ýýýý .ý ,-ýi ý: - QMRO Home

/-

i

ýýýý .ý

,-ýi ý:

rtpia, IZI- r r`. tJIL Q C. YCIJ. 3, OF Ok, ýW"

fý°' T II Alv Yý"'ý rte r" OF ;'., w,. ýJ FtC.: )i., CLJ. x)11 OF

PL . t_ýE'. i. )ýýIC BT, ý. ý.; _; --GI{E_: ý1 X1: 1 Gi IL sei º` 1' LOPI. M iiÄ1+: Rý. ý$

0

. 4. thesis -muhmitted by

J'ULCTIIT"1 . AUGJi392

for the degree of Ph. D. in the University

of London.

Department of Bo ta i-y anti 13iýci_erzi., tr; 7 ýteýt*"ý_eld

Col. ee, I1o2. d. ýri .

June 1977

U'IV.

LONDON 0ýi Ot LS-

ßp f--R- I

14T

The p'i ica1 end, chomical factors ti hick affect

sporu at oil, and tu e ý-ro 'th and. eur\rivai of vegetative

populations of blue-, green algae were examined in two

eutrophic kataglacial lakes of the Ellesmere (Worth Salop)

group of meres. "A'he im . aortance of individual factors were

examined under controlled conditions using natural

populations or uni-algal isolates from the mores.

The induction of akinete formation, which, in all the

cases examined, proceeds once the elga f. ori: is a surface bloom,

is thought to be due to the extreme conditions, iparticularly

of solar radiation, which prevail at the surface during; the

summer. Winter blooms did not sporuj. ate. Nutrient

deficiency, particularly of orthophosphate, is not

considered to be a critical factor. This is supported by

the finding that blooming ulgal material is not depleted of

phoriphorue, that alkaline phospheta, e activity was low, and

that nutrient doficicncy under controlled conditions did

not induce sporulation of the species pre6 omi. nc±nt in the

Mores.

Nitrogenase activity (acetylene reduction) and the

photosynthetic rate (oxygen evolutiozi) were observed to

fall in the period subsequent to bloom formation. The

reduction of rnoaaured not photosynthesis may be correlated

with the increase of bacterial numbers. The activity of

light-damaged material was lower than that of unaffected

populations. It appears that there must be a low cellular

carbon to nitrogen ratio before akinete differentiation

xithetic can begin, indicating that a reduction of photos-y

activity relative to that of nitrogon assimilation is

r. equiýred . Akinete:: probably do not form a significant over-

wintoring r: iechanism in the lakes investigated. Numbers of

ak. inetes found during the winter were low. The bloom

PPO:; t:. _ations prob>_bly develop frort growth of the over-

wintering planktonic vegetative popul. ationa during the

"sprina- and early E3uarier. Cornparisoni3 of planktonic,

sedirmentinf; and benthic algal material indicates that

-3- germination of akinetes ohortly after their maturation

may give rise to the greater part of the ov erwinteiiing

vegetative populutioni.

Physical rather than chemical faetoro appear to

control the population sizes and 11Th cycles in the

enviroiirnents ; studied.

Iý" tw etw ". . rý1}spy. .

. i(. I. ýýtI. 1ý. j. iýý... i....,.. 3

X trinh to a. j: roau r3' nincoro thºjrikti to Dr 3? utox Tny

Par hits r; uitlana0 and oolntinuouti unaourni(. anont throughout

this study.

:u cry aoUunglioU at Waatfiold i: o: +, 1o o, London, IA

nlno erntotul :. ̀or diuounnion and help. In ; ptirtiouln I

would li3cu to than}: Brondu Thur'ro, Itr It. Plitoliall nncl 'Dr 11. hvnnr.

Vor nn iaitroduation to thcc : 3, cloppicm ruircaa, rind or

mcny holp ul owmrýc ntaý nrua tt ', r.. : tons ý ý. wa inc1tbted to

1)r t:. 13. Itayaio1d&.

In ithlttiuix, I rin inclobtocl to t ho :. 'rýictkooti or tho

Ororvonor l: ntatuo for pormintiion to work tit Lthito Morn, to

Nr L, It. Jobb for allowing, no0an0 to thu oliorcº and for

utorcaco fuuilit . ua, t, nd to Hr it. I:. Pininwcirinr sind ?! t Jor

K. U. 1'ottorcircco £or purrtiuuicm to work nt F: uttlo Moro,

B1t ku I uru and Newton r. oru. I nm ciltan grrntofui to thu

Prunhw: ttor Iiiologicn1 Aonoointion for uoo of thu Moran

Laborittory it Truoton Pýontfoxd Viuld Cuntru und to

1)r A. Ilrtyluy, thu itnrdun tit 3'r,, titon iºont1'orct.

Thin Fork hers buoix oup1 ortocl by it i; rtint from tho

Itetturni J: nvi, onriont 1(tn tj, trah Council, whocio nncintlinao

.ýj ctttýSýz. i.? nckrou?. odj; o.

('t' 'rr J.. S91 .

Page

....... Otto .................... ' ..... 2

ACr:; IOi; 7ýI: ýGEI ::::; ä ...........:.................:..... 4 JOIST U. a. ' 1'AD7:, I: 5 ........:............................ g

.................................... 12

Tlil: FIELD DATA

Introduction.... ' ....... ............................ 15 . The Mer. er ............. .................:..:........ `. 24 Licit of : ir)ecies Encountered in the tier. ee During the 29 ', er. iod of thý. s nve ßtß. rr . tion... ...................: . Zoonlankton ........................................ 31 The äitec Znyea:; tir-nted

l: ettlo Aiero .............................. 32

White 11er. e ............................... 32

23lake here and Newton ilex© :.............. 33 fateritis and P"Tc iiio(. f3

Field Sampling

k'Tankton..... ".,.. ." ."............... 36"

De thor.... ".......... "......... ". "" 36

äedimentation ....................... 39

The Enumeration of Field Material

Planktonic .... """"""".... """""...... 42

Benthic ................... "....... "" 44 Total particulate sedimentation ..... 45 sedimentation of algal material..... 45

Th e Chemical Ant. lyeis of Field Jctmples

Orthophosphate ................. **0*** 47 2litVate 1.11 d 11itritc7) " ....... " 48 ....... . UXgE)ll»..... ". ". "",.. ". "......... " 51

.......... p11

...... .. . 52 . ............. .

....... .. ". ""..: Conductivity 53 ..... .. " The Iie; -zsu2i o naht of l'Iiysical_ Par. Fir. i tero

J tý : E. pe 2' ilttLre. "..... . .... .".. . ..... """ 53 l+ i g- lit ...... .... ". """... ". ".. " ......

53

Total particulate euz3lperndcd solids.. 53

-6-

i

Re su? _t. s

The: Cheriica and 21r. oical l nvironrient `temperature .........................

Lieht ............................... Orthophosphate ...................... I'; itrat e ........ " ..................... .

T! ie Annual Cycle of Growth arwd Succession

of the Blue-green Algae

The plankton ....... ................. Sedinentation.......................

)enthic populations ................. Dismission

The Growth and Abundance of Planktonic

Populations .., ............................ Sporu]ation and Germination........... '... Ov rw i nt or ing

""""""""""" . 000000 """"""""""

JLC ION 2

GROWTH AND CELL I)IPIJREIITIATZUTN UNDER A VARIETY OF

PHY ICAL AND CHL'I. ZCAL GO DITzozrs I21tý"pduct?

_021. .... "..... """""".. "...................

I1 ter. ia? _s and P"'ethodB

Isolation ............................... Culture D: edict ............................ Preparation of Utock-. 'jjolutionr, and Media.

The Culture of ýil gae """""""""""""""""""""

3ui±ors..... """"""""""""""""""""""""". """

L .L ht

High intensity ....................

Nonochroriatic, action spectra.......

Ultra-violet ........................

Asse crent of Al al Materiale """""

F1 E: ül I ! -, -E;

tTI"Oý'7 h, """""""""""""""""""""". ".

Page

54 54 65

71 71

71

83 95

104

110

115

121

122

125

125

1 27

128

129

129

130

132

134

135

136

1DI 7

-7--

Page

iTutrients and `or. ulatioii ................ 139

3ýz lit Quantity and $porulation'........... 147

Light Quality and üporulation............ 143

Ultra-violet Light and Sporulation....... 149

Diseus:, ion

Growth and Sporulation .................... 152

'Inorganic Nutrients and 2poruilation...... 153

Other Compounds and Sporulation........ .. 155

Light Quantity and Sporu? ration........... 156

Light Quality and Sporulation.... ........ 157

" Ultra-violet Light and. : 3porulation....... 158

SBCÄICPJ 3 T1ii 7rHY3IOLC(fl CAL ACTIVITY LP IU TUI: A,, L POPULATIONS

OF ALGAL Introductic, n. "... """"".. ".... """" """""""""""""" """ 160

Materials RflcI. Methods

Collection of 'Material and Preliminary 7. reEitrliP. llt """"""""""""""""""""""""""""""""

165.

Alkal. ne Phosphatage..................... 165

Labile Orthoj)hosphato.................... 167

Nitrogena5e (Acetylene fleduction)........ 169

Photosynthesis (Oxy-en Production)....... 170

Carbon, Hydrogen and Nitrogen Analysis... 171

Assessment of Bacterial Plumbers in Algal

Samples .... ..... 171

Results

Yho phorus Availability .................. 172 NitroL ezi Metabolism...... .... ............ 177 Oxygen Production.... 09... ............... 185

Discussion

Yhosphorus ............................... 192

Nitrogen and Carbon ...................... 193

Change of Tletaboliom and üporulatior_..... 198

Page

............... ......:.............. 204 t] rý"-l :; T` trrcr

" 1i. L' : ý. rte. 4L 1ý a3 """"""""""""""": """"""""""""""""". """". "" 213

11

Reprint of published paper bound in at back: -

Rather, J. A. and Fay, P-0977) Oporu? ation and the

developne: nt of planktonic blue-green algae in two

Jalopian rneres. Yroc. R. Soc. ?. rond. B. 195,317 - 332.

i

LL3 Ute' 1ABIL J

; ýo . 1'ago, Title

1 28 Uhan ; es of nutrient concentration of surface

samples from the merest

2 66 Depth (m) of 10;; and 1 of sub-surface radiation.

Concentration of orthophosphate-in the terse.

3 6ü Kettle I: ere and White I-Tere, 1975.

4 69 Kettle Mere and White Piere, 1976.

5 70 Concentration' of nitrate in the mores (Kettle

Tiere and White Mere: ) , 1975 and 1976.

6 71 Conductivity, transpar9ncy and pH of the surface

waters of the mores, 1976.

Amount of algal material in the* plankton.

,7 73 Kettle Here, 1 974 "

8 74 White Mere, ' 197 . 9 75 Kettle Tiere, 1975. -

10 76 White More, 1975.

11 . 77 Kettle Mere, 1976"

12 78 White Mere, 1976.

Akinote percentage of algae from the meres.

13. ß0 Kettle Mere and White Mere, 1974.

14 81 Kettle Mere and White Mere, 1975.

15 82 Kottle Tiere and ýIhite : lore, 1976. *

Integrated algal standing crop.

16 105 Kettle bier. e, 1974. - 17 106 White Piere, 1974.

18 107 Kettle Tiere and White Fiere, 1976.

: IECTýUI3 2

19 124 List of the species obtained and maintained in

culture, their source and method of isolation.

20 126 Cocaposition of ASI'I 4 medium. 21 136 Growth rates of isolates of AI)h. flus-mono.

22 138 :. 'f3 ect of iI11ji'E. i buffer on the gro%rth of

planktonic t3.1 Etl isolates,

-10-

To o. Page itlc y

23 140 Effect of orthophosphate, and nitx'ate on cell

differentiation. of a White I: ere strain of AT)h. f1 or, --n2uao.

24 141 Effect of orthophosphate and nitrate on cell

differentiation of a I: ettle Here otrain of

Aph. flos-ncruae.

25 143 Effect of HI: PES buffer on heterocyst frequency.

26 144 Effect of I3I: YES buffer on sporulation. 27 146 Nporulation of . 4nabaena sp. ' (? ) under different

nutrient conditions.

28 149 Effect of u. v. light-'on algal growth.

29 '150 Lfi'ect of short u. v. doses on the sporulation of

! Dh. flos-HQUHO isolates from Kot-', -, le Mere and

White Mere. '

30 151 Effect of u. v., and fluorescent light inteneity,

on the sporulation of A. flo s-tequoe collected

from MJhi to More, spring 1976.

SECTION 3

Labile orthophosphate (boiling water extracts). 31 173 Kettle Mere, 1975 and 1976.

32 174 White Mere, 1975 and 1976.

3; 175 Newton Mere, 1975 and 1976.

34 176 Alkaline phoophataEe activity of algae from the

raeres, 1975 and 1976.

Nitrogenase activity (ac. etylene reduction).

35 173 Kettle Mere , 1976.

36 179 White Mere, 1976. 37 1F30 No'iton here , 1976.

Het er. ocyst frequency of algae from the rneres. 38 181 Kettle Here and White Here, 1974.

39 182 Kettle Mere and White , "fiere, 1975. 40 183 Kettle here and shite Mere, 1976.

41 184 Carbon : nitrogen : hydrogen ratios of bloo-i-

forr_iing algae, 1976.

- 11 -

No. Pago Title " Oxygen production.

42 l a6 Fettle 1 976 . 43 1 F37 : ihitc T". ore, 1976.

. 44 1813 Newton Mere, 1976.

45 190 Bacterial numbers in algal samples, 1976.

No. ýaý o

1 25 2 37 3 40 4 '41 5 50

Vertical profiles of tern] erature.

6 55 Zettle IJere, 1974. 7 56 White Mere, 1974.

8 57 Isettle AZere, 1975. 9 58 White Mere, 1975.

10 59 Kettle Mere, 1976#' 11 60 White Mere, 1976.

Vertical distribution of oxygen. 12 61 Kettle Ilere, 1 975 " 13 62 White Ziere, 1975.

14 63 Kettle I"lore, 1976.

15 54 White. Mere, 1976.

16 67 Absorption of filtered water samples from

nettle Here and White Mere.

Total particulate sedimentation.

17 84 Kettle IIore, 1974.

18 85 kettle T. ere and White liiere, 1 975 "

, . 19 86 Kettle D? ere and White here, 1976.

Rate of sedimentation of algal riaterial.

20 " 88 Kettle Ilore, 1974.

21 89 Kettle Iiere, 1975.

22 90 white Mere, 1975.

23 91 Kettle here, 1976.

24 92 White *1ere, 1976.

"u: 3I)ended sot id. 3.

25 93 Kettle IIere nnci White hero, 1975.

26 94 Kettle here and #+ýiitu Liere, 19lß"

LIJT ul" i a.

GUt.: U

i1t]. e

The Pieres of the ' 11esriere Region.

: JaxipLing sites on I: ettle llorc and ý3hito Mere.

Di asrara of sedimentation trap.

Positioning of r; ediraent traps.

Column used for reducing nitrate to nitrite in

waiter samples.

-13-

Iyo. Page Title

Amount of algal riaterial in the surface eedir-cuts. 27 96 Kettle Nere, 1974.

28 97 1Jhite : Tere, 1 974 . 29 98 Kett? e here, 1 975 " 30 99 White here, 1975. 31 100 Kettle Mere, 1976.

32 101 ºlhite Mere, 1976.

SEC'T'ION 2 33 130 Spectral distribution of energy output of

"Daylight" fluorescent tubes.

SECTION 3

34 168 Digestion apparatus for the extraction of labile

orthophosphate.

35 169 Partial removal of as phase frogs serum bottles

using "Vacutainers".

SECTION 1

THE FILLD D. ýTA

IIýTIWUDUCýICI4'

The succession and growth of algal species has been

the subject of much research. The potbntial object of this

research is the control of excessive growths of the "nuisance

species" (Fogg, 1969) of algae. - The effect of this excessive

-growth, commonly termed "blooms" or "water blooms" vary

from unsightly scums on the surface of the water or an un-

wholesome soupy appearance, through the clogging of filters

in water treatment works,, to the poisioning of livestock

through the release of toxins. Fish mortality may result

from deoxygenation of water as the algal mass succumbs to

bacterial decay.. The contamination of water supplies is

produced through the release of organic compounds which the

blue-green algae excrete in abundance (Fogg, 1952). Although

recently brought into prominence through concern over the

environment, algal blooms have long been recorded. One of

the first printed records appeared in Smith and Sowerby's

"English Botany", 1808,. in which Gloeotrichia echinulata (Conferva echinuleta) was observed in a lako in Anglesey.

I1uch earlier is the record by Giraldus Cambrensis, who in a

tour of Wales during the twelfth century, writes of what,

' from the description of colour, probably was a Cyanophycean

bloom in Llangorse lake, Breconshire (Griffiths, 1938).

The problem has become increasingly apparent with the

enrichment of lakes and rivers through extensive urbanizat-

ion and the proliferation of the use of inorganic -

fertilizers. This process of eutrophication has produced ''a

general concern over the health of water bodies, particularly

with increasing demand for supply and recreation.

It is apparent that any effective control over the

ecology of aquatic organisms must be based on a comprehens-

ive lwowledge of the behaviour of the separate species to

the range of conditions which are likely to be met, the

interactions between species, and the effect that each

species may have on the environment. Of equal importance,

and only slightly less complexity, is a detailed compre- hension of the ph; 'sical and chemical factors of the system

under investigation.

-16-

The dominance of blue-green algae in many lakes and

reservoirs in both temperate and tropical regions has been

studied in relation tb many factors. Of particular importance in temperate regions appears to be the amelior-

ation of conditions during the. spring. Increasing insolation

results in a warming up of waters and a more favourable

light climate for algal growth . (Tailing, 1971). A 'decrease

in'the amount of wind will result in an increasing stability

of the water column, which favours motile and buoyant forms

(Moss, 1969) at the expense of the denser algae, particularly

diatoms, which rely on turbulence to maintain the cells in

suspension (Lund, 1'966; Knoechel and I: alff, 1975). The

transition from the spring to the summer assemblages is the

result of complex interactions between temperature,

turbulence, solar radiation and the physiological capacities

of the species concerned. The amount of time spent below

the photic zone will affect' different species to varying

extents as a result of the compensation point requirement

for active growth. Temperature may have a similar effect in

the selection of different species through differences in

the optima of photosynthesis and' respiration.

The close correlation between temperature and the

ascendancy of the blue-green algae has been demonstrated by

Hammer (1964,1969) in Saskatchewan lakes. ITinimum temp-

eratures have been determined for the growth of different

species which, together with varying optima for the concen-

tration of orthophosphate, determine the order of occurrence

of different species. A similar relationship has been

observed by Reynolds (1971) for the Shropshire (Salopian)

meres, but with variations in the order of the species

common to both areas. Lin (1972) ex, mined the iYifluence of

a reduction of nitrate concentration on chdnge of populat-

ions of algae, from a diatom community to a blue-green

algal one. -Interrelationships between the blue-green algal

species have also been investigated with a view to.

explaining the sequence of succession. It was suggested

-(Lin, - 1972; ilacuner, 1964) that the decay of the previous

species stimulated the growth of subsequent species. Vance (1965) also stresses the importance of extracellular

-17- metabolites in the control of growth of natural, populations.

The inverse relationship,, the inhibition of growth of the

proceeding species by the succeeding' species has been

demonstrated by Keating (1975) in cultures and also using

filtrates of lake water collected when the various species

were dominant. - With the relative, stagnation of the epilimnion,

nutrient supply and deficiency produce, further selective

pressures. Available carbon dioxide has recently been

discovered to be important (Ruent. el, 1969; King, 1970). A

supply of gaseous carbon dioxide results in a change of the

'dominant species from the, blue-green algae, which are more

efficient at removing carbon dioxide at low concentrations,

to. green algae (Shapiro, 1973). A similar effect has been

"observed by , Haynes (1975)

. on the artificial destratification

of a small lake. However, ,

Schindler et al. (1972) has shown

that sufficient carbon dioxide enters the water from the

atmosphere ' through diffusion to permit eutrophication, ifý

concentrations of nitrates and phosphates are sufficiently

high. Destratification would also supply other nutrients

from the, hypolimnion and stimulate growth (Iverson at al.,

1974). If completely mixed, however, _a nutrient limited

bloom will be replaced by a light limited one of smaller

size (Lorenzen and Mitchell, 1973). The depth available for

mixing and the attenuation of, light, the "critical depth"

of Sverdrup (1953 would be decisive.

The ability of, luxury consumption and storage of

phosphates by blue-green algae (Battex-ton, and Van Baalen,

1968), the enzyme mediated relea8e of orthophosphate from

organic phosphorus compounds. (Fitzgerald, _and

Nelson, 1966;,

Heath and Cooke, 1975) and the-ability of aerobic nitrogen

fixation in the heterocystous species (Fogg, 1971) are

probably important factors in supporting bloom densities of

algae. Although the rapid appearence of a dense scum of algal

material gives an impression of a vast bionass and a

phenomenal growth rate, this is often nisleading.. Reynolds (1971,1972,1973a) has shown that a bloom is merely the

accumulation of plan,: tonic material at the surface , of the

_is_ water, "t ie suspended 'material becomes' 'crammed' within a few

centime tiers at the surface. This buoyancy is imparted by

the pr es: enoc of gas vex iclos within the cell leading to a

reduction of density. 'The relationships between cell turgor

pressure, photosynthesis, 'gas vacuole number and buoyancy

are described by Walsby (1 971) and Dinsdale and 4. alsby (1972).

With the incredise of cell turgor pressure brought about

through the accumulation of osmotically active compounds

produced by' photosynthetic activity, the weaker gas vacuoles

collapse. The density of the alga thus increases, and'the

cells'sink through the water column to a region of lower

light intensity. ,A

reduced rate of photosynthesis, ' which

may also be brought about by carbon dioxide deficiency

(Waleby and Booker; " 1 976) , and 'the conversion 'of - simple

sugars. to osmotically inactive storage pröducte, results in

a reduction of cell" turgor pressure and enables the further

de novo synthesis of vesicles. These factors were also

investigated by Reynolds (1975a) for a natural population

of Änabaene circinnlis. Surface blooms result from an over-

reaction of the potentially sensitive and accurate buoyancy

control mechanism (Walsby and Klern. er, 1974) with a sudden

decrease of turbulence (Reynolds, 1972). Thu' overbuoyant

algal material rises rapidly to the surface, where it

becomes--trapped by surface tension. Gentle wind then removes

the scum to the lee shore, but mixing into the water is not

achieved unless the. wind is, sufficiently strong to break

the surface of the" water.

In this position the algal populations are highly

vulnerable to extremes of temperature and light. Bacterial

decay would proceed at an optimal rate at the temperatures

generally' encountered at the surfpLce-during calm conditions

in the summer. Alga-lysing bacteria were found in many

eutrophic freshwater lakes. Though growth requirements of

bacteria and condition, for lysir are similar to that

which prevail in stagnant surface waters, -extensive lysis

is rare in natural ecosystems (Daft et al., 1975). The

"nue bore of bacteria found in the natural environment was

never enough to lyse an algal bloom within 60 hours. If

there is an input of more bacteria (eg. from a sewage works)

-19-

or if the' alga-is attacked by other pathogens, the conditions

would favour lysis by the bacteria.

The depletion of-carbon dioxide and the supersaturation

of oxygen, produced, as a"result of the high rate of. photo-

synthesis in productive waters and the-increase of

temperature, leads to photo-oxidativo-conditions (Abeliovich

and Shilo, 1972; Boyd et al., 1975). Photo-oxidation has

been shown to occur under natural conditions even at

relatively low temperatures (Shilo,,, 1975), though, natural

populations, have a greater resistance against photo-oxidation

than laboratory. grown material. This resistance is, conferred

by high levels of super-oxide disrnutase (Eloff et al. ,, 1 976) . Since the"greater portion-of the planktonic algal

population is compressed within a shallow depth of water at

the surface during blooms, the survival of some of this is

critical for the development of future populations.,

A , factor of considerable importance for the periodicity

and development of large planktonic populations, which has

so far received scant attention when compared with the

number of investigations performed while algal material is

abundant, is the distribution, abundance and behaviour of

the algal populations during the greater part of-the year

when they are not an important component of the phyto-

plankton.

An important adaptation of many heterocystous blue-

green algae toadverse conditions is the formation of

akinetes, resistant "resting spores". which are protected by

an elaborate envelope (Fogg of a., 1973). Each akinete is

produced by the transformation of. a single vegetative cell,

and-this is accompanied by cell enlargement,. deposition of

storage material . and` envelope layers, and in the planktonic

species, by the disappearance of Gas vacuoles. The increase of cytoplasmic density resulting from the

loss of gas vacuoles and the massive deposition of cyano-

phycin (structured) granules permits the akinetes to sink

and ovorwinter on the mud sediments (Wildman et a1.., 1975)-

Thin-has been observed in many water bodies, particularly the deeper continental lakes, and it is generally assumed that the tikinetes form the : 3tock for future vegetative

-20-

growth of the Anabaenn species . (Vladimirova, 1968). A

similar pattern has been ob6erved for G. echänulata (Hoelofs

and Oglesby, 1970; than, 1974) and 4DhRnisomenon floe-accuse

Rose, 1934; Chernusova-et al., 1968). -Some doubt has,

however, been expressed as-to the relative importance of

sedimented overwintering akinetes, and whether they

represent a significant source of vegetative material. Although germination has been observed to occur with improv-

ing conditions of. light and temperature in the. spring while the lakes are, still unstratified (Reynolds, 1 971 , 1972)t

the numbers encountered are never very large and probably do not contribute significantly to the population maxima (Reynolds, 1972,1975a). Sufficient vegetative material

survives during the winter within the. plankton, and the

. increase of populations can be^ccounted. for by the,

vegetative gröwth of this planktonic material. Reif and Fine (1967) also observed planktonic populations of an Anabaena species.

-. Non-sporulating species, particularly Microcystis, may

survive the winter, in a viable vegetative , state in the

bottom muds (: iirenko et al., .1 969; Reynolds and Rogers,

1976). An increase in the depth of mixing, as summer

stratification breaks down, initiates the hydrostatic

collapse of gas vacuoles and the sedimentation of colonies. Investigations by-Reynolds and Rogers. (1976) on Rostherne

Piere, one of, the deepest of the meree, have clearly shown

the sedimentation of, colonies into the'mud, where'they

remain during the winter.,, At the onset of summer stagnation in the following year a marked migration of colonies takes

place from the sediments to the epilimnion. The transition of a vegetative population to a resting

stage may be expected to be initiated by a deterioration of the conditions neccessary for vegetative growth. Nutrient

deficiency, particularly that of orthophosphate (Wolk, 1965)

and nitrate (Glade, 1914) have been reported to be controlling factors of akinete_formation in laboratory cultures. Experiments of Fisher and- tlolk (1976) showed that extracellular products taken from an old stationary phase culture stimulated the immediate formation of akinetes when. added

-21-

in small amounts to juvenile cultures-of Cy lindrospermun

licheniforme. This finding may be- corroborated by the

observation of Rose (1934) on natural. populations of. Aph. flos-aquae where the decay of-surface blooms during

the summer induces eporulation, of filaments below the

surface. This'is probably a coincident'-rather than a

causal relationship, as this species has been observed to

produce akinetes during the winter months (Lemmerman, 1900;

Wildman et al., 1975). Akinete production in G. echinulata does not appear to be stimulated by adverse conditions (Roelofs and Oglesby, 1970), as they were observed in

rapidly growing cultures and in planktonic populations before the population maximum was reached. Fritsch (1945)

suggests that the filament size in this genus and in

Cylindrospermum may determine akinete production, =but no

indication of potentially-respon&ýble factors were given. With few exceptions, akinete production is'limited to

heteroc. -stous (nitrogen fixing) species. Singh (1942a),

Hill (1972) and Cronberg (1973) have described species of

the genus Raphidopsis, a non-heterocystouc, alga in-which

akinetes have been regularly observed..

From experiments in which the differentiation ofla

vegetative cell into an akinete is inhibited by. surgical

rehoval of the adjacent heterocyst, 'lolk (1966) postulates

a control over sporulation by heterocyzts. This is supported

by general observations of a consistent relationship

between the relative positions of heterocysts and akinetes

within a filament. The relationship appears tobe species

specific. Departures from-this were. also described. No

regular relationship was found between the position of

akinetes, and heterocysts of iLnnbaena planktonica (Burger,

1974). Sinha and-Kumar (1973) obtained noii-heterocystous

mutants of Annbnonn doliolurn which were capable of

sporulation. Tyagi (1974) has demonstrated an inhibition of

sporulation by inorganic nitrogen compounds and a stimulation

, by glucose in the wild-type strain of the same species. A

similar inhibitory effect of nitrate was shown by Singh and Srivastava (1968). These observations' tend to suggest that the position of akinotes within the filament depends on

-22- criticMMl concentration levels within the cell of the

prothicts of nitrogen and carbon fixation.

Factors which could be of importance in the control of developmental stages of akinetes are light quality and

quantity. A light induced' developmental cycle has been

described for I"Jostoc muscorum '(Lazaroff and Vishniac, 1 961 )

in which low light intensity, or the addition of culture filtrate from light grown cultures initiates the formation

of the typical-Itostocacean morphology from the aseriate

stage of dark grown cultures. The effects of light quality

on the Nostocacean developmental cycle was inveotigated by

Lazaroff (1966). A non-photosynthetic control mech"anism"

appears to function. Red light (650 nm)'stimulates the

development of'filaments from the aseriate stage. This

effect may be photo-reversed byý exposure of the alga to a

broadband in the green region of'the spectrum. The action

spectrum'of the photo-induction. of the developmental cycle displays a single sharp. peak of activity at 650 nm (Lazaroff

and Schiff, 1962).

Light is also a factor required'for akinete germination.

Harder (1917a) established a linear correlation between

light flux and the rate of germination. A similar relation-

ship was observed between light intensity and germination. No germination occurred under dark conditions. The same was

also observed with A. doliolurl by Singh and Sunita (1974).

Red light was found to be most effective in the stimuj. ation

of germination (Harder, 1917b; *Reddy et al., 1975). The

effect of red light can be reversed by far-red. The green

and blue regions of the spectrum have no effect (Reddy

et a?., 1975). Kauchik and Kumar (1970) found that

germination could occur in all regions of the spectrum, indicating a requirement for non-photosynthetic light and

an absence of developmental photo-control of the type

described by Lazaroff (1966).

No light requirement was observed for the germination

of a'_; inetes of d:. i1_on-neune although this species appeared to require cold pretreatment before germination (.: ose, 1934). Germination immediately after akinete maturation has been observed under favourable conditions with many

-23-

species of Anabnena (Fritsch, 1904; 3pratt, 1911; Fay, 1969a).

Glade (1914) also observed the imrriediata'germinatiori'of

akinetes of Cý, indrosi)brmum on the re-addition of 'fresh

medium, and Singh and Sunita (1974) of akinetes produced in.

light grown cultures of A. doliolun.

Although some data have been gathered on factors

affecting akinete production and germination, these are

almost exclusively confined to'laboratory studies and to

algal species obtained from culture collections. The species

represented are originally and principally coil-borne algae,

the planktonic forms being relatively-difficult to culture

successfully until recently. ' Little work has been done on

the importance of-akinetes as overwintering or resting

structures in natural planktonic populations.

In this study an attempt was made at'the'assesment of

the relative importance of akinetes and vegetative material

in the'perennation and prdpagation of planktonic species of

blue-green algae in two contrasted lacustrine environments,.

and also of factors which affect population development.

This was coupled with laboratory experiments; -using isolates

fron the study-sites and natural plankton samples, designed

to isolate'the potential factors responsible for the

induction of sporulation and akinete germination.

THE P"i1: RE

The mere: of the Salop-Cheshire Plain are a series

of water-filled hollows in the mantle of glacial drift

which covers the solid rock of the region. The thickness of the drift varies, but in the Ellesmere" region the Triassic

sandstones and marls lie under about 100 rm of the recent deposits (Sinker, 1962). The hollows may be either enclosed between hummocks of glacial deposits, or kettle holes formed

by the melting of large blocks of ice left during the

retreat of the ice front in the last (Weicheelian)

glaciation. Some of the merest particularly in Cheshire,

may have resulted from subsidence through the dissolution

of the underlying strata. The mores have a clustered distribution, one of the most important of which lies in

the region around illesmere (Fig. 1). This also marks the

southern boundary of a minor re-advance of ice, resulting

in a terminal moraine connecting Wrexham, Whitchurch and Bar Hill in a bilobate form (Boulton and Woreley, 1965).

