Interaction between clay minerals and hydrocarbon-utilizing indigenous microorganisms in high...

Interaction between clay minerals and

hydrocarbon-utilizing indigenous

microorganisms in high concentrations of

heavy oil: implications for bioremediation

SITI KHODIJAH CHAERUN1 ,* , KAZUE TAZAKI

2, RYUJI ASADA

2AND

KAZUHIRO KOGURE3

1Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192,

2

Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, and3Ocean

Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164-8639, Japan

(Received 10 February 2004; revised 23 July 2004)

ABSTRACT: This study focused on whether the presence of clay minerals (montmorillonite and

kaolinite) in marine or coastal environments contaminated with high concentrations of heavy-oil

spills were able to support the growth of hydrocarbon degraders to enable bioremediation. The

bacterial growth experiment utilizing ~150 g/l of heavy oil (from the Nakhodka oil spill) was

conducted with 1500 mg/l of montmorillonite or kaolinite. Bacterial strain Pseudomonas aeruginosa

(isolated from Atake seashore, Ishikawa Prefecture, Japan), capable of degrading heavy oil, was

employed in combination with other hydrocarbon degraders inhabiting the heavy oil and seawater

(collected from the Sea of Japan). The interactions among microbial cells, clay minerals and heavy

oil were studied. Both clays were capable of promoting microbial growth and allowed

microorganisms to proliferate (to a greater degree than in a control sample which contained no

clay) in an extremely high concentration of heavy oil. Observation by transmission electron

microscopy of the clay-oil-cell complexes showed that microbial cells tended to be bound primarily

on the edges of the clays. X-ray diffraction analysis showed that the clay-oil and clay-oil-cell

complexes involved the adsorption of microbial cells and/or heavy oil on the external surfaces of the

clays. How do the interactions among clay minerals, microbial cells and heavy oil contribute to

environmental factors influencing the bioremediation process? To our knowledge, there are no

previous reports on the use of clay minerals in the bioremediation of the Nakhodka oil spill in

combination with biofilm formation.

KEYWORDS: clay minerals, montmorillonite, kaolinite, hydrocarbon degraders, heavy oil, bioremediation,

Nakhodka, oil spill, Pseudomonas aeruginosa, biofilm formation.

Microorganisms are the major agents mineralizing

organic pollutants in terrestrial and aquatic environ-

ments, and their adsorption on soil mineral colloids

(e.g. clay minerals) is of great importance (Ortega-

Calvo & Saiz-Jimenez, 1998). Laboratory and field

investigations have shown that the biodegradation

of organic pollutants is enhanced or inhibited in the

presence of minerals, clays and organic soil

constituents (Weber & Coble, 1968; Ogram et al.,

1985; Robinson et al., 1990; Miller & Alexander,

1991; Scow & Alexander, 1992; Guerin & Boyd,

1992; Knaebel et al., 1994), and that clay minerals

have a role in the sorption of organic and inorganic

compounds as we l l a s mic roo rgan i sms

(Schiffenbauer & Stotzky, 1982; Lipson &

* E-mail: [email protected]

DOI: 10.1180/0009855054010159

ClayMinerals (2005) 40, 105±114

# 2005 The Mineralogical Society

Stotzky, 1983; Lipson & Stotzky, 1984; Boyd et al.,

1988; Lee et al., 1990; Khanna & Stotzky, 1992;

Tapp & Stotzky; 1995; Vettori et al., 1999; Lin &

Puls, 2000). Nevertheless, no data exist on the

simultaneous use of clay minerals as adsorbents and

carriers in degrading heavy oil. A mechanism

describing the interactions between clay minerals,

heavy oil and microorganisms is also required.

