMMERS, A. 0., CHOTTEL, J.,CLARK, D. and SILVERS, S., 1975. Plasmid-

borne Hg(II) and organomercurialresistance. In: Schlessinger,

D. (Ed.) Microbiology-1974 pp 219-226. The American Society for

Microbiology, Washington, D.C.

SUZUKI, I. and WERKMAN, C. H., 1957. Phosphoenolpyruvate carboxylase

in extracts of ThiobaciZZus thiooxidans, a chemoautotrophic

bacterium. Archives of Biochemistry and Biochemistry 72: 514-515.

SUZUKI, I., and WERKMAN, C. H., 1958. Chemoautotrophic fixation of co2

by ThiobaciZZus thiooxidans. Iowa State College Journal of

Science 32: 475-483.

SUZUKI, I. and WERKMAN, C. H. , 1958-b. Chemoautotrophic Co2 fixation

by extracts of ThiobaciZZus thiooxidans. Archives of Biochemistry

and Biophysics 77: 112-123.

269

TABITA, F. R. and LUNDGREN, D. G., 1970. Glucose metabolism in

FerrobaciZZus ferrooxidans. Bacteriological Proceedings Pl7, pl7.

TABITA, R. and LUNDGREN, D. G., 1971. Utilization of glucose and the

effect o f organic compounds on the chemoli thotroph ThiobaciUus

ferrooxidans. Journal of Bacteriology 108: 328-333.

TABITA, R. and LUNDGREN, r. G, 1971-a. Heterotrophic metabolism of the

chemolithotroph ThiobaciZZus ferrooxidans. Journal of Bacteriology

108: 334-342.

TABITA, R. and LUNDGREN, D. G., 1971-b. Glucose-6-phosphate dehydroge­

nase from the chemolithotroph ThiobaciZZus ferrooxidans. Journal

of Bacteriology 108: 343-352.

TAKIMOTO, K., MINATO, T. and HIRONO, S., 1970. Relation between

the unit-cell dimensions and minor elements of sphalerite from the

Chichbu Mine, Sitama Prefacture, Japan. Suiyotaishi 14: 186-189.

[ Cited from: Chemical Abstracts 8060 g]

TANO, T. and IMAI, K., 1968. Physiological studies on ThiobaciZZi.

VI- Certain properties of NADPH-cytochrome C oxidoreductase of

ThiobaciZZus thiooxidans.

32: 401-404.

Agricultural and Biological Chemistry

TAYLOR, B. F. and HOARE, S. L. , 1969. Thiobacillus denitrificans

as an obligate chemolithotroph. Bacteriological Proceedings p63.

TAYLOR, B. F., HOARE, D.S. and HOARE, S. L., 1971 . ThiobaciZZus

denitrificans as an obligate chemolithotroph: Isolation and growth

studies. Archives fur Mikrobiologie 78: 193-204.

TAYLOR, J. H. and WHELAN, P. F., 1943. The leaching of cupreous pyrites

and the precipitation of copper at RioTinto, Spain. Transactions

of Institute of Mining and Metallurgy 52: 35-96.

TEMPLE, K. L. and COLMER, A., 1951. The autotrophic oxidation of iron

270

by a new bacterium, ThiobaciZZus ferrooxidans. Journal of

Bacteriology 62: 605-611.

TEMPLE, K. L. and DELCHAMPS, E.W., 1953. Autotrophic bacteria and the

formation of acid in bituminous coal mines. Applied Microbiology

1: 255-258.

TILTON, R. C., COBET, A. B. and JONES, G. E., 1967. Marine Thiobacilli.

I- Isolation and distribution. Canadian Journal of Microbiology

13: 1521-1528.

TILTON, R. C., COBET. A. B. and JONES, G. E., 1967-a. Marine

ThiobaciZZi. II-Culture and ultrastructure. Canadian Journal of

Microbiology 13: 1529-1534.

TOMINAGA, N. and MORI, T., 1974. A sulfate-dependent acid phosphatase

of ThiobaciZZus thiooxidans Its partial purification and some

properties. Journal of Biochemistry 76: 397-409.

TOMIZUKA, N. and TAKAHARA, Y., 1972. Bacterial leaching of uranium

from Ningyo-Toge ore. Proceedings of IV International Fermentation

Symposium- Fermentation Technology Today, pp 513-520.

TOMIZUKA, N, 1976. Microbiological oxidation of lead sulphide.

Report of the Fermentation Research Institute No. 48, pp 51-66.

TOMIZUKA, N., YAGISAWA, M., SOMEYA, J. and TAKAHARA, Y. 1976. Continuous

leaching of uranium by ThiobaciZZus ferrooxidans. Agricultural

and Biological Chemistry 40: 1019-1025.

TORMA, A. E., 1971. Microbiological oxidation of synthetic Co, Ni and

Zns by ThiobaciZZus ferrooxidans. Revue Canadienne de Biologie

30: 209-216.

TDRMA, A. E., 1972. Biohydrometallurgy of cobalt and nickel.

TMS-Paper section, paper No. A72-7.

TORMA, A. E., 1975. Microbiological extraction of cobalt and nickel

271

from sulphide ores and concentrates. Canadian Patent 960 463.

TORMA, A. E., 1976. Biodegradation of chalcopyrite. In: Shapley, J.M.,

Kaplan, A. M. (Eds.) Proceedings of 3rd International Bio­

degradation Symposium pp. 937-946. Applied Science Publishers

Ltd., London.

TORMA, A. E., 1977. The role of ThiobaeiZZus ferrooxidans in

hydrornetallurgical processes. Advances in Biochemical Engineer­

ing 6: 1-37.

TORMA, A. E. and GABRA, G. G., 1975. Effect of wetting agents on

chalcopyrite oxidation ability of ThiobaeiZZus ferrooxidans.

IRCS Medical Science, Biochemistry, Microbiology, Parasitology

and Infectious diseases 3: 228.

TORMA, A. E. and GABRA, G. G., 1976. Effects of surface active

agents on the oxidation of chalcopyrite by ThiobaeiZZus ferro­

oxidans. Hydrometallurgy 1: 301-309.

TORMA, A. E. and GUAY, R., 1976. Effect of particle size on the

biodegradation of a sphalerite concnetrate. Le Naturaliste Canadien

103: 103: 133-138.

TORMA, A. E. and ITZKOVITCH, I. J., 1976. Influence of organic solvents

on chalcopyrite oxidation ability of ThiobaeiZZus ferrooxidans.

Applied and Environmental Microbiology 32: 102-107.

TORMA, A. E. and LEGAULT, G., 1971. Contribution to characterization of

a rnicroorganisrn- ThiobaeiZZus ferrooxidans. Paper presented at

54th Conference of Chemical Institute of Canada, Halifax, Nova

Scotia, May 30 - 2 June.

TORMA, A. E. and LEGAULT, G., 1973. Metal sulfide biodegradation by

ThiobaeiZZus ferrooxidans. Effect of their total surfaces.

Anneles Microbiologie l'Institut Pasteur 124A: 111-121.

272

TORMA, A. E., and SUBRAMANIAN, K. N., 1974. Selective bacterial leach­

ing of lead sulphide concentrate. International Journal of

Mineral Processing 1: 125-134.

TORMA, A. E., WALDEN, C.C., and BRANION, R. M. R., 1970. Microbio­

logical leaching of zinc sulphide concentrate. Bitechnology and

Bioengineering 12: 501-517.

TORMA, A. E., WALDEN, C. C., DUNCAN, D. W. and BRANION, R. M. R., 1972.

The effect of co2 and particle surface area on the microbiological

leaching of zinc sulphide concentrate. Biotechnology and Bioengi-

neering 14: 777-786.

TORMA, A. E., LEGAULT, G., KOUGIOMOUTZAKIS; D. and OUELLET, R., 1974.

Kinetics of biooxidation of metal sulphides. Canadian Journal of

Chemical Engineering 52: 515-517.

TRIBUTSCH, H. , 1976. The oxidative disintegration of sulfide

crystals by ThiobaciZZus ferrooxidans. Naturwissenschaften 63: 88.

TRIVEDI, N. C. and TSUCHIYA, H. M., 1975. Microbial mutualism in

leaching of Cu-Ni sulfide concentrate. International Journal

of Mineral frocessing 2: 1-14.

TRUDINGER, P.A., 1955. Phosphoglycerate formation from pentose

phosphate by extracts of ThiobaciZZus denitrificans. Biochimica

et Biophysica Act 18: 581-582.

TRUDINGER, P.A., 1956. Fixation of co2 by extracts of the strict

autotroph, ThiobaciZZus denitrifican. Biochemical Journal 64:

274-286.

TRUDINGER, P. A., 1961. Thiosulphate oxidation and cytochromes in

ThiobaciZZus X. I- Fractionation of bacterial extracts and

properties of cytochromes. Biochemical Journal 78: 673-680.

TRUDINGER, P.A., 1971. Microbes, Metals and Minerals. Minerals

273

and Engineering 3: 13-25.

TRUDINGER, P.A. and KELLY, D. P., 1968. Reduced nicotinarnide

adenine dinucleotide oxidation by ThiobaciZZus neapoZitanus &

ThiobaciZZus strain C. Journal of Bacteriology 95: 1962-1963.

TRUSSELL, P. C., DUNCAN, D. W. and WALDEN, C.C., 1964. Biological

mining. Canadian Mining Journal 83: 46-49.

TSUCHIYA, H. M., 1977. Leaching of Cu-Ni sulfide concentrate from

the Duluth Gabbro. GBF Monograph Series No. 4, pp 101-106.

Conference Bacterial Leaching.

TSUCHIYA, H. M., TRIVIDI, N. C. and SCHULLER, M. L., 1974. Microbial

mutualism in ore leaching. Biotechnology and Bioengineering 16:

991-995.

TU0VINEN, 0. H., 1972. Microbiological aspects in the leaching of

uranium by ThiobaciZZus ferrooxidans. Atomic Energy Review 10:

251-258. (International Atomic Energy Agency, Vienna)

TU0VINEN, 0. H. and KELLY, D. P., 1972. Biology of ThiobaciZZus

ferrooxidans in relation to the microbiological leaching of

sulphide ores. Zeitschrift fur Allgemeine Mikrobiologie 12:

311-346.

TU0VINEN, 0. H. and KELLY, D. P.1973. Studies on the growth of

ThiobaciZZus ferrooxidans. I- Use of membrane filters and ferrous

iron agar to determine viable numbers and comparison with 14co12 -

fixation and iron-oxidation as measures of growth. Archives

fur Mikrobiologie 88: 285-299.

TU0VINEN, 0. H. and KELLY, D. P., 1974. Studies on the growth of

ThiobaciZZus ferrooxidans. II- Toxicity of uranium growing

cultures and tolerance conferred by mutation, other metal cations

and EDTA. Archives fur Mikrobiologie 95: 153-165.

274

TUOVINEN, 0. H. and KELLY, D. P., 1974-a. Studies on the growth of

Thiobacillus ferrooxidans. III- Influence of uranium, other metal

ions and 2:4-Dinitrophenol on ferrous iron oxidation and co2

fixation by the cells suspension. Archives fur Mikrobiologie

95: 165-180.

TUOVINEN, O. H. and KELLY, D. P., 1974-b. Studies on the growth of

Thiobacillus ferrooxidans. IV- Influence of monovalent metal

cations on ferrous iron oxidation and uranium toxicity in growing

cultures. Archives fur Mikrobiologie 98: 167-174.

TUOVINEN, O. H. and KELLY, D. P., 1974-c. Studies on the growth of

Thiobacillus ferrooxidans. V- Factors affecting growth in liquid

cultures and development of colonies on solid media containing

inorganic sulphur compounds. Archives fur Mikrobiologie 98:

351-364.