An a result of their mode of formation, many of the

meres have neither natural: inflows or outflowe of any

significance. In some cases the mores are connected to each

other and to the adjacent low lying areas by ditches, such

as the Crose P'Iero-Sweat I"lore complex (Sinker, 1962). The

water is supplied principally by percolation through the

drift, the water level marking the intersection of the

water table and the surface of the ground. Reynolds (1975b)

concluded from studies on the supply of silica in Crose

Mere that the inflow of water is through the rim of the

viere, no overflow,, _ rather than through the lake basin in

general, which in rendered impermeable through the

deposition of secondary sediments. Despite the buffering effect that a phreatic water

supply would have during periods of drought and on the

chemical environment of. the mores, considerable fluctuations

of water levelo in the meree have been produced as a result

of drainage scheme during the lust century. Hardy (1939)

"r!. entione a tall of 2.5 in in the level of Crone here and further instances tAre given by Sinker (1962). That

fluctuations in levels have occurred before written records

-25-

Fig. 1 The Peres of the Ellesmere Region.

SCALE: - One'inch to one statute mile = 1: 63360

Iianmer, Mere

Grid'

north

The More

Ellesmere

Newton Mere

Blake Mere Kettle 1Mere

Cole Mere

White Here

0 Sweat Mere

Croce Mere ,.

-26-

may be inferred froca the sE. parati6n 'of 'open bodies- of`

water by peat deposits, such-as the separation of Blake

Mere and- I. ettle- Mere by a peaty bank. Fluctuations of

water level, as well as the steepness of, many'of the banks

and a lack of-input'of'. sediments in enclosed' lakes, may-ý°'

account for the general paucityýof marginal vegetation (Sinker 1962). ,

Through the leaching of dissolved' compounds in the

glacial drift by the ground water which supplies°the meres,

these lakes became eutrophic. - However a range of

concentrations of dissolved material'exist in the different

meres, which Gorham (1957) correlates with differences of

local geology. He also attributes the high' level of sodium

and chlorine ions to evaporationresulting'in a-concentration ofthese ions. Although thV. 'Ellesmere area has a

precipitation to evaporation ratio of 1.4: 1 (Sinker, 1962)

this would' result". in a 3.5-fold inoreäse'of dissolved

compounds when compared'with the input through precipitation" (Gorham, 1957).

As discussed above, blue-green algal blooms are not

wholly'a recent ' phenomenon: Neither are they to the.

inhabitants of the-shores'of'the meres. "The manifestation

of surface blooms, -eloquently described by : linker (1962),

has been preserved in the local folk-lore, -which has

produced its own term for blooms: - the "breaking of the

nier. es". Another colloquial expression, although less well known, is-the "cruddling of the Tieres" , by analogy with

the separation of-milk into curd and whey on the addition

of rennet (Wardle, 1897). Phillips (1884,1893) was the

first who described the Cyanophycean origin of the breaking

of the meres around Ellesmere. Further descriptions are

given by Griffiths (1925), and by Reynolds (1973b, 1973c)

who also compares the annual periodicity of Grose Viere with

that of the other mores and of the Cheshire mores described

by Lind (1944). The similarity of the composition of

species of algae in many mores led to the concept of a

-'regional type' of succession (Reynolds, 1973b). Departures from this are discussed in terms.. of the typo of agricultural

ac tivityy in the area bordering the mores, taxe nature of

I

-27- the catchment area, morphometry of the mores and ionic

composition in relation -to the ecological ranges of the

predominant species.

Although the merea represent, a chemically stable habitat, as indicated b; r the absence of major qualitative

chances in the phytopl anl: ton, an increase in the use of inorganic fertilizers on the farmland within the catchment

areas are probably the cause df the largo increases of

nitrates in recent years (Table 1). An increase of 4 to'

10 - fold has been observed between the investigations of Gorhan (1957) and Reynolds (1973b). This is not restricted to the meres in'Salop, an Rostherne Mere in Cheshire also

appears to have undergone increasing eutrophication (Pearsall, 1923; Lind, 1944; -Grimshaw and Hudson, 1970),

although the increase of nitrates is leas marked.

Concentration of orthophosphato, already high when first

analysed in 1955 in samples of water from the mares (Gorham. 1957), have changed little when compared wlthýthose of

nitrates over the same period, (Reynolds,. 1973b).

The monitoring of changes in the phytoplankton

assemblages which result, from changes in the nutrient

concentration and-composition of the mares, particularly'an

-alteration from limiting concentrations of nitrate to those

of phosphate, would be of Great value in determining the

environmental requirements of planktonic algae, and their

interrelationships. The Salopian mer. es, with a wide variety

of sizes and morphometry within a relatively small area,

appear an ideal site for such a study. The mares are deliberately referred to in the singular; their importance

lies in their value as a group of lakes, and it is the

Croup which should be studied and conserved.

0 4) U U

H

"0

F4 O0 El

O H

H El 0

N N

EHI (11 1i Uý ^

a^

Uy

O"

Or a ßr-4

to

C) $1 U'

N 4) i

"ri "r1 El St P'O

cl UO

O U] ý, ) v C)

9 GS 4

C. )

x p4 C) .n r-3 E+

t4 0 4.3

F-4

. -. -. OD o c- c, ON

1 f

.0 L(\ rn

C) Q>

Lr\ i S4

r £ ý

Q) " rl "r1 X 3 t1 .

0 0 0 ö. 1 1

to o

fJ i+

Co Co

0 01

oo .o v- , +ý o

i N

e O

O O

"I

-Co N ul O c- N

11

Lý Nrr

t(1 O

11 tz

, -.

A

.' N" "ý ý\ r C .' '-, r 't. )

v C! ) 7 ti . 4. ) r1 Iß

cO

Q. Rr E"t

-28-

.ý ... CC) %lo 'n N ON ON

ON a (IN Ir- T-

... . V,

ßs1 G) U tA3 13

r J ý v t! J cd

S4 G\ U c'

Q) ,0 "ri "r1

ý? F c3 ß

'co CJý N' ao rr\ i " r O O O Q O t

O 'D 0

O O aý U N Sa ý ý 9) Q fV " " ;.; u1 0 O r 0 0 Q) t !

+) O O "r1 r O

N

0 0

Ill o N CT NN

wO rN

N

C I. .Ii

. ý i hl

ON v

ro 4) r. ri t7 cj 0

r

O r' C3

. W-4 E4

a'

u-ý a3 v LA G

v1 "a 0) r

[, - C-

C)

ö ýrt 1-3 :;: ti

r1 U1 O0O 0Ö11

O

NO w Kl%

N 0

LA OO M OOii

co co 1O

ci O

rn a' tt11

oo

03 "11

o-% 1 11 Co

C'

0

0 O 0'

0 0 ä

CO

U .U o

O

co Q N N t\ C1 Q " "

:: r- U CJ ý

ß) " ! 1 1 U + 0 0

U%

0

M +11

Co O

'i C' CO

ti ON N C" u' G' r-

'- f L1 cr T-

. .. I r- "a r- -

2I r-{

.4-) Lý V r"1

4-1 GZ

Ci .Z

0 1.0

rii .4

P Q

4 0 G 0

4

) r

a R: E+ 1 0 Pý Eý i

LIST Uff' . ̀i''1 Lei r)-yCOU1 T1: HLV IN TH I'ii 1ti 3 DURIIdG THE

PERIOD OF THIS INVI. "'iSTIGATICN. (1974 - 1976 ).

Kettle White Newton Blake Mere Mere Agiere Mere

MYXOPÜYCEAB: Chroococcalea

Coeloq haeriuri ku. tzing; nnuri Ilttiieti. +

Coel osphaeritun naeglianum Unger. + +

I'? icrocystis aeruj±nosa K{itzing. + +

i"Iicrocystis floc-Eigurte ('Iittrock) +

Kirchner

I"iicrocystis wesenbergii Komarek + + ":

' Hormogoniales ,

Anabaena circinalis (Kiltz. ) + +

Hansgirg - Anabaena flos-aquae (Lyngb. ) Ereb. + + *#

Anabaena solitaria-Kleb. + +

Anabaena spiroides Kleb. .+

+

Aphanizonenon flos-aquae (L. ) Ralfe. + +

Gloeotrichia echinulata +

(J. E. _Srith)

Richt.

Oscillator. is Opp. + +.

Predominant genera of other Classes. .

13ACILLARIUYHYGEAE

Melosira. spp. +

Stephanodi. scus epp. +

Asterione- 1a formosa- Hass. - + +

Fragilaria cr-otonensis Kitton

" CHZUF. O1HYC1! AE: Volvocales

Ludorina spp. +

YPnd. orina App. +

Volvox , l, )bator Ehrenb. +

Chiorococcales

Anl: istrodesrlus spp. +

Scenedesmits upp. + +

Yedirnstrum boryranur"º (j'urp. ) Heneeh. +

I'edinstrt: m (1111-)l -nx Iisyen +

-30-

Kettle White Iiewton Blake Mere' fiere : Tare Here

Conjugaloa

Clo3teriurn spy' +

DINlOP: HXCI.. B

Ceratium hi. rundinella 0. F. I-I. +

YericUniur' sp. +

CRY. T UPI YCEA

Cryrptorzona , spp. +t

Notes.

Presence recorded. , Recorded, but collections mad©

only in limited periods during the eu ier.

No record does not indicate absence of the species

concerned in Blake Mere and Neuton Mere.

The M xopiayceae were identified and named from Geitler

(1925) and Komarek (1958).

Where information is given, in the descriptions of the

raeres investigated, of . the species which were found by

other workers, the full name an3. authority is given of that

species quoted in the paper. cited. This is because the

authorities used by the various investigators are different (including the authorities quoted-here). However, where the

same species was found by. the same author indifferent

meres abbreviations are used, after the full name and

authorit; ts are given where first encountered. Where

further reference (e. g. in the Results to Section 1) to

the species found in the meres during; this study are made,

abbreviations are also used.

hh-

-31-

ZOuPLANKTOII

The data given here were supplzed by rosemary J.

Nicholson (perscnal' corzmunictition) froi work done on''

Kettle Mere and White Mere during 1974.

Predominant speci0s:

CLADOCERA

Daphnia hyaline, Loydig

Daphnia lonpisnir 0. F. fuller

COPEI'ODA

Cyclone strenuus Fischer.

It was observed that the population maxima , of both

the cladoceran and copepod species tends to co-incide with

high densities of Aphanizomenori and Anabaenx species. There

is no indication, however, of significant grazing of the

filamentous blue-green algae by the zoopJ. ankton. Changes of

zooplaxkton numbers may be associated' with increases of

bacterial populations during summer blooms.

From studies using algal strains from the Cambridge

Culture Collection it was found that logarithmic phase

cultures of A. floc-aquae, Anh, floc-F duce and M. aeruginosa (non colonial form) were capable of supporting Daphnia

Growth. Aphanizomenon gracile Lemm. and G. echinulata

were not grazed under similar laboratory conditions.

khý-

THi: Sl'S`. E 3 INUM2IGATiil)

Kettle. i"iere.

Kettle Iiere (National Grid reference SJ 418 341)#-

which, as the name suggests, is probably a kettle hole,

is one of the smaller mares, having;, a surface area'of 2 ha

and a maximum depth of 7 m. It is well sheltered from winds

in all directions, `being `at 'the bottom of a deep 'conical"

depression and fringed with' woodland'. The form-of the lake

basin is bowl-shaped, with steep banks, 1 to 2m high except

where eroded by the livestock which drink at the mere and

probably contribute to the already hi, gh'concentration'of

dissolved compounds. Iargin'al vegetation is poorly developed,

and is. -limited to a small area of Nurpher, there being-no -

change since the investigation of Griffiths (1925). Due to`

the surrounding. deciduous woodlands and to the peat, banks-,

around the mere the input of organic material'is considerable.

she water is coloured a peaty-brown and the=bottom mud is

principally an unstructured. black organic-ooze (gyttia).

Although having a relatively low conductivity when

compared with other mores, the concentrations of orthophos-

phate and'nitrate are high. -The concentration. of a range

of ions are given by Gorham . 0957).

This mere. has - long been famous for ' "breaking"

(Yhillips, 1 854), `the dominant species at that time being

Anahaeria cirdinalis Rabh. Griffiths (1925) records.

Anhan: izomenon f1_os-sguoe (L. ) Ralfe as the- dominant

together with Anabaena a'ffinig Lemri:, T4 icroc, %stis floc-aquae

(Uittr. ) Kirch. was rare. The predominant species found by

Reynolds (1973b) between 1966 and 1968 were Anfibaena

circin ilis °Rabh. , ex Born. et Plah., An. abnena f'! os-acturne

Breb. ex . Born. - et Plah. -' and Anhanioonenon floc, -aaune Ralfs

ex Born. et Plah. IIicro'c stir or coccoid species wore not

recorded.

Uh to Toro.

White f'iere' (Rational Grid reference : 3J 415 330), with a stü., face area of 26 ha and a. muxiczum depth of 15 n, is

considerably rlora exposcd than 1: ettle ! ier. o. The deeper

uree. s are relatively enal:! and are collfiried to a steeply

-33-

shelving portion öf the east end of the. mere. In general

the depth is betw'en 4 and 8 m, gradually deepening from

the shallow west end -up to the lip of the trough in the

east. Marginal vegetation; `predominantly `iýenyanthý s and Nuphrar., is confined to the shallow areas with gentle

gradients in the leeward regions of the mere. Fine organic

sedimcits accumulate in this region, the sediments in the

more'exposed areas consisting mostly of sand. This is

probably a result of wave action, which can be considerable,

removing the smaller'particulate deposits.

The concentration of dissolved salts in higher than in

Kettle Ziere and a wider range 'of algal species is found.

Only A. circinaiis was observed by Phillips (1884). Of the

filamentous species"a variety of A. "affinia was recorded as

dominant and Anabaena cir. cinaDis Kuetm. Hansg., Aph. flos-

aquae and Rivularia echinulata l?. Richter as rare by

Griffiths (1925). No I"iicrocystis species were recorded.

Reynolds (1973b) records A. circinalis, A. flos-aquae,

Anaheena Espiroides Klebe, Anabaena solitaries Klebe and

Alph. flos-saune as well an Microcystis aeruginosa Rutz.

emend Blenk. and Coelosphaaeer. Turn -r_aegelianum (Unger. ) Lemu.

Blake 'Piere and Newton 'Mere.

During 1976 a'! gal material from Blake P"Iere and more

espocially from Newton Acre, was collected and used for

expeririental purposes (see Section 3). Thus brief xV

descriptions of these meres would be useful, although no

regular field-work was done at these sites.

Make I Piere. (National Grid " reference SJ 416 339).

This ziere, which occupies the same basin as Kettle

Mere and is separated from the latter by a-peaty bank, is

as sheltered as Kettle Viere through being completely

surröurided by woodland. The two meres are connected by a

shallow ditch. The surface area (8 ha) of iOlakle ; -: ere is

considerably treater than that of : settle T"lero, and has a

rnrxin m depth of 13 n. : ": arginal vegetation is relatively

sparse although is abundant in the shallow bays.

slic; rare aquatic p_ ant, Lunhrgr puniest, was reported to

-34-

occur in Blake Ilere (+3in1: er,, 1962).

Apart from Nowton Nero, Blake Piere is the most dilute

of the meres examined by Reynolds (1'973b). The boatmen of

the Shropohire Union Canal, which runs along the western

chore of the mere, were aware of this aspect of chemistry.

The soft water of Blake I-? ore was used-in preference to that.

of other eueres on account of it making such good tea

(Phillips, 1884). No significant increase of dissolved

orthophosphate was observed between Reynolds' (1973b)

investigation and this study (Table 1).

As well as the softness of the water, Blake Mere was

also distinguished by the absence of breaking (Phillips,

1884). Griffiths (1925). stated that the mere was not known

to break until the. last few years. He records A. affinis,

and A-. circinal_is in smaller quantity.. The following species

were recorded by Reynolda (19-73b): - C. naej elianuri,

A. circinalis, A. floc-Hnuae and Anh. floc- ice.

Newton P-sore. (National Grid reference SJ 425 343).

In terms of the degree of shelter this more is

intermediate between Kettle Mere and jihite Mere. It is of a

similar size and depth as Blake Mere (i. e. a surface area

of 9 ha and a maximum depth of 10 m) but is more exposed

due to the sparsity of surrounding woodland. Marginal

vegetation is also very scarce. Newton I'"lere is the most dilute of the series examined

by Reynolds (1973b) and, like Blake Fiere, no increase in

the concentration of orthophosphate was observed (Table 1).

Attempts to reduce the breaking-of this mere by the

addition of copper sulphate were made for several years (Griffiths, 1925).

The formation of an early bloom (during February) has

been reported by Phillips (1884), the species involved

being A. circinglis and Cocaosj)ti, -, ieriun klit int ianum NL1C.

Doli. cro! -, nernurt ralfo ii (Ktltz,. ) [= A. affinis according to

Griffiths (1925)] was observed during the summer. Species

recorded by Griffiths (1925) were: - Rticrocy! stis aerut-iinosaV Kfltz. (dominant) and in smaller quantities A. circa rialis

and t; nrttýlict: ýplýaý, rý tzrt 1ict ý .L rt:: ur1 (Unger) Lem-r-1. (rare).

-35-

Reyno? _ds

(1973b) z. "ecord - A. cir. cinali s and Anh. los-&iine,

but none of the coccoid : ipecies.

3L.

MA ER GALS AND METHODS

Field 'Sampling.

Plankton. - Plankton samples were collected at regular intervals

of depth, generally of 1 m, using a Ruttner sampler of 11

or 21 capacity. The sites from which samples were collected

are indicated on fig. 2. When first lowered into the water

the sampler fills with surface water, which, due to the

lids which obstruct the free flow of water through the

sampler tube, will be carried down to deeper layers. To

reduce contamination the sampler is raised and lowered

through 0.25 ma few times to flush the sampler at the depth

from which the sample is to be collected. Although this is

undesirable as far, as stratification is concerned, it

appears to be effective, in that little blue-green algal

material is collected in regions where none would be-

expected to occur, such as below the thermocline in stably

stratified lakos dur: iný7. surface blooms. This has also been

verified using alternative sampling methods (such as a pump)

which are not prone to this error.

After bringing the sample to the surface the water.

was partitioned for various analyses described below.

When blue-green algal material was sufficiently

abundant, large volumes (5 to 10 1) of water were collected

for analytical and experimental purposes and for the assay

of physiological activity.

As far as possible, -preparation and preservation of

the samples were carried out in the field. Material for

visual exam4ý. nation and counting were preserved by the

addition of Lugol's iodine to 'a final concentration of 1i",.

Samples for chemical analysis (dissolved com-ounds) were

filtered sterile through 0.45 µm Millipore filters, or

analysed immediately after collection.

Benthos.

Mud samples were collected using the Buttner sampler, or, during 1975, a Jenkiii surface mud sampler (Mortimer,

19, t2). It was realised that the Kiuttner sampler is not the

most suitable instrument for accurate ouraplirig; of muds, as

-37-

Sampling position

0 Sedimentztion trap

200 m'

-38- the bottom lid disturbs the surface sediments as it is

lowered. The type of sediments generally encountered in the

lal: es-studied was a soft organic ooze within which the

material is zairlry evenly nixed'by turbulence (Davis, 1968).

An exception to this would'be the recent sedimentation of

algal material during periods of thermal stratification. The-softness-of the surface deposits enabled apparantly

undisturbed samples to be collected. The weight of-the

sampler alone is-sufficient to'make it sink into the mud,

once lowered gently'to the bottom and kept vertical by

maintaining, "tension in the line. -These problems were avoided by the use: of the Jenkin sampler. This sampler is designed

to minimize the disturbance of-surface sediments produced- by turbulence as the sampler is. lowered or as-the sample is

collected. An un-obstrtiicted. Perspex tube is>held vertically

in a four legged frame, thus enabling the sampler-to be

stood on the'lake bottom after slowly lowering itt. through

the water. The lids which seal the top and,. bottom'of the

tube are. held back, by levers. Powerful springs clamp the

lids onto the tube once the catch-is released. The movement

of the lids is damped, using a dashpot, to reduce disturbance

of the core sample. The cores were separated into-three portions: - the

water just above the mud surface, the surface sediments,

and the deeper-sediments. -Bach portion represented-approx-

imately 50 mm depth of the core and were seperated by 50 mm

from-each other to prevent cross-contamination. The most

effective method of separation of the core was also the

crudest. The sampler was carefully tilted and, the excess

supernatant water first decanted off., hnown volumes of each

portion were'- collected by continually pouring into calibrated

sample bottles. The loose and flocculent nature of the mud

-did not allow the cutting of sections, and the presence of large numbers of3: leaves and other pieces of organic material

made collection of mud by pipetting difficult. The samples

were preserved in formaldehyde, sufficient being added to

produce a final concentration of 4. %

-39- Sedimentation.

The rate of sedimentation of the suspehded particulate

= material was determined by the use of sediment traps. Each

trap (Fig. 3) consisted of a polythene oontairier. which held

14 or 16 smaller plastic tubes of 14 mm internal diameter

" and 100 ram high. Each tube acted as a small sediment trap

and also served to reduce turbulence and erosion of the

sedimented material within the trap. The traps were anchored 2m above the mud surface in the positions indicated in

fig. 2, with a submerged buoy 2 ra. above the trap itself.

The trap was located by a small surface buoy attached to

the submerged buoy by a loose., line-. (Fig. 4). The use of a

submerged buoy reduced the effect of surface wave action to

a minimum. The positioning of the trap 2m above the mud

reduces excessive eontarninatiorl from the surface deposits.

As each trap was placed at least 4m below the water surface (ie. in a total depth of waterýof'6 m)'and`below the summer

thermocline, there was no growth of epiphytes on the traps.

When samples were collected the entire trap was lifted

out of the water and the tubes, containing the sediment,

removed. They were replaced with-n set of clean tubes, thus

reducing the potential accumulation of bacteria to a

minimum. Significant interference of this kind was not

observed, but on some 'occasions during the autumn a leaf

may come-to rest on top of the tubes. The now tubes were

filled with'alga-free (tap) water and the trap lowered, for

a further period of collection, gently in position so as

not to disturb the sediments. The trapped material was

preserved in Lugol's iodine.

The rates of sedimentation, using this type of trap

with straight parallel walls, has been found by Kirchner (1975) and Pennington (1974) not to be significantly different for traps of different sizes, unless the surface

area to depth ratio is high (i. e. in the case of shallow

traps). During isothermal conditions resuspension (erosion)

of material in thene traps is higher than in traps with a

collar, such as the Tauber trap (Pennington, 1974). The

linear correlation between the amount of particulate matter trapped and the total mouth area indicates that the "snow

-40-

Fig. 3 Diagram of sedimentation trap.

SCALE: - 1: 3. Submerged buoy,

2m above.

Metal > ring. ---=-

Polypropylene

container holding

14 or 16 plastic tubes.

Anchor, 2m below.

N

line. 'a

-41- Fig. + Positioning of sediment traps.

Surface marker buoy

SCALE: - , 1: 25.

Loose line, 1 to 2m slack. ti .

Submerged . buoy, 2 to

mbelow the water, - surface.

Taut 'line, 2 m.

'Sedimentation trap. -----

Line to anchor, 2, m.

Anchor.

ýý i"'

-42- fence effect" (the formation of non-turbulent conditions at the rim of thetrap which would increase the amount of -

material sediuented) is not'sißnificant, since this would.

be expected to increase with increasing ratio of cirounfer-,

ence to-surface'ar. ea (Pennington, '1974).

The Enumeration Of Field Material. '

Planktonip. Plankton samples were examined using the-Utermohl",

inverted-microscope technique as described-by-Lund et a3,0-' % (1958). The preserved samples, which were"storedrat, 10 0C,

were thoroughly mixed and sufficient volume sedimented to

provide enough material for statistically significant counts.

About 100 organisms per sample'is required-to"give an--error

of not more than' 20h" (Hobro and" Willen, 1975;, -see also Lund

at al., '1958). The algae were'sedimented either in"calibrated

sedimentation tubes used' for''oounting"or, when a larger

volume was required, in measuring`cylinders. To provide a

more rapid sedimentation which is'less prone-to resuspension,

particularly of the larger colonial gas""vacuolate, 'speoies

of blue-green algae, the gas vacuoles were collapsed"-uoing

the""häimer, cork'and bottle" technique (Klebahn, 1895).

The formation of bubbles was avoided by-allowing the sample

to warm up to room temperaturg before treatment.

The period required for settling-depended on the--

height of the vessel used, '1 or 2'h was sufficient for the

5 or 10 ml sedimentation, cells used for counting, " 2 or 3

days für a-250 ml'measuring* cylinder. After the sedimentat-

ion of large volumes the supernatant was siphoned off using

fine bore glass tubing to limit the flow rate. The siphon

was carefully placed in the cylinder, after the material

had sediriented, such that no turbulence was produced. The

loss of blue-green algal material tend'other large species

of algae was 'found to be low (less than "1 iö) , as determined

by filtration of the siphoned off supernatant through a Millipore membrane filter, followed by drying, clearing in

immersion (cedar wood) oil and e:: amination as described by

Mc llabb (1 960). A known proportion of the settled material

was then re-sedirientod in a cedimcjntation cell. A problem

kh, I

-43-

often encountered iihen algal material. had to be sedimented

in this way was the relative scarcity of blue-green algae

. in relation to other particulate material. Thus it was

sometimes neccessary to use several .` sedimentation cells per

sample so that what. was being counted was not obscured by'''

-other material. -Separation by differential buoyancy, '' before

the gas vacuoles were collapsed', did, -not provide consistent

results.

The technique. of sedimentine a , known amount of algal

materialland the scanning of the-entire surface area of the

sedimentation cell avoided the-problem of non-random distribution. on the-floor of the sedimentation cell. =The time required td examine'each sample was not excessive-

since the low power objective was sufficient-to identify

and enumerate the blue-green algal species studied.

- The results are expressed as-numbers of. filaments or

cells per ml. The standing crop per unit area (1. m2) of

water was calculated, by trapezoidal integration of the depth

profile of algal distribution (Lyon, 1970)°. - Frequencies of heterocysts, and akinetes were also determined. For this`a

minimum of 1000 cells were examined sequentially along "

filaments within' randomly chosen, transects-across-the sedim-

entation cell. Frequencies were expressed as i-2 of the total

cell number. 'a

After-use: thecells were rinsed with distilled water

andthe_internal surfaces were brushed, using a small paint

brush, to remove any adhering algal material-. - This method

was efficacious, as examination of cleaned cells, after

being filled with water and allowed to sediment in the

usual manner, did not , reveal the press ence of any algal

material. I. lore severe cleaning techniques, such as the use

of concentrated nitric. acid,. have been used sparingly when

required, since the cover glasses used for the base of the

cells were sealed with the epoxy recin''Araldite (Ciba-Geigy

Ltd. ), which appears to be resistant to acids for a short

period.

hhý

k -44-

Benthic.

The quantitative examination of mud samples was Rosenthal'!

performed by means of "a ° Mod-Fuchs,, ýhae. mocytometer. To

achieve any degree of precision a large number of sub-

samples had to be examined, due principally to the obscuring

of algal material, particularly akinetes, by detritus.

The samples were thoroughly mixed before subsampling

and diluted if necessary. Gas vacuolso in a small volume of

sample could be collapsed by filling a 10 or 20 nil plastic

disposable syringe and exerting pressure while holding the

thumb over the spigot. Enough pressure can be generated by

pressing with the palm of the hand'(Walsby, 1971):. Usually

it was necessary'to examine up to 10 to 15 ' complete grid

areas (18 to 27 mm3) to find sufficient material. Clumps of

akinetes were broken up by-shaking by hand a small sample in

-a serum bottle with 10 small (2 mm) glass beads for 30 s.

This relatively mild treatment did not damage the cell'

material present. '

In order to facilitate the identification of algal

material against the background of detritus, a Zeiss Standard

18 microscope with epi-fluoreecence condenser Model IV%F

was used, utilising the natural fluorescence of chlorophyll

. -a. This was effective; but many akinetes, particularly

those which appear to have been in the mud for a long

period, did not fluoresce' brightly. This may be explained {

by the observation that chlorophyll may be replaced by {

phaeophytin in akinetee of A. cylindricrti (Fay, 1969b).

Transmitted light microscopy was used to enumerate non-

fluorescent material such as empty akinete envelopes.

The larger algae, such no the colonial coccoid'species,

were counted using a Sedgwick Rafter cell, the use of which

is described below (Page 46 ). Although the mud had to be rs

considerably diluted a large volume can be scanned quickly

with low power magnification. Due to the difficulty in sampling from a particular

stratum of the mud core, the results are express>ed as

numbers per ml o± the sample. The linear correlation between

the amount of algal material and the proportion of solid

material in the sample enables a correction factor to be

-45-

applied. This permits a more precise comparison between

sanples. collected at different times. The proportion of

solid material was determined by settling the samples under

gravity for two or, three days in tapered, graduated,

centrifuge tubes.

Total Particulate Sedimentation.

Ten tubes from each sediment trap were randomly

selected and their contents tipped into a beaker. The tubes

were rinsed. out with distilled water and the'combined sample

filtered through a coarse gauze (1 mm'mesh size)`to remove

the chironomids which were sometimes present. These organi=-

sms are benthic, "the traps, which are below the thermocline,

constitute a suitable habitat. The filtrate, after rinsing

the gauze, was washed into a 250 ml centrifuge tube and the

gas vacuoles collapsed by a sudden increase of pressure as-

described above (Page 42 ). The material was then centrifuged

at 1000 xg for 15 min. At the and of'this time the speed

was reduced gradually over a period of 10 min to avoid

resuspension due to the vortex produced if the speed is

reduced too quickly. The supernatant was decanted and the

residue carefully transferred into small preweighed

porcelain crucibles. Ito'further'washings of the sediment'

were done, since the loss of''material becomes significant.

The remaining liquid zwar evaporated using a hotplate and

then the crucibles, öontäining'the dried sediment,, were

dried at 110 °C to constant weight. After weighing the

sediment was ached at 500 °C for 24 h in a muffle furnace

to determine the loss on ignition (ash weight). Conventional

methods for accurately weighing hygroscopic substances were

used throughout. 'Yreparation for the next batch then

commenced with-the cleaning of the 'crucibles, first in

distilled water and then in concentrated nitric acid. After

drying in the specified conditions they were weighed.

Sedimentation of Alea1 'Material. The. -remainder-of the tubas (4 or'6, depending on the

size of the trap) wore used for the assoosment of algal

material. The contents of t1ie' tubes were mixed, producing a

-46-

single sample-for each trap. The concentration of algae-was determined using a Sedgwick Rafter cell. Like. othexTvisual

examinations the gas vacuoles were collapsed to speed

sedimentation, using the syringe technique. The Sedgwick

Rafter cell, which had previously been washed in distilled

witer and thoroughly dried, was filled with slightly more

than 1.0 ml of the ' sample .' The, chamber was covered with the

cover glass such that no air bubbles were trapped. After

allowing the material to settle for 1 min sufficient

transects were examined so that at least 100 individuals of

the most abundant species were encountered. The-type of cell (Maxta Graticules, London) used is engraved with a grid of 1 mm squares, each square representing a volume of 1 mms- (1 Ili) which' facilitated rapid counting and simple

calculation at whatever magnification or microscope was

used. This was particularly useful when the field counting

technique (Woelkerling et "a ., 1,976)

. was used when algal

material was relatively, abundant.

It is assumed in the methods used above that the

entire content -of the sediment trap tubes was in the

process of sedimentation. Thua, in the assessment, of.

particulate sedimentation the entire-volume was centrifuged; and in the enumeration of algal-material a known proportion

of the contents of the tube was examined. The total amount

of material within the trap can thus be calculated, and

knowing the area of the'trap"mouthrand the time interval

, between samples, the rate was calculated in, terms'of

numbers (or g) per m2 per day.

The Chemical Analysis Of Field Snnnlec.