Microorganisms are generally able to grow at low

substrate concentrations. However, not all micro-

organisms are able to grow on, and consume, high

concentrations of substrate (i.e. heavy oil) as the

carbon and energy source, since high substrate

concentrations may cause inhibition of some

microbial enzymatic reactions (known as substrate

inhibition (Shuler & Kargi, 1992)). Hence, a study

was conducted to address the interactions between

clay minerals and microorganisms in high concen-

trations of heavy oil (~150 g/l).

The primary objective of this study was to

investigate the interactions between clay minerals

and hydrocarbon-utilizing indigenous microorgan-

isms in high concentrations of heavy oil (collected

from the Nakhodka oil spill). These interactions

were studied by X-ray powder diffractrometry

(XRD), scanning electron microscopy (SEM) and

transmission electron microscopy (TEM) in an

attempt to determine where and how microbial

cells are bound to the clay minerals (montmorillon-

ite and kaolinite) that are abundant in nature. These

techniques were also used to evaluate the interac-

tions between microorganisms, clay minerals and

heavy oil. Such understanding might provide

information about the factors influencing the

bioremediation of oil spills.

MATER IALS AND METHODS

Clay minerals

The montmorillonite standard JCSS-3102 (from

Mikawa, Japan) and kaolinite standard JCSS-1101

(from Kanpaku, Japan) used in this study were

obtained from The Clay Science Society of Japan

(Okayama, Japan). Both clays were used in batch

experiments without pretreatment.

Bacterial strain

Apart from microorganisms inhabiting natural

seawater (NSW) and heavy oil, a bacterium was

also added as an inoculum in order to promote the

degradation of the heavy oil. The bacterium was

isolated from Atake seashore, Ishikawa Prefecture,

Japan. Using 16S rDNA sequencing, this bacterium

was affiliated to Pseudomonas aeruginosa (98%

similarity). This bacterium was selected for study

because it produced high extracellular emulsifying

activity after 2 days of incubation in the presence of

heavy oil (2% v/v) and 1 g/l of yeast extract

(Chaerun et al., 2003).

Bacterial culture and batch experimental

conditions

Batch bacterial experiments were conducted with

montmorillonite standard JCSS-3102 and kaolinite

standard JCSS-1101. The growth medium contained

the necessary components needed for bacterial

growth. The following were included for each

litre of medium: 25 g of NH4NO3, 0.5 g of

FeC6H5O7´nH2O (ferric citrate), and 0.5 g of

K2HPO4/l of distilled, deionized water (NDW).

Natural seawater without filtration (collected from

the Sea of Japan) was also added to each flask as

additional medium (Table 1). Briefly, the batch

reaction vessels consisted of acid-washed 1000 ml

Erlenmeyer flasks containing 600 ml of growth

medium: 480 ml of NSW was mixed with 120 ml

of an autoclaved NDW. The montmorillonite or

kaolinite (1500 mg/l) was added to the flask and the

pH of the medium was adjusted to pH 7.8 with 1N

NaOH solution. Immediately after sample prepara-

tion, before introducing the inoculum which

originated from the heavy oil and the NSW

medium into the flasks, all five flasks with their

contents were autoclaved, allowed to cool to room

TABLE 1. Elemental composition of natural seawater

(NSW) collected from the Sea of Japan that was used

for medium analysed by ED-XRF.

Element Wt.%

Na 8.27

Mg 1.49

Si 0.26

S 2.94

Cl 79.94

K 2.99

Ca 3.57

Co 0.05

Br 0.42

Sr 0.06

106 S. K. Chaerun et al.

temperature (24ëC), and then inoculated with NSW

medium, the stock liquid culture of bacterial strain

Pseudomonas aeruginosa (4.5% v/v), and heavy oil

(collected from the Nakhodka oil spill) to a final

concentration of ~150 g/l, serving as the sole

carbon and energy sources (Table 2) (Chaerun &

Tazaki, 2003; Chaerun et al., 2003; Tazaki, 2003).