TUOVINEN, 0. H. and KELLY D. P., 1974-d. Use of microorganisms for the

recovery of metals. International Metallurgical Reviews 19:

21-31.

TUOVINEN, O. H., and KELLY, D. P., 1978. Metabolic transitions in cult­

ures of acidophilic Thiobacilli. In: Murr, L., Torma, A. E. &

Brierley, J. A. (Eds)Metallurgical Applications of Bacterial

Leaching and Related Phenomena. The Academic Press, N. Y. (In

Press).

TUOVINEN, 0. H. and NICHOLAS, D. J. D., 1977. Transition of Thiobacil­

lus ferrooxidans KG-4 from heterotrophic growth on glucose to

autotrophic growth on ferrous iron. Archives of Microbiology

114: 193-195.

TUOVINEN, 0. H., NIEMELA, S. I. and GYLLENBERG, H. G., 1971. Effect of

mineral nutrients and organic substances on the development of

Thiobacillus ferrooxidans. Biotechnology and Bioengineering 13:

275

517-527

TUOVINEN, 0. H., NIEMELA, S. I. and GYLLENBERG, H. G., 1971-a.

Tolerance of Thiobacillus ferrooxidans to some metals. Antonie

van Leeuwenhoek Journal of Microbiology and Serology 37: 489-496.

TUOVINEN, 0. H., NIEMELA, S. I. and GYLLENBERG, H. G., 1971-b.

Toxicity of membrane filters to Thiobaaillus ferrooxid.ans.

Zeitschrift fur Allgemeine Mikrobiologie 11: 627-631.

TUOVINEN, 0. H., KELLEY, B. C. and NICHOLAS, D. J. D., 1975. The

uptake and assimilation of sulphate by Thiobaaillus ferrooxidans.

Archives for Microbiology 105: 123-127.

TUTTLE, J. H. and DUGAN, P.R., 1969. Inhibitory effect of organic

substances on autotrophic growth of Thiobacillus ferrooxidans.

Bacteriological Proceedings GP131, p.64.

TUTTLE, J. H. and"DUGAN, P. R., 1976., Inhibition of growth, iron and

sulfur oxidation in Thiobaaillus ferrooxidans by simple organic

compounds. Canadian Journal of Microbiology 22: 719-730.

TUTTLE, J. H., DUGAN, P.R. and APEL, W. A., 1977. Leakage of

cellular material from Thiobaaillus ferrooxid.ans in the presence

of organic acids. Applied and Environmental Microbiology

33: 459-469.

UMBREIT, W.W. and ANDERSON, T. F., 1942. A study of Thiobaaillus

thiooxid.ans with electron microscope. Journal of Bacteriology

44: 317-320.

UMBREIT, W.W., VOGEL, H. R. and VOGLER, K. G., 1942. The significance

of fat in sulfur oxidation by Thiobacillus thiooxid.ans. Journal of

Bacteriology 43: 141-148.

276

UNZ, R. F. and LIEBERMAN, M. T., 1973. The microbiology of acid mine

water treatment in packed bed columns. Research Publication No. 77.

Institute of Research, Land and Water Resources, the Pennsylvania

State University, Pennsylvaniz.

UNZ, R. F., and LUNDGREN, D. G., 1961. A comparative nutritional study

of three chemoautotrophic bacteria, FerrobaciZZus ferrooxidans,

ThiobaciZZus ferrooxidans & ThiobaciZZus thiooxidans.

Soil Science 92: 302-313.

VAINSHTEIN, M. B., 1976. Systematic position of ThiobaciZZus

trautweinii. Mikrobiologiya 45: 137-141.

Van CAESEELE, L. and LEES, H., 1969. The ultrastructure of autotrophic­

ally grown ThiobaciZZus noveZZus. Canadian Journal of

Microbiology 15: 651-654.

Van der WALT, J. P. and De KRUYFF, C. D., 1955. Anaerobic metabolism

of thiocyanate by ThiobaciZZi. Nature 176: 310-311.

Van GOOL, A. and LAUDELOUT, H., 1967. Spectrophotometric and kinetic

study of nitrite and formate oxidation by Nitrobacter

winogradskyi. Journal of Bacteriology 93: 215-220.

VANSELOW, D. G., 1976. Mechanisms of bacterial oxidation of the copper

sulphide mineral covellite. Ph.D. Thesis. University of N.S.W.

VELDKAMP. H., 1970. Enrichment cultures of prokaryotic organisms. In:

Norris, J. R. & Ribbons, D.W., (Eds.), Methods in Microbiology

vol 3A: pp. 305-361. Academic Press, London & New York.

VISHNIAC, W. and SANTER, M., 1957. The ThiobaciZZi. Bacteriological

Reviews 21: 195-213.

VOGEL, A. I., 1962. A Text Book of Quantitative Inorganic Analysis.

3rd Edition, pp. 343-357. London, Longman.

277

VOGLER, K. G., LE PAGE, G. A. and UMBREIT, W.W., 1942. The

respiration of ThiobaciZZus ferrooxidans on sulfur. Journal

of General Physiology 26: 89-102.

WAKSMAN, S. A. and STARKEY, R. L., 1923. On the growth and respi­

ration of sulfur-oxidizing bacteria. Journal of General

Physiology 5: 285-310.

WAKSMAN, S. A., WARK, C. H., JOFFE, J. S. and STARKEY, R. L., 1923.

Oxidation of sulfur by microorganisms in black alkali soils.

Journal of Agricultural Research 24: 297-305.

WARREN, K., 1973. Mineral resources for the future. In: Mineral

Resources, pp. 241-244. Penguin Books, Ltd., Harrnondsworth,

England.

WATANABE, A. 1968. Hakko Koyokaishi 26: 115.

f Cited from: Gupta & Sant, 1970 ]

WEBLEY, D. M. , DUFF, R. B. and MITCHELL, W. A., 1960. A plate method

for studying the breakdown of synthetic and natural silicates by

soil bacteria. Nature 188: 766-767.

WEBLEY, D, M,, HENDERSON, M. E. K. and TAYLOR, I. F., 1963. The micro­

biology of rocks and weathered stones. Journal of Soil Science

14: 102-112.

WELCHER, F. J., 1962. Standard Methods of Chemical Analysis, vol. 3B.

Van Nostrand, New York.

WENBERG, G. M., ERBISCH, F. H. and VIOLIN, M. E., 1969. Leaching of

copper by fungi. American Institute of Mining and Metallurgical

Engineering Meeting Feb. 1969; Washington D. C., Reprint No. 69B43.

WHITE, A., HANDLER, P., and SMITH, E. L., 1973. Principles of

278

Biochemistry. 5th. edition. MgGraw-Hill Kogakusha Ltd. Tokyo.

WHITTENBURY, R. and KELLY, D. P., 1977. Autotrophy: A conceptual

phoenix. Symposium- Society for General Microbiology 27:

pp. 121-149. Microbial Energetics.

WILLIAMS, R. A. D. and HOARE, D.S., 1972. Physiology of a new

facultative autotrophic ThiobaciZZus. Journal of General

Microbiology 70: 555-566.

WINOGRADSKY, S., 1887. Uber schwefelbakterien. Botan. Zeitung 45:

489-526.

[ Cited from: Vishniac & Santer, 1957.]

WINOGRADSKY, S., 1888. Uber eisenbaketrien. Botan. Zeitung 46:

261-269.

[ Cited from: Tuovinen & Kelly, 1972.]

WONG, C., SCHARER, J.M. and REILLY, P. M., 1974. A discrimination

among microbial iron oxidation mechanisms, Canadian Journal

of Chemical Engineering 52: 645-653.

WOOD, A. P. and KELLY, D. P., 1976. Triple mechanism for glucose oxid­

ation in ThiobaciZZus A2. Proceedings of Society for General

Microbiology vo. 4 (Part 1): pp.23-24.

WOOD, A. P. and KELLY, D. P., 1977. Heterotrophic growth of ThiobaciZZus

A2 in sugars and organic acids. Archives for Microbiology

113: 257-264.

WOOJ, A. P., KELLY, D. P. and THURSTON, C. F., 1977. Simultaneous

operations of three catabolic pathways in the metabolism of

glucose by ThiobaciZZus A2. Archives for Mikrobiology 113:

265-274.

WOOLLEY, D., JONES, G. L. and HAPPOLD, F. C., 1962. Some metabolic

differences between ThiobaciZZus thiopa.rus, ThiobaciZZus

279

denitrifiaans and Thiobaaillus thioayanoxidans. Journal of

General Microbiology 29: 311-316.

YOUATT, J. B., 1954. Studies on the metabolism of Thiobaaillus

thioayanoxidans. Journal of General Microbiology 11: 139-149.

ZAJIC, J.E., 1969. Microbial Biogeochemistry. Academic Press,

New York and London. 345 pp.

ZAJIC, J.E. and CHIU, Y. S., 1972. Recovery of heavy metals by

microbes. Developments in Industrial Microbiology 13: 91-100.

ZAJIC, J.E. and NG, K. S., 1970. Biochemical uranium leaching.

Developments in Industrial Microbiology 11: 413-419.

ZAVARZIN, G. A., 1972. A heterotrophic satellite of Thiobaaillus

ferrooxidans. Mikrobiologiya 41: 369-370.

ZAVARZIN, G. A. and ZHILINA, T. Z., 1964. Thione bacteria from

thermal springs. Mikrobiologiya 33: 753-759.

ZIMMERLEY, S. R., WILSON, D. G. and PRATER, J. D., 1958.

Cycling leaching process employing iron oxidizing bacteria.

u. s. Patent 2,829,964.

B APPENDICES

NO.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

I

APPENDIX 6.1

ISOLATION OF NON-AC IDOPH ILi C SULPHUR OXIDISING

BACTERI/1.

P = PELLICLE T = TlJRlllllTTY + = GROW111 nm l CA TED

MEDIUM A MEDIUM B

·------ - --------- -- -------------

SAMPLE 2 DAYS 7 DAYS 2 DAYS 7 DAYS CODE. ~-..---- --- . -· -·-- ----- - -~----, -· - - .. - --

% S203 t. s2o3 r-- -----pH GROHTH

% S203 pH GROWTH

% S203 OXIDN. pH OXIDN.tROWlH OX ION. pH I OX I ON. iROWTH

--·-

2 6.5 0 0 5.0 21 p 9.0 0 0 9.0 10 +

3 6.5 0 0 6.0 10 + 9.0 0 0 9.0 0 0

5 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

6 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

6a 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

7 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

9 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

9a 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

11 6.5 0 0 6.0 10 p 9.0 0 0 9.0 0 0

12 6.5 0 0 5.5 15 p 9.0 0 0 9.0 0 0

13 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

14 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

17 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

18 6.5 0 0 6.5 0 0 9.u 0 0 9.0 0 0

19 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

21 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

24 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

26 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

29 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

31 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

33 6.5 0 0 5.5 25 T 9.0 0 0 9.0 0 0

36 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

38 6.5 0 0 6.0 16 T 9.0 0 0 9.0 0 0

48 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

49 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

Enrichment medium A cont a incLI sodium thi!Vitil phatc and its jnitail pH was 6.(,. (Starkey, l!l:itl-a)

EnrichffiC'nt medium ll was devi~;cd by lleijcrnick (1904). It also contained sodium thiosulphatc and its initial pll was 9.1.