Those water samples which were to be used for the

analysis of dissolved'subctances were sterile filtered

through washed 0.45 Lm Millipore membrane filters either at the site immediately after collection or shortly afterwards (the maxima L delay of 5h had little effect on the

concentration of analysed compounds if the algal material

was not abundant). If these filtered samples were not to be

analyued within 4 days they were deep frozen, otherwise

khý

-47- they were kept at 10 °C until analysis: Using this method

the effects of algal and bacterial uptake of orthophosphate (Heron, 1962) or the release of orthophosphate through the

use of chloroform as a preservative (Fitzgerald and Faust,

1967) were minimised. All containers and glassware were

soaked in nitrated sulphuric acid (100 ml saturated

solution of sodium nitrate in 11 concentrated sulphuric

acid) and rinsed several time$ in distilled water.

Orthophosphate.

The, method of-analysis in that described by Golterman

(1971 ). In a hit,. hl. y acidic environment orthophosphate forma

a yellow complex with molybdate

to a blue compound by ascorbic

by antimon'y'.

(Reagents')

ions which is then reduced,

acid in a reaction catalysed

All reagents were made up from Analar (BDH, 'Poole,

Dorset) grade chemicals.,

Orthophosphate standard solution; ý40 µg`ml P04-P.

0.1757 g of dry (1 h at 1150 0C) potassium di-hydrogen

orthophosphate was dissolved in distilled water and diluted

to 1 1. A few drops of chloroform were added as a

preservative. "Fresh dil. utions. from this stock were prepared

for each calibration curve or standard test solution.

I1olyjbdate - antimony solution.

4.8 g ammonium molybdat e, (NH4) 6No7024.412O,

and 0.1 g % sodium amtimony tartrate, NaSbOC4H4O6, were dissolved in

400 ml 4 11 sulphuric `acid and made up to 500 rml with the

same acid.

Ascorbic acid, 0.1 M.

2.0 g ascorbic acid dissolved

water. Solutions were not used for

(Procedure)

Into a 50 ml volumetric flask

the filtered sample diluted with

nece: sary. 5 ml of the r o1'bdate - 2 ml bf the ascorbic ¬ cid solution

in 1 CU ml distilled

rsore than one week.

wao pilpetted 40 ml of

listilled water if

antimony solution and

were added and the

volume made up to 50 nl. The contents were thoroughly mixed

and the absorbance of the sample and standards measured at

s

-43-

least 10 min after -mixing , at . 882 nm against reagent blanks

using 10. min cuvettes.

she absorbance of the. lake «ater, zeroed against

distilled water, was insignificant at the wavelength used.

No interfering corpoundh3 (organic compounds, i arsenate)-

appeared to be. present, as determin ed by using internal

standards. The. concentrations of orthophosphate met with

were generally high enough for direct determinations,

requiring no, further concentration.

Nitrate and Nitrite.

Nitrite concentrations. wera determined by the

sulphanilamide and It-(1-näpthyl)ethylenediacline diazotizat-

ion reaction described-by Bendschneid*er. and Robinson-. (1952).

Nitrate was reduced-to'-nitrite usinC. the cadmium - copper

-reduction column method described Eby : Wood ct-a. (1967) .

(Realteints, nitrite analysis)

Sulphanilamide..,

5g sulphanilamide, I1i2S02C6H4NII2, were dissolved in

500 ml 1.2N hydrochloric acid and stored in an amber bottle.

= N-(1-napthyl)ethylenediamine dih. ydrochloride.. s -

0.5 g-N-(1-napthyl)ethylenedi6mine dihydrochioride,

C10H7NHCII CH2NH2.2HC1, " was. dissolved in' 500 ml distilled, 2

water and stored in the dark in an amber bottle.

Standard nitrite solution', 70 µC N02-M ml-1.

0.345 g of, dry (1 h at 110 °C) sodium nitrite was,

dissolved in distilled water and a few drops of chloroform

added as preservative. The volume was made up to 1 1...

Dilutions were prepared as required. (Procedure, nitrite analysis)

1 ml of the sulphanilamide reagent Was added to 50 ml

of sample in a 100 mzl stoppered measuring cylinder. This

was followed ' within 2 to 6 min, by 1 ml, of I1- (1-napthyl)

ethylenediamine dihydroohlöride and the absorbance of

standards and samples measured- at 543 nrn in 10 cup cuvettes.

A Bausch and Lomb Spectronic 700 spectrophotometer was used. (Reagents, nitrate reduction)

Copper sulphate solution, 0.08M.

20 g cop,. -or cull. hate in 1.1 distilled water.

--4 9-

Ethylenediaminetetraacetic acid (MTA) solution,. 0.11,1. 38'g of the tetrasodiura salt of EDTA dissolved in

500 ml diestil led water, diluted to 1'1.

Hydrochloric acid, 211.

85 ul of 12N hydrochloric acid diluted to 500 ml with distilled water.

Nitric acid, 0.311.

10 ml of 15.411 nitric acid diluted to 500 ml with distilled water.

Hydrochloric acid, 0-001511-

0.125 ml of 12I1 hydrochloric acid diluted to 11 with 'distilled water.

Column wash solution.

1 ml EllTA solution in 50 ml 0.001511 hydrochloric acid.

-Mitrate standard solution, 70 Ilia 1403-H. ml-1. 0.1264 g dried (1 h at 110 °C)

potassium nitrate

dissolved in distilled water and diluted to 250 ml. A few

drops of chloroform were added as a preservative. Dilutions-

were prepared as required. (Procedure, nitrate reduction)

Coarse cadmium turnings, already prepared, were washed

in 211 hydrochloric acid and rinsed with distilled water, "

washed in 0.311 nitric acid, rinsed in distilled water, and finally washed in 211 hydrochloric acid and rinsed in

distilled water again. The filings were treated with 100 ml

of the copper sulphate solution per 40 g of the filings in

a 250 ml conical f -lask stoppered with a bunk; through which

passed a wide bore glass tube. The treated filings were

washed with distilled water such'that they were not exposed

to the air. she column (Fig. 5), plugged at the bottom with Pyrex

glass wool, was filled with distilled water and the filings

tipped in via the tube, again so that they were not exposed to the air. During this process the column was tapped to

settle the contents. 50 rnl of the wash solution was run through the column and was then allowed to stand for 24 h,

renewing the wash solution several tines. Before use the

prepared colurina were conditioned by passing through 31 of

solution coiitnininj; 840 Eta; NO3-N 1- and 20 ml 3DT,, solution

-50-- Fig. 5 Column used to reduce nitrate to nitrite in water samples.

Redrawn from ', food et al. (1067).

SCALE: - 1: 2

50 ril reservoir

Metering /stopcock

" .-..

spy

Copperized cadmium

filing3

-ý Measuring

cylinder

Capillary Pyrex glass wool plug tubing

-51 -

1_1 To each 50 ml of-sample to be reduced iras added 1 ml

EDTA solution. Excess"wach solution was"-removed from the

colurin by a pipette to about 5' mra above the top of the

filings. A small-aliquot of the sample was added to the

reservoir to rinse out the previous sample, and"1 to 2 ml'

allowed to pass through the column before removing the

rest using apipette. The remainder of the sample was

added and ameasuring cylinder put under the discharge-tip.

About 5 ml was allowed to pass through the column to rinse

the cylinder, and 151m1"ýcollected. A consistant flow rate

of approximately-1-drop per second, adjusted using a stop

clock, was maintained for a seriesofsarnples, standards

...,. and ý blanks.,

The reduced samples were gnalysed for nitrite as

described above. 'Por each"eeries'of samples standards of

known concentration of nitrate were also run through'the

column to determine the efficiency-'of reduction.

It was found-that the ' nitrite` levels -were very low in

the water"bodies sampled and'therefore analyses of nitrite

were only done-when the concentration"would be expected to

be higher 'than, normal, such- as *, in the "anoxic reducing

conditions in the hypolimnion. Contamination of water

samples"'by nitrate contained-in cigarette tobacco (Taylor,

1975) would be minimal.

Oxygen.

Oxygen concentrations-were determined using the

Winkler uethud described by Golterrian (1971 ) or by using

an Electronic instruments Ltd. (Chertsey, Surrey) model

1520 oxygen probe. (Reagents, Winkler)

Potassitun iodate, 0.100: 1.

3.567 g, dry (1 h at 110 °C) potassium iodate dissolved

in 11 distilled water.

Uodium thiosulphate, 0.02511.

6.2 C sodium thiosurphate dissolved in distilled

water. A pellet of sodiun hydroxide wan added to stabilise the solution and the solution diluted to 1l und stored in

-52-

an. ariber bottle.

Alkaline iodide solution. 250 c sodium hydroxide dissolved in distilled water

and 75 L; potassium iodide added and diluted to'-500 ml.

Polyviol indicator.

A saturated solution of polyvinyl alcohol in distilled

water. Manganous sulphate, 50iß.

250 g : iancanous oulphate dissolved in 500 ml distilled

water..

Orthophosphoric acid, a. g. = 1.75.

(Procedure,. Winkler) "' Samples were collected in calibrated 60 ml stoppered

glass bottles such that no contamination by air or trapping

of air bubbles occurred. 1 ml" of the manganous sulphate

solution and 1 ml of the alkaline iodide solution were added

in rapid succession to each bottle using syringes. -The-

stoppers were replaced and the contents mixed, and mixed

again after the precipitate-had-settled-to the lower third

of the bottle. Before titration 2 ml orthophosphorio acid

was added and the bottle resealed such that none of the

precipitate was lost. The entire contents of each bottle

was speedily titrated against 0.02521 thioe-ulphate. using a

rzicroburette (10 ril) and Polyviel as the indicator. A few

drops of the indicator (orange to colourless) were added

as the end-point (pale straw colour) was approached. The thiosulphate spas standardised weekley by titration

against the standard potassium iodate solution. 0.02511

thiosulphate appeared to be relatively stable over n

period of up to two months.

Calculation: - U

8000 x rlthio x titre

2 Vol of bottle -2 nil

Nomograms (Hart, 1967) were used to determine ý--, saturation.

pH. she pI-i of the surface waters was measured on site

using aI etrohm battery operated" pII meter. Olotrohm. Type

L'280 n, IlerKoau, 1,4witoorl-rid j.

-53-

Conductivity.

An Electronic Switcht. aar Ltd (iiitchin, Englund) type

r: C-1 conductivity bridge was used and the conductivity of

the surface water expressed as µciho cm-1. - .

During' the summer of 1975 a Bach (Ames, Iowa, t7. S. A. )

Direct Reading Engineers' laboratory, model D: l- L/2, was

used in the field for the chemical analysis of: water

samples. The results were comparable but less precise than

those obtained using laboratory methods. The analytical

procedures are derived from those described in-the American

Public Health Association ; tandn, rd'rilethods (1971) .

The Measurement Of Physical Parameters.

Temperature.

The Ruttner sampler contained an accurate mercury in

glass thermometer and the oxygen probe was automatically

temperature compensated; the temperature readout could also be obtained but to a lower degree of precision than the

mercury thermometer.

Light.

Light penetration was estimated uainCj a Griffin

Environmental Comparator (Wembley, IIiddlesex, England), the

sensitive element of which comprised yf a cadmium Sulphide

photocell encapsulated in a glass bead. It is most

sensitive in the red region of the spectrum. During the

summer of 1976 a 300 rim diameter matt surface aluminium

Secchi disc was used to determine transparancy.

¶olal Particulate Suspended Solids.

An aliquot, t eneral? y 100 to 400 m_l, of the preserved

plankton -sample was filtered through a washed, pre-weighed

25 cam Gr/C 4hatnen (made by It &R Baleton, Englund) Glass fibre disc and washed with 100 nl distilled water. These

were dried at 110 00 to constant weight. The length of

drying period did not affect the weight significantly once

constant wei ht had been refached.

RESULTS

The Chemical Aid Physical :: nvironr, Lent.

`. temperature. - Distinct effects on thermal stratification and on

water temperature of the different degrees of exposure and

of the different surface area: depth ratios, in thetwo

meres studied, are shown in figs. 6 to 11* Thermal

stratification in Kettle Mere is already well marked-, by

April and renains-stabily, stratified until October (rigs.

6,8 and 10). The period of stratification i"s considerably

curtailed in White Mere where it prevails generally' only

between June and September (Fins. 7,9 and 11). The

difference in stability of the, water'column, between the two

meres is also refldcted by the higher surface temperatures

reached and the more sharply defined thermocline in Kettle

Mere. Further, the epilimnion'is shallower than in White

Mere. Although both'meres are exposed. ,. to, periods of, windy

weather causing the cooling of the surface layers of water,

mixing . in White-Mere generally extends deeper, -, and may

sometimes disrupt stratification during the summer, -as

happened for example' in August, -1975 (rig. ' 9). --

The water temperature during isothermal periods is

similar in both meres, although on the breakdown of

, stratification in the autumn, there is a more marked

cooling in Kettle lýaere when compared' with White More, due

to the mixing of a relatively large volume of cold

hypolimnetic water with the warmer waters of the shallow

epilimnion.

Oxygen.

A similar relationship between exposure, turbulence

and the vertical distribution of oxygen is also obtained

for the two years (1975 and 1976) that data are available (Figs. 12 to 15). The water below the thermocline in nettle

Paare remained alriost totally anoxic for several months

during the sLUiver (Figs. 12 and 14). The 1,; ) saturation

"isopleth may approach to within 2m of the surface during

calm, periods, which, will result in . considerable

, oxygen

depletion of the surface waters, should a relatively minor

-5! -

co

C' r1 " (U c- ". l C) 0

4-t tr 0 i g

Cri ii 4- "rl P

CJ C, C4 '. 0 C) V E CL)

r1 ". -t G) -W 4J 4-)

tý aý S 0 "r1 Q) Cu 4ý C7

ý1! 1f

ocý

NO CN co

.

tu "uI(I0Q

O

H Ü

C7

ti

a:

tl u.

-56-

o'

co

c- $..

H C)

lam- vC 4.3 +) +) +3

"ri . i: 4-4

iW :=:. 0 H

:2

J

G7 41 4) r. 0

7

Y H N

2

H

}

a zý r4

CIZ

rn -

V1

r" co

r"

Mr

cc

c- C: r

r- 00 I'D

It cl-

/ Lr, L! C'-- u C\ r4. " C) r "-l a) 0 ti

C) F+ 4- "rt s4 w r n) $.., c)

Ca r . "r4

" 4-) . a> . 4-) - #.. 5 .. ._.. t: . 43 S. 0 ". -I CI C L4 U. W :4 . 0 N

-a--

CN ýl s\

v

rid

uj

tiv

. C:

0 1

r=.

ý7

-D

-58-

N1c"

Lrl%

0

N

N--

O

" C7

- 0 c - " tJ

T- t«. to O : $3

o G. S

Q) r4 U E

C3 "ri $ý n

.ý. c w Q :4y ' 0 I-i

Ia

.. a

U

F+ U

C: J

4-1

0

F:

. =7 w

0 c- c" iM -2- U1 %D L` Co C" cz-

4) t9

m r4 " Ca c- "ý U0

G ;3 C) S4 d-) "r4

C) S, to r4 N £3

Cl v C) r-i "rf +C) .C

b: +ý Lr O "r4 4) G> 4ý L7 GV 's : -' OH

A ýO rO co Cý! RI CJ rr

Nr

Lrl%

O 00

L'\

0 T- (IQ 1 .*

u

-59-

1

a

0 <s

a

ti a

a

cý

c1 ö

7 ti

:L

ct

ýi

0

Pi

rD

0

9"

w 11

c^ý

Fý. P)

CN

a

io try

PQ

ýBJI \ý

o u> N

0 L3 "r4 .r CW

C r. -+ a a, ". T- fm 0 (0 u; '

.r Li +) 0 - " to H r.

ý4 IC " 0= ºP4 r. C P P4 O

r-1 C) Q> "'i cis 13 . 4a £: . º)

r W 0 u cn

t. 3 Z. r+ Cý Z, 4+ 0 Z' cm.

0

U O

O

G=,

O e- N tr1 .ý LrN lG

-61-

-62-

0 n

0

0

o

Lr\

o to "-, .1 cý m y r) h Z r a 4-4 co

0 0

r 4-) i-) 0 ce >C U

ý 2' r 0 r' 0 + F s " . 0

C) r+ C C) "r4 C C) KN «_ C L 0 -) r U U C) C3 t, c0 U C3 Cj r+ X C) V L. U c3 L.

+) 41 0 i: ". a 4. ", S. c a zý ti u ±. Q) 44 0 X cri a C) s .) (L4 > 0 U 4) Li 4 " -4 LC to

i

i

a

c c

v W 0

a z

s, a

5 t

0

ac r

co w LL-

Z ti

orn: - rn a .. ý uý ;. cx C. c z-

-63-

L

o W . r4 . C) .. 3 .. 3 co

o, r3 p 0 C'

. o co C) aý U; U

Cj "ri "r1 C) "

c, r_ P. o E c t +31

- 4) 0 r r. 4 "rq

X C) ý + f

4. ) +0 0 S. Lo

I

V. a)

. rj 44 0 s'.

0 o

o

ýÜ

C r

0 0

n

C \` Jý

71- W

i

N

41 r- 0

. T!

C7ý W

a ._

a H

ti

QrN trl I--" tC

U' %4U n

-64-

Q

" 4) 4, c0 Q> tai lý 7 ri 0 4) A Cb

ON i4 0 CO a) t/! £i 0 r 4) 6) +. U <. G) "r"ý N

L! N 0 f+ N ,> U "" "rA "ri Q) " a) Cs R. "

C) 'D " 0 l. v f:. , C: E Q) fr P. 0 +) P, O 0) H N 1 "rl V. 4) rl

hfl :z tý hJ +. 3 U - O +> d 4-1

" 4) +3 O 1:: c. "'i 44 0 V. ) "rl St c; C) +) 'L7 F. 4-)

"ri C N 4-+ 0 . S. cß Z 0. 0 cd j : i: > 0 0 i-) Q "ri "rl U) to

m

a- h

c

_i x

0 00

; -ý .ý ý*.

ýý

0 r fU týl -t (. vJ ß" c

r,

I

H y

L

-65-

amount -of mixing occur. This is also observed during the

breakdown of stratification in the autumn, when a large

volume of oxy`en depleted water is mixed with n relatively

small- volume of saturated or dul y-satursited water, which

results in a low percentage saturation of the water body

as a whole. Saturation gradually increases during the

winter º1onths to about 7U; j', before stratification is renewed. Periods of anoxia in White !, ere are limited to the

deepest areas during the most calm conditions (Figs. 13 and 15). The depth of water column which is fully saturated is

greater than in Kettle Mere, and the reduction of saturation during the breakdown of stratification is less marked.

Although periods of supersaturation occur in both

mores during the summer, in White Mere these periods are

longer, a greater depth of water is affected and the

maximum levels of supersaturation are higher.

Light.

There are considerable differences in the penetration

of light between Kettle More and White Mere even during

periods when populations of planktonic algae are low (Table

2, Secchi disc data for 1976 in table 6). The considerable

light absorbance of the water in Kettle Tiere is principally

due to the high level of dissolved organic compounds, which

impart a brown colour to the water. The absorbance is

particularly high in the u. v. and blue regions (200 to 400

nm) of the spectrum and is twice as high in Kettle Hero

than in White Mere (Fig. 16). Kirk (1976) reports that

. dissolved yellow organic compounds ("gelbstoff") greatly

reduces the amount of photosynthetically active radiation

and that the blue region of the spectrum is extinguished at

moderate de, »ths. The reduction of the depth of penetration of light

during periods of algal abundance is more marked in White

Tiere than in Bettle !,: ere. The greater absorblince of the

Kettle ere water tends to damp down any further fluctuations

of light penetration due to chancef-, in tilt-, till,

which, in any cane, are generally higher in White Fiere.

kh,

-66-

.". ""s

4l

"r+ -NNN \O .'r

l c- N, \ MN K1 N N, % N --d" n ON rN

fý N . 1- ON 00 _: t u1 ttl O 0

N c- NN K1 N. - Ký

N

+) O 00 O tt'N 0% O- ur . a-) ". """"""

C) rrOrrrr r- r "

0 . r4 43. ct4

. rq

CIS 4-) Cl

\"\N\Ö

N 0. ̂ N t11 N 00 N a) C. ) cö 4i Q) P4 f+

. LrN

t0 C` ONN0

:3 a) 0n ***"tt1 ýO t! 1 N'NNN

a) co +2

a' u1 O rý OOOO1 O- c- N -7 M Kl rrrrrr IA N

a\ C- ONO Ln ON co ulý -t ao OnN

f: rMNM4 c- 4NN. NNN N

OO .- . I- ON OOOO u1 OOO o".. NrrNNr r- O r- -r4 cV "r

4-4 0

E vd N1 ýO N- co co CO 00 O cam- N . s: 4-3 \ ý1 ýO \ C` \\\\\ r- \\ ý- 4) Cl -I- \\ Ul \N 03 t+1 \` u1 \

AN IA NNNN c- c- NN /O LN

03

N $4 IA co 0M0N u1 0"0

N GD ý: "t""". """"I" Ul U. ' U. ' Kl N. ' ýO %Z Uý

H C) v 4-1 8 u1 0 L1 [- t, 00 L` Q01! 1 Elr- Pý N c- rr- ý- rNr

Q. ' r 0)

u1 N0N0OO UN ýý ""t"""t"t""" c- N e- 1\ ý' NNNý

Q: H 4) 6 to OOOOO u1 OL-\ Q', OO

rrr c- 1- c- c- 41:

rO c- c-

o e- cv

A r' rN Qý r K\ r (V Q ýN Nr

0 N 0 0 rl v4

a

-67-

O C)

O O Co

U 0

0 0

8 .+1 E'

F:

. L: ; -,

s~

.l o ^J C) : -' _:: t. :J

p 0

0 0 n1

C)

kh,

ý! ) ý,,; -r, ' ". 10! -, "tIy

-68-

U1

a'

4-

H to rz w

to N Si 4)

C)

. Li i) 0

. r{

C) 4J

A

G7 O

,Si

O

P4 O

4-4 O

91. O

cd

M 0

co H

I

O1r C'" r 00 OOO LIl r %. 0 Co [` C' M0 K\ N [` Ol%

8 Co Co

4) OOOrrNN -3 Nc r' O

OMO ul CO cO \. O O %, O - Kl ONr N Ln 03 ire rn 0 ý-o 00 (Di CO c- 4.3 :

". -1 OOc; O c; O. ONOr l"- l l! O j2 s

CU CO w co N rin CO Z- N . ̀moo 0 0ý 00 ,0

OOoOOO r" rrrrOO

OOWO Co OOON %IU u1 L` i~ NMr [- L` U1 N 10 Q' NN Lý N

Nrr c- N Pal -OcfrOO

C) 0 U1 O\ C% 4- 0r0 (` \O %IO U-\ L'-

+ý rOOOOr-rrJOOO C) '

OO- U\ O oO "ý- OL\ ýU l` C'- LI\ öN K1 r\r\- O' C- ýO uý ul C-N

rr (5 OOOc; 2r r" CGOO n e-% v

cs- rl t

H9 Ä+

"H1

ru O LI- AS 0v ti

9-4 Ei 4-4 d) 0 O -P S

, sl O

, ll Äi 0y

N0 AS

rti -rf

11.0 C`- CO CO CO W 0' Or cr- r

d) 41 WN. %Z ce \ \,

M\Nc h\1 \\

c ul ON C- NrrNNNNr 03 ,t pr

-69-

N rn

4-

H to E a ca O

(D

n) 43

a) 4-3

P, ca 0 p4 0

s-4 0

0 0 0 4) Cd

0) U

O U

_: t 0 H

cn N

Nz

., l

e

a) C)

4-3

C)

0

0 H C4 s

4i 0

. c: N

"r{ 0

W\ 0 U1 NM -t 03 00 N- M0 r' t\ NN _e LA r4, \ _e n -i- UN

000O00000 c- c- %- r- c-

Lr\ N c- 0 ý, O WN /D 000 Lr\ %, D 0 L- 00 _t .: ' -i' In NNN- -4- Lf f\ t! 1 C` \-C co OOOOOOOOOOOO3O6

- C\ N c- ur n00N (v 0 h' t4\ M r1 rn NNN -4- -7- -L U\ NN 'N OOOO0OOOOOOOO88

ON Cý LII- _zt- 0 Nl 0 [` - CO Co rn C' cv ýD \. 0 w �O \. 0 N r- CO ý CO CO CO rn rn

............... 00000000000000 v2

n ul N L1 N1 NnU 'N 0N IN .-0 ý-O C` 00 *1O N I'0 N ,D'. O %Z C` \O [` Lam- '. O '. O '. O

" .0.0.0.0...

0.0.0.0.0.0". 0 0000

cl-O 'O c- OO co *r NOI '1 00 O w CL) t` ur ul% '. o L\ u\ u-N 4- -I- . -- . .0.0.0.0. o. O. O. O. O. o. O. O. O. O 0

CO NWN1O CO KN

cam- N

u1

I-N

0

H o el- U

E d) 0

{'3 30

"0 4-i o N0 Id 10 (is ti

w

4

f t

-70-

n cý uý ýo '. o rn 0 0 GO

O O 1

0 1

O 1

L w Oý N N r O '. O Co r , z, r O

1 0

1 "O 0 O O O

Z "ri

co ýD O Co £ O

O "0 1 1 1 " O

H

EO N O Ö Ö 0

E O O 0 O O

O N

ý' N

- I\ N Ö

N Q) N\ " " " 1 1 1 " O O O 0

x N N ý r

N F

O . " 1 1 P

"

r

. " O O O O O

s, a) H 4 O

ýi U) O

"-I +H

"F) O cj

-P " Q) - G) "r4 d) U cQ Ga O

U

\ - 'ý

n "0 N N

Co r- N

rl tý \ Ul

\ o - , cd

S] Q N c N r CO

H

OD 1= ö 0 " " I 1 1 1 " i " O O O O

N CM r ý " 1 1 1 1 1 " 1 " .. 0 O O O c-

CO CO CO -I" r4, \ r- c- N r- c; r O O O O 0 O

O

0 A M CO N N N

u n �

Ul Kl [- O O 1 O O O O O O O

0

M tr D

0 0 0 Q O w

r n N ý N tt $

i 1 ÜIN O O O O O O O

I+l

fü

O U

iy G

.r

4-1 O

O

rd

7

\ M \ N \ C cý oc, O" N c0 J ! rl N - N c r ýý

k

i

-71-

t i N. rn u1 ur\ CO O N - t: c y\ I 1 1

C` ýJ Cý 00 Co all Co Co 03 CC (J\ Cý

U L` 0. ON >-a

vi r U r SI

" M ßr1 O 1. tr\ c'- N N r d Cý CU vý N' CI ýO N d) S-i rP I 1 1 1 I I I I " " " " " " " zý S-( N U) , PCl 4- 4- PA N r r g

O v 0) -P H

S: "ri

G) O "r1 tý ýt -d- l- L` N- O '. O 0 O oo 0 l! l\ 4- P. o 4-1 0 Cl- CO O' tS\ M O' O\ l` 1 CO l- I CO lý- U 0 N N N N N cat N N N N N N N

H N O U rý

4H t- F"t

1 Ul

9 N Ul Nl 00 O tf N U1 r4, \ - o 4 1 1 1 I + N Cl- lý l- Cl- C'- CO lZ O CO CO t7 ý O 4-1

C o v

'T' SI

p"+ Q: 7i O S-i N- tI\ ü1 r- u' U"\ lý-

++ - c3 O\ '. D L'\ Co 4- 0 lý "ri SI d) t. 1 I I 1 1 1 1 I " " " " " " "

W :, c c- c N N Cd c r{

U)

-p G) U U H i"i

u) +' H 2S "ri i-> $ q aý o x Y,

U "rl -P . L' "rt

b ri u1 ul cam, ) N to a. ) N N h N lc\ U'

\Jp 0 +-2 (x) o O" O N UN '-0 C'- 1 .O 1 %Z I lý- (0 c-- N r N c- v- r- c- r r 'c U

0 H cý 'ü

ci ?a O H .D U

" L) O \ N 03 N lt1 CV IJl r N ui c- % N N Ul

-72- Orthophospii. at e.

Concentrations of orthophosphate were'high throughout

the period of investigation, although during periods of

stratification there were signs of depletion in the surface layers of water (Tables 3 and 4). Concentrations in general

were slightly higher in Kettle Mere than in rihite Mere. The

high concentrations sometimes measured near the bottom,

particularly during 1975 (Table 3), are probably a result

of contamination by sediments of the sample taken.

%

Nitrate.

Nitrate (Table 5), like orthophosphate, appearsto be

present in abundance, and no marked depletion was observed during population maxima. The concentrations, however, are

generally lower, and since mdrq nitrogen than phosphorus is required for growth, particularly of non nitrogen-fixing

organisms, this element is more likely to become limiting

than phosphorus (Gerloff at al., 1952).

The Annual Cycle Of Growth And 3ucceeeion Of The Blue-Green

Algae

The Plankton.

The sequence of appearance of the principal bloom

forming species of blue-green algae (abler, 7 to 12)

generally conforms to that observed in other temperate

eutrwohic lakes (Hammer, 1964; Reynolds, 1'971 ).

A. circinalis and A. floc-a u. ̂ Eo frequently produce large

populations, particularly in White Ziere, in the period

immediately preceeding thermal stratification (Tables 8,10

and 12). There is some indication that the populations of

A. cireinalia develop before those of A. floc-aquae. This is

particularly noticeable in 'Iihite ;. ere with the collapse in

mid :,: ay, 1976, of the unusually large overwintering populat-

ion of . º. ci rci nalis (: `nable 12). . 24nall differences in the

succes:; ion of algal species are frequently obscured by the

bimodal distribution of algal numbers with time, a recovery

of p? nnktonic ponu1ati onH oec: t: rri ný; a month or so after the

initial oollFajlse of a hloori population. The increase of

numbers in Kettle Nero npT ears to be halted with the onset

i6,

-73-

Pl w `

"ý 4-3 cý rýl

v 0

/i 0

W U r1 ci O 0j

rl O M O rl U F4 " O

d N

-N U]

N 04 d) £

rAi c$ d %lD r1 H " vý

4H (u c3 4-+ r1 +3 0 4-H S., "r+ O 0 0 M fA 1 x- Q) i-4 0 UJ

U .Q . "

:J 4-1 1 Q 0 0

z ý- " fti C` N ON

0 1 O C.

Pl P4 H 4%

O O a) Pi Q) f-4 M

P-1 ý rt H

4-3 E N +) O -p 01

H Z to 0 "r-4 0 a) s4 4-3 'LS r M R r+ 0

. z. ro

co 0 4-i H O 4-i

4-) "

O O

Q r-i

%10

N1

to . rq

U kj

Ui

s

ni r- _zr CO OOOOOOO c- r t- O

ONOOr cU c 000 c- OOO c- N r-

o0 u\ 000 cv o .f -J-

C(O)N COO ö ni

OOO%. p NrM c- COO00000

80 NNOOrOOOOOOO

M Cam- u01 r Iý1 N000000

000000000000

N 000O, 00000O00

000000000000

o00000O00000

OOOOOOOOOO ,OO

OO9O -f OOO 0-: I-0 CO r 4- ON r t! 1 00

U-N T- o0000o0Ooöc; o

co No oOOOOOO O- OOO

ý1 t! 1 O OOOOOOO0ONO6

0 N %lO QOOOOOOOOOO

OO 1 ý'

rOOOOOOOOO

O Cý Ü tIl r ý: . C)

cU ul O N Pal 0 C0 Q c- 0 0

ý - c- N CO co Q-, r j \ \ \ \ Oý \ \ \ a) \ \ ('D C) N' t: A \ C-. Q V) C) r r N CTS r I' c- Chi Oti N N r

-74-

Im r-H E N C)

0 H 0 U

0

Q) ii r C)

"r{ " Qi "r 4 Ui

4-i N 4-i ri 0 tM ä.

O 0 i H N $4 0

"0 Q) U

0 0 . - z

O ". .o 4-) a4 E n c co

$4 a, H I ý.