Overall, five experimental flasks were established:

two main flasks contained medium, bacteria, plus

montmorillonite (coded as R1) or kaolinite (coded as

R2); two abiotic control flasks contained medium

plus montmorillonite (coded as R3) or medium plus

kaolinite (coded as R4); and one biotic control flask

contained medium plus bacteria (coded as R5). To

avoid microbial growth and to ensure sterility during

the experiment, abiotic control flasks were auto-

claved three times and heavy oil solution was

sterilized by filtration through a 0.22 mm pore-

sized filter. Cultures were incubated for 105 days at

room temperature (24ëC) with shaking at 125 rpm,

allowing microorganisms to grow. Samples were

removed periodically at sampling periods of 0, 36,

64 and 105 days. Separate sets of samples were

made up and prepared for analysis by XRD, SEM

and TEM. In addition, the liquid medium of three

experimental systems (i.e. R1, R2 and R5) was

supplemented with 1 g/l of yeast extract after

36 days of the experiments.

X-ray diffractometery

Relative changes in the basal spacings of

montmorillonite or kaolinite were measured by

XRD. Samples were prepared by spreading 2 mg

of the suspended clays over a 2.5 cm2area of a

glass slide. The slides were air-dried, placed in a

desiccator containing silica gel to prevent rehydra-

tion, and observed using Cu-Ka generated at 40 kV

and 30 mA by using the 2y/y method and a scan

speed 2ë/min (Rigaku Rinto 1200 X-ray diffract-

ometer). To determine whether the montmorillonite

remained expandable or not, samples of mont-

morillonite-oil and montmorillonite-oil-cell

complexes were treated with ethylene glycol and

then dried at room temperature before being

analysed by XRD.

Microscopic techniques

For observation by SEM, freeze drying was used

for sample preparation (Suzuki et al., 1995).

Briefly, the suspension samples were fixed with

2.5% (vol/vol) glutaraldehyde, pipette-drawn,

mounted on 0.22 mm membrane filter, washed and

fixed with t-butyl alcohol, subsequently frozen in

liquid nitrogen, and dried up with low-vacuum

SEM. After freeze-drying completely, samples were

transferred to the brass-stub with double-sided

adhesive carbon tape, coated with carbon, and

then observed by using a scanning electron micro-

scope (JEOL JSM-5200 LV). For TEM observation,

samples were fixed with 2.5% v/v glutaraldehyde

for 2 h at 4ëC, mounted on copper specimen grids,

allowed to dehydrate at room temperature, and then

viewed using a JEOL JEM-2000EX transmission

electron microscope.

Analytical techniques

The pH in solution was monitored using a pH

meter (Horiba) at set time intervals. To determine

the total cells in the biofilm and planktonic milieu,

1 ml of the homogenized solution-containing cells

(free-living and attached to clay surfaces) was

placed in vials, sonicated at 4ëC for 3 min and

vortexed for 10 s to disperse the microorganisms

attached to clay surfaces. The resuspended biofilm

or planktonic sample was serially diluted with fresh

minimal salts media, and plated in nutrient agar

(NA) plates supplemented with 1 g/l yeast extract

and 0.85% (w/v) NaCl, then incubated at 25ëC for 3

to 5 days. For statistical purposes, sample analyses

were performed in triplicate. The data are presented

as the arithmetic mean Ô standard deviation of the

mean.

RESULTS AND DISCUSS ION

Microbial growth

During the first 36 days of incubation, there was

no significant increase in the number of microbial

TABLE 2. The chemical composition of heavy oil

collected from the Nakhodka oil spill that was used as

the sole carbon and energy source of microorganisms

analysed by NCS analyser.