NO. SAMPLE 2 CODE.

pH

26 50 6.5

27 52 6.5

28 54 6.5

29 55 6.5

30 5G 6.5

31 57 6.5

32 58 6.5

33 59 6.5

34 59a 6.5

35 60 6.5

36 60a 6.5

37 61 6.5

38 62 6.5

39 63 6.5

40 64f 6.5

41 65. 6.5

42 66R 6.5

43 67 6.5

44 68 6.5

45 68J 6.5

46 69 6.5

47 69K 6.5

48 140 6.5

49 142 6.5

50 144 6.5

II

APPENDIX 6.1 (c-0nt.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S203 ,ROWTH pH % S203 GROWTH pH % S203 GROWTH

OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 5.0 30 p 9.0 0 0

u 0 6.5 0 0 9.0 I 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 5.5 25 T 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

B

7 DAYS

pH % S203 GROWTH OXIDN.

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

NO. SAMPLE 2 CODE.

pH

51 145 6.5

52 148 6.5

53 151 6.5

54 152 6.5

55 153 6.5

56 154 6.5

57 168 6.5

58 169 6.5

59 171 6.5

60 172 6.5

61 173 6.5

62 174 6.5

63 175 6.5

64 176 6.5

65 177 6.5

66 178 6.5

67 179 6.5

68 181 6.5

69 182 6.5

70 183 6.5

71 184 6.5

72 186 6.5

73 192 6.5

74 193 6.5

75 194 6.5

llI

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM [3

DAYS 7 DAYS 2 DAYS

% s2o_ GROWTH pH

% S203 GROWTH pH

% S203 GROWTH

OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 5.5 35 p 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

7 DAYS

pH % S203

GROWTH OXIDN.

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

NO. SAMPLE 2 CODE.

pH

76 195 6.5

77 196 6.5

78 196a 6.5

79 197 6.5

80 198 6.5

81 331 6.0

82 333 6.5

83 334 6.5

84 339 6.5

85 340 6.5

86 341 6.5

87 342 6.5

88 343 6.5

89 344 6.5 I

90 345 6.5

91 346 6.5

92 347 6.5

93 348 6.5

94 364 6.5

95 365 6.5

96 366 6.5

97 367 6.5

98 368 6.5

99 369 6.5

100 370 6.5

IV

APPENDIX 6.1 (cont.)

ISOLATION OF NON - ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S203 GROWTH pH

% S203 GROWTH pH

% S203 GROWTH

OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.l 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 o 0

10 p 5.5 60 p 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 I 0 0

0 . 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 5.0 30 p 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.0 25 p 9.1 0 0

B

7 DAYS

pH % S203

GROWTH OXIDN.

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.0 0 o 9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

V

APPENUIX 6.1 (cont.)

ISOLATION OF NON - ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM B

NO. SAMPLE 2 DAYS 7 DAYS 2 DAYS 7 DAYS CODE.

pH % S203

GROWTH pH % S203

GROWTH pH % S203

GROWTH pH % S203I

GROh'TH OXIDN. OXIDN. OXIDN. OXIDN.

101 371 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

102 372 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

103 373 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

104 374 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

105 375 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

106 376 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

107 377 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

108 378 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

109 379 6.5 0 0 6.5 0 0 9.0 I 0 0 9.0 0 0

llO 380 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

111 381 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

112 382 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

113 383 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

114 384 6.5 I 0 0 6.5 0 0 9.0 0 0 9.0 0 0

115 385 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

116 386 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

117 387 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

118 388 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

119 389 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

120 390 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

121 391 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

122 392 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

123 393 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

124 394 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

125 406 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

NO. SAMPLE 2 CODE.

pH

126 407 6.5

127 408 6.5

128 409 6.5

129 410 6.5

130 411 6.5

131 412 6.5

132 413 6.5

133 414 6.5

134 415 6.5

135 416 6.5

136 417 6.5

137 418 6.5

138 419 6.5

139 420 6.5 I

140 421 6.5

141 422 6.5

142 423 6.5

143 424 6.5

144 425 6.5

145 426 6.5

146 427 6.5

147 428 6.5

148 429 6.5

149 430 6.5

150 431 6.5

VI

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S203 GROWTH pH

% S203 GROWTH pH

% S203 GROWTH

OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

B

7 DAYS

pH Ix s2o3

GROIHH OXIDN.

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

NO. SAMPLE 2 CODE.

pH

151 432 6.5

152 433 6.5

153 434 6.5

154 435 6.5

155 436 6.5

156 437 6.5

157 438 6.5

158 440 6.5

159 441 6.5

160 443 6.5

161 445 6.5

162 446 6.5

163 447 6.5

164 448 6.5

165 449 6.5

166 450 6.5

167 451 6.0

168 452 6.5

169 453 6.5

170 454 6.5

171 455 6.5

172 456 6.5

173 457 6.5

174 458 6.5

175 459 6.5

VII

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S203 GROWTH pH % S203 GROWTH pH % S203 GROWTH OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.C 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

15 T 4.3 100 TT 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

B

7 DAYS

pH % S203 GROWTH OXIDN.

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

7.0 30 T

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.1 0 0

VIII

APPENDIX6.l (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM B

NO. SAMPLE 2 DAYS 7 DAYS 2 DAYS 7 DAYS CODE.

pH % S203 GROWTH pH % S203 GROWTH pH % S203 GROWTH pH % S203 GROWTH OXIDN. OXIDN. OXIDN. OXIDN.

176 460 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

177 461 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

178 464 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

179 465 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

180 466 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

181 467 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

182 468 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

183 469 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

184 470 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

185 471 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

186 472 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

187 473 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

188 474 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

189 475 6.5 0 0 6.5 0 0 9.(J 0 0 9.0 0 0

190 475a 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

191 476 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

192 477 6.5 0 0 5.0 50 T 9.1 0 0 9.0 10 T

193 478 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

194 479 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

195 480 6.5 0 0 5.5 40 p 9.1 0 0 9.1 0 0

196 481 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

197 482 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

198 483 6.5 0 0 6.0 27 p 9.1 0 0 9.1 0 0

199 484 6.5 0 0 5.5 33 p 9.1 0 0 9.1 0 0

200 485 6.5 0 0 5.0 49 p 9.1 0 0 9.1 0 0

NO. SAMPLE 2 CODE.

pH

201 486 6.5

202 487 6.5

203 488 6.5

204 489 6.5

205 489a 6.5

206 490 6.5

207 491 6.5

208 492 6.5

209 493 6.5

210 493a 6.5

211 494 6.5

212 495 6.5

213 496 6.5

214 497 6.5

215 498 6.5

216 499 6.5

217 500 6.5

218 501 6.5

219 502 6.5

220 503 6.5

221 504 6.5

222 505 6.5

223 506 6.5

224 507 6.5

225 508 6.5

IX

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S20 % S203 % S20 3 GROWH GROWTH pH GROWTH pH OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 5.5 35 p 9.0 0 0

0 0 5.0 45 p 9.1 0 0

0 0 5.3 30 p 9.1 0 0

0 0 5.4 40 p 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.0 15 p 9.1 0 0

u 0 5.0 50 p 9.1 I 0 0

0 0 6.5 0 0 9.1 0 0

0 0 5.5 40 p 9.1 0 0

0 0 5.0 55 p 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 -- 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

B

7 DAYS

% s 0-1

pH 2 3rROWTH OXIDN.

9.0 0 0

9.1 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

8.0 40 T

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

9.0 0 0

X

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM B

NO. SAMPLE 2 DAYS 7 DAYS 2 DAYS 7 DAYS CODE.

pH % S20:

GROWTH pH % S203

GROWTH pH % S203

GROWTH pH % S203

GROviTH OXIDN OXIDN. OXIDN. OXIDN.

226 509 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

227 511 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

228 512 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

229 522 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

230 523 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

231 524 6.3 18 p 5.5 53 p 9.1 0 0 9.1 0 0

232 524a 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

233 525 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

234 525a 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

235 528 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

236 530 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

237 536 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

238 549 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

239 550 6.5 0 0 6.0 33 T 9.0 0 0 9.0 0 0

240 551 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

241 552 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

242 553 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

243 554 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

244 555 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

245 556 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

246 557 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

247 558 6.3 10 T 5.0 65 T 9.1 0 0 9.1 0 0

248 559 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

249 560 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

250 561 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

NO. SJ\MPLE 2 CODE.

pH

251 562 6.5

252 563 6.5

253 564 6.5

254 565 6.5

255 566 6.5

256 567 6.5

257 568 6.5

258 569 6.5

259 570 6.5

260 571 6.5

261 572 6.5

262 573 6.5

263 574 6.5

264 575 6.5 I

265 576 6.5

266 577 6.5

267 578 6.5

268 579 6.5

269 580 6.5

270 581 6.5

271 582 6.5

272 583 6.5

273 584 6.5

274 585 6.5

275 586 6.5

· XI

APPENDIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS ·-,.._

r~ s203 GROWTH pH

% s203 GROWTH pH

% s203 GROWTH

OXIDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.0 30 T 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 5.5 42 T 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

C 0 6.5 0 0 9.1 I 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

0 0 6.5 0 0 9.0 0 0

0 0 6.5 0 0 9.1 0 0

B

7 DAYS

pH % s203,

PROWTH OXIDN.J

9.0 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.1 0 0

9.0 0 0

9.0 0 0

9.1 0 0

9.0 0 0

9.1 0 0

XII

APPEt~DIX 6.1 (cont.)

ISClATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM B

Ml. SAMPLE 2 DAYS 7 DAYS 2 DAYS 7 DAYS CODE.

% S O i % S203 % S203 % S203 pH 2 3 GROIHH pH GROWTH pH GROWTH pH GROWTH

OXIDN. OXIDN. OXIDN. OXIDN.

276 587 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

277 588 6.5 0 0 6.1 25 T 9.1 0 0 9.1 0 0

278 589 6.5 0 0 6.0 30 T 9.1 0 0 9.1 0 0

279 590 6.5 0 0 5.5 45 p 9.1 0 0 9.1 0 0

280 591 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

281 592 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

282 593 6.5 0 0 5.8 29 p 9.1 0 0 9.1 0 0

283 594 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

284 595 6.5 0 0 6.5 0 0 9.1 I 0 0 9.1 0 0

285 596 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

286 597 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

287 598 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

288 599 6.5 0 0 5.5 45 p 9.1 0 0 7.5 29 p

289 600 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

290 601 6.5 0 0 5.5 39 p 9.1 0 0 6.0 49 p

291 602 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

292 603 6.5 0 0 6.3 15 p 9.1 0 0 9.1 0 0

293 604 6.5 0 0 6.5 0 0 9.1 0 0 8.5 20 p

294 605 6.5 0 0 5.0 59 T 9.1 0 0 7.2 32 T

295 606 6.5 0 0 6.5 0 0 9.1 0 0 9.1 0 0

296 607 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

297 608 6.5 0 0 5.4 49 p 9.1 0 0 8.0 41 p

298 609 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

299 610 6.5 0 0 6.5 0 0 9.0 0 0 9.0 0 0

300 611 6.5 0 0 5.4 61 p 9.0 0 0 8.0 40 p

NO. SAMPLE 2 CODE.

pH

301 612 6.5

302 614 6.5

303 618 6.5

XII I

APPI:N!JIX 6.1 (cont.)

ISOLATION OF NON-ACIDOPHILIC SULPHUR OXIDISING

BACTERIA.

MEDIUM A MEDIUM

DAYS 7 DAYS 2 DAYS

% S203 GROIHH pH

% S203 1,ROWTH pH

% Sz03 GROWTH

OX IDN. OXIDN. OXIDN.

0 0 6.5 0 0 9.1 0 0

0 0 6.0 24 T 9.1 0 0

0 0 6.5 0 0 9.0 0 0

B

7

pH

9.1

9.1

9.0

DAYS

% Sz03 GROIHH

OXIDN.