P, 0 ri P. 4) E U) r1 0

c' ri "r1 "(l

c ,^ VJ

H . c, RS P U rl O

Q) 'C7 U d- "r-1 Cd d

: r-I

Q

co c^

4.1 <4 0

-FP

0

00 cl, H . fl cj Eý

ý. O t<l try o C. -

to M O" O N O c- 0 0 O c- N r O

u, cT ac 0 ýD 7

U) CO m " . 4. O . .% O O NN O a (\ 0 cv -ct O 0

U 0

O c " L` N w . c ;V K%

O O O o0 -ý N O c-- c- 1 t+\ O O

aý f o N O

pq O O O O O O O 0 o %Z 0 C0

ü; 0 -d- --J- o N

Z O O O O O O O 0 u\ O 4-1

0 rý1 N 0 O O O O O O O O cC rý1

Ö

O O u1 110

cd pq 0 0 0 0 0 0 0 (z 4 O O O

N O O O O \O O

r+ O O O O O N w u\ c" O ý- O

0 O 0 O N 1.0 K\ a 0 o 0 o O o N N. CO U\ c 0 0 4

ul\ N N ý-

a) PQ 0 0 0 0 0 0 0 0 0 0 0 0

"r1 v " O ý-4 0 O C3

O O O O O N c- r \O O O O O O

(ý rl r o

l` O O O N Cý 1

O O O O O N

w N- V- O O O "

3 Ö 0 0 0 N- 0 4)

. . . . " %o cl\ 4) "1. ) O Gq O O O . '- c- CO O Lr, \ O O O cu . 4) ")

4-1 v 0 c. Lo

1 "' + ;. O O O rN r-

O r

c r 4 N N

O H c1) 4

i Lr\ 10 0 r- rl(N 0 -H

" N- O O 0 " r tý r t6 O O O O V- M N O N U1 L`\ C- C'J

(D .) "i") r

O lr O w Q O . ' " M

"14 m 0 0 c- c- N ß" 0 0" \, D O" N\ r 4i 4.4 H

Irj 23 k-0 " Cý w

N %lD N- N. r UJ 4-, 4. ) v M 0 C. Gý N\ cc 4' N 0 " N O U ý-i . i: 0 0 l\J VD CO c- c- N 4. CC .± H 0 C>

0. r-i H U E- H H

pý co 0 0 " LI\ lD r(\ 00 i) '. D O\ lf\ G) U U

0 rTh 'O o - o' o r: O 0 O O -a rý tý\ \-O r\ -Z N c c c° -, t U ü] Q U (1) (L) m H H

[-_ co CO C) G1 CQ C` ý \ \ \ Oý '\ \ \

ý C) cj \ . .

L-1 i-a c- r- N

U" c- N

l r i1 G\ N Cv r

0 Pd

-75-

t- oo P4 00 _: t N Uh N r U

' C(D ý Ä c- O O O r 1 N

c N O O O

` 11-0 N N +p C N of c- n C D" c - 0) " " . " CO C% -j- c- c- " "

r+l O O O O c- -t M rýl rfl K\ O O O O U O

0 r- 0 rn 0 " ". n . - -t t4, \ 00 0

>; O r 0 c c- N u\ -4' C` M cJ O O O O

r-1 E

cn ýo a o 0 0 o a o ýn o o O o o O Ü N

0 : 0 1,0 0 c- 0 ö

rrý c- O O O O O Ö rýý O O O O O O

U

ö ,ý O M

ý uý v) ý: G cý c-

N 'O G a O O

RJ N O O O O

a) N

N r r-i . 1. *ri

)U r4 '. 0 0 0 0 0 0 0 0 0 0 0 0 0

CH CH

w 4-1 r 0 0 0 ". + M cV ý- O

fý 0 c

rn O O Ö O O O O Ö o M O O O O Q) Q) U C7

Z 0 ýý M Cý S N O i1 ý

z N l 1

lf l >~ A 0 O O c- r- -t c- N Z- N N O O O O

O - N Pl

C) 1 '-O O O O O O O O O O O O O O O 1 A °

a) u> c M " (A O M o o cý ö 0 0 o rý ö v o o O o N

gi r1 ri C) M " CO

M U , 0 Orn M 0 N U1 ä (D O O N O M O Ul O W O c- O O O

Cl) 'i3 V

Z %

\Z O c- d O O 0 0 0 0 0 o 0 0 O r-4 9: Z

C) 0 r M O ul O O O O O O 0 O O O O G

4a rI 0 4t

+> " C- Q

O O n 15 O N G O O O d O O O O 0

v1 " "-+ w 0 0 0 0 0 0 0 0 0 0 0 0 0 0

O' C)

r-I "ri ýO c- N

vi $

M O O O O 0 O O O O O O O O O H

U

O c- cm CJ G` 0 O O N O G O d O t7--

, "i. 0 +' C) \D (ý. CO Cr 03 CO C) ` r" CV

ca " t Cff G" (\! C"- ;u %- c- iv tv tG N w

" -7E-

p4 ... . . . .. . UI pq OOO O- O OM c- lý ýD O

4 T- O .. 7' O O L1 4. O

OOOO '- O M Oý ßf1 N O O U

o N 4. O O %D M

;i0 O0O0O 0O M C` a0 O O 0

r

to 4) Q

O to r-4 0

Ö

t! ) " d +-

ri c4 cd rq r4 4.4 -r-4 to i4 4-4 a) Cd

4.4 ri "1-) O O H

f~ rl O N iq O 1A

, fl N U

4.4 ,:

5 0 IA

Z " .l ON

1 N c- O "" Sa +1 9 "ý n

u i z

M O r

1 Z

' L' P, 0 ". ., ý

ä ' 0

aý 4. ' $-l ul PL " 0 N to to ý' "rl ri

cJ N d

S 0

C) 'C7 U "4) cl "r4 c

Cd (d N c

cd CZ 40 O 0

4-i d r°"1 0 N"+

"+. "

0 0

0 "j

E-4 "rl v

o ö as 0 0 0 0 0 _e 0 0 0 0 0 0 0

M tom. U O O O O O 'D O O O O O O O O O

UN O 0 O

O O O O N -

O N O O O O O O N- v

E O

Cq O O O O O c- 0 0 0 0 0 0 0 +ý O

%. O W N a) 0 0 0 0 O -

0 O r (3 0 0 0 0 4)

UN 0 ,D ö

O O O O O tt1 lt1 Ö c- O o O O

i

Lr Pý i I

W O O O O o r o O O O O O O "

r- OOOOÖ-00000

,OOO �0 \O _e -t M

-01 O O O O O %0 r O O O O O O O O

0 M O O N

Ö

n Fq M c; O O cc r O O r O O O

Lr\ ' J (3 (Z z1- c; 0 0 A

0

eý * N

`: 0 a -1- o N N ° 0. 0" t- ý 00 0 4-4 O r N %M [` N1 N O, 2 Cam. Ö N O e- 0 0

ri N

u1 O O "ºi PQ c- O O O O 0 O O O O O O (3

a N -d O co O O O M 00 O O O O O O O H

a Ei N l- 00 Co -

O O O N r

O O O O O O O O 0 U

a) M lýD N. CO 00 00 00 O r r

N to Cai cý \ 1 \ N 1 N- A

A A \ º CO t \ l ". N u1 Q' N C- N r- - Cu N '. D N ý- CO O

-77-

U) N . r{ 0

H 0 O

0

4J 4.11

U)

4-1 4.1 N N

4-i 4-4 "r4 O O 0

0 Si fa H N N 0

,Q U

, EI 4-1 ;t ; z. 0

" Fi '. O 0 O .O 0" tý Q, F

1 pi

d) N P.. . 54 c9 ü) +) 0 r+ N tQ +p

r1 "ri -I-) ". ý +. P c) f1 U

"r-C --t O $4 N ^o O

U

td H SI fQ N EO ai

SI 4-t cý O

-I-3.

O F

a, r1 03 Ci

fi

Uff. \O

U)

U) M U O

U . ri 0

c3

0 ri t'\

p . 41

c4 "r ý, o

4) M

0

"ri iý

Qo

vw C:

O

4-1

O

. r-, H n; r

. r{ U S-4

0

i'ý u\ uý UN ti l% OO -2-r- OOOOOO rn 0 ........... 0. OOOOOOO-NOO r2 000

00 -ý c- CO N c-- C` O tll c- OOO V- 0O 00 ,NN 00

OOOOOOOOOOOOOOOr

T- IA ltd c- N w0 uý r -t 0rr . d- Nl I'D N\ 00C.

0088600 t- 000 I'D c-

N K' OO0000000000O0OO

NNNN

OOOOOOOOOO8O O" O N\ O

ONN Ký Co 0OU0O00000N uo KN

N L- r-

0000000000000000

0000000000000000

N " OOOOOOOOOOOUOOOO

4 0000000000000000

NNN

00000000000000000

NM ýO 000000000000OM _ä

%lo 0

0000o00oOÖaQoo0o

r- OOOOOOO0O6NOOOOO

0o co OOOOOOOOON8 OOOOO

OÖ0 NO

0 ul% Kl 0NN %lo OOOOOOOOO'-9OOOOO

T- T" G T- 0000u, N CO tt\ N OOOO0OOOO c- OO c- O Uý C

c '- OOvc0N T- N "D O 4-

OOO O) OO c- U c- NO CU O jt c-

%. D

rn

0 . -.: E

aý 4)

C) (ii ýý A

N -S UN u\ ý10 kOw 'U rv \ r+1 \ ýf \L\\'\\\\

Cxlý N CO (\l u\ J P\\ cl, )N)cNN

r\ 4-

-78-

t(N P. O N- 0 oo O UN

to q O O M o Ö Ö Ö O O O O O O to i - Co N

0 0 U1 '. O 4- '. D

Z: O O O O O O O 40 O O O - O O Ö O

0

t O O -* r O O O O O O O a- O r O O N . ,

1 , -I 0 %0

d c d q O O O O O O O O O O O O O O K1

tý O d

544 1

M C- O N "

0 to r-i Ö " O O O O O O O O O O O O c- -e O c- Q

4 v -ý O

00 t- -2" 0 O

- 0 O O O O O O O O O O O O N

- 4

c ya v

d 0 I r cý d q O O O O O O O O O O O O O O O O +1

ý tH rl tý

f. 0

w U . to 1ý 4.4 "r+

93 0 0 ö Z O O O O O O O O O O O O O O O O e

$4 r-4 G) $4 0 1Q

ý 4-+ " a o o 0 0 0 0 0 0 0 o 0 0 0 0 0. o o O ° N- to

(ä Z 9.4 W q O o 0 o O O o 0 0 o O o 0 0 0 4 cä r-A $Z 1 :Z 10 P. Ö _ r.

P. d N _' la 9) (D p4 JL4

E W r4 O O O O O O O O O O O O O O O N L('

O u

3 U] 0

gl el ri 4j . -p "

l 03 O

N -: g am ä O O O O O O O O O O O O O O O

"0 ö

94 to o

4) $ r4 t+\ O 00 00 O O O ri d d fr., " . 1 . " " " " O

N M O O '- N 0 0 0 0 0 0 c; 0 c- 0 rý U 0 d 92 G) 0 to O 4- 4- " Co 0 P r4 cS cn z O O N N1 O O O O O O O c-

r L-

N N r O 0

4-4 d, ri 47 0 M +3

0 0 0 N O 4- O uN +? " " " " " " " . '. O Ký N- r N

4 O O O N1 \ O c- O C r tN c- c- N N C` 4-4 0

0 E m

b Co r

0 %0 1 ' i = N\ r 0 0 IM N O O O O O O O O O E N

IK- t Z 0 P e- N

r- ' N O Ü U'\ M c-

� . " (0

� s. r' ºý1 ý- N ý- r 0 0 0 0 0 0 0 0 0 H ý

0 C) a %Z am m

8 ' O - c Ö

M c 4 ý %M ' 93 O 0 q- M e- r+\ N c - C' O O O O O O O O .ý C)

U d

4r -P

d t+1 l1ý l! ý lfý \D IU IO %o % D O p 41 cm NN + \ Lr\ \ \ \ '. D \ \ \ \ \ ti N d \ \ \ O \ QO r \ r OD 4- 00 N-

A a 00 CO N to cm t+\ r N N -ý - c- ý- N N U Ö

-79-

of thermal stratification (able:; 7,9 and 11). Accumulation

at the surface tends to be more rapid and intense, a

reflectioxi of the greater stability of the water column of Kettle Tiere.

The initial appearance of A. st)iroides and A. solitaria is typically during mid summer and there is some evidence to support Reynolds (1971) contention that A. solit=; rim is

a summer forra while A. Bpiroicles f. spiroides Kleb. may be

classed as a spring form, together with A. circinalis. These differences are more clearly visible in Kettle Here.

The growth of Anh. gIos-naguAe generally mirrors that of A. cir. cinal: ie. This similarity. - is more distinctly seen in

Kettle 1-? ere, the appearance of populations in . Ihite Here

tends to be more variable. - The last of the blue-green algae to form blooms of any

significant extent is Microgstia (principally M. aerufrinosa,

DI. wese*ert*ii is also present in small amounts in both

meres). These blooms are formed before thermal stratification

breaks down in the autumn. Relatively high overwintering

planktonic populations of Microc;, stis are found.

Although not included in tables 7 to 12, G_ ochinulqta

made brief appearances in the phytoplankton of White Mere

during June and July. Surface blooms of this alga were

observed to occur almost siruultaz, eously in Newton Nere,

where it frequently becomes dominant, and in White Mere.

No occurrGncco were recorded for Iýettle Piere.

During periods of surface bloom formation, with a

large proportion of the algal population within a few

millimeters of the surface, a sudden change in the

physiology: ical activity of the heterocystous species occurs.

This change is revealed by the extensive formation of

akinetee, and is often so rapid, particularly in Kettle

Mere, that sovie stages are eaoily missed altogether (Tables

13 to 15).

Akinete production (sporulati(On) is initiated at the

formation of a surface bloom of the t; pecieo concerned, irre: pent the of the size of the pop; i1, ati. sri. 6: ': q11

po,, u1 tic: tis of . ý. _ iroi, i.: ý Ae_ f oi±tFi rý, 14it}7 little

e113e in the P1ci1::: ton st the ý: u: re tzr.: e, Esporulý, to riss rorad7ly

-80- -

CT t i t

to

" I 1 I

(D rj UN U'% ' Ö

co c N (D 4-) ri t

10 H 4. ) 4-) H co

O F3 N

1 1 1 1 1 1 N 0 4-4

y N O r l0 " . --ti�yý

a c d

Q 3 O F\ - - 1 1

ý rl (D ö "

0 o t 1 O 1 cff " CT' ýi r

4.3 V F ö

" 4-4 IL4

c N O N

N rte. " " " "r4

P4 o O r- r 1 + a Q a)

" a 4-3 to 10 2 0 0 4i

0 X 4-4

`ý 1 t N I I + tý

Q A W "ri 0

Kl N ý{ ý "r i r r ý lý H

H cJ 3 I M O O p 'd 4)

"rl N LO -P + I t O 1 + 0 CO d)

" Ö

(D C) it G) rq 4-ý 4)

to

4.3 4-4 -H r,

0 O -H 4-) IH -Y4 H 4)

C- \

C- \ \

00 \ Oý " .)

4 Z DC 3 Ä - O r N Q

N l N Z

+ 1 0 x 3

-81-

Lf's

C-

C,

4) El 0

r4

CH 0 N m co 4, ss U

C) P,

d

N

. r{

C) r1 H

H 0)

1 I I 1 1 0 t I 1 1 0 I " 0

p 4)

CH

" 1 1 O 1 1 I 1 O O 1 1 1 1 1 0

Ir: N

4-) cd

C: 0 b O I I I 1 O I 1 1 t 1 1 1 1 P a)

4) H H

r i O 0 ". "

4 t I O I O O O O N 1 1 1 t I N " k O t

t o O

N rI C I I I I I I I I I I I I I I

. rj (D r-4 0 > to P4

f\l O N

1 O O 1 O 1 1 1 1 1 1 1 I 1 O C

4-)

1 O O 1 1 O O O r O O O I O 0) 4J

I T3 N ýi W Hi

x RS H

LLO (Z 1 O O t 1 1 1 1 1 1 t 1 1 1 0

" H 0 U

! L, t -I CO +) r-I 1

N y 0 t H "rl

" H

a) H 4) 54 SL4 "

. s . O' ro

_ 1 O O 1 1 1 1 I 1 1 t I 1 t E+

O C) +) +s G)

to 4-) 0) O 4-3

4 O ++

ri C) K\ '-0 C- 00 CO CO 00 O r N 4) H G)

c5 \ \ ºn \ N V3 C trl .D \ N L\ \ O + A N L\ ' N N- N c- v- N ý N T- CO Z " l .a

-82-

N a'

d C)

13 0 w m to 1

4-4 0 a) bo c0 -p U

C) P4 Q) +) a) . r.,

u*%

a) ri Cd H

0)

c6 r, ý' 111t1tt1I111OOO

tO O

O

"1t1I11$1I1IOOONN . ct

cß

11111i1111111111 li§ O N111111111111II11

. 41

to d

1IIIIII1OO1 t\ O 1r

. r1 co

111111I11I 1OOOON

Q

O

"5 1OO1Oi0000000001

O H

t1111IOON111111O

tr] wOOOOOOOOII1I1OOO

U

v1f1111O1OO1OOO11

O O K1 -f' !! 'ý ltd ýO ýO ý ýD c\0 r

c3 \\N\O\ 00 ý' \I I-

ý' 00 \ cV C` Q o0 CC) N U\ Nr ý- N /O NN4rN

b

ti

m

d ri A2 cat

H

0

_C j }ý

as the gene: rally 1nnr[, er populations of A; circinrt1is and

A. flosýaclizze, although for these species too, population

size is no requirement for sporulation. The clearly

demarkated succession of species in i, ottle Ller. e leads to

blooms predominantly of a single species, and this situation

further results in a distinct succession of sporulation

throughout the summer. The more or less simultaneous

blooming of several species in White Mere, though present

in different proportions, results in a more or leas

simultaneous sporulation. Unlike the AnabHona species,

AJ)h. - flea-Rnuae rarely produces akinetee in large numbers.

An absence of akinetes of thin species in Kettle Mere was

reported by Griffiths (1925), and none were observed either

during 1974 or 1975" Under the unusual weather conditions

of the summer of 1 976 , however, large numbers were produced

in Kettle : era during October.

Sedirientation.

With the differentiation of akinutes the alga concerned

is effectively removed as a component of the phytoplankton.

This is through rapid sedimentation rather than a

redistribution within the water column. The loss of gas

vacuoles and the formation of cyanophycin granules as

akinetes mature leads to an increase in. density.

Sedimentation is also has-toned by the clumping of large

numbers of ak., i. netes and fragmented filaments in a matrix of

mucilage incorporating bacteria. Rates of sedimentation of

total particulate natter are given in figs. 17 to 19.

Although there is some correlation of this with the

eed _mentation of algal material (figs. 20 to 24), this is

frequently obscured by the greater contribution of resuspen-

ded matter, particulsrlj in isothermal conditions. This

source of interference would be lower in Kettle fiere than

in White i"iere. This may be reflected by t; ', e lower values

for siwpended solid:,, except during periods of surface

blooming, in 1 976 25 and 26) . Tli(: total ctrnount of

riaterial trapped in Kettle Mere is consiratently lower,

even during periods of atzblo otrutifiontion. she

coiltributxwic of the, differ : nt i; ec: ieEi to the total

-84-

Cý

7 0

f". 0 C)

C7 '

ký

cý

J

a

.ý ýS v

Y ). w

c

i

_iU

Ic cl lrU

n) iV t, r ýý

-85-

13

13

A.

a

nQ

"oý

rý I c3m

co ®1%Q

tfý " ON Ö" r- "ri Q

R) tja

Q1 7ý

cQ

-3: 0

H c 4-3 a

:_ cc «c. N 00 Pi

r ri r-H C)

:ü4. ) 4) . +. ) ".. i

(hto °T44a; d) Lr, U u\

Ll r.. r,; T--

cy

c

8

U L-ý1

G7

. 94 4-i

O

C)

; T. Li) 2

-86-

r)

b

U U

a sI

U)

0 Y="

r,

H

i

(9. Aalq 'I

L, " _-r

-87-

sedimentation rates are clearly seperated, which reflects

the succession of the species in the water and of the

sequence of sporulation'(Figs. 2u and 21). The contribution

of Ilicroerstis to the total sedimentation rates in the

autumn is particularly marked.

Resuspended detritus forms the main component of the

trapped material in White E ere during; the winter months.

Sedimentation of diatoms, mostly Asterionella, as the

water column becomes less turbulent during the spring,

forms the major proportion of sedinented material during

April and May. There follows a marked decline in the amount

of material trapped. Although this decline marks a five-

fold reduction of the amount of sedimenting material, the

values are twice as high as those in Kettle Mere. This is

mainly due to the sedimentation of larger populations of

algal material, but the greater degree of turbulence within

the whole water body may be an important factor.. The

sedimentation rates obtained (between 0.5 and 2.0 U m-2

day-1 for kettle Mere and 1.0 to 5.0 gm2 day-1 for. White

ilere) are similar to those reported for other small hard-

water lakes (White and Wetzel, 1975). The inorganic residue

left after combustion was higher in material sedirnented from White Mere than from Kettle Mere. In the case caf White Mere the percentage ash varied from 45,,, to 60w of the

total weight, in Kettle Mere from 20, to 30/0. The greater

proportion of organic material in the seston of Kettle

Mere is probably a reflection of the differences between the

sediments of the two reres. , The rates of sedimentation of the principal forms of

blue-green algal material are given in figs. 20 to 24.

Peaks of sedimentation of algal material correspond with

periods of sporulation and/or the decline of . surface blooms.

nigh rates of sedimentation of i iicrucyritis colonies,

particularly in Kettle More (Figs. 20,21 and 23), may have

been brought about bfr a greater depth of mixing, as

indicated by the deepened epi?. imn: i. on in the autumn. A

similar response of i iicroc-:: tis 1, rß: 7 - been observed in

Rostheriiu 1%. ere b; i Reynolds and Ro,; ers (1976). The high

rates of ssedi mentation of 1Lr. ihT iizý in 4i'lhite tiers in Apr. il

-88-

8

1-`

113

:$

~J

I I

,y

, I ( co s.

c LZ "r1 ý-"

iJ k Q, O r-i C: S U

"r4 x 4-4 cl, ni

C9 G7 V J N N CJ

aý 4 J Ll

C' L

'A in r-4 r-4 ýA " 4-1 r4

". ý! C c, O O O

. f, .ý

r-

ý) W (ý ý" .1 aC: m rt j

-89-

D

ýr

O "

91

13

ýD rn

L1

L7

[ F ý'

i

1

rf

G

il

r-1

V

'e'1

l

. -ý-

fý

"r"t

Cam : c7 E

4.4 E- U v

U .1

cZ

C,

fý U (-)

ýý c"ý

ä .ý

ýý r. U

F. '_-ý

ý, ý , S,

ýr ý'. )

1. ̂. C,

t- ý

D

". ýDý`

V .ý t

1. \

()

I

-go-

®\I %-

v

p,

1

ti uý Dq

D cý

CIO

Dn

r4 co . ri 4-3 rt Cl D DK ra ro V H tom: 4. c: t

o c) F4 to U

+i C. 11 N P. DPa. ® r;

. d) :J1O N u] 1 i. 1

N G=i tU U] rI t' W r-i

ri () O0 Fi C. 4 ai C-. a

L. ý_ý fl

(`j nl ) r'+ l' li

p :a ýýL". z. t: i,: n'j

-91-

U f. ý

0

E4 C-)

Cl)

c,

ti to

0

ti

'3

N

ä

cý

in c ýa t-ý

71 'L r..

-'s i; �

PJ (cG c_)

-92-

co U, t4

4-4 0

Ci

fti

UV

41 0

Qf "r4 ý U) U

N . 14

%10 i: i It Q In 9), C- c -s

Ii+

"" Cý c'y 1O

V. C)

C> !n .ý-! i. !

ý ýN r-1 - +ý Li "n Lý '

NOU "'

. 1"

N W' rA r-1 t= CJ riC. ' " -0 G CZ 0 .0 tý: MO

P4 = !ýO E-4 41 tb EU

aD

F'j

I--

i

f 'el

Ll a

Dp

(sue : ý, s - ý -t. ýrý Luýý: 1ý, üý, ) p uº Oi. ". zaýý: nTi

(Sv'rýi: lýýtii L': 1,. . i{t:.. j

5Ti)'LUt_ý UJ ý~`_ý ~.. ý"ý `,

I, LI IT 1

-93-

B Dý7

a cl

A®D

G, G7 "-"

Z 0 Lr\ U ýJ N

r1 4) ij ri O

cý x ii) cn <ý c: a

bý

DQ

Q

\I/D` i9

pD

®ý p Uýp

ýlnj `N:. 1

w 1/ D tJ , ýDý 0

ot

1 äý©D ^

C- C .

1 1 ti

ý, 't a3 C V v t'

c; :, r-1 ti

.V G1 YY {ý

,4 co

'iJ ., L

r)

r! 2

4-1

ti

fýG

-94-

0 L> F3

( ©

I D ! 3

I I ( N

" C) 1 v

4-4

r N

ri Q u;

1 ..

r.

1 ""

. - )N c- r-'

0 (C

Ll

1

'

I

0

C) hJ

N

t

N H

+ý

'(C s.: C)

N V 0. i

N H 'cý

£ 0

U

Q) C)

J

Q)

QI C6

r1 El 0

V

W

Vl

f`I

to 14 CL)

ý: .' u) (C H 0 'H . C;

l '' ul

1-1

cn J) -4 > 0 i

0 `` D of C'i ýD

/7

ri D0 \\

Eý D41 ""'Aoc>

Q

N

rý

0

4ý

U/ 0

r

Cl)

N

Gý

®a ýQ ý"

® Dý

D ti/ýý

L-- \,..; Li \ _r '''i fJ r- Cý \ ,_r

qi

Ea

. 1..

.u U

: r:

>,

r, ý, s

r, .ý

t__-1 ; ý..

4ý

-95-

Land May of 1 976 (Fig, 24) marks the rapid decline of the

planktonic population during mid May.

From the large amounts of material sedimenting through

the water column it would appear that most of the population,

including gas vacuolate filaments and colonies, had

suffered a reduction in buoyancy. It has been suggested

that the activity of proteolytic enzymes in crude cell

extracts allowed to stand increases the fragility of gas

vacuoles (Jalsby, 1971), thus a slight increase of turbulence

could bring about hydrostatic collapse of the vaouoles and

induce sedimentation. This is particularly noticeable in the

1976 White Mere population of A. circinalis (Fig. 24) which

had not undergone sporulation at the time of sedimentation.

In the winter, and also-at intervals between the

sedimentation of surface blooms, little algal material is

trapped. This indicates an absence of resuspension from. the

sediments of large amounts of algal material in the period

subsequent to sedimentation, as well as the lack of

further large planktonic populations of the species

concerned.

Benthic Populations.

Fluctuations in the amount of algal material in the

surface sediments are given in figs. 27,29 and 31 for

Nettle Mere and figs. 26,30 and 32 for. White Tiere. Large

increases in the numbers of intact akixietes were found in

both meres in 1974 and 1975. The peak in early June is

produced by the sedir_ientation of ak. inotes of A circina_li^

and that in August mainly by akinetes of A. floc-amuse. The

late sure-mer forms contribute to akinete numbers to a

smaller extent, particularly in Kettle Mere, where akinetes

of A. F$1)iroidos are found in early Au,; u. -3t and of

A. solitaries in late August or early Lieptember. A

particularly large: number of akinetes of A. s, piro: ide was

observed in the surface mud of tiihite I"iere in $epteriber,

1 974E (Fig. 28). The unusually hot and drys weather during

the surlrier of 1976 produced a different pattern of

succession and spor. ulation. 1 cirý:: ýýi Lio Was aiccomp . trtik: d

by an e: urJ_y bloom of A. s lon- 3ý ýr-w at Lhe c? nd of T: Fty in

-96-

C3 tU ý' : -y rý

(J

r. ý

a rg ynA

Qri

r.. ®0

0

..:.

n-ý H U: t " 4) (j

. aý TJ ." ® rj ai G uý c ý

41 v (to " ti 'O CJ I -ý Q% rI n

Q Rý

(r: C) CJ C1 G. l ci

r f. I J' "r "1

111) 171 N y' +) rl .

J

H C. C C) ri U H4 Ný " f61 .ý

+ý J u L

E: "S & )

^3 :, 6 0 et L % :s . Q U)

4- H Q0

Nl ýl U1 Cý U1 c- c-

-97-

0

0

0

UC

Q

ý3

7 C ýA ýý

0

Ci

00

h4

4.3

I I co

( ! . f I C

a-1 13

Ö 1- r

ýý. -c ";, w a., E' ci

L1\

a.

a .y

ýý 'ýý tý.,

ý;

iý

-98-

i3

13

I

/1 o

00

lpl

0 -. `ý

. r`_jý

(J.. L;. zo IO3

el, ý

tit C r4l NIN

T-c, Ir-

,ý\ V

Q

6ý ri L7 "

I cd 4) H

" :+ a) C lU °' b 'ä cý

" Q

u £ 0 4 I L - rn o u ý r1 C -

r-+ - c) 4

Q T C: w c! :. )

s ,ý Cr r I

4-1 r- 0

nr r{ N r-i i J rS U ri G

4J Q r t,

j CJ 0

ri al F !~ O 4 i:

4-ý- 0 r-4

0 ter : ýc: o .a N ". -i E -4 0

to

4-4

0

i a w

0 M rý

nj . i4

.r

If', C:

-99-

? "ý _ý

ý 9%

C3

0 c» K-- D

O

a 0

a0 b>

ý: /: .ý

ýý <ý

A '-'

0 r1 4.. )'. "C ^J cc 0 i> . 14 U S. U t' N Q) Cr t.: "r4

E i ý2,

. -, .., d c, I J G; " i ý' r' f'. 1 ýý

I

a c, c a) c ) i;

rl c; ra I

c4 j, r c' 1 .. -. ý. 1 _`4 U

O Pu O v: _ v .J ,. 1 t4 VI A

N f~ :> H C) t H C"

e:: " ra C) 11 1 ,; f ., -' H Ö 0 0 f_

Lt \ C) !. \ tf

tý

Y t=1

J-y

fJ

6L

-100-

' ý.

.. j, ... ; ý,

N- c"

K1 tý

w

S " co

--I tr

i, t v N

4-) -r1 'LC U ýT J CJ Q)

"1 4-3

cL` 0) N C, G) t', J "

' A C3 W Cil t. )

as 4-, r c: c

U rQ E C c; ", -i r')

1ý Q) ý4 -1ý "r 1 S; .L 4) H V H

"0 cL n3 Ci O 43 -0 Aý3 r-A

El $4 11 0 C, 0 . "r1 f. E4 r+ H u

r-ý

c-) u

U)

c5 -r

0Q

13

13

C, 13

Cl''"r 1

tf\ wl t' RI ý-

t_ ll -()ä t

N

. t: 4J P., 0

;., .r

r

. r,

rz:

;ý ,. I

j': '

-101-

p "1

0 ® ̂ . ýa t-I : " C)

"-3 - 'i U] U N tý C) C)

" }. a) H P. a) W t, "N 0 4) -r i C- r-, -P CI + c\ ') c) U ;t a C) "1 U r r1 .. ý

ri ' Q; 4; ýiJ V

C) z+ 1 S' fti <U S4 4-I 71 r! 0. Cl <. "r1

4-3 N 1. ý' C) r! 1 ri ý-i J

-4. ) . -, 1) of ri 4a C c)

Gv -t --1 E-

;a 0 F -.

Cý C, J 0

0 a

rý

iA .C Fý O

Cý

G$

0ý

iýý Qý t'3

,n .' rt\ ýý c: J' ýý U114 'l C_)

L

-1 U2-

Kettle Mere. The other f3Pecief3 eniunierated did not start to

develop largr populations until the end of June. The only

contribution to Hkinete numbers in the mud, until the

sporuln. tion of A. spiroides in October, was the sporulation

of a relatively small population of A. fflos-a7uae at the

end of May. Iihite Mere, like Kettle fiere, had relatively

small populations during June, following; the collapse of

the A. cir. cinalis population in Hay. No akinetes were

produced by the latter population and none were observed

until mid July and August, when the planktonic populations

had recovered and again produced surface blooms.

Although vegetative material of the sporulating

species was identified in the sediment traps, the length of

survival in the anoxic mud of Kettle Mere is short.