Chemical composition of heavy oil Wt.%

Carbon 82.9

Sulphur 1.2

Nitrogen 0.22

Clay minerals and oil-utilizing indigenous microorganisms 107

cells in three experimental systems (R1, R2 and R5)

(Fig. 1). Presumably, the high initial concentration

of heavy oil (~150 g/l) did not favour the

proliferation of microorganisms or perhaps the

internal machinery of microbial cells went through

a prolonged period of adaptation to the new

environmental conditions due to the very high

concentration of the substrate. After 36 days of

incubation, the liquid medium of three experimental

systems (R1, R2 and R5) was supplemented with

1 g/l yeast extract. The supplement of yeast extract

resulted in an abrupt increase in the number of

microbial cells for up to 30 days in both R1 and R2

but not in R5, indicating that only in the presence

of clay minerals (i.e. montmorillonite and kaolin-

ite), cells had adjusted to the solution with high

concentration of heavy oil and were able to

multiply abruptly resulting in an exponential

increase in cell mass and cell number density with

time. Subsequently, after 64 days of incubation up

to the end of the experiment (at 105 days), cell

numbers appeared to decl ine markedly.

Microorganisms appeared to tolerate better the

high concentrations of heavy oil when grown in

media containing montmorillonite or kaolinite

rather than when grown in media alone, probably

indicating that the clays mitigated the toxic effects

of the high concentration of heavy oil used in this

study (Fig. 1). A sharp increase in the number of

cells ensued in both R1 and R2 which contained

clays, but not in R5 (no clays) (Fig. 1). A possible

explanation for this behaviour involves microbial

competition.

Alteration of pH by microbial activity

associated with microbial succession

No alteration of pH was observed in any

experimental system during the first 36 days of

incubation after which time, microbial growth

significantly reduced the pH of the aqueous solution

of samples R1, R2 and biotic control (R5) to ~3ÿ5,

while the abiotic controls (R3 and R4) maintained

the initial pH of ~7ÿ7.8 (Table 3). It seems that

changes in the pH of the aqueous solutions of R1,

R2 and R5 were most likely to be brought about by

microorganisms themselves (Table 3).

X-ray diffractometery (XRD)

Figures 2 and 3 represent the basal spacings of

clay minerals (i.e. montmorillonite and kaolinite)

and of their complexes with hydrocarbon-degrading

microbial cell and/or heavy oil. The formation of

clay-oil complexes of either montmorillonite (R3)

or kaolinite (R4) did not lead to a large increase in

the basal spacing of either montmorillonite or

kaolinite. For the clay-cell-oil complexes, the

binding and adsorption of coincident heavy oil

and microbial cells did not bring about an increase

FIG. 1. Microbial growth (bacteria and fungi) as viable cells in colony-forming units (CFU) per ml over the

course of the experiment. Error bars represent standard deviations.


in the basal spacing of kaolinite (R2). This was

expected since kaolinite is non-swelling (Stotzky,

1986) with external binding surfaces.

In contrast, there was a small increase in the

basal spacing of montmorillonite (R1) after 64 days

of incubation (there was a slight shift to 16.0 AÊ

spacing). The reason for this behaviour is unknown.

It is supposed that three processes were dominant:

(1) the biodegradation product of heavy oil might

result in metabolites that had diameters which were

small enough to migrate into the interlayer space of

montmorillonite; (2) the extracellular emulsifying

agent (biosurfactants) produced by bacterial strain

Pseudomonas aeruginosa used in this study

presumably also had a diameter which was small

enough to enable expansion of the montmorillonite;

(3) the extracellular polymeric substances (EPS)

produced by bacteria, as indicated by TEM images

in Figs 6 and 7, might also be responsible for the

expansion of montmorillonite. However, these

options were not confirmed by direct analysis.

Relevant literature (Knaebel et al., 1994) showed

that the linear surfactants (dodecyl linear alkylben-

zene sulphonate, stearate, and stearyl trimethylam-

monium chloride) have diameters which are small

enough (<10 AÊ ) to migrate into the interlayers

TABLE 3. Alteration of pH in association with microbial succession (bacteria and fungi)

predominating in the liquid culture of each experimental system over the course of the experiments.