0 0

0 0

0 0

NO.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

FF=

ML=

SC=

SM=

XIV

APPENDIX- 6.2

ISOLATION OF ACIOOPHILIC IRON OXIDISING BACTERIA

9K medium of Silvennan & Lundgren (1959) with 1% ferrous sulphate was employed

as enrichment medium.

SAMPLE OXIDATION OF Fe ++

CODE 2 DAYS 7 DAYS 14 DAYS

FF-SW 0 0 +

FF-9W 0 0 0

FF-lOW 0 0 0

FF-llW 0 0 +

FF-12W 0 0 0

FF-13W 0 0 0

ML-lW ++ ++++ ++++

SC-10 0 0 0

SM-lW 0 0 0

SM-2W 0 0 0

SM-3W 0 0 0

SM-4W 0 0 +

SM-SW 0 0 0

SM-6W 0 0 +

Fairfield Sewage Works. 0 = "d . f F ++ No oxi ation o e

Mount Lyell Mine Water. + = 15 20 % oxidation ++

of Fe .

Sulphide Corporation Ltd. % ++ ++ = 40 so oxidation of Fe .

Saint Mary Sewage Works. ++++ = 100 % oxidation of Fe:+

xv

APPENDIX-6.3

ISOLATION OF ACIDOPHILIC SULPHUR OXIDISING BACTERIA

NO.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

FF=

ML=

SC=

SM=

9K Basal salt medium of Silverman and Lmdgren (1959) with 1% elemental sulphur was employed as an enrichment medium.

2 DAYS 7 DAYS

SAMPLE CODE. pH GROWTH pH

FF-SW 2.5 0 2.0

FF-9W 2.5 0 2.5

FF-lOW 2.5 0 2.5

FF-llW 2.5 0 2.5

FF-12W 2.5 0 2.5

FF-13W 2.5 0 2.5

ML-lW 2.5 0 2.0

SC-10 2.0 ++ 0.7

SM-lW 2.5 0 1.9

SM-2W 2.5 0 2.5

SM-3W 2.5 0 2.5

SM-4W 2.5 0 2.5

SM-SW 2.5 0 2.5

SM-6W 2.5 0 2.5

Fairfield Sewage Works. 0 = No growth.

Momt tyell Mine Water. + = Not more than

Sulphide Corporation Ltd. ++ = Not more than

Saint Mary Sewage Works. +++ = Not more than

GROWTH

+

0

0

0

0

0

0

+++

+

0

0

0

0

0

106 cells/ml.

107 cells/ml.

109 cells/ml.

APPENDIX 6.4

CALCULATION OF DNA BASE COMPOSITION

(Hirschman & Felsenfield, 1966)

XVI

1- The absorbance at 5 mµ intervals from 235 mµ to 290 mµ was

measured. The absorbance values are set out in appendix 6.4a.

2- Appendix 6.4b was consulted for values of ai, Si, yi, and the

three sums were computed.

µl = I: Ai .ai = Ai235 ai235 + ----- + Ai290 . ai290

µ2 = I: Ai . Bi = Ai235 f3i235 + ----- + Ai290 . f3i290

µ3 = I: Ai .yi = Ai235 yi235 + ----- + Ai290 .yi290

Values of µ1, µ2, µ3, are set out in appendix 6.4d.

3- Appendix 6.4c was consulted and thevalues of the following

expressions computed:

ccp = µ151 + µ252 + µ353

C = µ1s4 + µ2 53 + µ3 5s

[l]

[2]

[3]

[4]

[5]

where cp is the AT fraction in the unknown DNA sample, of concentration

C, in mole nucleotides per litre and 51, s2, s3, s4 and 55 are

the constants shown in the appendix 6.4c.

4- From equations 4 & 5 the value of cp, the mole fraction of AT

was calculated as follows:

cp = ~ C

µ151 + µ252 + µ353 =

µ154 + µ253 + µ3 5s

5- Having calculated the mole fraction of AT the mole fraction and

XVII

and hence % of GC was calculated as

GC = 1 - qi

% GC = [1 - <P] • 100

Using the values of µ 1 , µ 2 ,& µ 3from appendix 6.4d, calculated the

values of Cqi as follows:

C<P = [(66.2 X 0.9329 10- 7) + (-7250.6x 2.0631 . 10-7)

+ (24441.1 X 0.6198 10-7 )]

= [(61.8 .10- 7) + (-14958.7 . 10-7) + (15148.6 . 10-7)]

= 251. 7 . 10-7

Cq, = 0.2517 . 10-4

Now calculated the value of C according to equation [5]:

C ~ [ (66.2 x 0.2792 . 10- 7 ) + (-7250.6 x 0.6198. 10- 7)

+ (24441.1 X 0.2124 • 10- 7 )]

= [ (18.5 10- 7) + (-4493.9. 10-7) + (5191.3 . 10-7)]

= 715.9 10 - 7

C = 0. 7159 10- 4

0 .2517 10-4 <P =

0. 7159 10-4

= o. 352

Therefore A+T molar ratio is 0.352

GC mole fraction ratio would be [ 1.000 - 0.352] . 100

c: 64.8%

XVIII

1\PPENUIX 6.4-a

NATIVE ONA SPECTRUM FOR BJR-451.

Ai >..i

-- ..--- --· MEAN

1 2 3

235- 0.275 0.285 0.280 0.280

240- 0.300 0.315 0.315 0.31 O

245- 0.380 0.375 0.385 0.380

250- 0.418 0.432 o.425 0.425

255- 0.485 o.490 0.471 o.482

260- o.486 0.475 0. 491 0.484

265- 0.440 0.435 0.430 0.435

270- 0 .410 0.400 0. 390 0.400

275- 0.320 0.350 0.350 0.340

280- 0.270 0.280 0.269 0.273

285- 0.210 0.210 0.210 0.210

290- o. 140 0. 140 0. 155 0. 145

APP_ENDIX 6. 4-b

PARAMETERS FOR THE THREE-TERM ANALYSIS OF

NATIVE IJNA SPECTRA.

Purameters fur three-term aruilysis uf native DNA Spectra

~,(rnµ) «1 /J, Y1

~--~--·-·

235 -2026 -656 3052 240 -1889 -1251 5031 245 -1390 -1917 6338 250 43 -2830 7480 255 -319 -1807 7016 260 -608 --1141 7307 265 2515 -3370 7052 270 871 -- l 409 5740 275 - :l86 -154 4587 280 ll59 --1 ;i58 3938 285 1797 - 2424 31G4 200 1187 --209!) 2188

APPENDIX 6.4 -c

l\111lti[>ly eflch tm·rn hy 10- 7

SI 0 03'.!9 S-l 0·2702 S2 2·0fi31 S5 0·2124 S3 0·6198

XIX

X X

)..i

235-

240-

245-

250-

255-

260-

265-

270-

275-

280-

285-

290-

Ai a1,

0.280 -2026

0.31 0 -1889

0.380 -1390

0.425 43

0.482 - 319

o.484 - 608

o.435 2515

o.4oo 871

0,340 - 386

0.273 1159

0.210 1797

0. 145 1187

APPENDIX 6. 4 d

CALCULATION OF µl, µ2 & µ3 .

Ai. ai Si Ai. Si

- 567 .3 - 656 - 183. 7

- 585.6 -1251 - 387.8

- 528.2 -1917 - 728.5

18. 3 -2830 -1202.8

- 153 .8 -1807 . - 871 .o

- 294.3 -1141 - 552.2

1094. 0 -3379 -1469.9

348.4 -1409 - 563.6

- 131.2 - 154 - 52.4

316.4 -1558 - 425,3

377 .4 -2424 - 509.0

172. 1 -2099 - 304. 4

µ 1 = 66.2 µ2 = -7250.6

y1, Ai. yi

3952 1106. 6

5031 1559,6

6338 2408.4

7480 3179.0

7616 3670.9

7307 3536.6

7052 3067.6

5740 2296.0

4587 1559. 6

3938 1075. 1

3164 664.4

2188 317.3

µ3 = 24441.1

XXI

APPENDIX 6 . 5

WAVE LENGTH, SLIT WIDTH, LAMP CURRENT AND GAS MIXTURES

USED FOR MINERAL ANALYSIS BY ATOMIC

ABSORPTION SPECTROPHOTOMETER

LAMP GAS ELEMENT WAVE LENGTH SLIT WIDTH CURRENT MIXTURE USED

0

A µ mA

Ba 5,535.4 100 5 N - A

Cu 3,247.5 100 3 A - A

Fe 2,483.3 50 5 A - A

Pb 2,170.0 300 6 A - A

Ni 2,320.0 50 8 A - A

Zn 2,138.6 100 6 A - A

A - A= Air acetylene mixture.

N - A= Nitrous oxide air mixture.

APPENDIX- 6.6

Gesellschaft fur Biotechnologische Forschung mbH Braunschweig-Stockheim A. M. KHALID AND B. J. RALPH

THE LEACHING BEHAVIOUR OF

VARIOUS ZINC SULPHIDE

MINERALS WITH THREE

THIOBACILLUS SPECIES

Edited by W Schwartz

Conference Bacterial Leaching 1977

Verlag Chemie · Weinhe1m · New York 1977

THE LEACHING BEHAVIOUR OF VARIOUS ZINC SULPHIDE MINERALS

WITH THREE THIOBACILLUS SPECIES

A.M. Khalid and B.J. Ralph

School of Biological Technology, University of New South Wales, Kensington, New South Wales, Australia.

Abstract

The availability of high-grade specimens of sphalerite, wurtzite

and marmatite prompted a comparative study of the leaching rates of

these minerals in the presence of Thiobacillus ferrooxidans, !· thiooxidans and T. thioparus. In this preliminary study, samples

of the finely-ground minerals, of equivalent surface area, were sub­

jected to attack by populations of equal magnitude of each of the

three bacterial species in shake flasks at 30 c. The microorganisms

were acclimatised to the particular substrates.

The results indicate that

(i) Wurtzite is much more slowly degraded than marmatite or

sphalerite by all three organisms.

(ii) Marmatite is leached more rapidly by T. ferrooxidans and

T. thiooxidans than sphalerite, in both the presence and

absence of soluble iron.

(iii) Iron-free synthetic zinc sulphide is leached more rapidly

by T. thiooxidans than by T. ferrooxidans or T. thioparus.

The hexagonal crystalline structure of zinc sulphide in wurtzite

appears to be more recalcitrant to microbial degradation than the

cubical form of sphalerite, and the substitution of iron for some of

the zinc in the marmatitic form of zinc sulphide appears to greatly

facilitate biodegradation.

Introduction

Interpretation of the behaviour of sulphide minerals as substrates

in biochemical reactions is frequently complicated by uncertainties

with respect to composition and the effects of some physical

characteristics. These minerals frequently do not display a simple

stoichiometry but exhibit a range of compositions, and their strict

166 A. M. Khalid and B. J. Ralph

chemical definition as substrates may be blurred by the presence of

other metallic ions in solid solution, by imperfections in the crystal

structure and by lattice substitution. Natural specimens of the

highest grade may contain inclusions of other minerals of sub-micron

dimensions, the detection of which can be difficult. Variations in

composition may be accompanied by changes in crystalline form as in

the pyrrhotites, and some sulphide minerals of identical chemical

composition exhibit more than one crystalline form (for example, iron

and zinc sulphides).