Filaments are only observed in the period immediately after

sedimentation. They appear to persist longer in the mud of

White-Mere, but here too there is a rapid decline in

numbers, particularly during periods of stratification when

the hypolimnion is depleted of oxygen. A decrease of

filament numbers in the mud during the winter may be the

result of mixing into the plankton. Thus the numbers found

in the mud result from an interchange of filaments between

the mud and the plankton. Despite the rapid input of akinetes into the sediments

of both meres, there is an equally rapid decline in their

numbers. This rapidity may be riasked by the sedimentation

of akinetes of another Anribaena species, which would

increase the total numbers plotted in fibs. 27 to 32. Each

species exhibits a similar pattern, and the whole process

of sporulation and sedimentation is generally completed

within the period of stable stratification. Resuspension is

therefore unlikely to be the major cause of decreased

akinete numbers in either mere, which is corroborated by

the low numbers of akinctes found in the plankton during;

the Iautuyul and printer. After the decline in akinet¬, numbers

of thc: last ap.: ciuo to "poruJ. fate (generally A. voll', tnria or

A. ^ iroidt 3) the nurahrr. s remain. low throughout the winter

ancl the following An exc e}! tiun to tlhi:; is the l LLr,; e

muib ;r Of fljýa71(ýEý: i found in Kottl(. ' sture bes. -wo? n ", arcli and

-103-

976. in contrast to the vegetative cells of Anabaena, there

are large overwinterinj; populations of I-Iiqrocncitie-ý in the

surface sediments, particularly in 1t tle '; ere. Lise the Anabaeni species, however, an increase of numbers in the

mud is reflected by a decrease' in the plankton, within

which during the winter few colonies are to be found. The

reverse situation can be seen in Kettle Tiere where an increase of numbers in the plankton during August and September (1974) and a smaller increase in June (1976) is

paralleled by a decrease of numbers in the sedirments. The numbers of overwintering; colonies of Microcystis

on the sediments of White Mere are smaller, which, like in

the case of filamentous species, is probably a consequence

of the greater degree of turbulence. The relatively high

number of pl-snktonic colonies, as compared with that in

Kettle Mere, supports this view. Decrease in numbers of material sodim©nted during

periods of severe oxygen depletion of the hypolimnion are

also appar t, although less precipitous than that of filamentous material and akinetes. Benthie population levels are supplemented by further sedimentation of

planktonic colonies during destratificatjon in the autumn.

DL CU axOl4 The C, rowtl-!.. And Abu ahce Q Plan oni, c Pot Actions.

Despite the similarities in nutrient composition and

concentration of the two mores investijated, the dominant

blue-green algal species and also the population levels

attained are significantly different. This difference is

more clearly seen in tables 16 to 18 in. which integrated

algal standing crops for 1974 and 1976 are given, than in

tables 7 to 12 which give algal abundance at selected depths.

The large and actively growing population o±

A. circinalis in White More during the winter of 1975 to

1976 supports Reynolds (1971) data on the lower temperature

tolerance for growth of this species, which favours its

early dominance in relation to other species. The most

rapid period of growth was, however, when the water had

warmed to above 9 °C. The response of pro-bloom populations

of Aph. fos-aquae is also similar to that reported by

Reynolds (1971), the increase of numbers being moot rapid

once the water temperature had reached 12 °C. The 1974

occurrence of Ajh. flos-aquae in White Mere was unusual in

that a planktonic population did not develop until the

termination of thermal stratification, possibly the result

of an increase of nutrient concentration in the surface

waters.

Although occurring in both meres, Anh. _

floe-ýzcýuae is

atypical of the filamentous species in that larger

populations were present in Kette Nero for the years

investigated (except 1976) than in White Mere. hammer

(1964) reports that this species did not increase until

the temperature had exceeded 20 0C. A high temperature

requirement for growth may explain the differences between

these two meres, with a more rapid wariaing-up and higher

temperatures reached by the epilit: uiion of Kettle Berl

encouraging more rapid gror:: tli. This inay also account for

the large populations found in White [,, ere during; the hot

weather of 1 97o . Large po, )ul atiozny Of this specie", eitre,

hoviever, reported for White here in 1967 and 1968 and also

for. other : LarE, e mc: xý. ý ýlieyzioJ_c? ,1 971 )

Gen lie Fand I,: uloney (1 969) reportod that a Phosphorus

L

-105-

N "r1 4-) C7 c- co

N c ` O 0 0 c - O 0 N

i cC

J

r f. e " U

N N

O C H 0 O " cy- IL) U) cö

0 0 N fý

si ) N

O Q E H

O N KN

U Cd 4-4 t! 1 r (`J r" v c- Q O O Q +- rl C 44

w r-I o " "r1 Cl) 4.1 S, a) cd

N "rl "rl 0 0

cC H +) S4 Z 0 -f rI N U Cam- H

ON 0 E 1 4-+ c- u) O O O Q O Q O Q O Q

"" 0 "rr P1 w "

" U] (1) j-i Pl .d C) 0 f-I El: (a S-r O 0 U f~ F. N 'Z3

(2) H r t0 r. 4") O SI 0 I 4-3 fti

-* O c- I'D U, \ C- 'Ü

i

0 0 p, vJ 0 0 a0 N Ctl O N O\ _; I- u1 0 l ý

-P r + SI " z4 -rl

'-1

O cý f-1 RS

r s~ cý !J rI Ul

co 0 4 O O O O O O O O O

r

Lo r. =4 "

0 H

U) "r1 H ýO z C-

"rl C 00 Q1 0 c- c- Vi " .. 7- Q "

r-{ ! -a C'N r- " VIN " ,a ri c- c- Pal c- c- 0 c- c- 0 T--

Q) w ". 0 Co cxi . ö \ \ a. '1, 0 c- r c- (T c- N r N

0' N

f

-106. -

N

0 V-

O ", i 0 0

r- O üQ

ON

to cd

4) H 9

CH HO"

H f-1 O 4) "ri

CH Q O "I~ 0

Si 20 d) U -4 C- E1 4+ Q\

Or

ß, " 01 00

ýo $-4

O F; ¬"; tU 0:

Ucj U G)

h7 4) 01 "ri

rl N ý' '0 -ri P-0

Pr c0 cß u)

y "rl ri 4)

cü O

TJ V JN

-i-3 co .. ý

to .

t, --

r4 c1 H

ä Pl

U) r1

U O Fa U

. r{

O

U) O

H 4i

. t;

Cd 4) . r. { H 0 to

Q

,d 0

. rj P-

L;

CT

0

H co ri

"r-I U

U

rn CO Kl% c- c

OONrrr N\ NN

OOOOOOO O- O

O 0000 U\

rNNOO

Mr Iý

ý: 0000 c-' c- VD Nr0

"C 00 tNr\ v- N\ O\ N rc\ c- c- O00 V- N O*N Oti rNN

u U1 l- c--

N N CC) UN Z- Lfl%

-N ýO K1 cam-- C`

Q) "0 "0 LI- Cl- 00 CO ol\ ý3 U ßc7 \\a; O r\ Cý \ t` 2 c-' N (7" r fý c- cU O\ N

-107-

P,

to .ý N 43 C.

a' N N . 00

ý" C) .t N r N T- O\ O o

" U ", 1

U] , c. d)

F, ' G) O co H 0 O " V Ui

4-) 1 0 1A

N chi 4

O O Q r

º3 ri -1 C) 4 -1 9

4-1 o .

.rj C 4-1 t-l ) C cd

ö ö 0 H " +)

z 0 n H C)

° 4 T o 0 0 0 0 0 P4 0. 0 P4 $4

o C m U Z O 'L3

N H ". 4 -P 0

0 1 ri 4 M " " CO r P P

(11 O O O M cV C`

W P

v] 4 ct "rý

9 > Ui V.

i fi Ui 10 0 o 4) i a " rn

H

6

: : : : : :

00 rg

p r1 Lr\ O r r r co V

H d

M ýD Cý" iM ONOONiN

00 - 0 cO cam- CZ L['

rn 0 0 0 0 0 0 0 r

C, ti C, a) 43 . rj

C o 0 0 0 0 0 0 rn

N ýO r Q'

N oO %0 L-

O N1 c- 1 C, - c-

0 U'S C7\

%0 %110 %10

MNNN

L

-108-

concentration of 1 .0 LM supports a population of 105 cells

r_il of Anh. floc-aquae and that low concentrations of

phosphorus can support bloom populations. Flowcver, in

continuous cultures of diatoms and bacteria Fuhs at al. (1972) found that growth is limited below 3 ý1g 1-1

phosphorus and that the atomic ratio of nitrogen to

phosphorus required for maximum growth is 16: 1. A ratio of

about 55: 1, determined from analysis of water samples from

Canadarago Lake, leads the authors to suggest that

phosphorus will be the limiting nutrient in the lake

studied (Fulis e: t al. , 197-2) . atomic ratio of nitrogen

to phosphorus was found to be much lower in the meres

studied (from 6: 1 to 1: 1), which would indicate that

nitrogen rather than phosphorus would become the first

limiting nutrient. Studies on Yhaeodactylum by Goldman

(1976) showed that enrichment of phosphorus had no effect

on growth, whereas increasing concentrations of nitrate

produced a linear response. Concentrations of phosphate and.

nitrate used by Goldman (1976) were high in comparison to

those found in the rneres. Despite this, however, no

significant depletion of phosphorus or nitrogen was observed

in the surface waters of the meres. Studies on the mineral

nutrition of M. aeriginosa by Gerloff and Skoog (1957) also

indicate that the requirement of nitrogen is higher than

that of phosphorus, while the converse nay be true for

those taxes which are capable of nitrogen fixation. The

concentration of nitrate in the mere.:: was not observed to

fall to very low levels (i. e. below 0.1 to 0.05 me 1-1),

even during periods when non nitrogen-fixing species were

most abundant. The contribution of nitrogen-fixing species

to the nitrogen budget of the lake may be important in

maintaining the concentration of nitrate during stratificat-

ion and surface b?. oorls (Fog; and Stewart, 1965). Nutrient

depletion, particularly of nitrate, occasionally approaching

limiting levels, was reported to occur in cruse : sere

o7 dts, 1971 ). In Kettle I, iere and White iiere nutrient

deficiency does not appear to limit the growth of the

planktonic re(n al,; uc.

J,. factor of particular : imj)ortnnc; J in 1 hc,, control uf

-109-

growth of planktonic populations in the meres is the amount

of light which is received by the orga: nisrn. Large

quantities of dissolved organic matter in Kettle here are

supplied principally through the autumnal leaf fall of the

surrounding woodland and its subsequent decomposition. This

material strongly absorbs photosynthetically active

radiation (Kirk, 1976), and therefore its presence in the

water will reduce the depth of the photic zone considerably. The potential for photosynthetic growth, particularly during turbulent conditions, is therefore greatly diminished in comparison to that of White Moro.

With the reduction of turbulence during the early

summer the buoyant algal material floats towards the

surface and frequently forms surface scums. This placoc the

alga under completely diff037ent conditions as compared with those that exist while suspended within the water. Light

intensity is high and carbon dioxide may become depleted in

the surface layers, factors which will tend to reduce algal

growth (Tailing, 1976). The earlier stratification of Kettle

Mere places a further imposition on population sizes. The

observed increase of pH values suggests that the proportion of freely available carbon dioxide to that of other inorganic carbon ions is decreased as a result of the

abundance of algae in the surface layers (Table 6).

The speed at which an algal species disappears as a

component of the phytoplankton is frequently more rapid than its appearance at the water surface. Under conditions

of depletion of free carbon dioxide as the pH increases,

and supersaturatioii of oxygen in the surface: waters and

under a high degree of insolation, photo-oxidative death of bloom forming algae may be one of the main factors causing their rapid deteý'. oration (Abeliovich and : ýhilo, 1972; Shilo, 1975). Although the temperature of the surface

waters in temperate lakes may approach, for limited periods during the suruier, that in the Israeli fish ponds examined by the above authors, high water temperature is not it requirement for photo-oxidmtionn (Abc: liovich and 6hilo, 1972). Photo-oxidation of blooming; blue-E; re,, ni algae under natural ooud±tjo"13 has been reported by Bluff et fll..

(1976) and by

-110-

Boyd et al. (1975).

Photo-oxidative death may not be the only cause for

the collapse of blooming population. of algae. Vaeil' eve.

and Levitin (1974) suggest a combination of factors,

including changes in the partial pressure of carbon dioxide

and oxygen, nutrient deficiency and bacterial and/or viral

infection. Algae-lysing bacteria have been isolated from

eutrophic lakes by Daft et al. (1975) and found that their

numbers are directly related to the algal biomass. Lycis

may also result from a change in environmental conditions

affecting the balance between the algae and bacteria.

Although bacteria were observed in large numbers within

clumps of senescent algal material, their appearance is

probably a consequence of algal breakdown rather than the

cause of it. The rate of breaks'own increased considerably

when algal material collected from natural bloom populations

was transferred and kept in the laboratory.

Although vegetative cell material of the summer

surface blooms frequently disintegrates and disappears

almost-without trace, this is not always the case. The

sudden disappearance of a large population of A. circinalis

in White Mere during mid May (1976) appears to be the

result of sedimentation of intact vegetative filaments onto

the sediments, This material did not reappear in the

plankton until July, and meanwhile filament numbero in the

sediments had greatly diminished. Populations of r1i'crooystis

are also secliraented and remained in the riud in a relatively

intact state, being more resistant than the filamentous

species to bacterial attack.

orulation And Germ-' n,.,., t ion.

: 3evcr. al rý:. )orts ro1rted the production of ak. in©tes in

1aborator. y batch culturoo to conditions of nutrient

depletion. is o1 : (1965) found that phot3phate deficiuncy

induced c orulation of A. c-j_zn«ricn. Deficicncif: o of both

phom-rhate and nitrate stir., ulated sI>orulution in

n (Glad, --, . 191, i). Observatimis on the

s )orulrit1on of planktonic sp, iciea in the mores, however, do

not indicatt: that nutrient rx critical role.

61

-111-

The very rapidity of the phenomenon, when compared with

algal growth, suggests that depletion of nitrate and

phosphate is not the main factor. These compounds are

always in abundant cuDply even in the water filtered from

algal scums. Other nutrients, such as the trace elements,

which are in sub-optimal concentrations in some lakes, may be of importance (Goldman, 1965). Ixtracellular products,

which accumulate in batch cultures, have been shown to

stimulate sporulation of C. licheniforrie (Fisher and Wolk,

1976). This could also be of importance in the natural

environment, when lyric and decay of part of the population,

enhanced by the activity of bacteria at elevated temperatur-

es, will result in an increased concentration of extra-

cellular products within the' scion.

However, when considerizlg. the range of species present

and the differing population sizes accumulated in a bloom

at various times of the summer period in two chemically different meres, and the similarity between the response of

organisms, it is likely that other factors, not present in

a laboratory batch culture, may also be involved.

The regular coincidence of akinete formation with the

occurrence of surface blooms for all the species studied implies that conditions at the water surface are the

stimulatory agents. A sudden increase of light intensity as

the algal material reaches the surface would explain the

observed suddenness of the onset of ak. inete production and its limitation to b__oo. je produced during the sur: imer.

Treatment with u. v. light (253 nn at U. 62 m'r; cm-2) has been

shown to induce earlier sporulation of A. _

doliolur (LAngh

et cl., 1967). Supporting evidence for the role of high

light intensity is found in the absence of sporulation of

surface blovris formed in on-In periods during the winter.

The density of the overwinterink; planktonic population

of A. ci rcinalis in White vIere during late 1 975 and early 197() r ,: £ic!: ed thoso at which thi ß specios would Eýporulat..

duri : L; the suii er, and considerably exceedod the ýo, ýu1 tiori: >

nt which the other -3peci, c_s would r3porul_ette, 3wrticular_l_. y in

Kettle ITnfortunutely this population had oudrienly dccr, oti. -ý,.

d iii i umboro (:, er .:, 'o '1110) bt iforo t}a normui1

L

-112-

"sur.: mer spurulation period" had begun.

Filamentous vegetative material aeldoni survives for

long period:. alter its 3Odinentation from e. declining bloom.

Although blue-green algae (i.. muscorun and A. ? i. ncir. icý-a. )

have been found to be more resistant to anaerobic

conditions than other taxa, the reported naxirlum length of

survival is ten days (Dokulil, 1971). It hao been suggested

that the resistance of planktonic species which form

surface blooms to anoxic conditions is the result of a

metabolic change from aerobic to anaerobic respiration (13ud' ina et a!., 1973). However, the unhealthy state of

post-bloori populations would curtail the length of survival. This frequently' results in the reduction of vegetative

material to an indetectable level, particularly in Kettle

Mere. Akinetes appear to be much more resistant structures

to the attack of bacteria and to anaerobic conditions

prevailing; in the lake sediments. This may be inferred by

the presence of appara. itly healthy and intact ekineterý in

the mud throughout the year, although often in considerably

reduced numbers when compared with the input during

sporulation. The rapid decline of akinete numbers in the sediments

would suggest that this was not so, that in fact akinetes

were as susceptible to the effects of anoxic coldi. tions and bacterial attack as vegetative material. Apart from the

presence of akinetes during periods when nortL-tlity would be

expected to be high, such as the interval between the

onset of stratification and the sedimerntation of a fresh

crop o Fan-f. nctee, the reduction in iiurnbere iu equally

rapid where alcine. tes have uedineiited in the period

immediately following this brcahdown of thermal. etratil: i c-gtion.

This can be seen in White I: erc , 'here the: . f1_uctutºtion. 3 in

nusabber. u of aki netei of thooc specie: 3 which , 3porul_atc 1,1te

4n the uum ner follow a :; iL�ilar pattern to that in I. ettle.

T": ere, wi: icii is still L; trrltified at that tir: e . fºprtrt i rcri bacteria other mßy 1)t3 of

`1 ()21; )) 0I-): ervE cI the, j: ri?: Jc ict. of : 1,

c';: 'tr. id on the rnl: _net es of f: _t cý_l'i tf. n1 , rt ; r_ 1ton i ci

., )--i i iiii la" (_ ffJ____ Lt: iu. " viir. J? )t: t11' tut Üt, f

L

-113-

I. ortarek, 1958) in : settle : Iere, White i.: ere and Blake Mere.

)? reserved mater. ial from these mores was examined l by Canter (1 954) who plact s this fungus in a new fillec1 es, Rhi2 o 'hon

akinetimm. This species, and also a similar one which

attacks the akinetes of e 'k. sr. iro: icie s were observed in

Kettle :: ere and White here. On some occasions up to ß01

of the akinctes of these species were infected by the fungi.

The infection appears to be limited to material which is

still in the plankton, those akinetea, which have sedinented

were found to be' free from attack, protected, perhaps, by

the anoxic conditions. Although fungal disease may be an important factor controlling akinete numbers of the above

species, and nay in part account for the small populations

of these algae found in the rieres, other An b enct species

seem not to be affected.

Hence, it is likely that the main cause of the rapid

reduction of akinete numbers is germination shortly after

maturation. Germination, without a resting period has been

observed in cultures of many blue-green algae, in the

original growth medium (Pay, 1969m; Khan, 1974), or on

transfer to fresh medium or following replenishment of

deficient nutrients (Glade, 1914). Immediate germination

would account for the observed recovery of many sporulating

planktonic po ulation: s about a month after the collapse of

a bloom. Al: illete germination is probably more important in

Kettle Fiere than in Whito ! lern, wheru the nur beres of

surviving veg.. tative filaments are generally higher . Although few actual instances of the proces,; of gornination

have bean obsc, "vcd, it was fou. zld th; tt up to 6O, of the

total number of skinlete:.; were empty while : till in the

plankton. ft_luments were sometimes ol)6ervod during

those periods, l, ut these could equally ro ; alt from

fi_l_ar: eiit f raga&; lltatioll and the roueding-off. of the terminal

cells . she germination of alk: inotes tmmuy be . stirultated by a

charts e of comc. itions in the water as th,; y t}lrou,, '. i the

water column,. 1htrtrieiit doneeirtratir'n: r could be of

ir.., ortnrce, b. zt r. -ctin t--(-t -r. iilr, rj_tioF- l; Eotlre(. nn t11H

2' : ý;; UIlU( 3 Of 'L. rLJ t;, 1) chei icaJ_Ly t,, nd f'11i211i1CýIi', r cmJ_1"ý ]. ±verI; L

L

-114-.

neres suggests that light intensity is, the critical factor.

Also the similarity between a range of species, admittedly

of a single genus, indicate that environmental rather than

genetically determined factors control the response of

akinetes. With the sedimentation of akinetes onto the surface

sediments in Kettle Mere and the deeper areas of White Mere,

the light intensity to which the material is exposed is

greatly reduced. Light has been shown to be a requirement

for germination (Harder, 1917a., b; üingh and :; unites, 1974),

and also that light of the red region of the spectrtun in most effective (Harder, 1917b; Reddy et al., 1975).

Although light does penetrate to the sediments in both

meres, the wavelengths above 600 nm are absorbed more

rapidly than the shorter wavelengths by water, the most

penetrating wavelength being at 560 nm (Kawana, 1975).

Wavelengths below 500 nrn, in the blue and violet regions of

the spectrum, are also strongly absorbed by wster (Nestlake, 1966). I. aushik and Kumar (1970) found that

germination of two blue-green algae, A. dolioliun and Fischerella mucicola, could proceed in light of all wave-

lengths. No light requirement was apparant for the

germination of kx)h. floc-anuag (Rose, 1934). The range of

responses encountered suggests that although light is

required, the quality and quantity of light needed by the

various species-is different. However, it would not be

prudent to extrapolate from observations made in the

laboratory to possible events in the field, or from one

species. to another. Conditions, whether they be light,

chemical or other, appear to be conducive to the germination

of those akinetes which have not done so while. stil"_ in

the plant-: ton. This would be a considerable . portion since

the length of time spent in the plankton appears to be

relatively short. The non-germination of a small proportion

of akinetes in the mud leads one to speculate that light

may be the critical factor. Some of the material would be

subjected to conditions of total darkness by being covered

with sediments through d sturbance of. the surface layers

during tli& auturmnnl overturn.

-115-

Resusp©nsion of recently sediraented material is not - considered to be an important factor in the reduction of

akinete numbers, which is Lenerali., º complete while the

meres are still stratified. Fuw anirietes were observed in

the plankton once sedimentation had occurred, even during

periods of maximum decline in numberä. Those akinetea which have sedirnented onto the mud surface in Kettle Mere are not lifted into the plankton during the winter, but appear to be mixed more deeply into the mud. In White Mere,

particularly in areas where the bottom is relatively hard,

very few akinetes are observed during the winter. This may be the result of mixing upwards into the plankton, possibly followed by germination under increased light intensity,

contributing thereby to the overwinterina vegetative

. populations. Numbers in the so'ter mud are generally higher,

which may be due to a thicker covering of sediments hindering resuspension, to a greater depth of sediments in

which akirietes are found as well as to the redeposition of

suspended akinetes removed from other areas of the mere.

Overwinterinj.

Viable akinetes of Anabaena and Alhianizomenon species

have been observed in the mud of many lakes during the

winter after the mass death and subsequent sedimentation of

algal populations (Rose, 1934; Chernousova et al., 1968;

Vladimirova, 1968; Wildman et alo, 1975). The presence of

akinetes, which are generally assumed to be formed under

unfavourable conditions, leads to the conclusion that these

structures will produce by germination new vegetative

populations once more favourable conditions return in the

following spring. Reynolds (1971,1972) observed germination

of akinetes of A. cir. cinalis in Crose Mere while the lake

was still unstratified in the spring. The, inj)ortance of this for population development is considered to be

doubtful (Reynolds, 1972,1973a) since numbers were never

very large and the incr. ease of the plnnktonic populations could be accouated for by the growth of the ovorwintering stock of planktonic vegetative; material.

Fror-i the present invustiL; Fition: s oil I: ottle 1-, ere and

L.

-11 6-

White Mere the same conclusions might be drawn. The

germination of akinetes appears to take place soon after their maturation rather than in the sprint, or early summer

of the following year. The filaments produced may contribute to the revival of the planktonic vedetative population,

particularly in Kettle Mere where little vegetative

material survives a surface bloom. It is this population

which overwinters in a vegetative state in the plankton

and is particularly abundant in white Mere. Further

germination of akinetes may occur during the winter and

spring, resulting in a gradual diminution in numbers of

akinetes in the mud. This pproc'escs appears to be insignificant,

the changes involved are small and may be attributed to

other factors such as attack by pathogens or the mixing into deeper sediments. No sianzficant increase in the

evidence of germination was observed during the spring in

either mere. If the vernal germination of akinetee

contributes at all to the plý-mnktonic populations, this

contribution would be expected to be greater in Kettle Afore,

where the overwintering vegetative populations are much

smaller, than in White Mere.

From this it would appear that akinetes, rather than

acting as an overwintering mechanism, seem to protect the

algal populations from extinction during the extreme conditions of a surface bloom. The thick envelope would

protect there from bacterial attack and they are resistant

to extremes of oxygen tension, light intensity and

temperature. Thus the response of the algal species to

surface blooming and the fate of the akinetes in the

subsequent period appear to be critical for the development

of future populations.

A few comparisons between the different specie, may illustrate the point further. In Kettle 1'ý; ere during 1974 a large population of A h. flog-ngune bloomed in early June.

The numbers ra,. idly declined thereafter and no akinutes

were ob: erved. Only a very minor recovery of the oluxiktonic

population was observed,, in corit'rf,.:, t to those species which did oporitl te, and the , )opulý, itjon levels in 1975 were los:;

than 1, j of the mn:: ii. lur1 Produced in 1974- yTklu, 1976 pC)j)Ll_'it1o11

-117-

had increased sor. iowhat when compared to that pr. - sesrt In

1975, and also, unusually for Kettle 1,: er. e, alcinetes were

produced. ilthough it. is3 not possible to draw firm

conclusions from this, particularly sinco other species

undergo similar fluctuations in the comparatively extreme

conditions of Kettle Fiere, it appears that species which

sporulate regularly on blooming are "buffered" against

further great fluctuations of their abundance due to a deterioration of conditions (Lund, 1965). This can be seen

in White I"Iere, where the population abundance of the main

akinete forming species A. circinalis and A. flora-aquae

exhibits a relatively regular pattern throughout the three

years of the investigation. This does not, however, remove

the influence of environmental factors on the growth of the

vegetative population, on the production of akineten and on

the subsequent development and abundance of future

populations.

Although resistant, akinetes are not totally immune

from attack. As mentioned earlier, a large proportion of

the akinetee of A. solitaries and A. spiroides were observed

to be infected with a chytrid fungus. This would reduce' the

potential overwintering stock bf either akinetes or

filaments (produced by. germination of these species when

con pared with unaffected algal species. If such a tenuous

extrapolation is-to be trusted, then one might expect

fucdre populations to be reduced in comparison to that

which might have developed in the absence of pathogens. The

Marge fluctuations observed in the numbers of these species

in both 1; ettle here and 'white Nero, is certainly in marked

contrast to those of other sporulating species of . 1inabfiena,

. and tends to support the idea of a reduction of the

nbuf ferine; " capacity of the ahinctes due tQ attack by

pathogenic organisms. This, however, is only one factor in

a complex interaction of riany factors, and thus must not be

viewed in isolation froii the rest of the ecology of the

species cor_('ernnc: d .

she colonies o Iiicrc)c-:, tip appear to be mach more

resistant : -cructurns than the vegetative stages of the

ti:; ii cn:: pccio" to the extr"e: ico of conditions, 1711. ieii prevail

-118-

during surface blooming and subsequent sedimcntfutiou. This i-iould be expected if riicroc sties Species are to

survive at all, since-: no Morphologically distinct resting structures are produced. Changes of enzyme coz: iposition and

activity of I'i. aer. ugiiiosa were found as populations

declined in the post-blood phase (: 'ud' ina et fei., 1973).

These changes resulted in a transfer from aerobic to

anaerobic respiration, which would enable colonies to

survive in the mud. Other species, including ADh. f. J. os-

a uae and A. variebilis were also found to be capable of

anaerobic respiration in pure cultures (tiud' ina at rel. , 1973).

Viable colonies, as determined by their fJ. uoresoenee

and their ability to grow in-the laboratory from riud

samples, were found in the Qedjments of both mores between

the time of sedimentation in the autumn and the reappuarence in the plankton during the late summer. A distinct annual

migration of colonies was observed in the deeper lakes: (Sirenko et al., 1969; iopachevskiy et al., 1969; Reynolds

and Rogers, 1976) in which populations of I"Iicrocystis which

sedimented in the autumn to the mud have overwintered there,

and then a proportion of these migrated upwards through the

water column to the epilimnion the following year. The

stimulus to upward migration is probably an increase in the

reducing conditions resulting in a stimulation of

physiological activity (Topachevskiy et a)., 1969) and gas

vacuole formation (Reynolds and Rogers, 1976). A similar

pattern of the distribution of colonies was observed in

Kettle Pifere, which, due to its sheltered position and low

surface area to depth ratio, will result in relatively

undisturbed sediments. she generally lower number of colonies in the mud

during the winter in dhite IIere is probably the result of

turbult. %nce maintaining a larger proportion of the

population in suspension. he same was observed by iýeynolds

(197a) for Grose Mere, which is a. 1 so a relatively shallow body of water in relation to its surface area. In both

Kettle hic-re and White i ere, Irowever, there is an increase

of colonies in the ; nud during; the autumn prod iced by

-119-

sedimentation. Later reduction in numbers in the mud of

Kettle here is mainly due to the mixing of "colonie: i deposited on the . iurface of the mud into the deeper layers

by turbulence insufficient to lift material into the

plankton.

S

SECTION 2

GO , IT"AND CELL DIFFEi3ETJ IATIUI1 UI DER A VARIETY

OF FHY$ICAIý AIM cm iICAL CONDITIONS

hL

TI: ': WDUCýTON

Contrary to previous. investigations, which are discussed fully in the Introduction to Section 1, nutrients,

particularly orthophosphate and nitrate, do not appear to be critical factors in the control of sporulation of

natural populations. Although deficiency of orthophosphate

or nitrate may induce sporulation of the planktonic species

studied, such conditions were not encountered in the meres,

even during bloom formation. The coincidence of akinete

production with the formation of a surface bloom indicates

that the conditions prevalent at, the surface during the

summer may conceal the critical factors. such factors would include a high degree of insolation, depletion of dissolved

carbon dioxide and the accumulation of extracellular

products. A change of light quality as the algal material floats towards the water surface could also be an important

factor. These factors may act directly on the mechanism

controlling akinete di ferentiation, or indirectly through

producing a change of the growth rate or physiological

activity of the algal cells.

Although some aspects of the physiological activity of natural populations will be examined in Section 3, some problems will occur in the intepretation of this type of experimental work. Despite the fact that the incubations

themselves were performed under controlled conditions, the

environment to which the material was previously subjected in the field was only approximately known. ]: ach iiicubc. tion is thus an individual experiment as the conditions and Material chances.

Despite the inherent problems of the selection of

strains adapted to growth in the laboratory environment, it

was considered that the constancy of the conditions of,

growth and the homogeneity of the algal material would

produce useful insight into the process. of growth and differentiation.

It was to toot the validity of the suf, Gestion. s made in the of ýiectiorr 1 that the experiments on isolates of Iplz: nktoniic al, trre from the nuru2r3 herein described

wore aevioed.

rIATnr IAL; i AND MiiTiIODS

Isolation.

The samples of algal material which were used for the

attempts at . c; colation were collected, if sufficiently

abundant, from the plankton, or from mud samples. The use

of damaged or unhealthy material, for example that present during surface blooms, was avoided. Large inoculations (about 10iß volume) of the natural populations were riade into 100 ml conical flasks containing 50 ml dilute culture

media (See pace 126 for composition). The medium used was free of combined nitrogen, which suppressed the growth of

non nitrogen-fixing species of'algae. The rate of growth of bacteria was also initially reduced. in the early phases of

growth after inoculation into the media free of combined

nitrogen. ümall amounts of the mud samples were also

enriched with nutrient media. Although growth would oocur in the natural, unenriched, samples, this was stimulated in

enriched cultures. In the. early stages of growth of these

mixed cultures, all the heterocystous epecies which were

originally present in the sample were still represented,

and this enabled attempts to be made to isolate and grow them all.