Experimental system 0 days 36 days 64 days 105 days

R1: heavy oil + microorganisms + M 7.8Ô0.0 7.8Ô0.1 3.9Ô0.1 4.0Ô0.1

The predominant microorganism bacteria fungi fungi fungi

R2: heavy oil + microorganisms + K 7.8Ô0.0 7.8Ô0.1 6.3Ô0.1 5.0Ô0.2

The predominant microorganism bacteria bacteria bacteria fungi

R3: heavy oil + M 7.8Ô0.0 7.8Ô0.2 7.5Ô0.2 7.0Ô0.2

R4: heavy oil + K 7.8Ô0.0 7.7Ô0.1 7.5Ô0.2 7.7Ô0.1

R5: heavy oil + microorganisms 7.8Ô0.0 7.7Ô0.2 3.7Ô0.2 3.4Ô0.1

The predominant microorganism bacteria bacteria fungi fungi

Note: M: montmorillonite, K: kaolinite, mean Ô standard deviation

FIG. 2. X-ray powder diffraction analyses of the heavy oil-microorganism-montmorillonite complexes (R1) and

the heavy oil-montmorillonite complexes (R3) during the 105-day incubation. Basal spacings of montmorillonite

in both R1 and R3 are unaltered. Only after 64 days of incubation does the basal spacing of montmorillonite in

R1 alter, suggesting that the extracellular emulsifying agent and/or the resulting biodegradation products of heavy

oil may intercalate into the montmorillonite. The analyses with ethylene glycol treatment (EG) show that

montmorillonite in both R1 and R3 still has the ability to swell.


(10 to 20 AÊ ) of montmorillonite. Harter and Stotzky

(1973) also reported that several forms of the

protein intercalated the H- and Na-smectites which

caused an expansion to 16.4 AÊ . Pepsin also caused

a slight expansion of air-dried Na-smectite to

15.2 AÊ (Harter & Stotzky, 1971).

Furthermore, the XRD spectra of the intermediate

complex (i.e. montmorillonite-oil-cell complex) at

105 days tended to have somewhat more irregular

and broader peaks than those in the initial complex

(0 days), indicating that some metabolic products

mentioned above might remain to intercalate the

montmorillonite. In addition, the analyses with

ethylene glycol treatment showed that the expand-

ability of montmorillonite in both R1 and R3 was

retained throughout the experiments.

Scanning electron microscopy

Figures 4 and 5 present the images of the unique

or attractive dense network of microbial cells and

several clusters of clay particles (R1 and R2) after

FIG. 3. X-ray powder diffraction analyses of the heavy oil-microorganism-kaolinite complexes (R2) and the heavy

oil-kaolinite complexes (R4) during the 105-day incubation showing that basal spacings (7.1 AÊ ) of kaolinite in

both R2 and R4 are unaltered throughout the experiments.


64 days of incubation. Both clays (montmorillonite

and kaolinite) apparently showed similar tendencies

in the formation of the clay-cell complexes. The

interactions among clay minerals, microbial cells

and heavy oil resulted in several dense agglomer-

ates consisting of microbial bodies (bacterial cells),

flagella (fungal hyphae and spores), fibrous

attachment features, and clay mineral particles

embedded in X-ray amorphous organic extracellular

exudate. These configurations could be observed

using SEM. Montmorillonite particles tended to

have X-ray amorphous surfaces on which the

microbial cells were mostly entangled inside the

biofilm matrix resulting in a flatter, smoother

matrix (Fig. 4c). Conversely, kaolinite particles

tended to have a somewhat rougher matrix with

rougher surface microtopography than mont-

morillonite, and most of the cells tended to

emerge outside the biofilm matrix, so that they

were easier to discern (Fig. 5c,d).