A limited amount of published information suggests significant

variations in the behaviour of different crystalline forms towards

microbial attack and further effects arising from substitutions in

the lattice. Leathen et al. (1) noted a high degree of resistance to

oxidation by museum-grade pyrite in the presence of acidophilic,

iron-oxidising bacteria whereas high-grade specimens of marcassite

were readily attacked. In a more detailed investigation of these two

iron sulphide minerals, Silverman et al. (2) confirmed the resistance

of coarsely-crystalline pyrite to oxidative attack by Ferrobacillus

ferrooxidans and Thiobacillus thiooxidans and observed that both these

species readily degraded marcassite. Trussell et al. (3) reported

that the marmatitic form of zinc sulphide was rapidly and completely

leached bacterially but that under similar conditions, iron-free

sphalerite showed only a minor release of zinc ions.

The availability of relatively high-grade specimens of sphalerite,

wurtzite and marmatite prompted a further investigation of the effect

upon leaching rate of variations in crystal structure and of the sub­

stitution of some zinc by iron in the zinc sulphide lattice. A

further comparison was made by the use of three different species of

Thiobacillus, each operating within its optimum pH range for growth.

Materials and Methods

Two sphalerites and a triboluminescent wurtzite containing some

sphalerite were obtained from Ward's Natural Science Establishment

Inc., and a marmatite from Zinc Corporation Ltd. Chemically prepared

zinc sulphide was obtained from Ajax Co. Ltd. The content of

principal metals in each sample is shown in Table 1. Semi-quantitative

analysis by emission spectroscopy for 28 elements on the same samples

is shown in Table 2. X-ray diffraction examinations confirmed the de­

scriptions of the minerals. Each sample was ground to pass 400 mesh

and specific surface areas were determined by the B.E.T. method (4).

Values are shown in Table 3.

Leaching Behaviour of Zinc Sulphid.e Minerals 167

Table 2

Semi-quantiative Analysis of Zinc Sulphide Minerals by Emission

Spectroscopy (%)

Element

Ag

Al

As

B

Ba

Be

Bi

Ca

Cd

Co

er Cu

Fe

K

Li

Mg

Mo

Mn

Ni p

Pb

Sb

Si

Sn

Ti

V

Zr

Zn

Zinc Sulphide

<0.0005

0.0200

<0.1000

0.0005

<0.1000

<0.0002

<0.0010

0.0200

0,0010

0.0600

0.0005

0.0010

0.0600

<0.1000

<0.0050

0.0200

<0.0010

<0.0010

<0,0010

<0.1000

<0.0050

<0.0050

0.2000

<0.0007

<0.0010

<0.0010

<0.0030

Marmatite (N.s.w. l

0.0010

<0.0050

<0.1000

0.0005

<0.1000

<0.0002

<0.0010

0.0200

0.2000

0.0050

<0.0005

2.0000

3.0000

<0.1000

<0.0050

<0.0030

0.0020

0.1600

<0.0010

<0.1000

0.6000

<0.0050

0.2000

0.0007

<0.0010

<0.0010

<0.0030

Sphalerite (Okla.)

0.0005

<0.0050

<0.1000

0.0005

<0.1000

<0.0002

<0.0010

0.0200

0.6000

<0.0010

<0.0005

0.1000

0.0800

<0.1000

<0.0050

<0.0030

<0.0010

<0.0010

<0.0010

<0.1000

0.0050

<0.0050

0.3000

0.0007

<0.0010

<0.0010

<0.0030

The major component is Zinc.

Sphalerite (Spain)

<0.0005

<0.0050

<0.1000

0.0005

<0.1000

<0.0002

<0.0010

0.0200

0.0700

<0.0010

<0.0005

0.0100

0.0300

<0.1000

<0.0050

<0.0030

<0.0010

<0.0010

<0.0010

<0.1000

0.0050

<0.00~0

0.1000

0.0007

<0.0010

<0.0010

<0.0030

Wurtzite (Utah)

0.0030

0.2000

<0.1000

0.0005

<0.1000

<0.0002

<0.0010

0.0200

0.2000

<0.0010

<0.0005

0.0600

0.0600

<0.1000

<0.0050

<0.0030

0.0010

0.0100

<0.0010

<0.1000

1.2000

0.6000

4.0000

0.0007

0.0050

<0.0010

<0.0030

168 A. M. Khalid and B. J. Ralph

Table 1

Content of Principal Metals

Zinc Iron Lead Copper Nickel

Zinc sulphide 66.2 0.01 0.05 0.005 0.05 Marmatite (Broken 50.4 12.2 1.35 0.48 0.05

Hill, N.S.W.) Sphalerite (Okla.) 67.7 0.3 0.05 0.03 0.05 Sphalerite (Spain) 65.7 0.2 0.05 0.005 0.05 Wurtzite (Utah) 46.9 0.7 5.6 0.12 0.05

Percentage composition (w/w) By atomic absorption spectroscopy Theoretical Zn content of ZnS 67 .1%

The three Thiobacillus cultures used were obtained from the School

Culture Collection. The Thiobacillus ferrooxidans culture (BJR-Kl)

was isolated from mine drainage waters at Mt. Lyell, Tasmania. It is

a strict autotroph. The T. thiooxidans culture (BJR-K0l) was isolated

from the sulphur stockpile at the Zinc Sulphide Corporation plant at

Boolaroo, N.S.W. The T. thioparus culture (BJR-451) was isolated

from a soil sample from northern N.S.W. The T. ferrooxidans and T.

thiooxidans were grown up on 9K basal salt medium (5) with ferrous

sulphate and elemental sulphur as the energy sources respectively, at

30 C in shake flasks. The cells were harvested during logarithmic

phase by centrifugation, washed with sterile sulphuric acid solution

(pH 2.5) and resuspended in the same solution for storage at 4 C. The

T. thioparus strain was grown in continuous culture under optimum

growth conditions (pH 5.5; temperature 30 C; dilution rate 0.lhr-1 )

on Vishniac and Santer's medium (6), harvested from the outflow by

centrifugation, washed with sterile basal salts medium, resuspended

and stored at 4 C.

Table 3

Specific Surface Area of Zinc Sulphide Minerals (B.E.T. Method)

Mineral: Zinc Marmatite Sphalerite Sphalerite Wurtzite sulphide (NSW) (Okla.) (Spain) (Utah)

Surfa~e Area: 30.00 9.14 7.60 5.70 6.40 m /g

Prior to the leaching experiments, the T. ferrooxidans and T.

thiooxidans cultures are acclimatised to the particular mineral sub­

strate either by growth in the appropriate medium with the mineral as

energy source or, in the case of poor or negligible growth rates under

Leaching Behaviour of Zinc Sulphide Minerals 169

Table 4

Leaching of Zinc Sulphide Minerals by Thiobacilli

Zinc Sulphide

Marmatite (NSW)

Sphalerite (Okla.)

Sphalerite (Spain)

Wurtzite (Utah)

T.ferrooxidans#

FeT (ppm)

Fe 2+ (ppm)*

zn 2+solubil­isation Rate (mg/1/day)

Max. Cone.

zn 2+ (ppm)

T. thiooxidans

FeT (ppm)

2+ * Fe (ppm)

zn 2+solubil­isation Rate (mg/1/day)

Max. con. 2+ Zn (ppm)

T. thioparus

FeT (ppm)

2+ Fe (ppm)*

zn 2+solubil­isation Rate (mg/1/day)

Max. cone. 2+ Zn (ppm)

<1.0

0.1

24.0

385

<1.0

<0.1

40.0

530

<0.1

<0.1

6.9

59.5

3. 5

<O.l

230.0

1800

425

415

130.0

1270

3.0

<0.1

4.9

80

* #

At conclusion of experiment

Significant amounts of ferric course of experiment.

1.1

<0.1

22.0

296

8.0

5.0

13. 0

204.0

<0.1

<0.1

3 .1

27.5

<1.0

<1.0

15.0

178

<1.0

<0.1

12.0

155

<0.1

<0.1

3.9

31. 5

2.5

<0.1

9.0

102

15.5

14.0

6.5

85.0

<0.1

<0.1

Ng

<6

Ng = Negligible

compounds precipitated during FeT = Total Iron

170 A. M. Khalid and B. J. Ralph

such conditions, with mineral plus the relevant soluble substrate.

The T. thioparus culture which had poor leaching capabilities against

all the natural minerals, was acclimatised against the zinc sulphide

sample. Acclimatisation was carried out in shake flasks at 30 C for

4-5 weeks, and the cells harvested by centrifugation after filtration

of the flask contents.

Determination of Leaching Rates

In the leaching experiments, an amount of mineral calculated to

provide 10 m2/100 ml. medium for surface area was added to a sterile

1000 ml. conical flask and the mineral sterilised by propylene oxide

vapour. Sterile basal salts medium (200 ml.) was added to the flask

(9K medium for T. ferrooxidans and Vishniac and Santer medium for!·

thioparus). Inoculation with acclimatised washed cells was at a level

to yield 10 7 cells/ml.

The inoculated shake flasks were incubated for 360 hours on a re­

ciprocating shaking machine at 30 C. The pH of the suspending medium

was maintained at the initial level by manual adjustment from time to

time. Samples for analysis of solubilised metal ions (5 ml.) were

removed at appropriate time intervals and the volumes adjusted with

sterile medium. Zinc and total iron in the samples were determined

by atomic absorption spectroscopy; ferrous iron was estimated by the

o-phenanthroline method (7). The results of these experiments are

shown in Table 4.

A further experiment was carried out to explore the effect of

ferrous iron concentration on the leaching rate of the chemically-pure,

iron-free zinc sulphide sample, in the presence of T. ferrooxidans

and T. thiooxidans. Similar procedures were followed, except that the

pulp density was doubled. The results are shown in Table 5 and

graphically in Figure 1 (for T. ferrooxidans only).

Discussion

The results of experiments of the kind described can only be de­

finitive if carried out with properly-annealed, high-purity synthetic

minerals. Nevertheless, the results obtained suggest that wurtzite

is much less amenable to attack by all three of the Thiobacillus

species employed than sphalerite, marmatite or chemically-pure zinc

sulphide. The hexagonal crystal structure in which its component ions

are arranged, in contrast to the cubical forms of sphalerite and

marmatite, may contribute to this difference in reactivity.

Leaching Behaviour of Zinc Sulphi</e Minerals

Table 5

Effect of Ferrous Iron Concentration on the Leaching of Iron-free Zinc Sulphide by T. ferrooxidans and T. thiooxidans

Ferrous Iron Concentration

(ppm)

0

5

10

100

SOO

1000

2000

4000

Marmatite

T. ferrooxidans

Rate of zn 2+ Solution (ppm/hr)

l. 50

1.65

l. so 5.2

15.8

20.8

29.0

28.0

31. 0

Tot-al zn 2+ Concn. (ppm)

550

570

600

1475

2150

2500

2575

2700

3575

T. thiooxidans

Rate of zn 2+ Solution (ppm/hr)

2.4

2.45

2.60

2.00

1.80

1.8

1.75

Total zn 2+ Concn. (ppm)

730

790

820

670

610

570

550

171

The substitution of a substantial proportion of zinc by iron in the

marmatitic forms of sphalerite leads to still greater amenability of

the mineral to microbial degradation. Whether the presence of iron in

the lattice directly influences the reactivity of the marmatite sur­

face or whether sufficient iron is released to initiate a ferrous­

ferric leaching mechanism is not completely clear. However, during

the course of the leaching of marmatite'by T. ferrooxidans, significant

amounts of ferric compounds were precipitated and the residual con­

centration of total iron was low. The concentration of ferrous iron

was negligible. In contrast, no precipitation of ferric compounds

was observed during the leaching of marmatite with T. thiooxidans

and at the conclusion of the experiment, the total iron concentration

was substantial and mostly in the ferrous form. The release of zinc

from marmatite by T. thiooxidans is considerable and presumably results

predominantly from a direct attack upon the sulphide moiety of the

mineral. The fifty percent higher release of zinc from marmatite by

T. ferrooxidans suggests that in this case a ferric leaching mechanism

is also involved. The enhancement of zinc sulphide degradation by!·

ferrooxidans in the presence of added ferrous iron and the negligible

effect of added iron with T. thiooxidans, support the participation

of a ferric leaching mechanism.