As well as the selection of nitrogen-fixing blue-green

algae through the use of media free of combined nitrogen,

temperature treatments were also used in some cases to the

selective disadvantage of other algae (Allen and itanier,

1968). This was only applied to the mud samples collected during the late autumn and winter of 1974, from which a

, wide range of algal taxa were cultivated. The samples were heated at 30 0C for 30 rain in a water bath and then

returned to normal culture conditions. This treatment

appeared to be effective in killing off most of the algae

apart from the blue-green species. Wierinea (1968) has

used this technique to remove non-sporing bacteria from

cultures of akinete forming blue-green algae. However, due

to the potential selection of resistant, strains this

"technique was not generally used here. Sirlilarly, chemical treatments such as the use of aiitibioties, and treatment

with u. v. light were also avoided. Despite these precautions

-123-

selective pressures would be applied as a result of the

choice of media and the culture conditions used.

Further purification was by means of micropipetting

and sterile washing, (Eberly, 1964; Gentile and, Maloney,

1969). From the culture flask in which the algae have been

growing a drop of the suspension was placed on a sterile Petri dish. The suspension was diluted with sterile medium if rendered necessary by the density of the algal filaments.

On the same Petri dish were placed up to six drops of

sterile medium. A micro-pipette was produced by the heating

of the tip of a sterilised plugged Pasteur pipette. Once

the glass had become soft it was rapidly pulled out, thus

producing a fine tube. This was broken to a suitable length

by bending.

Transfers of small volumes of algal suspension were

made from drop to drop. A Bausch and Lomb (Rochester., 11. Y.,

U. S. A. ) stereozoom binocular microscope (up to x 60) was

used to identify and remove single filaments. After five or six washings, a single filament was transferred to a sterile 7 ml serum bottle containing 1 ml medium. This was then

incubated under the conditions described below (page 128 ).

this procedure was repeated for each species which was isolated. Also for each species, at least 20 separate filaments were isolated and incubated into serum bottles.

The manipulations were carried out under aseptic conditions in a Iii croflow Limited (Fleet, runts. ) inoculation chamber. Because of the continuous current of sterile air which

passes out through the ckbinet, care was taken that the

drops of liquid did not dry out. When necessary additions

of sterile distilled water were made. In those isolations in which growtrl occurred further

transfers were made into sterile media in 1 C)U nl conical

flasks. The material was also es. +mined microscopically for

Purity. Altholzßli with this isolation procedure unial. gal isolates were obtained, no axenic cultures were produced. gable 19 gives n list of strains isolated and cul turod.

While st: veral species survived for liraited periods in

laboratory culture, it was nut pe.:: 3ib]. e to maintain them for lcrig periods in culture. The mo: 5t uL1 ptc:, ble L;, pcciuo,

.L

-124-

Table 19. List of the species obtained and maintained in

culture, their source and method of isolation.

Collected and isolated in 1974.

Species Site Source : Micro- manip.

E'utrient enrichment

Temperature treatment

Anh. f1 os-Flr}uae !P1 Mud + + +

Anh. floc-educe WTI Mud + + +

Anahaerin up. (? ) Wm Mud + + -

A. floc-nguae 111.1 Pl + + -

A. : niroides NM Pl + + - A. circinalis WPI Pl + + - A. sol itaria E. ß Pl + + -

Sites: - aI = Kettle Mere, WN White Mero, rIT-I = Newton More,

EM = The Mere , Ellesmere.

Source: - Pl = Plankton. (? ) = tent ativo ident ification.

which grew well under the conditions used, appeared to be

Aph. floc-aquae, followed by the smaller celled Anabasna

species. This is reflected in the range of species that it

was possible to maintain for more than two years in culture. The larger celled Anahaena species, particularly A. soiroides

and A. solitaria, but also A. circinalis were, by contrast, difficult to maintain for any length of time. This type of

planktonic algae has, however, been successfully isolated

and grown. Volk and Phinney (1968) report the culture of

A. mpiroides in Gerloff et al. 's (1950) modification of Chu's (1942) number. 10 medium. Mc Lachlan and Gorham's (1961) ASTA medium was also successfully used by these

authors.

A factor which may be of importance in the survival

of single filaments of planktonic algae is their buoyancy.

Eberly (1964) suggests that the trapping of single filaments

in the meniscus and the subsequent drying of the medium may be a factor contributing to the low percentage of survival

of inocula of single filament: 3. The effect of this factor

was reduced in the isolation inocula by twice or thrice

daily mixirr;; continuous agitation rapidly breaks up the

filaments of the planktonic species Inver tieatcd . These

factors may contribute to the low degreo of survival of the

-125-

large celled species. These species, however., appear to be

inherently more fragile in any case, filaments and cells

were often observed to break up during the process of

micromanipulation and sterile washing, despite careful handling. Gerloff et al. (1950) observed that cultures of

planktonic blue-green algae are only established with large

inocula. The importance of optimal concentrations of

glycollic acid, an extracellular product, in the medium

for the rapid growth of cultures is discussed by Fogg (1965).

Culture Media.

Of the various media tested, ASPI 4 supported the

active growth of the greatest range of the planktonic

species. Other media tried, in decreasing, order of success,

were: - Allen and Arnon's (1955) medium, A. floh-actuaa

medium (Slalsby and ichelbarger, 1968), ABM 1 (Gorham at R_ ., 1964) and G©rloff at al. 's (1950) modification of Chu' E; (1942) number 10 medium.

The composition of the ASPI 4 medium used in this

investigation is basically similar to the ASPI 1 medium

used by Gorham at al. (1964), (Table 20). The major ions

are present in the same concentrations but the trace

elements are generally increased four-fold. Ferric chloride is increased ten-fol in concentration and sodium molybdate is an extra constituent. Sodium EDTA is also increased

five-fold in concentration to the AUM 1 medium.

Preparation Of Stock Solutions And-r-ledia.

All the conetituente of the medium were of analytical

grade cheriicale and were dissolved in Pyrex giae distilled

water. Sufficient of each salt was dissolved in one litre

of distilled water

over that used in

make up 11 of the

dic3eolved in water

hydrochloric acid.

dark.

to produce a thou$and-fold concentration the medium. Thus 1 r1 of each stock would

medium. The ferric chloride wac

acidified to a Iii of about 1 .5 with

The stock solutions were stored in the

äufl, i c" mt volume: of each of the otock -; Olutions t7t r. o added to hair the final volume of distilled ': r+ti. r to

-126-

Table 20. Composition of AB:.. 4 medium.. (Smith and Foy, 1 974 )

Compound

A Full-ytrenj; th rid 1 µrao,.

1

I)iluýe mg 1-

i3 (10,. ) E1rnol

1-T

Podium nitrate (NaII03) ** 850 10000 85 1000

Magnesium sulphate 49 200 4.9 20

(I"IgSO1 . 71120)

Magnesium chloride 41 200 4.1 20 0"IgC12.611

20) Calcium chloride 29 200 2.9 20 (CaC12.2H20)

Di-potassiurz hydrogen ortho- 17.4 100 1.74 10

phosphate (K2IiPO4)

Di-sodium hydrogen ortho- 14.2 100 1.42 10

HPO ) phosphate (Na 2 4

Ethylobinediaminetetraaeetie 35 100 3.5 10

acid, disodiuri salt (Ila2 EDTA)

Ferric chloride (FeC13)' 6.5 40 0.65 4.0 Boric acid (H3BO3) 8.0 16U 0.8 16

Manganous chloride . 4.0 28 0.4 2.8

(MnOl2 . 411 20)

Zinc chloride (ZnCl2) 1.2 12.8 0.12 1.28

Cobaltouß chloride 0.08 0.32 0.008 0.032 (CoCl2.6H20)

Cupric chloride 0.004 0.0032/0.0004 0.00032 (CuCl2.21I20 )

Sodium molybdate 2.4 10 0.24 1.0 (ITa2DIo04.2h20)

**, Sodium nitrate was generally omitted.

produce the final concentration of the constituents ne

given in table 20. Section A of the table (full otrenCtti)

givos the concentrations usually uuecl for this rmedium,

which is similar to that often uced for laboratory batch

cultures. The concentrations given in section B of the

table, which are 10, j of the full , strength conct ntrrations,

WFG:: that , nenernlly used for the culture of algae j, -, plated

-127-

from the raerr: e. The dilute raediura WEM used throughout for

most of the experimental work on there isolates.

The additions of stock solutionb were made in the

order given in the table, and between each addition the

medium was mixed. The orthophosphate salts were not

generally added at this stage except when dilute media were

prepared. After making up almost to the final volume with

distilled water, the pH was checked using a Pye Unicam

Limited (Cambridge, England) Model 290 pH meter and

adjusted to a value of 7.5 with dilute sodium hydroxide

and hydrochloric acid solutions. Initially the pH of the

medium was low, due to the acidified solution of ferric

chloride. After adjustment of the pH the medium was made

up to the final volume, taking into account any further

-additions which may be required.

50 ml of the medium was dispensed into 100 ml conical

flasks (or 100 ml in 250 ml flasks), which were then

plugged with non-absorbent cotton wool. The plugs were

capped with aluminium foil. These small volumes of medium,

and other apparatus, were steam sterilized at 120 oC

(15 p. s. i. g., = 10.35 kN m-2) for 15 min. Larger volumes of

medium (above 1 1) were autoclaved for 25 min at the above

pressure.

After cooling, appropriate additions of orthophosphate

were made: This was done to avoid the precipitation of

polyvalent cations which would occur if the whole medium

had been autoclaved (Good and Izawa, 1972). Precipitation

was particularly noticeable in the full strength medium. In

the dilute medium, however, precipitation was not apparent,

or only of ight, if all the constituents were mixed before

autoclaving. The prepared sterile medium was not used for at least

24 h after <: utoelaving, and before use the p11 of a spare

aliquot was checked.

The Culture Of Algae.

1t) ml conical ftn: skc were used for the maintenance of

stock cultures and also for some of the experiment:, i1 work.

lnoculnti, -n$ into frw:: iii raediu111 were carried out under aseptic

-128-

conditions usin sterile plugged Pasteur pipettes. An

inoculurm of about 2,, volume was used . Stoclc " cultures were

grown under "Daylight" fluorescent tubes (Ecko, I"'. azda or

Phillips) at a luminous flux of 1000 to 2000 lux. (For

further details on light conditions, see the section on "Light, Fluorescent" below, page 129 ). The temperature was

ambient laboratory temperatures, which fluctuated during

the year from 16 to 26 0C. The'naterial was grown under

static conditions, as it was found that many of the species

isolated did not grow when agitated. This was also observed

for A. sxpi roides by Volk and Phinney (1968). Many blue-green

algae do not Crow if shaken tog- vigorously (Pay and Fogg,

1962) and -others show optimal shaking speeds (1'ocg and Than Tun, 1960). A degree of mixing is, however, generally

considered beneficial for rapid growth, and an evenly

suspended culture and homogeneity of treatment, particularly

of light, of all the cells, The species which were isolated

appear to be highly sensitive to agitation, but are

generally maintained in a suspended, evenly distributed

condition in static cultures duo to the presence of gas

vacuoles within the cells and an absence of large quantities

of mucilage. A limited degree of mixing was supplied

through gentle swirling of the cultures three or four times

a day. The isolates of Aph. f_los-ßryuae appear to be an

exception to the above. Although the filaments were evenly

suspended in static culture, this species could tolerate

mixing, shaking or gentle aeration through a sinter. This

enabled these isolates to be grown in the type of

continuous culture apparatus described by Pty and

Kulasooriya (1973).

Buffers.

hero buffered nt dia were used , the buffer was added

after adjusting the p: i of the medium before fiutoclavinC.

Buffers used ', are tris (} ydroxynýeth;, ý1} acainornethene (THIS)

sied ýý-2-hydro yothylpitýera7ine-Tý' -? _-ethailot. ulýýhvnic acid T}le cation binding cajtci. ties of these compounds

are ne 1if, ibý_ý: (Good e al., 1. x)66) and they apppear, through

not beirr; effected by autocl"iv: iur-, to be heut eteble (smith

-129-

and Foy, 1 971,4). Three dipeptide- buffers, DL-alai l- lycine,

DL-alanrl DL-alrtnine and T, -glycil-g? ycine, were also

te. ited. A ranee of concentration:, from 0.5 to 4.0

were used for =iiýPi::: 3 and : iltlä, and a range of 0.05 to 0.5

g 1-1 for the peptide buffers. All the buffers except TC{IÜ

were obtained from Signa Chemical Co. Ltd., äurrey. ri"IZIS

was obtained from B. D. 11. Ltd.

Teml2erature.

Much of the initial experimental work was done under

ambient temperature conditions in the laboratory. Although

the temperature varies-significantly throughout the year,

fluctuations were generally small for the duration of a

short-term experiment of a few hours. Ranges are given with

results of each experiment where controlled conditions were

not used and the fluctuations significant.

Experiments on growth rates were carried out under

controlled conditions in a Grant (Cambridge, LnCland)

thermostatic water bath. A refrigeration coil was required

to maintain a constant temperature below 25 °C, particularly

during the summer months. Experiments were carried out at

20 ±1 °C .

During 1975 a Gallenkamp (London) cooled illuminated

incubator, model IIi 287 was used for the storage of stock

cultures and some experimäntal work. Either continuous

illumination or a 12 h light - 12 h dark cycle was used.

For the maintenance of stock cultures a temperature of

10 0C was selected. for other experimental work 15 oC

was

used.

Li t; ht

Fluorescent.

stock cultures were grown under lateral illumination

by, "Daylight" fluorescent tubes. she light output had a

peak e1lergy at 58? 0 nn, falling off rapidly at increasing

wavelen, -t11s to 25, -' of the peak cutout at 650 nr. 1 and 5/, ') at

7Ui, rill. At shorter wavelengths the decline i": ras l e. 3,; rapid,

with a shoulder at 4 30 I'm of 40,, of the peak output. Energy

at ý: a\re t: ". t; t}13 below 3 j0 In was norligib e \ý'ig. 3--J)

ý.

-1: 5U-

Fig. 33. Spectral distribution of energy output of

"Daylight" fluorescent tubes. Redrawn from Thorn Ltd.

Technical Pocket Book.

100

75

50

25

0 300 400 500 600 700

Wavelength, nm

Vertical scale: - mll nm band width for a 1500 mm tube at 65 W

The light intensity used for stock cultures was 1000 to

2000 lux, and for general experimental purposes 2500 lux.

For some experiments using fluorescent tubes intensities

of up to 10000 lux (10 klux) were obtained. Data will be

given where relevant. An Lvans Electroseleniwn Ltd. (Halstead, Essex portafle photoelectric photometer was

used- to measure luminous flux.

High Intensity.

tilhen a very high light intensity was required to

illuninatu algal cultures, a G. L. C. Ltd. (London) tungsten

halogen lamp (from I"Ialhari Photographic, London) was used.

The energy output from the lamp, which was housed in a

reflector, was 1415 W m-2 at 500 ram from the filament. A

value of 45 klux was obtained under the same conditions. The wattage was measured with a Kip-) and Zonen (Delft,

Holland) model AL 4 F: icrova galvanometer and a cocnpen:; ated

therr: onile type CA 1.

Due to the high output of heat from this lamp, the

culture ve ; el i were protected by a water, cooled Per: pev

shield internored Bett een lartip Ctwi cultures "1 11:, was in

-131-

the form of a covered shallow tray with provieiori for a

rapid flow of cooling water between the eurfucess. The upper

surface of the shield was not lamm than 500 rrrr from the

lamp fi. lamexit, to prevent local overheating;. Cultures for

incubation were placed immediately below the shield. A

mall fan was used to circulate the air between the shield

and the cultures and also above the shield. Temperature

control was effective, with no more than a2 0C rise in

the temperature of water placed under the shield during a 5h period of the maximum intensity used.

Approximately 7O, 'of the total power output of the

lamp is absorbed by the water-filled shield. That this was

primarily in the red and longer wavelengths of the energy

spectrum can be determined from the greater degree of

transmission of luminous fluX., About 4Uij of the light

measured using a photometer is absorbed. The maximum

luminous flux at the liquid culture surface was 15 klux,

and the wattage 270 11 m 2. The cultures were about 600 mm.

from the filament.

To produce a more even distribution of light throughout

the culture and a more precis Ly determined light climate,

the incubations were done in plastic Petri dishes. 15 ml of

the algal suspension was pipetted into each dish, producing

a shallow pool 2 mm deep. The cultures were covered with

the plastic lids to reduce evaporation and the volumes were

made up to a pre-calibrated mark with distilled water if

required.

A12h light - 12 11 dark cycle was used. The temperature

did not vary by more than 1 °C (from 20 °C) throughout the

incubation period. This was for a maxiriur1 of one week. As well as using natural populationo of planktonic

algal material collected from the mere., cultures of the

laboratory gr. own i solateoo were used. This material war, not

buffered by using any of the compounds described in the

section on buffers (above, page 123 ). The algal isolates

were initially pr. epL red as described in the section on the

culture of algae (Pace 1,27 ) and for each strain throe

replicate-, were treated. Two "stages of 1'rowth of the stock

Culture?:; wc.: re'. u feel LAB U Zporinnertal ciatorial. These were

-1ý2-

actively groFzing material in the mid logarithmic phc. aso ,

and post logarithmic, stationary phase cultures. The

re;; pect-i ve ages of the cultures, under the conditions used

for the growth of stock cultures, were 8 and 17 days. From

cultures of these algae material was harvested and

incubated further under the conditions of strong irradiation

described above.

Small volumes (about 1.0 ml) of the incubations were

collected daily from each replicate for microscopic

examination.

A luminous flux of up to 10 klux wa obtained using

fluorescent tubes without heating of the cultures. A fan

was sufficiently effective in mni. ritainin¬; the ambient room

temperature in the vicinity of the culture area, so that

no further precautions were required when fluorescent tubes

were used as the light source. Apart fron this detail the

procedure for high light intensity experiments was the

same as that described above. Due to the reduced -fire risk,

experiments were also performed under continuous illumination.

Monochromatic, Action Spectra. . To obtain light of a sufficiently high level of energy

and of a precisely known and narrow wave-band, a range of

interference filters were used in conjunction with a Rank

Aldis Tutor 2 transparency projector. The interference

filters, of 47 mm effective diameter, were obtained from

Carl Zeiss, Jena (C. Z. Scientific Instruments Ltd., London).

The half width (the range of wavelengths transmitted at

half the peak energy tratnsm_zission) for the range of filters

used was 6 to 9 nni. A li Lhtpr. oof filter holder was fitted to

the condenser of the projector such that the rim of the

filter was in contact with the concen er. This ensured that

the filter was normal to the incident light. The condenser

was adjusted so that it would be focussed at the maximum

distance allowed. oiith this Bettir. the beam would be

approximately parallel. Shift of the peak wavelenCths to a

shorter wavelencth would be minimal for angles of tilt up

to 1 Uv. Thhi s shift in proportioria]_ to the square of the

tilt, fi71gle and would be F bout , of the wuveleIl1 th for

I

-1_ý

; lf-10C:. nor: l_t1 to t}'; (E: inc . de :t

light hC: am, an Ct': t. rý, ure of 2". C t'i7"UCýuce r c'" ., no rlý_ý... ýý,: 1r1ko

c'r_:. _ ge in the on.

f1J_ ýer c ial'i? Cte: ri Ectico was obtained fro t}'. e I':: f. 21i1 i(ic. t: ltrc ': 3

data :.. }1<ii: ts '_. Ct Cýit£iý Uf; ue (and,

u?.: -, o from of

Gr(t105 1. lrJY . Yet'"(, 'iu. J

'-ý upon f?

/ 1I( ý' C4 11,. _....

c". 4 V_ upon

The Y)o`. Jkex, of the p-)Y") j ector Li: of a. 3 Vi i) !! Fi'+; ý.: 3 Ai1. J_G}1

produced an en: -, rte,:, flu, ": of 3 ': 1 ri-` at 5C() to 6C'() r. il: l from

the light source with the filters in. plcac . : °hE3 J>o^: i Lion of

the incuý)atio_i vessels was varied . io cis to maintain a vu

cono trtnt energy flux throuühJ, the experiment for each

wave? _e}zg; th. 7)u(: to the fru*ittmcy of lamp

automatic record was ýýept of the length of bu. rn_ý. ný;. An

electric ctoc.: we.,, -, svvlitc_1(. d on or Off vile :t : L: it; }lt oy)c: r: ýteti

relay. A cadImiuam üulp''}tide photo-conductive cell wee used

and the circuit wired no that the relay was activated when

the light ' eveý_ increased above pan. tarbiý: ar,, value set u: 3in

a T)otentior! eter. The recorded tii, c in therefore the time

which has elapsed since the projector was switched on to

the time when -witched off or t' r. ou h bu:! 1) failure. All l_ the

componexit a were obtained through Radio Oý1),, Arct3 Cor1poI'? Hnt S

Ltdd' London.

The a ý_ t^ilter7 :ý ý_ used were the algal . solute grown

under the condition dC: EscribeCL in the section on the

Pn -fe 1 27 . T"! --o .: cat: x_ : 11 was tii:; e: t when in

the early o; iri t: rzic pha e of growth. Three re;, lici tern of

each _E?: 3 at each wavelength wter(. -, incl. ii:, Itede The

incubation Yt:: E 1; W -rc 10 mm UZF±�; ic .:? iE: (: . rO1)h )t 01 :1 ter

This E3?.: 'ü? ^ed call even iIIII. -lirlatiou

over the , >11rface

of the ctl-Iture. Although -the cul tsrc. s w(. 1vu not C'<; 1 ß. 1f i Clý

tue Ut most of the isolates : icl. ectod for this

evo: L7_yv sus, ? 7t'. nd dl " 2)4Me, ho-o(-, vor, became

})L1U; '_1: _ý; aid floated to the Hltr1aco of the ']i : ý1Lf[1" N: l: 'tý 1.

to h": II. -ICdI due to the f=!: all area of il on.

. '. ra11.. v'. rý_rl

wrmý poSitiuno d nor. ýr. -i1_ to t1iE-e I. nciüc2ilt l_. a betr: a aad- : ueh

-1: )-

-11'3: a "Ic; rL.; ' flu, -, ö.! 1Lta r: a rucuiv"d. Y', '-. Cl.

waE 'L-,. ouseci in a 1±. F; ht 'tight bo; c tiritli t11(:, projector sIrii ii. rij,

t}: rough :a holt it onto ý--n . 2hß to:,, pcrrituro dIid not e: --reed

the ambi nt tý . "Ytt. ]Rýitu ? out:; i d6 the ox, and the e: c. -crir, ent. ^. a

Wore executed at c: ter 7F? rature of 23 OC. (, nu. W r. "o' eng th Q

l4'7xýnj :a alr$o dolle n; 10 1 C. 5) C, thfý npp ra us, at

that time, being houst. c. in n C(lü-roori.

mt the termination L )-f incubation ;. period, t_unorally

after about 7ý h, the reJ-licrýter were cýxrý. i±ncci ciicroz copica--

1ýy.

Ultra-violet.

or irradi&itiorl of ulaa1 raut.: ria1 withi u. v. 1i lit, tý 0

Hank-via (31our; h, Berk,: hiru) fiuorcscenco lamp I: Qdul 19 way

used. Tki ru-tcd to-ýu l out put at 5C0 im i fror ti, i: -2 -2 lamp Was l; vi ra , o" which 1V 'ki m vi w-3 in t. ho rci; iori of

3'o 370 27x2. di, flor3nce i: l the rt:: )1.1ý. t of heatink, up

of the ? amp during, operation,, which in also measured 'by the

-therrio ie us-AA

she cu? tares word prepared in the swne way as the,

material used for the action spectrum (above), i. e. they

were in the early logarithr iic phc ie of growth. 15 ml I.

al_± quots were dispensed into pla. Jt: Lc Ye. tr: i. dishes ac!. d. were.

thee: Of 't-i,. -, U iI'"i. i i to

2 lr. Dur- rrf the z) r aci of ixr.: d"on the eovrs 'Jere 2 -(,

ft

Ci "1'" tº__tc r the i: Y' at 1e 1. ý, ]. Et J`± d t': G Cam:, t? C :. r:: p-lc.. Ci 'E'lld

she prates p: :c":: 1 under

'For .. QC'ig - er x Uji to 1 l) ;! of lo 1,. -vE; '_: i of

L1. V. Fl : 3inmi1_rir ilrOCC. 1. ure was i. tcio tool ,

h1. C1^It, VUT for

t jý C7 1C ýU ;L ý_ciý "QZ_.

i ý'ae'_^C' l1: if Cý" . ý_

Il j- .,, @X r'2 rý `-lE: iv I. ýit11]^2i1

'2ho atlUtlilt on 011(2.119.

V'1^f tiT COVF, 2'. __r c the E

of layers of 0,2mc _, of nittji1, '. A "(-a"iccd t1

t3; 'tG11: 11; O rncli tiori by . boll- 5U.,,. A' iii. i

YE? `ý. j ? (_ý Cü2. ttL1 t? s were further incubated tind (? r normatl.

i-"_1.1-uaJ. n. at 3_U11.

iýL1L' tw'7C? Tc !i C11. J 7i( LLtJ: t 1()Z1 Ci(3: iCr. l )l': ý i! L)C)VC tJ41`k?

perfor!,. ed at I-oi':, ': L11 lilÜZt Zlý ý. '! ti L)7 : story 1 C'; 1Y t; Z'; l't t1]'L'.; of

-135-

Lxper. i hunt a at controlled wt; ro dont3 in :. l

water bath at 22 0C. In t1ies"e experir,: entE the norc', zl

f uoreocent 1. J_. 11r1inaticni of 800 lux was ll?? iý_f; C: i?. ̂ . tOCl with -2

u. v. radiation of-10 ,+m. The culture-, were; crown izi

250 : "11 conioul 1a. 31: s which were otopperc: 2 wi to non

absorbent cotton wool harms. The tray or. vwhiah the f1s0lts

were put in the crater bath was moved : slowly -uh rough itbout 15 linear oruci]. 1 Ltions per in co that rý1Jr_ the fla., Acs

rece-5_ved an equal Eiuaunt of light but thu cooaitcnts were not

unduly' Sha1zon" ý''he'' algal $u$poncii0 n: i v ere illuminated

continually throughout the duration of t; he ex1jar. irnent .

x a_ Iýýiteri ? Asse :: anent Of

T: icroscopic ex imination Of each of the three repl is teD

were made at (usually daily) interv: tiF; during nn experiment

and at the end of the experä^zeýýtý7_ period.. 'mints were

made of heterociyst and akinete frc cjuency Fis described in

the, Methods to 2oc tion 1 (rage 43 ;. Obuervatio: s were

also made on the appearence of the r-illral cultures and of

the filaments and cells.

Determinations of Cell density, and from this

calculat: +. ono of the growth rate, were made uuing an L`vci. nm

klectroc; eien±um Ltd (Halstead, Essex) colorimeter. A red

filter was used. The mean doubling time, G, in hours, was

calculated fron the relative growth constant, k (Fogg,

1965). The relationship between o. )t-i_cal density readings

and cell density ', zcs found to be linear (Nef±crt, 1 971 ).

loG1 v ODt -1 log10 ÜJ0

t ihere t=t Lrn intorwil in }hcau. rG )etween

r*ieasurcr1ent of OD vag u ,: ̂ .

ÜD Optic il don Ly a±ter. t hours.

0. UJ() - Optical cien:; ityy when t --

To cnlou1at' G: - G-0.301

16,

X36

Rl: jULT:;

Growth.

'the 'pattern of growth exi: ibiteci by the algal isolates

is of tree lc;, rpi (. EL]. . '"i. E C: 'ioidal form. The two strains of

Arph. isolated re:; pectively from Kettle Mere

and ýI_hite ',.. ere, we. 'e the crost extensively used of the

iF, o_! atoms from the rieros for growth experiments. This wren

due to the ease of culture., rapid growth, t)od suspending

qualities and lack of clumping, and a close correlation

between cell nurý. 'aers and optical dentmit4-. Data for the

grow l; h constant, k, in log10 units day-1 and the mean

doiib7 in g time, G, in clays, for different conditions; fire

given in table 21 . The data presented here, and in other

tables, are the mean of three replicates.

Table 21 . Growth rates of isolates of .4hf. los-aS uae.

leraperature: - 20 0C. Light: - 3700 lux, continuouf.; i11,. r ±natinn. 10jo inoculuri.

Piedium (ASM 4) Kettle

k hero

G Wh: itcj

k Mora

G

10; x, +1,103, +P04 0.207 1.45 0.128 2.36

" -I' J3 " 0.1 60 1.88 0.164 1.83

50iÄ, +110 ," 0.156 1.93 0.196 1.56

to _1; 03, it 0.119 2.54. 0.252 1.19

Teinperatuuro :- 23 to 28 qC (Moan = 25

continuous illumiiiý-ition. 2ý) i. rioculum.

10iß, +I? C: 3, +PU4 0.176 1.71

_1iO3 11 0.261 1.15

5C,; +11t 3, it C). ', U1 1 . G0

,: -I]0 ,"0.272 1.11

00) . Licht: - 2500 lux,

0.207 1.45

0.176 1.71

0.326 0.92

0.301 1.00

: note: - Values represent the maximum rates achieved, over a

two day period.

ýýhr. i rates obtained were similar to tLo. -e:, of other

fi? _'':. lentoucý blue-green algae under similar conditions (l''ny

and F: ul asooriya, 1 97,45; Smith and 2P'oy, 1')74; Foy et 11.. , 1 ; 76). Gro r li in riedie co.: t: sir _rýt a :; ourcc: of co ibined

the 7 : ý7 i1- nt. ý_y lower, than

-137-

groiith in media free of combined nit. °ogen in the Kettle

.. ere ct ' in. The reverse tends to be true for the '. hhite i-iero strain ,

--0_

thcupJI the d ffereiice! 3 in both otrn r, were sr alý_. : 3oth strz"i.: _ns e h1 bit Q similar response, to an

increase of 'CE; L1perature, with an increase of growth rate,

despite the lo or light intensity used. Increase of biorna: s

of both strains proceeds at a sir filar. rate. Cultures in dilute medium (1Q,; full strength, column

B, table 20) had similar maximum rates as those in the more

concentrated (5C;

' of full strength) media. The , row, h

curves in the two media follow similar patterns until the

'10th to 14th day after inoculation, when nutrients

orthophooDhate) in the dilute mediuri are exhausted. Tho

final yield is from. t: ro to three times higher in the

concentrated media than in the dilute. $irailarities of

gro. ith rmtos in media of d;. ff. er. ent concentrations was shown

by Shubert (1974) for äconede: 3rrius.

'ºi±th a 2; c inoculurn, u lag phase of 5 or 6 days wen

observed. This was reduced to 4 or 5 days when the size of

the inoculum was increased to 10,,, ". No change in the runximum

growth rate or final yield was produced.

Buffers.

During the period of active growth there was a marked. increase of pH. Several buffers were used to maintain a

constant ph throughout the growth cycle. Of those tested,

only TR: f proved to be to:: ic to the two strains of

Apl,. flo-R. , Iu ,e used. High concentrations (above 0.02,: ß w/v)

of the dipoppti'e buffers were also dctrimmýmtal to grswth. In the case of the dä- ýeý_, +; ide buf. ' ̀ 'ers this was probably not

a result of --direct toxicity, but through the encourcgement

of excessive bacterial growth. Concentrations of below

O. O1; ý w/v provod to no effective in maintaining a stable p-'I

throujllout the. growth of the alga and also reduced the

amount of bacterial g;. rowth. Macro:: copically the cultures

Zookerl healthy, and viewc: d micro;;, copical7. y the mater.? n1

ýaýppeared nor: n. al. Filaments in cultures buf'fcsred with

were, however, unwiually long. This v. a;,

probably due to a reduction of frc1; _. 'nentation as a renul. t of

-1 33-

the absence of h. eterocy. sts, even in media

which suggests that U 1ycyl-ý 1, ºcine, the

di-popticle , is utilised by the filga as a

combined nitrogen. This is interesting in

that relatively high concentretiona of ni-

free of nitrate, F: i:! lplet3t of

Source of

view of the fact

Irate do not

completely inhibit differentiation.

HEPI. S buffer, used by ämith and Foy (1974) was

observed to have the least c! ffect on algal growth and

morphology of the range of buffers tooted (But see table 26).

Growth constants and mean doubling times in 10/'; AM4

medium buffered with a range of concentrations of lIET i

are given in table 22. "

Table 22. Effect of IL: Yl'J. i buffer on the growth of

planktonic algal isolates.

Temperature: - 24 to 25 0C. Light: - 3500 lux, continuous fluorescent. k in log, 0 units day-1, G in days.