The abundant fungal hyphae were clearly seen on

montmorillonite and appeared to form the attractive

thread-like networks attached to the clay

(Fig. 4b,c,d). Numerous fungal spores were also

seen on the kaolinite-cell complex that has a rough

rose-like structure (Fig. 5d). Tubular halloysite,

fungal hyphae and bacterial cells appeared to

connect with one another as a somewhat roughened

matrix (Fig. 5b).The cell globules of different sizes

were distributed relatively randomly and densely

over the surfaces of the clay-cell complexes as

aggregates, suggesting that adsorption and binding

sites for the cells were distributed over the clay

surfaces. The cells appeared to develop on both

FIG. 4. SEM images of microbial cells associated with biofilm formation on montmorillonite in R1 after 64 days

of incubation showing the attractive network formation between montmorillonite and microbial cells where

abundant fungal hyphae are clearly seen (a and b). (c) The magnified view of montmorillonite-cell complex in

part a where microbial cells appear to be aggregating with montmorillonite as biofilm matrix. (d) A magnified

view of the montmorillonite-cell complex in part a where microbial cells appear to be aggregating with

montmorillonite which is surrounded by abundant fungal hyphae.


clays as biofilm matrix, indicating that the clays

acted as materials supporting their growth.

Observation by SEM showed that both clays

served as growth-supporting materials supporting

the data on microbial growth given in Fig. 1. The

results obtained here confirmed our previous results

that clay minerals such as bentonite and kaolinite

could have a role as microbial growth-support

media (Chaerun et al., 2003).

Transmission electron microscopy

The TEM images show more clearly than SEM

images the binding and adsorption of the bacterial

cells on the clays (Figs 6, 7). The unique and

attractive network formation of bacterial cells was

clearly seen as biofilm formation, consisting of

bacterial colonies embedded in extracellular poly-

meric substances (EPS) and appearing to attach

firmly to the clays. The cells appeared to colonize

clay surfaces, aggregate and/or grow into multi-

cellular colonies, and embed themselves in EPS

matrix (Figs 6a, 7a,b). The matrix of biofilms is a

complex mixture consisting mainly of exopolysac-

charides, protein and nucleic acids (Sutherland,

2001), and bacteria residing within biofilms

probably play a key role in the degradation of

organic pollutants (Davey & O'Toole, 2000).

Furthermore, the cells appeared to be predomi-

nantly bound on the edges of the clays.

Montmorillonite appeared as a dispersed flake-like

shape (Fig. 6a), whereas kaolinite displayed tube-like

(tubular halloysite) and hexagonal forms (Fig. 7a,b).

CONCLUS ION

The results show that when microorganisms are

associated with naturally occurring inorganic

FIG. 5. SEM images of microbial cells associated with biofilm formation on kaolinite in R2 after 64 days of

incubation showing the network formation between kaolinite and microbial cells. (a and b) The features of

tubular halloysite and kaolinite in aggregation with bacterial cells and the fungal hyphae and spores. (c and

d) The kaolinite-cell complexes showing the development of fungal-bacterial cells on kaolinite as biofilm matrix.


particulates such as montmorillonite and kaolinite

in oil spill-contaminated marine and coastal

environments, both clays are capable of stimulating

microbial growth in combination with biofilm

formation (i.e. the clays act as microbial growth-

support materials), thereby accelerating the bior-

emediation rates significantly. It seems reasonable

to conclude that the clays, especially kaolinite, are

capable of maintaining the pH at levels adequate for

sustained growth, and are thus considered to act as

natural buffers in marine and coastal environments.

These clays may provide protection against the

toxic effects of high concentrations of oil spills and

can play a very important role in the bioremediation

of oil-polluted sites, especially marine and coastal

environments.

ACKNOWLEDGMENTS

We are grateful for the cooperation and assistance of

all students of the Tazaki Laboratory and the Sato

Laboratory of Kanazawa University. We thank

Professor J.M. Adams and an anonymous reviewer

for constructive comments. This study was funded by a

grant from the Japanese Ministry of Education,

Culture, Science and Technology to Dr Kazue Tazaki.

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