172

4

3

-i ~ 82 ... --!

0 0

A. M. Khalid and B. J. Ralph

•

100 200 Hour,

eNo ferrous; • 5 p.p.m. lerrous; a100p.p.m ferrous; .soop.p.m. ferrous; 02000 p.p.m ferrous:•4000 p.p.m. lerrous;

Figure 1

·-t- -

•

0

I

300 400

a 10 p.pm. lerrous; .1000 p.p.m. lerrous

• Marmatite

Effect of Ferrous Iron Concentration on the Leaching of Iron-free Zinc Sulphide by T. ferrooxidans

The effect of iron substitution in zinc sulphide appears to be

quantitatively much more significant in respect to leaching rates

by T. ferrooxidans and T. thiooxidans than the differences in

crystal structure. A good deal more experimental data on the effects

on leaching rates of crystal structure differences and of lattice

Leaching Behaviour of Zinc Sulphide Minerals 173

substitution is needed before generalisations can emerge. The avail­

ability of a more extensive range of synthetic minerals would enable

such investigations to be carried out.

References

1. Leathen, W.W., Braley, S.A. and McIntyre, L.D. The Role of Bacteria in the Formation of Acid from Certain Sulphuric Constituents Associated with Bituminous Coal. II. Ferrous Iron Oxidising Bacteria. Appl. Microbial. ~: 65-68 (1953).

2. Silverman, M.P., Rogoff, M.H. and Wender, I. Bacterial Oxidation of Pyritic Materials in Coal. Appl. Microbial. 9 491-496 (1961).

3. Trussell, P.C., Duncan, D.W. and Walden, C.C. Biological Mining. Car.~d. Min. Jour. 85 : 46-49 (1964).

4. Brunauer, S.P., Emmett, P.H. and Teller, E.J. Adsorption of gases in multimolecular layers. J. Amer. Chem. Soc. 60 309-319 (1958).

5. Silverman, M.P. and Lundgren, D.G. Studies on the Chemoauto­trophic Iron Bacterium Ferrobacillus ferrooxidans. I. An Improved Medium and a Harvesting Procedure for Securing High Cell Yield. J. Bact. 77 : 642-647 (1959).

6. Vishniac, W. and Santer, M. The Thiobacilli. Bact. Rev. 21 195-209 (1957).

7. Welcher, F.J. (Ed.) "Standard Methods of Chemical Analyses". Vol. 38; Van Nostrand, N.Y. (1962).

APPENDIX- 6.7 POSTER PAPER PRESENTE,'D AT: The Third Inter>national Syrrrposiwn on EnvirOY11T1ental Biogeoohemistry,March 27,19??,0ldenberg.

MICROBIAL ECOLOGY OF OVERBURDEN HEAPS FROM URANIUM MINING AT

RUM JUNGLE, N.T., AUSTRALIA

A.M. Khalid and B.J. Ralph

School of Biological Technoloyy, University of New South Wales,

Kensington, N.S.W., 2033, Australia.

Abstract

Microbiological studies have been undertaken to contribute

to the understanding of the basic physical, chemical and biological

mechanisms concerned in the continuing heavy metal (primarily copper and

zinc} pollution arising from uranium mining operations in the Rum Jungle

area, Northern Territory, Australia, over the period 1953-71. Release of

heavy metals from overburden heaps owing to leaching of low grade metal

sulphide ores is observed. Climatically, the area is distinguished by two

distinct seasons, an almost rainless Dry from May to September and a Wet

from November to March. The population levels of principal autotrophic

microorganism types and some heterotrophs occurring in effluent waters and

in overburden heaps have been determined and are discussed in relation to

the climatic cycle and other factors. The results of a preliminary study

of the distribution of microbial types within one of the larger overburden

heaps, at the end of a dry season, are recorded and discussed.

Introduction

Large scale uranium mining and processing is proposed for a

number of locations in Northern Australia and one proposal is the subject

of the comprehensive Ranger Uranium Environmental Inquiry at the present

time. The only major uranium mining operation hitherto carried out in the

Northern Territory was that in the Rum Jungle area which lies about 64 km

south of Darwin and 80 km east of the Joseph Bonaparte Gulf on the Timer Sea.

The area is highly mineralised and uranium, copper, lead, nickel, zinc and

cobalt ores occur (Roberts, 1960). Extensive sub-grade copper mineralisation

occurred with most of the uranium ores. The rich uranium ore deposits were

extracted by opencut operations and processed on site for'the recovery of

uranium oxide and some copper over the period 1953-71. Some copper was

2

recovered by the heap leaching of two small heaps constructed in 1966 from

low grade copper sulphide and oxide ores.

During the period of operation of the processing plant, con­

siderable acid and metal pollution arose from the eventual release of process

effluents into the East Branch of the Finniss River system, owing to over­

flows from holdings ponds and tailing dams during the wet season, and by 1966

further sources of pollution had arisen from the autogenous leaching of

sulphidic material in the overburden dumps and low grade ores in stockpiles.

The termination of commercial operations in 1961 left two stockpiles un­

treated, an extensive tailings dam area containing considerable amounts of

residual minerals and, in the Rum Jungle Mine area proper, three substantial

overburden heaps and the leaching heaps used for copper recovery.

In 1973, the Australian Atomic Energy Commission, jointly

with the Department of Northern Australia, initiated the Rum Jungle En­

vironmental Studies with the principal objectives of assessing precisely

the extent of environmental pollution in the area and of gaining under­

standing of the mechanisms underlying the generation and spread of con­

taminants. A secondary objective of the studies was the obtaining of

information which could be relevant to proposed developments of uranium

mining and processing in areas of similar climate in other parts of the

Northern Territory. An impressive body of information has already been

amassed by the A.A.E.C. Research Establishment in the course of the R.J.

Environmental Study (Davy, 1975). Microbiological studies were commenced

late in 1974 by the University of New South Wales, in order to assess the

nature and extent of the microbial associations and to evaluate various

procedures for the control and suppression of microbial activity. Rum

Jungle lies in a very sparsely populated area of Australia and is over

3000 km fro~ the investigating laboratories in Sydney; the geographical

remoteness has imposed some limitations on all aspects of the environmental

studies.

Some General Considerations

The relatively limited information on the microbial ass-

3

ociations in rock piles containing sulphide minerals and in the effluent

water systems suggests a considerable degree of complexity which matches

the heterogeneity of chemical composition and physical characteristics of

the milieu (Ralph, 1977). Further, the microbial populations are likely

to vary both in nature and in magnitude under the in~luence of a diversity

of factors which include the age of the heap and climatic characteristics

such as rainfall patterns and temperature. The situation in overburden

dumps is likely to be more subject to climatic factors than those in

leaching heaps with recycle of percolating solutions and other controls.

Relatively simple models of the mechanisms operating in

leaching heaps have been proposed (Harris, 1969) and have some utility

for metal recovery operations. More effective control of mineral de­

gradation in rock piles might be possible if the intermeshing of geo­

chemical and microbial populations in specific locations does provide

information on the physico-chemical characteristics of the milieu and

can yield clues to the events which generate such conditions.

The first steps in the studies here reported have been

(i) The enumeration of the autotrophic microorganism types known to

be associated with the degradation of sulphide minerals (acidophilic,

iron oxidisers such as Thiobacillus ferrooxidans; acidophilic sulphur

oxidisers such as T. thiooxidans; less-acidophilic sulphur oxidisers

such as T. thioparus), together with heterotrophic types.

(ii) The correlation of population level data with available in-

formation on the physico-chemical characteristics of the sample location

(pH, moisture content, water-solubles content, soluble metal ions,

organic carbon content, etc.), and with climatic factors.

(iii) The nature, distribution and magnitude of microbial populations

within the overburden heaps and their variation with climatic conditions.

The investigation to date has highlighted the difficulties

of adequately sampling rock piles and the uncertainties of current

techniques for the enumeration of some microbial types.

Methods

The layout of the Rum Jungle Mine area is shown in the map

(Figure 1). Sample sites are identifiable by the map co-ordinates.

Samples were collected in September, 1975 (Dry season), March, 1976

(Wet season) and September, 1976 (Dry season).

Water samples were either transferred directly at the

field location into pre-sterilised containers or concentrated onto

membrane filters by syringe. Water samples for chemical analysis were

pre-filtered and membrane-filtered and the filtrate stored in pre­

sterilised containers. All samples were stored at 4 C as soon as

practicable. Solid materials were sampled with heat-sterilised imp­

lements and immediately transferred into pre-sterilised containers.

4

Prior to examination, solid samples were ground with a sterilised mortar

and pestle, under clean air conditions, to a particle size of approximately

120 microns.

Standard methods were used for the analysis of solid samples

in respect of moisture content, water-soluble components and pH (Belly and

Brock, 1974). The content of zinc, copper and iron ions was determined in

water samples and the water-soluble components of solid samples by atomic

absorption spectroscopy.

For the microbiological examination of solid materials, a

weighed amount of the finely-ground sample was agitated with a known

volume of 9K basal medium (Silverman and Lundgren, 1959) and aliquots of

the supernatant, with appropriate dilution, used for enumeration of the

various rnicroorganism types. Water samples were either examined directly,

after appropriate dilution, or, in the case of samples concentrated by

membrane filtration in the field, the organisms were washed off the mem­

brane and re-suspended.

Acidophilic, iron-oxidising organisms of the Thiobacillus

ferrooxidans type were enumerated by concentrating from the sample sus­

pension ont~ a washed membrane filter to a silica gel plate impregnated

with 9K-ferrous sulphate medium (pH 2.5), and incubating at 30 C for 2-3

weeks. The reddish colonies developing from each cell were counted under

a stereo microscope. Nine plates were counted for each sample; the

results were reported as a most probable number (MPN) and standard

deviation recorded.

5 Ac:· aophilic, sulphur-oxidising organisms of T. thiooxidans

type were enume'rated by direct streaking of various dilutions of the sample

suspensions onto 9K-thiosulphate agar (pH 3.5), incubating at 30 C for one

week and counting with a colony counter. Sulphur-oxidising organisms of

the T. thioparus type were enumerated by similar procedures, using agar

plates impregnated with the thiosulphate medium (pH 6) described by

Vishniac and Santer (1957).

Heterotrophic populations were evoked and enumerated on

nutrient agar medium (pH 7.4), Czapek-Dox agar (pH 6.8), and glucose

Scott Yeast extract medium (pH 3.5), (Manning, 1975). For example:

T. denitrificans A modification of the method of Hutchinson

et al. (1965) •

Metallogenium spp. Medium & procedure recommended by Walsh &

Mitchell (1972) were used for isolating and

enumerating these microorganisms.

Anaerobic, sulphate-reducing spp. Method of Pankhurst (1971) involving

Baars medium was employed.

Nitrogen-fixing activity has been sought by use of the acetylene

reduction test {Burris, 1972).