Ash* f]os-a ime Anaba'ena sp (7 IL PB 1I isolate WZ1 isolate ttilhito More g 1-ý kGkGkG

0 0.261 1.15 0.238.1.26 0.249 1.21

0.5 0.261 1.15 0.261 1.15 0.318 0.94

1.0 0.162 1.85 0.219 1.37 0.382 0.78

2.0 0.21 21 1.41 0.1 98 1.51 0.301 1.00

4.0 0.199 1.51 0.131 2.29 0.471 0.64*

8.0 0.079 3.76 Very little 0.500 0.60*

*, Of doubtful accuracy duo to the low population densities.

The molecular weicht of 230.3, and a concentration

of 0.0211, used by Smith and Foy (1974), is equivalent to

4.77 g l-1.

With increasing concentration.; of F1EY;?.: ý up to 4a 1-

the maximum growth rate of the Aph. floc-a iiae: was reduced

slightly. Thies spociei appears to be nensitive to higher

concentrationO. by contrast, strains of A. flos-LIauae and Ap, h. flo. 9-F3_n_Liae exactined by 3riitti and Foy (1974) rho: ed a r -duct-3-on of tho clean douralizig tic: io in raediti buffered with

corpared with unbuffered controls. The 'White:

, --,, re icio_ati3 of A'. 1}'1. iýý Oý-ýt( llflt? Wa; i riore Cnol1F31ti. ve to

I 'Z

-19-

concentrations of buffer above 2g 1-1 than the Kettle Ii ere

strain, which showed a Tradual decline of growth rýto with

increasing concentrations of At 2g 1-1 and below,

there was ? ittle effect on the growth rate throughout the

growth cycle, when compared to control cultures. In the

Kettle Isere strain, however, the final yield was increased

by up to 501, of the control (unbuffered) cultures, for

cultures buffered with 0-5 or 1.0 g 1- 1 HEPL: 3.

Unlike the A, h. flos-a uae strains, the growth rate of

the Anabaena sp (? ) showed a marked increase with increasing

buffer concentrations. With increasing concentrations of

'HEP: sS the lag phase was lengthened. At 8g 1-1 significant

growth did not occur until after 12 drys in culture,

whereas in the unbuffered controls and in the cultures with

-low concentrations of buffer, the lag phase was lec, 3 than

three days. A similar extension of the lag phrase of growth

was not observed in the Aph. floh-a uao cultures. The

yields obt ned in the buffered and unbuffered cultures

were similar at the same stage of growth in the different

treatments except at the highest concentrations of buffer.

pH control using this buffer was effective. The pH did

not fluctuate by more than 0.2 unite from the pä value of

7.55.

Tautrients And t$: iorulntion.

The response of the Kettle : lere and 'dhitß I4ere strain.,

of Aýýh. f. 1n: ý"'quae to varf. ous concentrations of nitrate and

orthophosphate are given in tables 23 and 24. No akinetea

were produced by the White : ere 'strain under any of the

conbinations of trea, t:: ients, even after the cultures had

l! orig passed into the stationary p}ia: ie of growth (Thblo 23).

Sj; orulation did proceed in the Kettle here strain,

part icul r: ly in old cultures (29 days after inoculation,

table 24). Akinetes were observed in younger cultures,

particularly in those with low coitcentrationo, of nitrate

(anca normal ortho: tho:. phate concentrations). !. *umbers vere

. generally low in terns of porcentaýe frequency, and only

one in 10 or one in 20 h©teroc: 'o-cc, were oboorved to have an

Ke. mciatecl ak: tnete. : L± iLer 1)ureuiitlt; e spw7uiatiOn HI. C&

ý. -

-140-

ca E

U UI "

O O

.N

ci -' cý r" 1

4i 4. ) O O 5

Fw Or r 9.1 "ý U O r-I H ö

1ý_ c3 U tO c$ Ll v

. rl ay " 4-3 0 rd co a) 4-) r Pi " i. Pi

C) d) U ü) 4-+ z U 0 O 4-4 :5 (1 "r 1 C d) Pi O C c3 0

1 0 rl 4l +) a) H O U 4.1

U1

O ,O O O

N Z z .N ". i " O cd 4.4 +3 V- F-4 0 54 i q

0 " -1 " r O c- t

cd 0

>~ +ý Pr c'3 U) r. uZ I O

r1 N L -P N N I

Q 4) "0 ri c- 0 4 1 "

"r4 C) " O

- tJ O (\j . 4. ) r4 0 " V L, M O cJ " O

4-t 0 s3 O O fti

4-) r"

U N ý. O Q)

4.4 $ O W C)

N 0

Q t: 0 a E-4 E-4

O O O O O

O O O " O O O

0 .0

0 0 0 0

IIIIII

rn OOO. O0

o N O O N

O O

O O to ý" O O O O

(v r-

M %lo Ü

N O O " O O rd H 0

Ö r4

0 0 0 0 H

U +3 " -5 Q

H

Ö U O U

t) 0

H pý

0

nc 4 .) V 4. ) N O O

, n V1ý, Y)

J\ r. lý)

U

Y'Z V1

UR L N\ c) 0 0 r11 U c) c)

. - "i

m 0 s4I L 0 c0 C4 0

.C z" 4 y 4 4'

:z "x ": i =: 's ý

-141-

H

WIN c to ;s 41 O " O O " O O " O O o c- N c- O

., -1 +) Cd o C 0 1 " " O O " O O " " + I. I) () WN N h1 N G O

o z.

0 c c- v7 U1 N - 0 -d" N

rd A I - U1 O O U -: I- " (NJ U1 rl W H " " " " " O 0 " - ýý O N O O -. t N c- O 4 z t0 a) " 4-3 -1,

V-

;4 a a) cc a ) 0 U1 (\J 0

0 0 4-( C 41 O C) " O O 1 1 1 -rf N O P-s 0 N N 'd I 1-( 0

u) 0 , c: i es

H H H F -, 0) 4+ 4-1 0 U

" C] 41 All o 0

Q ý

4-1 4-1 -H 0 c O --) t- t4r\ O

' ON 0 ö . -I N O o O O I 1 1

ti "d ,o .. ý C ct H u: 1 0

Q p O O 1 1

O 0 9 '- N N O O "

U ý ;. ( 4 I 0 c2 0 O ýn ( ý c o .. 1 0 " O 0 0 O ti

, S-ý a. ) a) cO O tll c- O O N P. { 4; +' U ti -I-' O h O

Is" O N 0 0 o ä

r\ ö 00 ül 0 (D O O " C) C) O Cl) rd

41 ýýl N

4.1 O 0 O O O iq

4-31 t- 0 4) rl O u1 O cV co O in, O O r C) N Z " 7 N . c- ON - O O r"

4-1 1 O rý n O "- o O 0 04 r-+ w c) a) fi U

. j" "N 4) +ý +ý r-1 C\J c3 .L G) U) 4ý Cw

0 " 0 'H ri P, U Ul C 0 0 0 t1 .Q £ f.. L It, C3 4) Q) h C) 4., E-4 £-+ a-) 4 -) D I1 n ,

4-a I 4. a N" O Uý I U I 4 z. e;

N -a C: V- r; C: i. , 1a c.. - V" V Q) U C", U N C. ('ý j U C) ti)

S"{ 0 f-(

-P 0) Q)

1 S (ý iý 4) U i+

0 ý+

4-' CJ -C-) 4+

Q)

C) 4) c: c; I ro r; { ýu . m r- 4 41 rJ r: "Y ý-, '

_ý 4ýj 4)

L

-142-

heterocyst frequenc-11111 ere observed at 17 and 29 days after

inoculation. The data for 21 days after inoculation,

however, does not show a similar pattern. Despite the

presence of akinotes, no increase in percentage frequency

was observed and the ratio between heterocyst and akin©te

numbers remained relatively constant throughout the

duration of the e°p-),.: ri. ment.

A similar pett:: rn was observed when the orthophosphate

concentration was varied, with a normal concentration of

nitrate. Few or no akinetes were found until 29 days after inoculation, when a marked increase was observed. Cultures

with low orthophosplint e concentrations had stopped growing before this time with no production of akinetes, Fand none

were observed in cultures grown at 10 mg 1-1 orthophosphorue. hIowever, for orthophoeT.; hate concentratione between 0.1 and 1.0 mg 1-1 about 1 of the total cell nudger produced

akinetes. A stimulation of sporulation was observed on several

occasions in media containing nitrate nitrogen, but this

response wee not consistent. Cultures grown in media

deficient in orthophosphate and nitrate, or at low

concentrations of these two ions, did not sporulrite. This

was true also of natural populations of planktonic algae (A. circinaalis and Aj, )h. floe-sause collected during the

early summers of 1 975 and 1 976) as well an the A ph.

uae isolates.

Other components of the medium, particularly trace

elements aid iron (che lated and ur_chelat(-:., d), were

px-amined in a similar manner to orthophosphate and nitrate.

:: one were found to induce sporulation to any greater

extent than that of control cultures, although growth was

occasionally greatly reduced.

The results described above were obtained from batch

cultures in unbuffered media. As mentioned in the section

on Results (Page 137) some of the di-peptide bu_°fer. o

tested had an effect on heterocy: st dif. 'erentiation. It was

thus t}iouj; ht unadvicable to use such buffers for experimento

in which the presence and frequency of hats>roc ya. ts could be

critical, is ich as the forr; ation of a'" irroter; Gent}:, 1965,

-143-

1966). ". i: ýP: ý: ý buffer was found to elir; htly ine "easo

heter. ocyst frequency during the period of active growth (Table 25). This was particularly noticai)ie for the

Table 25. Effect of HAPAJ buffer on hotorocyet frequency.

Frequency of heterocysto given arg a percentage of the total

cell number. Observations made 10 and 16 days after

inoculation. See table 22 for culture condition c.

. flos-acixr-v: Anabac. ne op. (?

HEP1 3 Kettle ; 'giere White Mere White Mere

g 1-1 10 days 16 (lays 10 days 16 days, 10 days 16 days

0 4.9 3.4 3.3 * 6.8 +ý*

0.5 6.0 4.5 4.6 2.2 6.2 5.1

1.0 6.6 .

3.6 5.0 2.1 7.1 5.4

. 2.0 5.9 5.4 - 3. ) 6.3 7.2

4.0 7.4 5.0 .-*5.6 4.2

8.0 - 4.3 - 2.0 - 6.5

** , Filaments fragment-ed. *, Breakdown of ale,, al material (lysis? ).

-, No data, little growth at time of sampling.

Aph-. flos-adac isolates, the differences between. the

Anab_zenn sp. (? ) cultures being less marked. A possible

factor may be the lower rate of growth in the buffered

cultures, although with the reduction of growth rate as the

buffer concentration increases there was no parallel

increase in heterocy: 3t frequency. Thus, after 10 days, the

buffered culture will be at an earlier, stage of the growth

cycle. This was more clearly seen in the nettle Mere strain

of Anh. floc-Lo: cluae, where, at similar stages of growth (measured by optical density), the heter. ocyot frequencies

were similar. The unbuffered culture after 10 days growth

would be comparable to the buffer, , -d cultures after 16 days.

This difference in growth pattern was not so clearly marked

in the White Mere isolate, in which the growth of unbuffered

cultures closely matched that of cultures with low

concentrations of buffer (to 1g 1-1

When akinete frequency was assessed a more distinct

-pattern was observed, particularly for the Kettle Nero

isolate of froh . f? _os-n. yune (Table 26).

-144-

Table 26. Effect of buffer on sporulation.

Frequency of aki_netes given an numbers per 100 heterocysts.

Observations made 10, "16 and 21 days after inoculation.

See table 22 for culture conditions.

g 1-1 os-

10 daIV76 16 acaua day6 21 days 10 da. -, 7o 16 days

? 21 days

0 0 0 0 13() 200 200+

0.5 0 0 18 31 65 200+

1.0 0 10 24 33 134 133

2.0 0 6 13 21 57 98

4.0 0 2 7 7 63 154 8.0 0 1 24 0 2 70

Notes. The White Miere strain of ATh. flo:. -rinuae did not

produce akinetes in any of the cultures at any time during

the experiment.

Due to the clumping arid- fragmentation of filaments,

accurate enumeration of chains of akinetee Vans; difficult.

M-1 = Kettle Mere.

Unbuffered cultures did not produce any akinotev in the

Kettle Mere strain of Aph. floc--pause throughout the period

of examination. However, even in the low concentrations of

buffer a marked stimulation of sporulation was observed

after 16 days. The density of algal material in this case

war, similar to that

culture. The latter was, by this ti

phase whereas the buffered cultures

active growth up to 21 days. Higher

buffer,. although producing cultures

in the unbuffered

ne, in the stationary

etili. showed Figno of

concentrations of

of lower bioraues at

16 days, still )roduces a stimulation of sporulatic)ri. The

pil of the media at this stage were similar (7.5 in the

buffered culture, 6.6 in the unbuf, 'ered culture, after the,

fall from a maximum of 9.5 at 12 days) . At 21 days after

_y similar, inoculation the pattern of response WHO basicall

but with ,; reatc r differences between the buffered and

unbuffered cultures. The optical density of the cultures

grown in ° .OL; 1 was only one : sixth of that of the

unbuffered cultures and the P11 Values were similar (7.6 in

-145-

the unbuffered culture, 7.7 in the buffered culture). A more "normal" pattern of growth anci sporuleition was

exhibited by the Eiiabaena ep. (? ) used. This algal strain,

of ur_de t ermined significance in the natural plar_ktonie

environment, sporulates prolifically in normal unbuffered batch culture after 5 to 7 days growth (i. e. at the end of

the logarithmic phase). In this respect it differs from the

Aph. floe-aquae isolates, which, in corirlon with many other

isolates of planktonic algae, only produces akinetes in old

cultures long in the stationary phase of growth.

After 10 days growth the unbuffered cultures, which had reached the statioziary phase, had produced large

numbers of akiretes. Akinetes. were also produced in the

buffered cultures, but to a lesser extent. The amount of

algal material in these cultt1res was less than in the

unbuffsred cultures. By the tie the buffered cultures had

reached a similar density, and also stage of growth, a

similar number of akinetes was observed to that produced in

the unbuffered cultures. Th; " effect of the retardation of

rapid growth can be clearly seen in the maximum concentration

of buffer used (8 g 1-1). In this culture little growth had

occurred by 10 days and no akinetes were observed. After. 16

days some growth had occurred and a few akinetos were

observed and by 21 days a similar number of akinetes were found as in the early stages of sporulation of the other.

cultures. The influence of culture filtrates was also examined on

the progress of sporulation in these algae (Fisher and Wolk,

1976). The data for the "baena ep. (? ) is given in table 27. Ho marked stimulation of akinete production was observed, sporulr-! lion proceeding in an approximately normal manner

after about 1 wevk of growth. Earlier differentiation of

akinetes occurred in media deficient of orthhophosphorus.

Earlier differentiation would also be expected to occur if

extracellular products produced by old cultures stimulated

sporulation. A greater, but not significantly earlier,

differentiation was observed in material inoculated into

older culture filtrates. This declined as the rrowortioii of

addition of fresh media increased. The extent of spor. iz_lation

L

-146-

Table 27. Sporu ration of Anabz<: nft tap. (7) under diffcrent

nutrient conditions.

Temperature: - 23 °C C. Light:! 4 klux, "I)aylieht" fluorescent,

continuous illumination. Tledin and addition:, sterilized by

aseptic membrane filtration.

Frequency of akinetes expressed as numbers per 100

heterocysts.

Unbuffered Buffered media

media (1 g 1- 1 1I}: 1, ES)

Media plus additions 5 days 7 days 5 days 7 days

ASM 4 (+PO4, -NO3) .0 30 0 0

ASM 4 (-P04 , -1103) 27 47 0 39

100.1 Aph. f-a (FNNI) filt. 0 88 0 50

50iä ++ It If 0 2 0 0

1iG it it It 0 0 0 U

oo, ü to (uri) If 0 32 0 0

500 it to 0 25 0 1 If it " 0 27 0 26

100j1 Anabaana sp. (? ) flit. 0 69 0 69

50io it .0 40 0 24

IS if 0 19 0 31

100iä Kettle Mere water " 0 0 0 6

50, o +4 0 19 0 9

1 ýu nu ýr 0 33 0 11

100iä v+hite Mere Water 0 79 17 65

500 to u If 0 30 0 40

1 j'ý "nn 0 24 0 20

20, E Kettle I"Iere water (-P04) 0 113 0 21

20, ä White Fiere water 0 128 0 0

20`, 0 ti h. f-a U11) flit., -1'04 0 25 0 1U5

20, E it (UM) "" It 0 18 .0 72

20; ö Arirabaena sp. (? )" " 59 E30 29 119

Notes. Where mixtures were use d, propo rtions were m ade up

; aitiz A; X-i 4 (10iQ) . This mediun containe d ort hophosph ate but

comlýý.. nF 11itrogen as nitrvate w as omitt ed. ( -Po4) ortho-

phospI: fate omitted fro!. medium. (-NO j) = nit rate omi tted.

iß4 = Kettle Mere. WM = White M ere. flit, = culture filtrate.

- 147-

in un--enriched culture., filtrates was similar to that

! produced in orthophosphate deficient media. Differences

between culture filtrates were possibly due to differences

in nutrient depletion by the different algae, although no

analy.. e: were done to test this a: -sunption.

Filtered water samples from the mores (Kettle Iere and

in the lowest (1'* %) White Here) produced similar responses

concentration used. Higher concentrations of Kettle fare

water produced a lower degree of sporulnntion than those

cultures grown in White Mere water. This, like the

differences between culture filtrates, was possibly A.

-result of the differing orthophofz?, hate concentrations. The

concentration of orthophosphate in these water samples was

0.7 and 0.4 mg 1-1 PO4-P respectively for Kettle Niere ctn.

White I ere.

When, instead of using normal orthophosphate

containing medium, phosphorus deficient media were used to

supplement-the cultures, a general increase of spor-ulation

was observed. Values we're lower in buffered cultures,

possibly due to the reduction of growth observed in cultures

buffered with H1, PES' (see above). Cultures which contained a

proportion of Anabeena op. (? ) culture filtrate produced a

considerable increase of sporulation, even after 5 days

of growth when the other cultures had not yet sporulated.

Similar experiments were also performed with the Kottlo

Meru strain of 1: Ah. . 'los-t. Lq e but no oporulation was

obeerved beyond that normally encountered in old cultures

or in media buffered with The White i Jere strain

typically did not sporulate.

Lit-, ht c izantityArid b-poruý_at. ion.

As described above, sore strains of alrac will produce

akinetes under certain nutrient conditions. The source of

light in all cases was continuous fluorescent illumination

by "Daylight" '! amps at betveen 2 and 4 klux. some cultures

were exposed to a cyclic light environment of 12 h light - 12 h da. rl: at 15 0C but otherwise r, rnilar conditions to that

describ: ad above. These conditions dich not produce an incrt: ase of akineto production when compared to a similar

-14 i-

length of e; xpooure, to continuous light. Under the

intt n: sitie: 3 used no increase of growth in the li4,, ht period

was observed over that produced under cent: ý. nuou. $ iJ lwiinat-

ion. The ariount of material produced 'war proportional to

the length of illumination.

Cultures of Anabaena cy'lindrica L. which were grown

under normal light conditions and then transferred to the

dark ceased to grow and did not sporulate. port term

re-illumination (fron 30 s to 2 h) of culturco kept in the

dark for 3 days and then returned to darknens produced. no

effect on gr-owth or sporulation. These experiments were

done in inorganic media without a source of combined

nitrogen. A similar cessation of growth and absence of

sporulation was observed when cultures wore : pubjected to

high light intensities. Cultures of the species listed in

table 19 did not grow when'placed under fluorescent

illumination of 8 to 10 klux. Exposure to high intensity

light from the tungsten, halogen lamp had a similar affect.

Return to normal light intensities produced normal growth.

A similar response was also noted in natural planktonic

populations. This material, particularly for experiments at

high light intensity, was collected from White Mere during

the early months of 1976. The dominant alga was

A. circi. na. lio and some A. . floc-aquae was Hlao present.

No increase of sporulation, apart from those akinetco

already present, was observed when post-lo; arithmic

cultures were incubated under high light intensity (also

8 klux). A similar lull of growth and other activities, to

that found when juvenile cultures tiyere used, was observed.

Cultures of all ages became bleached when exposed to high

light intensity.

Tai. ; 1Lt t'uolity And Jnorul-ition.

At the wavelengths used (475,575, E25,675 725 and

875 nri) only one case of induction of sporulation bias

observed under the conditions applied and for the list of

specit. ý: 3 given in table 19. At the end of the incubation

period of about 7> h coat±nuou.; 7.7 liar ination the nic-al

-149-

material was in an apparantly healthy state. Little growth had occurred, but no determinations were made.

Of the material used only the Anaba nn op. (? )

produced any akinetes. A omall number, 29 per 100 heterocysts and 19 per 100 heterocysts, were observed in incubations at 625 and 675 n.: i respectively. This was

probably due to growth and develoymeiit during the incubation

period since the stock material used for these oxperimente

was in the early logarithmic phase of growth (4 days old). Thus at the time of examination (i. e. after a total of

about 7 days) sporulation would have occurred anyway in

normal cultures.

Ultra-Violet ? d, ht And porultttion.

One of the most noticeable effects of continuous irradiation with relatively low energies of u. v. was a

marked diminution of growth (Table 28).

Table 28. Effect , of u. v. on algal growth.

Temperature: - 22 '! ' 0.5 °C. Light: - 800 lux "Daylight"

fluorescent and 1.0 w m-2 u. v., continuous. Optical

density measured after. 10 days ¬; owth.

Control U. D. Treated U. D. Algal material (- u. v. ) (+ u. v. )

A13h. fl oa-aquae (El; ) 205 ý.. w.

0.9 " (wI2) 2.0 0.2

A. floc-aquae (V{; 1) 1.0 O A. circinalis (Tai) 0.6 0.1

12"I, Kettle Mere isolate. UN, White Piere isolate.

In common with other factors (such as high or low light

intensity) which reduce the growth rite, the treatments

used with u. v. light did not stimulate sporti?. ation. she effects of short term doses of higher levels of

u. v. were investigated for the two otraino of A )h. floc-aguac (Table 29). Although there was a slight increase in numbers with increasing doses of radiation, this was relatively small-and the result;, were variable. Via effect of u. v. was not consi: steat between experim,. ntc;. äim filar experiments

-15U-

Table 29. Effect of short u. v. doses on the eporuiation of Animo. f? os-ac,, zae isolates from Kettle i"Iere and White Nere.

Dosage rate :-1U 6i rj-2 . After treatment the cultures wure

grown under the conditions described in the section on The Culture of Algae (Pate 12.7). Observations were made 2

wee s after treatment (no sporulation before). A: cinete frequency expressed as numbers Per 1 UL) 2ietorocycrts.

Length of u. v. dose (min)

Akinete frequency

Iongth of u. v. Akinete dose (rain) frequency

0 72 8

0.5 45, 16 83 1 132 32 64 2 70 64 171 4 102 128 194

Note. ho akinetes were observed i n any of the treatrtento

for the White Mere strain of Aiph.

performed with the other isolates did riot stimulate akinete

production. Both short and long term exposures to high and low levels, respectively, of u. v. light also failed to

produce a response in the natural A. cireinalis populations

collected from White Piere during the early months of 1976.

Observations were generally made at daily intervals in

the period after treatment. In those species which normally

sporulate (such as the Anabaerna sp. (? ) from White Mere)

after a period in culture, akinetes were observed in the

treated cultures after the normal period of time. In the

case of the Anrbaena sp. - (? ) this would be after about 1

week. However, in those treatments which had been subjccted to a greater dose of u. v. (30 min and above) fewer akinetee

were observed. he amount of material produced by these

cultures throu. 'ri growth ciao aluo lower.

These culzures, includinni; the natural nlariktonic Populations of ý ci rcir a1_i from White i"lere, when transferred to low light (10 to 100 lux) Titer incubation

with u. v., did not eporulate. At 3 and 10 klux. increasing,

numbers of akinete. s were observed, particularly in the

untreated cultures of A. o---, 'able 30) . Although the. IL. flc `3ýlCll1,

ýst , which was, F1 minor compollont of the

L

-151-

gb1. e 3C). Effect of u. v., and fluorescent ? _J-,

ht intensity,

on the sporulation of A. =los-Hgtije collected from White

Mere, spring 1976.

u. v. treatment: - 5 rein at 10 w m- 2. Temperature: - 23 to

25 0C. Observations made 5 days after treatment. After

treatment with u. v. the cultures were grown under the light

intensities indicated. Akinete frequency expressed ao

numbers per 100 heterocycte.

Light intensity lux Control u. v. treated

10 to 100 00

3t)OO 51

10000 32 7

Note. No akinctes before 3 days after treatment.

phytoplankton at the time of collection, produced akinetee, the dominant alga, A. cir. cinalis, did not. The material

appeared healthy, although the amount of growth in the u. v. treated cultures was less than that of the control. The

cultures, particularly in the high light intensity treatment,

were taken over by A. flos-ftguae. The filaments of A. oireinatis became fragmented after only 1 or 2 days

exposure to light of 3 and 10 klux intensity.

DI$CUS3ION

Growth. And Bporulation.

'With few exceptions, sporulation of laboratory cultures,

was not observed until the late stationary phase of growth.

This, or the complete absence of sporulation, appears to be

a feature of isolates of heterocystous planktonic algae.

Eberly (1966) did not find akinetes of t h. flos-£«iuae

after a period in laboratory culture although Wildman et al.

" (1975) observed akinetes in old cultures of the same

species. Schwabe and . strange - Bursche (1964) observed that

. although akinetes were produced in natural populations of

Aphanizotnenon gracile Lerrn. during the sutntner, there was

generally no sporulation in laboratory culture.

A eigrificar_t departure from the eenoral behaviour was

observed in cultures of the Kettle hero strain of Aph. floc-

aquae buffered with HEPES. 5porulation started earlier in

the buffered cultures, while no akinetes were observed in

the unbuffered control-cultures after 3 weeks of growth.

Although the control culture had, by this time, reached the

stationary phase of growth, the buffered cultures were

still actively Crowing. Culture density did not appear to

significantly affect the differentiation of akinetes. Those

cultures gro n __. the proecnce of higher concentrations of

which had suffered an extended lag phase qnd

cane=eq, zent: ya lower biomass after a giver p(-. rind of tirle,

had sporti_fiýL- G_ý to an equal extort as the denser. cultures >d.

in l_o,, rcr conccntration. c of

A1. thour h it would appear that IIEYES has some

stimulating effect on the sporulation of Aph. floS-Rguae,

other factors need to be considered. Nutrient depletion is

expected to be similar in cultures of similar densities and

the n. r during the period of akinete formation and

immediately prior to it were similar in the control and

buffered culture . Also, in eulturu.: e buffered at a similar

p11 using other buffers, no stimulation of sporulation was

obeorved. Thus, in those cultures in which little growth had occurred, such ne iin high co: cetvations of a

lower deL; reo of snorulation would he expecte(i if nutrient

deficiency i3 tile rinin caua factor.

-153-

These observations, however, are relavrnnt to only one

strain of one species. The other strain of Anh. flog=-aaune,

from Weite Were, did not produce any akinetea at all, in

any of the treatments. The Anabaena op. (? } provided a more

typical response. Oporulation was lower in cultures in

which there was less growth. This was the case in cultures

buffered with increasing concentrations of HEUS, in which,

although the growth rate tended to increase, the lag phase

was extended. Ater a period of growth subsequent to

inoculation the amount of algal material in the buffered

cultures was lower than"in the control culture. This was

reflected in the lower degree of sporulrstion, which,

however, increased to an amount equivalent to that observed

in control cultures. This occurred once the optical density

had increased to a similar leve*. in the buffor. ed cultures

as that of the control cultures. Like with the Aph. fl. oe-

actuaae strain, no akinetes Were observed before the

stationary phase, although sporul_ation in this isolate

proceeded at a somewhat earlier phase of the growth cycle

than in the Aph. floe-ac uaQ cultures.

Inorfanic Nutrients And Sporulation.

Cultures which were inoculated into full inorganic

media (except of the omission of combined nitrogen

compounds) and allowed to grow under normal conditions

occasionally produced akinetes at some stage in the poet-

logarithmic phase of growth. It is generally considered

that eporulation is induced by nutrient deficiency. Ortho-

phosphate deficiency has been found to induce early

differentiation in cultures of A. cvlirdrica (t1olk, 1965)

and Aph. ! os-aquae (Gentile and Maloney, 1969). Although

orthophosphate was reduced to indetectable concentrations

(below 5.0 N; 1-1) in old cultures of Anh. floe-Aquae

(whether or not they have sporulated), no induction of

sporulation was observed in those cultures which were

inoculated into orthophoc: phate deficient media. The

Aniibaena : p. (? ) on the other hand did chow a significantly

earlier production of akinotee in those media deficient in

orthophosphate. This strain of alEa, although isolated from

-154-

White hors, is not 'one of thu typical planktonic forms. Its

response to nutrient deficiency more resembles that of the

terrestrial isolates of algae (such as A. Rorliidrica,

A. doliolum, Cy' indrospernum etc. ), general? _y used by

previous investigators, than that of the planktonic species. While low orthophosphate concentrations did not

stimulate the sporu1r: tion of Arch. floc-c une, high

concentrations (10 mg 1-1) appeared to be inhibitory. Thus

it would appear that orthophosphate plays some role,

although the response was not as marked as observations on

other species. The failure of induction of aporulation of

cultures inoculated directly into phosphate deficient

media suggests that other nutrients may be of importance.

Another factor may be the requirement of a certain amount

of growth and development per se before differentiation of

vegetative cells into akinetee can occur.

Ariong the other inorganic nutrients tested was nitrate. Previous work has shown that nitrate deficiency induces

sporulation in C lindrosZermum (Glade, 1914) and that in

A. doliolum sporulation is inhibited by nitrate (Tyagi,

1974). Singh and Srivastava (196¬3) also suggest that

combined nitrogen is the controlling factor of morphogenesis, including sporulation, -of A. doliolum. In the Ah. floe-

aquae cultures there was an inverse relationship between

nitrate concentration and *heterocyst frequ mcy, which was

more marked in the White Mere strain. This is typical of the heterocystous species (Fogg, 1949; Kr1le et 1970).

Akinete formation appeared to be neither stimulated nor

inhibited by the presence of nitrate, although at the stage

of akinete production the concentration of nitrate would be

expected to be low. (This, however, was not measured).

Despite these observations, however, it was found that on

some occasions a marked stimulation of eporulation was

produced in cultures of Aph. floh-aeutiey (from Nettle Here)

which were grown in media coutaininf, nitrates.

From these observations, and also from the failure to

find -another single nutrient element (such ßc iron or any

of the trace elements) which would. cif*, nificrintly stimulate

sporulation, it would app(--ur that thto factors involved are

-155-

rnoro cor: l: lu than that as., ociated with the depletion of a

single nutri ent. The respoima of the terrestrial forms,

however, a, ->>ears to be, somewhat more' straight-tor award than

that of the pltinktoriic species. Although nitrogen and

phoophorus metabolisn appear to be critical factors, the

stimulation of akinote differentiation in the planktonic

species is probably a complex interaction between these and

some other factors. Possj, bl© candidates for ouch other

factors may be other nutrients, the age and development of

the cultures, and the presence, of extraeellular. products.

-Other Coripound: a And . norulation.

Organic co. pounds, either as extracellular products

produced by the algal cells (Fogg, 1965; Huntsman and

. Barber, 19'75) or through the activity of aseoeiated

bacteria (raunt, 1 961 ; smith, 1973) are often essential for

the'growth of algae in cultures. This is particularly true

when small- inocula are used and during the early phaeos of

growth. Apart from the stimulatory effect on the early

phases of growth, extracellular products have been found to

stimulate the production of akin. etos in C. licheniformo (Fisher and 'lolk, 1976). Although a similar type of response

was not observed for the strains of algae isolated from the

meres, one compound, the buffer k LPL6, was found to

stimulate siorulation in the Kettle Here strain of

Ash. floc-a(iuae. This process seemed to be independant of

the concentration of the compound (HOPES) for the rang:

tested.