Results and Discussions

The water and solid samples were collected in September, 1975

{dry season) and March, 1976 (wet season) from closely similar locations,and

enable comparisons to be made between some physico-chemical character­

istics and the population levels of some groups of microorganisms. The

sampling of solid materials was restricted on these occasions to less

than a metre from the surface. The population levels of various micro­

organism types in water samples collected during these wet and dry

seasons are-shown in Table 1, and similar data is shown in the same table

for rock samples from the 15-30 cm. depth zones. Table 2 contains in­

formation on the variation of populations with depth during the wet season

(March, 1976) on White's Heap and the Sulphide Heap.

In general, the population levels are lower in the wet season

samples, the effect being more marked in the spring outflows than in the

opencuts. The reverse effect is the case of tne acidophilic hetero­

trophs in Dyson's Opencut suggests an influx of organic nutrients in the

wet season. The reduction of population counts in spring samples by a

dilution effect is likely to be more marked than in opencut samples. The

reverse effect is shown in the counts for rock samples from the heaps at

depths of 15-30 cm., suggesting that at these near-surface depths during

the dry season, microbial activity is inhibited by lack of water.

In Table 2, the results of a first attempt to correlate

population levels and types with depth within the heaps are set out.

The samples were secured from holes dug with pick and shovel and pen­

etration to depths greater than 70-80 ems. was not practicable by this

method. It is evident from the scatter of the counts that any depth

effect is over-ridden by the heterogeneity of the heap materials and that

aerobic conditions persist to at least the depths reached.

6

During the last dry season (September, 1976), a more in­

tensive sampling of White's Heap became practicable, and mechanical equip­

ment was used to cut sampling holes to a depth of 4 metres in the top of

the heap and at its base. The locations of these holes, which are

designated A to G, are shown in the map (Figure 2). Composite samples

were taken from the walls of these holes (from 15-20 cm. in), in a

vertical sequence from the bottom at 30 cm. intervals (Figure 3). Sub­

sequent examination of these samples yielded the data shown in Tables

3.1 to 3.6. Direct sampling into appropriate media was carried out for

the detection of T. denitrificans and Metallogenium spp. These species

were not found in any sample, nor was any evidence for nitrogen-fixing

activity found in subsequent examinations. No growth was obtained from

any sample on the medium described by Manning (1975) for the different­

iation of colony types of acidophilic iron oxidising bacteria, but·this

author's medium and method for acidophilic heterotrophs proved most use­

ful.

The full evaluation of these results will not be possible

until similar sampling can be carried out at other times within the annual

climatic cycle, but some general comments can be made.

Moisture levels in top of heap samples (Holes D.E.G) are

7 ci1dracteristically variable but similar in pattern to those measured in

near-surface samples during the previous dry season. Wet season samples

are characteristically more uniform in water content with values generally

within the 15-20% range. The moisture levels of the base of heap samples

(Holes A,B,C) are clearly affected by the drainage patterns; the A hole

is near the point of e::it of a wet season spring and Band C holes are

near the highest points of the basement of the heap. As might be an­

ticipated, the water-solubles content of all samples are comparatively

low; in fact, about an order of magnitude lower than in wet season

samples.

There is a rough correlation between the pH levels of samples

and the occurrence of various microorganism types. For example, the hole A

samples (av. pH 2.77) contains significant levels of acidophilic iron­

oxidising and acidophilic heterotrophic organisms, and a similar situation

is evident in hole G samples (av. pH 3.0). There has been extensive

leaching in the vicinity of hole c, with considerable breakdown of rock

masses to clays and other finely divided material. The samples have a

much higher average pH (5.94) and the less acidophilic sulphur-oxidising

bacteria make up the largest microbial populations.

The complete absence of acidophilic sulphur-oxidising

organisms is an interesting feature, since earlier observations indicate

that this group is characteristically the most abundant of the auto­

trophic populations in the wet season samples. The occurrence of

anaerobic sulphate-reducing organisms in the sampling holes at the base

of the heap, in the zone 70 ems. up from the basement, is of great

interest (Dugan, 1975) and suggests an anaerobic zone running through the

base of the heap. The overall distribution of aerobic microbial types,

however, suggests that the penetration of oxygen into the heap at the end

of the dry season is at least 4 metres deep over the higher parts and may

be more extensive.

Acknowledgments

The studies reported have been carried out under a research

contract with the Australian Atomic Energy Commission. The authors are

most appreciative of the financial support provided and the continuing

help and advice available from the staff of the AAEC Research Establishment.

References

Belly, R.T. & Brock, T.D., 1974. Ecology of Iron oxidising bacteria in pyritic metals associated with coal. J. Bact. 117 : 726-732.

Burris, R.H., 1972. Measurement of biological N2-fixation with 15N2 and acetylene. In : Sorokin & Kadota, Ed. Techniques for the assessment of microbial production and decomposition in fresh water, pp 3-14. Blackwell Scientific Publications, Oxford.

Davy, D.R., 1975. Run Jungle Environmental Studies. Australian Atomic Energy Commission Report, AAEC/E365.

Dugan, P.R., 1975. Bacterial ecology of strip mine areas and its relationship to the production of acidic mine drainage. Ohio Journal of Science 75 : 266-279.

Harris, J.A., 1969. Development of a theoretical approach to the heap leaching of copper sulphide ores. Proc. Aust. Inst. Min. Met. 230 81-92.

Hutchinson, M., Johnstone, I.I. & White, D., 1965. The taxonomy of certain thiobacilli. J. Gen. Microbial. 41 357-366.

Manning, H.L., 1975. New Medium for isolating iron oxidising and heterotrophic acidophilic bacteria from acid mine drainage. Appl. Microbial. 30 : 1010-1016.

Pankhurst, E.S., 1971. The isolation and enumeration of sulphate-reducing bacteria. pp 223-240. In: isolation of anaerobes. Ed. Shapton & Board. Soc. Appl. Bact. Tech. Ser. No. 5. Academic Press, London.

Ralph, B.J., 1977. Oxidative Processes, Chap. 6. To appear in : Biological Factors in Mineral Cycling. Eds. P.A. Trudinger, & D.J. Swaine. Elsevier.

Roberts, W.M.B., 1960. Mineralogy and genesis of White's ore body, Rum Jungle Uranium Fielq, Australia. Neus Jahrbuch f~r mineralogie, Abhanlungen. Bd. 94 : 868-889,

Silverman, M.P. & Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans. 1. An improved medium and a harvesting procedure for

securing high cell yield. J. Bact. 77 : 642-647.

8

9

Vishniac, w~ & Sauter, M., 1957. The Thiobacilli. Bact. Rev. 21 195-209.

Walsh, F. & Mitchell, R.C., 1972. An acid-tolerant-iron-oxidising Metallogenium. J. Gen. Microbiol. 72 : 369-376.

):> C: ......... VI :;:o c+ (!)

-s "'O QI -s __. 0 -'• 0. PI C: ::I n

(!) ):> 0. c+ 0 ~ 3 -'• -'• c+ ...,, n ;:r ......

G) c+ C:

rn ':::T :;:o ::I (!) rn (!)

-s "'O -',

~ (!) -s 3 -'•

n VI 0 VI -3 -'• 3 0 -'• :::s VI VI -'• 0 0 -t) :::s ........

I : 5000 THE RUM JUNGLE HINE OIIAWN. II f . LOW SO'I AP'IUL 1914

~ ~ TO MO!("'' ,nc .. IIIINI ••• § §l

05 00 07 08 09 10 II 12 i, 14 15 16 17

! . T II , I

9l 94 9~ 90 i 98 _ 99! 1 oi _ 02 05

-- r j I

i-i

'1\J- I 1-- ! ':, . -+- -,- . ~~- --+-1-r-··1: -~--l--~--1---

I-

18 19 20 21 22 25 24 25 20 27 28 I - . I I

I

1 -I

I I

rH-l 29 lO

7 00 (']

) 9

57

50

55 I

§!soi-- -- __ :__ --

! ~

5

I ! . ~

~

48 >---------+-~ 41

ff

45

44

45 ,_ -

35 I I

34 1

' ni 32

51 I I

{!] 30 L

- , VI . . a1

n~1· -t-----+-+----J_ II

- 1 ·-;-I

I I

/

' 1 ·--·-- -t

- i---l- ---~ ,, t' / : . . -/ -- .

I

·1 I ••• •·~"., ... "M'" T"

I - , - - - ---+ -L-- -_, - I i

I I I -r::-:=::::

I .. , ussu , ,u" 1 Ill t ttSU ltl tUUS I I ., ,,,m " nun

"' tln)I

I ' i i I

__ -_ -~-.t,- _ --,I ~ 2~ ~ f!l I '

, 58

57

56

55

I 54

- .53

52

---- 51 I

--j 508

-----; 49

-4 48

47

46

145

- 44

-I 45 I

J 42

-- i 41

7•0B ---. 59

I __ __j 39

_I 57

36

35 .! - .. ,, .... , L' i 41· I , ,i.\f/ .... -i ,--, f I I '

--'---L--'----'---'-----'-----'-----'--...L...---'--'---'----'-----'--' _ ·· c..' _.,_-..J._....!1_...,______JI....--' _ : lli _l : . ,, ,,

- I 54

l l

l2

-1 51

90 91 92 Ol 96 97 98 99 00 01 02 05 04 05 06 07 08 09 10 If 12 ll 14 15 16 17 18 19 20 21 22 2l 24 25 26 27 28 29 50 E!J

50 8

flo•• o.,, llo••~T,.c;t.

,.,,. o .. ,,..." 1,1, ,11, .. , c .. , ..... n

@:) C)

~ ~ ~ ~

o, ...... u,_ loJ1.­

S1Kwv P'oh1t

fl lr t Woll

l,..eN OlrtWol

-;. ;..n.,;1i.,·

"' ::.===---

Loou """ ' •: llwbtli. f'ii., $ ,',•;)!

llilcinl"" l""iln t. lloch 1!•

,.1.__.,,._n~ Our-"Q'-0

:~~ ... ~'1.,!!-=~-1 U -dlf~•;,.-,.,.,tr_ s,,1,., l•uMOf''l•lDrtf .,._ Holl

Lan, OoM, ..... d = C)

l ll • Nf111,rd '--111• ,: - . tOOO 'f•rd c;,i, + Oiu ,ti. Jt..-t

100 • 0 - MIO JOO ~ ......

.. 01111o•hL ,nu"' ,,!!'.~:·~~.H:\<'~~~~T~l~l.:,".~;!1~T:~, ~ .... 11' n ' ..... ,~O• to1UOI ,,, . . .. • • • . , ; l. L...._. 1110,C.,.T'( fHI IOO ,-.10 fl•W,\<'l•U llt..C:•11• 1111111, 100,1 • 1-..,,. , •• .,. HIIIO I, U>•• t .. ,, IP H H O ~

- l • ot .... • •tlto o f - . ... h4'0NO .. , _ ,,_

c-,· ·E~r-, : .. -.... ~=~·,5i,,,,,,,"~'-~ .. ,_., ....... u,,.... • ......... , •.• , ·--·--· . . • _ _ JtUiS•_ _ _ _ ___ , ___ _

....L

0

11

05 06 07 08 09 10 11 12 44 I \ 44

I I I - - - - - - ---.t '----- ---. - -- -· _.,.._,,

43 I 43 I

42 42 I I

I I I I

I

41 1 41 I

35 __________ '--______________ _.35 05 06 07 08 09 10 1'1 12

0 Indicates sampling positions . .

WHITES OVERBURDEN HEAP.

Figure 2

Top

Basement

~ Indicates sampling points.

WHITE"S OVERBURDEN HEAP­

SAMPLING PATTERN.

( Not to scale J

Figure 3

12

13 TABLE 1

DRY AND WET SEASON POPULATIONS

Acidophilic Acidophil ic Non-Ac i doph i l i c Heterot rophs Location Fe-Oxidisers S-Oxidisers S-Oxidisers

DRY WET DRY WET DRY WET DRY WET

Water Samples Dyson' s No .4 1.6 0.4 1.5 0.25 0.-5 NG 60.0 NG Spring.

Dyson's Opencut. 0.33 o. 35 43.0 0.53 0.67 NG 20.0 391 .o

Stockpile No. 8. 0.28 0.20 23.0 13.9 0.33 25.4 160.0 NG

Inter-mediate NG 0.05 0.27 0.38 NG NG 3.7 0.45 Open cut.

White's Opencut. 0-.38 0.65 NG NG NG NG 1.6 NG

White's Spring 1.2 0.03 81.0 0.03 NG NG 29.0 0.54

Rock Samp 1 es

White's NG 0.06 NG 2.7 o. 15 NG 0.04 O .24 Heap.

NG 0.04 NG 1.6 NG NG 0.05 11. 6

NG 0.2 NG 0.03 NG o. 14 0.37 3,2

Sulphide o. 12 0.38 NG 3.04 NG 0.24 NG 0.72

& Oxide NG o. 17 NG 0.08 NG NG NG 0.04

Heaps. 0.38 0.90 NG 0.02 NG NG NG NG

Enumeration of organisms expressed as No. per ml or gram dry \-Je i ght. NG= No growth.

Location

White's Heap.

Sulphide Heap.

TABLE 2

VARIATION OF POPULATIONS WITH DEPTH IN HEAPS DURING WET SEASON

14

Depth Acid-Fe Acid-S Non-Acid Heterotrophs (cm.) Oxidisers Oxidisers $-Oxidisers Nutrient Czapek-Dox

Agar Agar

5 0.04 1.62 NG 11.6 0.37 25 o. 10 29.2 391.0 11.0 0.02 50 0.12 34 .0 2.7 3.6 1. 12

5 0.06 2.72 NG 0.24 0.37 25 0.29 0.87 0.63 14. 7 20.8 50 o. 1 13,7 0.05 5.8 11.8

5 NG NG 0.21 5,9 8.3 25 0.20 0.03 0. 14 3.2 2.4 50 0.03 0.02 0.58 6.9 2.9

5 0.27 0.99 NG o. 19 0.28 25 0.38 3.04 0.24 0.72 NG 40 0.35 0.51 NG NG NG

5 o. 14 11.9 NG 3.24 20.6 30 O .17 o. 18 NG 0.04 0.02 60 0.04 NG NG NG NG

5 0.45 0.23 NG NG NG 25 0.27 NG NG NG NG 50 NG NG NG o. 14 NG 80 0. 1 t NG NG 0.22 0.02

Enumeration of organisms expressed as No. per ml or gram dry weight. NG= No growth.

15

TABLE 3. 1

SAMPLING HOLE A (106-368) WHITE'S OVERBURDEN HEAP.

Distanc;e From Basement. (cm).

10 40 70 100 130 160

Moisture Content. 15.6 13.1 9.5 13.3 13.9 17.4 (%)

190 220 250

7.5 11.8 2.8

Water So 1 ubl es. ( ) 4800 2300 3600 4000 4600 3600 2300 2700 8000 p.p.m.

pH

Ac idoph i 1 ic Iron Oxidising

(x 103). Bacteria.

Acidophil ic s-Oxidising Bacteria. (x103).

Non-Acidophil ic S-Oxidisng

(x103). Bacteria.

Acidophil ic Heterotrophic Bacteria. (x103).

Nutrient Agar.

(x103 .

Czapek-Dox Agar.

(x l o3) .

Anaerobic Sulph-

3.0

NG

NG

NG

NG

NG

NG

2.7 2.7 2.6 2.5 2.7 2.9 2.9 2.9

NG NG 1.23 0.30 0.29 1.22 0.32 1.89

NG NG NG NG NG NG NG NG

NG NG NG NG NG NG NG NG

NG 2.56 6.56 7.52 60.32 10.53 10.63 0.50

NG 0.32 2.30 0,53 10.53 0.53 0.53 NG

NG NG 2.30 0.01 10.50 NG 1.00 3,05

ate Reducers. 0.55 0.35 0.20 NG NG NG NG NG NG

(xl o3).

Enumeration of organisms expressed as No. per ml or gram dry weight. NG= No growth.

16

TABLE 3,2

SAMPLING HOLE B (075-364) WHITE'S OVERBURDEN HEAP.

Distance From Basement. (cm).

Moisture Content. (%)

Water Solubles. (p.p.m.)

pH

Acidophil ic Iron Oxidising

(xl o3). Bacteria.

Acidophl ic S-Oxidising Bacteria. (x 103).

Non-Acidophil ic S-Oxidising Bacteria. (x 1 o3).

Ac i do phi 1 i c Heterotrophic Bacteria. (xl o3).

Nutrient Agar.

(x103).

Czapek-Dox Agar.

(xl o3).

Anaerobic Sulph-ate Reducers.

X 103).

10 40

8.6 4. 1

37600 4850

3,5 3.5

NG NG

NG NG

NG NG

5,42 2.50

NG NG

NG NG

0.25 0.11

Enumeration of organisms

70 100 130 160 190 220 250

4. 1 2.9 4.o 1.7 7,4 6.5 8.6

5150 4950 5920 4530 7000 6950 7550

3.9 4. 1 4.3 5.0 4.o 3,5 3,7

NG 0.75 NG 0.65 NG NG 0. 11

NG NG NG NG NG NG NG

0.20 NG 0.65 NG NG NG t.45

NG NG 10.52 16.87 40.63 60.27 15.23

12.25 6.50 3,53 NG 15 .32 10.23 11 ,32

NG NG NG NG 5.72 3.22 NG

NG NG NG NG NG NG NG

expressed as No. per ml or gram dry weight. NG= No growth.

17

TABLE 3,3

SAMPLING HOLE C (071-368) WHITE'S OVERBURDEN HEAP.

Distance From Ba semen t • (cm) . 10 40 70 100 130 160 190 220 250

Moisture Content. 14.3 15.1 14.8 14.4 15.2 16.5 17,4 16.5 16. 1 (%) •

Water Solubles. (p.p.m.) 3500 5100 2500 3051 5183 3915 5261 5578 6098

pH 6.5 6.5 4.5 4.8 4.5 6.5 6.6 6.9 6.9

Ac idoph i 1 ic Iron Oxidising

(x 1 o3). NG NG NG

Bacteria. 0.03 NG NG NG 0.02 NG

Acidophil ic s-Oxidising

(x 103). NG NG NG NG NG NG NG NG NG

Bacteria.

Non-Ac i doph i I ic S-Oxidising

(x103). NG NG 0.57 1.65 2.57 3.75 NG 12.58 11.58

Bacteria.

Acidophil ic Heterotrophic Bacteria. (x103).

NG NG NG NG NG NG 0.01 NG NG

Nutrient Agar. NG NG NG 1.58 0.76 NG NG _o. 31 0.10

(x 1 o3) •

Czapek-Dox Agar. NG NG NG NG NG 0.03 0.02 0.21 NG (x103).

Anaerobic Sulph-ate Reducers. 0.01 0.23 NG NG NG NG NG NG NG

x103

Enumeration of organisms expressed as No. per ml or gram dry weight. NG= No growth.

18

TABLE 3.4

SAMPLING HOLE D (097-367) WHITE'S OVERBURDEN HEAP.

Distance From Basement. (cm). 10 40 70

Moisture Content. 5.7 6.0 5-7 (%) •

100 130 160 190 220 250

4.5 13.5 13.9 10.3 13.5 13.8

Water Solubles.

(p. p.m.) 1848 2832 970 1086 8296 4745 3740 2797 7732

pH 4.0 3,9 3-7 3.7 3.0 3.4 3.4 3,3 3,8

Ac idophi 1 ic Iron Oxidising

(x103). NG NG NG 0.25 NG 0. 11 NG 0.20 NG

Bacteria.

Ac idoph i 1 ic S-Oxidising NG NG NG NG NG NG NG NG NG Bacteria. (x103).

Non-Ac idoph i 1 ic S-Oxidising NG NG NG NG NG NG NG 1.16 NG Bacteria. (x 103).

Acidophil ic Heterotrophic NG Bacteria. (x103).

0.26 0.33 o.45 0.53 NG o. 14 0.23 NG

Nutrient Agar. NG NG NG NG NG NG 3,25 NG 1.23

(x 103).

Czapek-Dox Agar.

(x103). NG NG NG NG NG NG NG 2.74 1.33

Anaerobic Sulph-ate Reducers. NG NG NG NG NG NG NG NG NG

(x 1 o3).

Enumeration of organisms expressed as No. per ml or gram dry weight. NG= No growth.

19

TABLE 3.5

SAMPLING HOLE E (063-385) WHITE'S OVERBURDEN HEAP.

Distance From Basement. (cm). 10 40 70 100 130 160 190 220 250

Moisture Content. 9.4 15.5 8.3 (%)

8.3 9.8 8.1 7.5 4.8 5.7

Water So 1 u b 1 es.

(p.p.m.)

pH

Acidophil ic Iron Oxidising

(x103). Bacteria.

Acidophil ic S-Oxidising Bacteria. (x103).

Non-Acidophil ic S-Oxidising Bacteria. (x103).

Acidophil ic Heterotroph ic Bacteria. (x103).

Nutrient Agar.

(x103).

C za pek-Dox Agar •

(x103).

Anaerobic Sulph-ate Reducers.

(x 1 o3).

Enumeration

9463 4265 8707 2741 4872 2323 6281 6737 11848

4.5 6.5 6.o 4.3 3.7 3.7 3.8 4.0 4.5

NG NG NG NG NG 0.53 NG NG NG

NG NG NG NG NG NG NG NG NG

NG NG NG NG 0.53 NG NG NG NG

NG 5,73 NG 3. 77 1.33 2.33 11. 32 NG NG

NG NG NG 1.01 0.54 0.33 NG NG 0. 12

NG NG NG NG NG NG NG 2 ,58 NG

NG NG NG NG NG NG NG NG NG

of organisms expressed as No. per ml or gram dry weight. NG= No growth.

20

TABLE 3.6

SAMPLING HOLE G (104-415) WHITE'S OVERBURDEN HEAP.

Distance From Basement. (cm).

Moisture Content. (%) •

Water Solubles.

(p.p.m.)

pH

Acidophil ic Iron Oxidising Bacteria. (x103).

Ac i doph i l i c S-Oxidising Bacteria. (x 1 o3) •

Non-Acidophil ic $-Oxidising Bacteria. (x 1 o3).

Acidophil ic Heterotrophic 3 Bacteria. (x 10 ) •

Nutrient Agar.

(x103).

Czapek-Dox Agar.

(x103).

Anaerobic Su 1 p-ate Reducers.

(x 1 o3).

10 40 70 100 130 160 190 220 250

7.6 8.6 7.5 9.2 5.4 3,7 3.7 5.1 5.4

4656 7097 9440 8497 7402 8471 8683 1267 1794

2.8 2.8 3.0 2.4 2.6 3.4 3.0 3.5 3.5

NG NG NG 0.73 0.33 o.64 0.01 0.02 NG

NG NG NG NG NG NG NG NG NG

NG NG NG NG NG NG NG 0.21 0.73

NG 2.53 1.24 1.33 0.73 0.98 11.73 13.63 10.25

NG 0.03 0. 11 NG 10.28 NG 5.68 NG 1.33

NG NG NG NG NG NG NG 0.52 NG

NG NG NG NG NG NG NG NG NG

Enumeration of organisms expressed as No. per ml or gram dry weight. NG= No growth.