Sometimes other morphological effects were also

observed in cultures to which certain organic compounds

were added. The complete suppression of heterocysts of

Aph. flus-nauae was noticed in cultures buffered with

N glycyl-gI. -vcine. (A similar effect was observed in

Anabaena o. -cil! ý,, rioides Bory by Demeter (1956) in a culture

media which contained sodium Elutamate). This is in

contrast to the finding that in cultures of Anh. flr.: -aquýio heterocysts were still present, even at high concentrations

of nitrate. It is pos. sLble that 11 E;? _ycy1--(; lycine is taken

up and assimilated b, - the exlE; rj, producing; an inhibit on of

-156-

heterocyst differentiation. That this is more complete than

the suppression of differentiation in nitrate containing

media is probably a reflection of the lower energy required

for di-peptide uptake and assimilation than that which is

required for nitrate reduction. However, since the cultures

were not axenic this must remain a hypothetical conclusion

as bacterial metAbolicm of N glycyl-glycine could have

produced conditions responsible for the described

inhibition of heterocyst differentiation.

As expected from the relationship' between hoterocysts

and akinete differentiation (Yolk, 1966) no akinetea were

found in cultures in which heterocyst differentiation had

been suppressed. This is in contrast to the observations of

Demeter (1956) that in the cultures growing with sodium

glutamate akinetes were observed, despite the absence of

heterocysts. This appears to be a direct stimulation of

sporulation by this compound, such as that observed with

HEYES buffer. Demeter (1956) found that both heterocysts

and akinetes were rare in cultures grown in media from

which sodium glutamate was omitted. The medium used by

Demeter (1956) was based on that developed by Allen (1952)

and included potassium nitrate, ammonium chloride and

di-potassium hydrogen orthophosphate. In media free of

combined nitrogen both het©rooysts and akinetes were present

in abundance (Demeter, 1956).

Li, -ht Quantity And Sporulation.

Contrary to the expectations from observations of

natural ponulations sampled during the summer, high light

intensity did not stimulate sporulation. \olk (1965)

observed a reduction of sporulation of A. c yiin. iri en, at

light intensities above 800 lux, and above 2500 lux few

akinetes were formed. A reduction of sporulation was also

observed under low light conditions. The observation by

Ylolk (1965) were made after a fixed period of time, and thus

it is possible that the cultures under different light

treatments had. reached different staken of growth at the

time of sar. °, pling.

Cu1. tu-" e: which were exposed to high light intennsity

l

-157-

did not grow. This was also true of cultures kept in the

dark or'unc? er low light interisity. As well as cell division

and enlargement, other metabolic processes api)ear to be

halted or reduced under these conditions. The result i5 an

absence of akinetes, even in the natural materiel used for

some of the experiments. It is, therefore, appar. Ant that

sporulation in laboratory cultures will only proceed after

a certain amount of growth, and if that growth be reduced

sporulation will be retarded. However, even when post- logarithmic phase laboratory cultures were used no

sporulation was observed.

The natural material used, collected during the spring

of 1976, probably represents actively growing, healthy,

logarithmic-phase culture3. If this was the o ee, than a

-similar response to that of the laboratory cultures used

while in the logarithmic phase of growth, can be expected.

Light Quality And Sporulstion.

Illumination of algal material with light of different

wavelengths produced a similar response to that discussed

above. When there was little or insufficient growth, no

akinetes were produced. The type of photo-inducable

developmental cycle observed for IT. muscorum by Lazaroff

and Vishniac (1961,1962) and for N. commune by Robinson

and Miller (1970) was not apparant. Induction of the

I4ostocacean developmental cycle was induced by a brief

exposure of the dark grown material to light of 650 um and

reversal brought about by exposure to greon light. Although

light of these spectral regions was used in the experiments

described here, the absence of response possibly indicates

a major difference between the life cycles and developmental

stimulikf the terrestial Nostoc species and the planktonic

Anabaena and. Aphanizor"; enon species. The a1 eence of a

response of the planktonic isolates to illumination with a

tungsten filament bulb after 2 days of dark trontment

supports this supposition.

Kau., hik and Kumar (1970) found that, apart from white licht, red light supported the creatost rate of growth of A. d liolum. The growtL-h in blue light wn lower, and jr.

-158-

green l id; hht miriirul. The transfer of cultures, grown in

white (tungsten) light for up to 4 days, into Green or blue

lig: it conditions produced no sporulation. IU: i netes were

observed, however, when cultures grown in white: light for

6 to 8 days were transferred to blue or green light. This

response is similar to that encountered with the Anabaonr-t

sp. (? ) used in this study, although Kau:; hilc rand Kumnr

(1970) did not indicate the age of cultures in which

akinetes would be observed when maintained in white light

only.

Ultra-Violet Light And-S; por. ulation.

Treatment with u. v. light was reminiscent of the other

light treatments in that extensive sporulation was not

observed in cultures in which growth was inhibited. Singh

et al. (1967) observed an earlier differentiation of

akinetes of A. doliolum and Qylindr. or; permum maius Kuetz.

when treated with u. v. light. In these e;: perirnc: nts, and also

in others in which u. v. was used to produce mutant strains

of blue-green algae (Singh, 1967; Singh and Dikchit, 1976;

Sinha and Kumar, 1973) a shorter-wavelength of u. v. light

(about 254 nm) w. s used than that applied in the experiments

described here.

The proportion in solar radiation of wavelengths below

300 nrl which reaches the earth's surface is very small (Collingbourne, 1966) when compared with that of other

wavelengths in the near u. v. and visible spectrum. A

considerable proportion of energy at a wavelength of 365 nm,

which was that used in this otud,, reaches the earths

surface during clear weather. Thus the u. v. wavelength

tested bears some resemblance to that encountered during

sunny days when algal material has formed surface blooms.

SECTION 3

THIS PHYSIOLOGICAL ACTIVITY OF NATUI A3

POPULATIONS of ALGAE)

II PRODUCTION

Although some physical and chemical factors have been

described and diocues d in t; cction 1 in terns of their

relevance to algal growth and the life cycle, the measured

values of these factors represent the external environment

to which the populations as a whole were exposed, rather

than the environment within a cell or for a particular

species. Knowledge of the concentration of certain disoolved

compounds gives little information of their availability to

the organieem. Marked local differences of the concentration

of nutrients may occur, particularly during a relatively

stagnant period. Such conditioiis may impose the limitation

of diffusive supply. Although plentifully present in the

general eater body, depletion of nutrients may occur in the

vicinity of an algal cell or colony, or within the cell

itself. Other factors could also produce local depletion of

nutrients. Bacteria, through their more favourable surface

area to volume ratio, take up and utilize nutrients ouch as

phosphates more rapidly than algae (Fuhr at n3-. 0 1972; Rhee,

1972).

Nutrient, particularly phosphate, availability may be

affected indirectly by the presence of trace elements.

Shapiro and Glass (1975) reported that additions of manganesee

as well as phosphates had a very marked effect on algal

growth when compared with phosphate additions alone.

Various other trace elements have been shown to be required

by blue-green algae, including cobalt (Holm-Hansen at al.,

1954) and copper (Telitchen:, o et al., 1970). Iron

concentration was found to determine which becomes the

dominant algal species in mixed batch cultures ([Torton and

Leo, 1974). Enzyme activity is often dependant on the

presence of trace elements. Molybdenum is a requirement for

the synthesis of both nitrogenase and nitrate reductn. se (Pay and Vasconcelos, 1974) and cobalt for the formation of

vitamin B1 2

(Holm-iiansen et al., 1954). As well as their

importance: in ; physiological processes, some tract) elecmontu:

have a vi: j4ble effect on the morphology of the species.

i: hitton et _. (1975) observed that terminal hairs are

produced in filaments of Ctllothrjx yiinlieri in iron

-161-

deficient culturfes. A similar effect was observed in

filaments of Gloeotrichin when grown in a medium free from

combined nitrogen. ün addition of an ammonium salt, the

filaments appeared uniform throughout (}: 'ay et al., 1963).

Phosphorus has been shown to have a controlling influence

on the colonial morphology of $cened. osrmus (Trainor and Shubert, 1974; Shubert and Trainor, 1974). The shape and

size of the "packets" in which algal cells are grouped may be expected to produce nutrient limitations through an increase in the length of the diffusive pathway in large

colonies or clumps. it has been suggested, on the other hand, that the gelatinous sheath of blue-green algae may form a favourable microhabitat for algal growth through the

concentration of limiting nutrients (Lange, 1976).

Alternative sources of potentially limiting nutrients

may be important during periods of stratification and

surface blooms. The hydrolysis of organic phosphorus

compounds in natural water samples has been shown to occur (Reichardt, 1971; Herbes et al., 1975), and an inverse

relationship between alkaline phosphatase activity and

orthophosphate concentration has' been demonstrated (Heaney,

1973; Healey, 1973; Ihlenfeldt and Gibson, 1975). Heath and Cooke (1975) observed up to a 30-fold increase in alkaline

phosphatase activity during a bloom of ADh. floe-a use in a

eutr: ophic lake. The authors suggest that alkaline

phosphatase activity could be an important source of

orthophosphate provided that a sufficient concentration of

phosphornonoesters are present in the waters. Fitzgerald

and Nelson (1966) also observed a large increase in enzyme

activity in phosphorus depleted cultures of natural

material over that found in culture: which were supplemented

with phosphates. This inverse relationship is only

applicable to phosphorus starved coils. In continuous

culture experiments using A. floc-figur-, e, hone (1971 ) ums

unable to determine any relationship between alkaline

phosphata; se: activity and labile call phosphates. In this

case phosphorus would be limiting au a result of activo and

rapid g, rowth, rather than the culls being starved. L itratu

was observed to stimulate enzyme activity in these

S

-162-

exper. j. r.: ents, as a consequence of the creator yield

resulting in an increased phosphorus demand. In the natural

environment no correlation between enzyme activity and

phoaphorus uptake or cell phosphorus was found during the

course of a bloom of xphh. floc-aquae (ilealey and Hend . cal,

1976). levels of activity were variabl. e, despite the

nitrogen and phosphorus deficient nature of the algal

material at the end of the bloom.

On the restoration of phosphorus sufficient conditions,

a rapid uptake of phosphorus occurs which is in excess of

requirements (Ketchum, 1939; Batterton and Van Ballen,

1968; Sicko-Goad and Jensen, 1976). Sufficient is

accumulated to support two further generation:; of growth

when the material is transferred to phosphorus deficient

medium (Batterton and Van Baalen, 1968). Stewart and

Alexander (1971) found that sufficierit phosphorus is

assimilated and stored within the cells of nitrogen-fixing

blue-green algae during one day to supply the requirements

of growth at 25 0C for one week. A long period of growth

of A. spiroides at the expense of stored reserves of

phosphorus has been observed by Volk and Yhixiney (1968).

These reserves are stored as polyphosphate (volutin) bodies

and may contain the major fraction of the total cell

phosphorus (Sicl: o-Goad et nl., 1975). The phosphorus is

stored in an osmotically inactive state as long chain

inorganic polymers (polyphosphates) (. >icko-Goad and Jensen,

1974).

In addition to the effects on growth, phosphoru.,

" deficient nay also affect the availability of other

nutrients. Stewart et ale (1970) and Stewart and Alexander

(1971) observed a rapid increase in nitrogenase activity on

the addition of phosphates to phosphorus starved cello.

Fixation of atrio: spheric nitroeen by blue-green algae

can be an important source of oombiiivd nitrogen. The

contribution of nitrogen fixation to the nitrogon bud ; et is

often nigh in tropical lake. (Hiom e sind Viner, 1971). high

rates of activity were found in many temperate lakem;,

including,, those xn the BritiE3h 10le, 3 (Iiorlio F1nd Pore;, 1 970;

itewart, 197.2). Go3iitile and IN, 'ulonoS' (1969) hove found t1l tt

-163-

that nitrog; on fixation under optimal conditions wao

sufficient to supply the nitrogen requirerleiits of

floc-aquae. A positivE: corrt; l, -ition botwe. an nitrogen

fixation and concentration of disoolvod or , rnic nitro, 3un

compounds uns observed (Horne and : ogg, 1 97C; home 'ýt ºl_. , 1972) and a negative (h orne at al. ,1 972) or not ET: r}J1ifjcant (Horne and Fogg, 1970) correlation between nitrato

concentration and nitrogonase activity.

Many of the metabolic procesces described above

require a source. of energy which, in photoautotrophic

organisms, is supplied through photosynthesis. Photo-

synthetic activity in turn is äffected by the availability

of the compounds mentioned above and thus any aspect of

physiological activity cannot be viewed without considerat-

ion of the whole physiology cif the organism. Many

observations have been made on the reduction of the rate of

photosynthesis in the surface layers of wFator under

-conditions of intense sunlight (Baker et el., 1969;

Reynolds, 1971; Tailing, 1971). Under prolonged exposure to

high solar radiation the rates of photosynthesis fell

(Harris ans? Lott, 1973). These authors suggest that

photorespiration is the controlling mechanism which governs

photosynthetic behaviour under continuous high light

intensity.

Thus during the formation of a surface bloom many

physiological changes could occur which may only be

apparant from the resulting alteration and differentiation.

Healey and Hendzel (1976) followed the course of changes

of cell nitrogen and phosphorus, chlorophyll n, ULNA content

and the protein to carbohydrate ratio of bloomo os

Anh. flos-Ha2uae. During the early otages of bloom formation

Growth was exponential and cell contents of RNA,

chlorophyll a, phosphorus and nitrogen were high. This was

reversed prior to the collapse of a bloom. Nevertheless,

the authors conclude that increauud nitrogen Mnd pho&iphoruL

deficiency is not the cause of bloom collapse. The

physiological state of the blooming algal population, is

reminiscent of the stationary phase of batch cultures.

Lhttnt; eE observed in the j)hySio oL; ie£il activity wero" t': oro

-1 Cuj

marked than those of the concentration of dit. iso v'-d ioni in

the water.

Changes of enzyrie compliment and activity were also

observed by Laud' ina et al. (1973) during the courco of

water blooms in the Dnepir reservoirs. such changes may

reflect an adaptation to chancing conditions or a change

from one stage of the life cycle to the next. Kostlan and

Nesterova (1975) suggest that the accumulation of storage

products during the process of sporulation in the final

stages of surface blooms indicates a decrease of the total

viability (activity) of the population.

In order to obtain further information of the critical

factors governing growth and development of the planktonic

blue-green algae of the meree some aspects of the

physiological activity of algal material were monitored

during the period of bloom formation. Through this it was

hoped to determine the effects of large scale environmental

factors on the activity of blue-green algal populations,

and of the changes of physiological activity on further

development of populations.

ids.

i"iATLR1ALä AND IlliT. IUD 3

Collection Of iiriterial And Yrolirliniirj Treatment.

Bulk living sarfples collected from the üur. face waters

of the meres were allowed to stand for 2h in '2 J. wide

mouth polypropylene containers or 25U ml mea: iurina cylinderes. When the blue-green algal material had rioen to the surface it could be easily collected from the meniscus by gentle

euction. By taking up different amounts of water, any desired concentration of algal material could be obtained. The larger zooplankton and particulate material was removed before the separation of the blue-green algae by pouring the water sample through a coarse net of 1 mm square mash.

Those which were not removed tend to swim rapidly to tho

surface of the water on standing for a few minutes. Thece

were rdpidly and efficiently 'removed by dieplacement ox'

100 to 200 ml of the sample with a beaker before the blue-

green algae had started to rise. These treatments resulted

in a relatively clean suspension of the buoyant blue-green

algae. Little was observed to be non-buoyant except

akinetes, dead material and samples which were beginning to

decay. The delay between collection in the field and start

of the assay incubations was ea, maximum of 2h if the

incubations were carried out at Preston PIontford, 6h if in

Lo21d oil.

Alkaline Phosnhatase e The D-nitrophenol colourirletric technique for the

determination of alkaline phosphatase activity was as described in :: iei,; a Technical i3ullctin, .i umber 1 04 (1974).

Reagents.

The reagents used, except soditua hydroxide and

hydrochloric acid, were obtained from Uig: ria Clieraicai Co.

Ltd, Kingston upon Thames, surrey, tnL; lund.

Alkaline buffer solution (glycine, pH 10.5) U. 1i":, with

chloroform preservative. Phor3phata e iubstrate (y-nitrophonol

phosphate, d. it3odiu ).

. jiipp ied me tablets, encu-; h . t'or 2 tests. One

tablet di cool ved i,: 1.2 ml ciiatillocä water .

; ý-zi ýtropk1L: nol : stwisurcl : ýolu viý: i. (16 Ilrnol rnl-1)

-166-

Dilutions (0.5 cal diluted to 100 ml with 0.02N sodium

hydroxide) prepared as required.

Sodium hydroxide (u. 02N).

0.8 g dissolved in 11 of solution.

Hydrochloric acid (concentrated., 12N).

Procedure.

Aliquots of intact cell suspensions were incubated at

37 oC. The enzyme alkaline phospha. tase is located at the

cell wall of a marine pseudornonad (Tho-ip , on and Nacl. eod,

1974) and within-the multilayered cell wall of a Gram

negative bacterium (Costerton, 1973). Berman (1970) has

shown that the activities measured in intact and disrupted

cells of Peredinim are the same. Bone (1971) used intact

material of A. flos-aC, uae. Chloroform, used as a

preservative, had no effect on the activity of the enzyme (Berman, 1970).

0.5 ail of the buffer and 0.5 ml of the substrate were

pipetted into 15 ml I-1cCartney bottles and allowed to

equilibrate thermally. The incubation was started by the

addition of 0.2 ml algal suspension or a blank of 0.2 ml

distilled water. After 1h5 ml of 0.02N sodium hydroxide.

was added to stop the reaction.

The absorbance of test, blank and standard samples was

measured at 410 na in 10 mm silica cuvettes. To determine

the absorbance of the sample alone (the algae were removed

before measurement by filtration through 1lhatman GIS/C

filters) a drop of concentrated hydrochloric acid was added

to each sample, which renders the P-nitrophenol colourless.

Calculation.

Due to the relatively low levels of activity in field

samples the method adopted provided an eightfold increase

of measured activity as compared with the original assay

description. This was achieved by the doubling of the

incubation period (from 30 min to 1 h), a doubling of the

volume of inoculurn (from 0.1 ml to 0.2 ml) and a halving of

the volume of the sodium hydroxide solution used (from 10

ml to 5 ml). This was taken into account when Preparing

standard curves as deecribed in the original method (sigma,

1 974) . The äncrkia t- in the amotult of L-n1. ýIýo, ýherzol

-167-

libi. rated appears to be linear up to 2h (see also Herman, 1970).

An aliquot of the concentrated sarnplo was diluted by a factor of ten and the dry weight deterriired as described in Total Particulate Suspended Solids (page 53 ). Activity of

alkaline phosphatase was expressed as äigma units per mg dry weight. (One jigma unit - the liberation of 1 µmol

Z-nitrophenol per h under the stated test conditions).

Labile Orthotphosn hate.

Boiling water extractable orthophosphate clot erminations

were performed according to the method described by

Fitzgerald and . +, elson 1966) .5 to 10 mg of filtered and washed material was used. 1 mg is the minimum quantity

required for a sufficiently accurate determination using the orthophosphate analysis method described above (Page 47 ).

Each filter disc (or several if necessary) was put in

a 30 ml flat bottomed glass tube together with 1U ml

distilled water. The mouth of the tube was sealed with a glass sphere (Fig. 34). This prevented contamination and drying out of the digestion mixture. The tubes were held by

a grid in a boiling water bath filled with distilled water.

After boiling for 60 min the' samples were cooled. Any

fibres detached. from the filter dimes were removed by

filtration through 25 mm diameter GF/C discs held in a

Swinnox (Millipore, Wembley, Middlesex, England) membrane

filter holder fitted with a 20 ml syringe. The filtrate was

collected in a 25 ml volumetric flask and analer; ed for

orthophosphate as described in Uection 1 (Page 47 )

reducing additions of reagents pro rata.

Contamination of the samples was found to be minimal. The orthophosphate content of Whatman F/. 0 filters (Pyrex

glass fibre filters with no binding agents) is low oven if

unwashed. No significant increase was noted when-several blank filters were boiled in the same tube.

Hydrolysis of organic phosphorus compounds, which

would occur in acid solutions, was not apparent. Phosphate-

starved algal Liat,. rial and lake water rich in organic

phocphoruc3 compounds produced no detectable orthophor. presto

-168-

Fig. 34 Digestion apparatus for the extraction of labile

ortho-phosphates.

Glass sphere (cap)

Filtered sample in

10 ml distilled

water.

000 ýý fl o70

Distilled water, 100°C.

Tube, 26 ml.

ffl-o CP

Perforated plate.

-169-

after digestion, which t. culd be expected if those were

hydrolysed.

ýi tlt3 ar ou.. t of labile orthophosphate is givon as mg;

orthophosphate-Pilo 0: thoruti per mg dry weight of alga.

I'litrof: enase, Acetylene Reduction 2.

Nitro en fixation was estimated ufiin. lg; the acetylene

reduction technique of Stewart at RI. (1 967) and. Hardy

e". ale (1968).

3 ml of the suspension was pipetted into a 7.5 ml

serum bott_e and the incubation started bfr injecting an

amount of acetylene equivalent to about 1O of the gas

phase through the rubber septum. Prior to the addition an

equal volume of air was removed so that the incubation was

carried out under atmospheric pressure. The suspensions

were continually mixed on a rotating wheel and the incubati-

ons terminated after 30 min by partial removal of the gas

phase urizig pre-evacuated Vacutainers (Reckton and

Dickinson Co., Rutherford, New Jersey, U. S. A. ) (Schell

and Alexander, 1970, see fig. 35). After collection the

F: i. g. 35. Partial removal of the as phase from a serum

bottle using Vacutainers. (From üchell and Alexundor, 1970).

Vacutainer Double.

Yerfori

Septum

Iricuba-

E'it? MrIe

iio_'_der

Thick rubber septum

)orur bottle

Vacutainere were equilibrated to atn, o ppiieric pros.. ure. The

incubations were carried out at laboratory temperature$

. (18 to 22 °c) which did not vary by more than ± 0.5 °C

throughout the incubation period. The light izitenuity at thc: ouri'ace of the incubation vec3. sG1 was 1 QUU lux, the

-1'l0-

source being an egiza1 mixture of tungFAtin -2i). }tmentt and fluorescent lamps.

The ethylene concentration was mere

chrorlatoCraphy u. 3ina a Varian AeroL; raph

Thames, Surrey, England) with a Toropak

each sample was removed an equal volume

1.0 ml) of distilled water was injected

s: urcncl by C as 2ueO (ýia1ton=on-

Ft aolurºn. BcjJA'ore (general? y 0.5 or

to maintain the as

sample at atmospheric pressure on removal of a sample. Estimations were made of dry weight of algal material

and the data are given as nrnol ethylene produced per rng per h. Counts were made of heterocyet and akiuete frequency (as jv' of the total cell number) of intact filarnante and

also the concent2^ation of heterocystss in the incubation

suspension was established. Determination of hoterocyst

density was aided and a more randorn distribution produced

within the hae. ^ºocytometer after breaking algal clumpo by

shaking for 1 min in a serum bottle with 10 small glass beads.

Calculation: -

Total nmol produced = x. a+b

b

X=b. nmol Hample volume used

For aizalysia a= Volume of gas phase of serum bottle

b= Volume of Vdcutainer (= 5 ml)

Photoc: vnthesi a (Üx7ri en : c'roduction

Gross oxygen production was measured using the light

and dark bottle Winkler techniqu e (Vollenucider, 1974).

Blue-green algal suapenc-ions were prepared as described

above (page 165 ). The concentrate was diluted . with

filtered lake water from the site of colleotion to within the range of 5 to 20 mg 1-1 dry weight. Light and dark (covered with aluminium foil) 60 ml bottles, together with bottles for the determination of initial oxyGen concentration,

were carefully filled with weil. mixed (by Btirrin(, not

shr-eking) ou3pension and iný, ubFited under the narre conditions

as cte cribed for the nit,, ogenase aeeave (pa(-, e 169 ). Tho

zero time bottle wao fixed at the start oz the: incurton.

-1 71-

An incubation period of 2 or 3 li way to

obtain significant differences in concuntrultion.

Gross o:;, i; en production (the diift ro, ico in o: cygcrn

concentration cIiangeL3 between light and. (dark bottles) was

e: _prassed as me oxygen -produced per xi dry weight of alga

per hour. Counts were also made of absolute call nunbers

and relative abundances of the species prccont. Not

photosynthetic activity was calculated from the difference

between the oxygen concentration in the light bottle and

the initial oxygen concentration.

Carbon, Hvdr. orýen And TL. itroggen Anl lvuis.

Analysis of total carbon, hydrogen and nitrogen wore

performed by the Chemistry Depart:: ient of the school of

Pharmacy, London. The instrument used, a Carlo irba model

1104, had an absolute error of about 0.3>ö.

Assessment Of Bacterial (lumbers In Algal Samnle . Bacterial numbers in algal samples were determined by

counting the number of colonies which grew on agar plates

from a known quantity of sample (and hence a known mass of

algal material). A small volume (2.0 ml) of the sample was

first homogenized by vigourous shaking for 5 ruin in a

sterile 7 ml serum bottle containing 10 small glans beads.

Dilutions were then made into sterile diluted AUI-I 4

medium. A known quantity, generally 0.5 or 1.0 ml of

suitable dilutions (10-3 to 10-6) were thouroughJ. y mixed

with about 15 ml of sterilized agar while the agar was

still molton, but not hot, in 1etri dishes.

The bacterial agar medium used contained 20 g iincto

nutrient agar (Difco Laboratories, Michigan, U2A), 1. U g

glucose, 0.5 c sodium caseinate, 1.0 g yeast extract and 50 ml soil extract. The constituents were made up to 11

with dilute ASii 4 medium and the p1-I adjusted to 7.3 . The plates were incubated for 4 days under lt+boratory

temperatures (about 24 00).

I i, 1 ýýiJ JJyiJ

]'hoo, horus lývailýhility.

ý'he concentrations of labile orthophosplia"te Oxtrr-toted from the wa: 3-zed algal r; aterial are given in tables 31 to

33. This component of cell phoe; phor. us represents the

stored surplus, taken up through luxury consumption (Fitzgerald and Nelson, 1966). The amounts present are

consistently high in all the mer. e. s studiud throughout the

period of blooming. Little or no orthophouphate is

extracted from cultures which have been grown in the

laboratory under phosphorus-starved conditions. There is no indication of depletion of stored orthophosphate, even during periods of intense blooming and epor-ulation, such as

of A. floc-aquae in 'Kettle T-lore (Table 31). Dil. Oerences

between the Anabaena species are small and the levels found

are similar for all three mores from which material was

collected. The amount extracted from A )Ii i. fl os-ac line (Table

31) is similar to that of the Anabaena species, but the

stored surplus in. G. echinulatf3, particularly in Newton

Mere (Table 33), is generally higher.

The concentrations r3presented are similar to those

found by Fit ziTerald and Nelson (1966) for natural

planktonic populations. The amount of labile orthophosphate increases in cultures supplemented with phosphate and

becomes raarkedly- depleted under phosphorus-sstarved

conditions. The concentrations found in natural phyto-

plank ton populations are intermediate between these

extremes but are closer to the values of the phosphate-

. enriched Culture: than to that mea snxred nttor phosphorus-

starved ti atraun s.

The abundance of phosphorus ir, also reflected in the

low levels of ttlkaline phoaphatftse activity (ýOublo 34).

Again there are no significant changen during the aura:, ier in

the amour meres from which material was collected. Ociao

species appear to have slightly higher levels of activi

such as G. cliiiiuifita in Ilowton hare, F; ol. ý. tttiria rind A. ýpiroid ''iýc: Activities obtained are i imi: tr to t11okue

reported !: or cultures frown in cuurpIus phoop1u tes

}'itzgeraid and ivle lson, 1966; Borne, 1 971 ).

-173-

p. a 0

m C) OOOOOOOOOOOOOOOO 0 $1

rn C)

"o ca cC t

UN v2 0 aOO oo [- N ai CH OO0000000 C% T- N C% v- rr, \

T- " .n +) B"

aýi "aýi

r-i ý

ý3 r-1

jam' cC

H 9-4 td

0M

"EÖOfOOOOOOOOO T-

OOOO 0 (0

vý Q +'

$4 Co "r4 O to

O0p

$4 g f. -H d ) to +' 0

p 4.3 0 52 9.4 R 0 -1-4 3 ß. URoOOOOOOOOOO

t- O 000

n 00 to

cu (C $ 0 a) d

vd $$4

ci

(Q d) C (D ma0 p f' rOO8O O$

ggOOOOOoOO

0 A9 00 CO , C: A

+) 4) "r1 54 92 O it cd

"ý v

$4 OOOooOOO O\ 0\

ON000

(C O C) r-i U

i-I 60 .a8 Ee CO N CO .# .7n Q' C' N tt\ O' c- WM 8

cm Q\ 0 CO /0 CO X1\ C- . -2 N cm t+l 0

_e R T- 0 r- 0NO0 c- N v' e- 'r NO r' 0. OOOOOOOOOOOOOOOO dD

Cý ýD C) 0

\= z27 \ cý NN rn ýN\\\ Qrr ýf OGH rNNM :ý

0 H 0

U

0 to

U U

U U P4 Co 43

. r.,

-174-

gd 43

rn rZ o `- . O O O O O O O O O O O O O O 'd U b CO

L r\ c' CO C)

00 " ý

ý N

i a 3

ý 04 0

O O O O O O O O O w O O O O O

O p

- 41 V

ii

42 0

c: "1

0 x

°' 0 ) ß4

P o 0 0 0 0 0 0 0 0 . ° o 0 $y Pt N G) W .0 +) 0 M1 " cö . r; $ 4

o C q

. 11 E t i

W ý 0

.0 m b +)

00

1 t0

O O O O O rn a\

C\ Co ( g

e- . : p

ýD p CT r r

CO 4

k ä ý p

t0 Ö

" Ö fr

i $V+ O " o 0 o

N r N O O e O O O O

r1 o " o

!m

N F-

raj

S r

N" O O

0. ul K\

0 u'\ c-

0 00 e- e-

rn 0' r

0 N

N g M N "-

Cý N r

CO CO N

0 00 K1

n r

r O r

A. o 0 0 0 0 0 o O o o a o 0 0 0

C)

N 0ý0

\ N1 u1 '. D

0 = coo N ems-

- N

N N N N

..

o C Y% a% o v- . 11

om ' "r4 V to () CL) a ., 4 to U Cl +s

10

E 0 b

O A V]

" r"

-175-

Cd 41

" cý

8 8 T-

A 0 O O O O O O O O O

E- ;4 fr cö

4J (D 0 .. .n

to 3

>a H 4-4 0 O O O O O O O O O O O O

ö U 3

oo- H z 1" . a oä 4 )

" El 0 .. .N ö 4

0 ) V 'vo 4 b

° O t , g X

14 -r-4 (1) 0 41 V- O O O O O O O O O O O 0

A to 14 P, 10 CO CO "d " -P 0 a ä

o am t b "ri C) H CO tO c4 "r1 tII ( I O to % ' 0 W ° 0 0 ": t 0 0 0 0 0 0 0 0 0 0 CO () 44 O CO Si

d-) () a) " C3 $4 a a w C) w o "r4

.O (0 H 9 o o o o

O ö T- S

O S

O a 0 ON T- T- O

- O O ö t i +. U

4) F' H N " "rl t) 6 .0 0

r"-1 U T-

M E K l 0 O' 0 0 M ON ko U"\ 0 U, \ C\j N

&

H ß, O O O O O O O O O O O O O bo

u1 'D

O + ' Ä

\

sue-

\ N N

\

M lý r

t` tý lý n = = = Lam.

-176-

+ t° to Cd to es to co rq "4 ( as 0 ro (0 ý

r. 0 r. :s 0 "ý V. " r.

d C. f+ "r1 U "rl "ý U

d L7 r4 "rl V

f"'. "rl fA U U U O

Ö 0 "¢ t I 1 1 1 I d C7 Q C7

4) A s 4

0 N v 0 [. S

" 4 a I I 1 1 1 1 O O O

r0 f 1 Ui 4-) " 4

r1 q " 4-) V. " " +ý C) U "r4 CO U "

" "ý :ý "fi

v W

N U

d 1 4 Ä d .. a 4 1 1

14 1

d a Lfl% ;4 CO L%- r .. . 0 ON 150 4 . 1

d " . 8 " a 14 Ö Ö ON " t0 " 4) 0 " to v a a o 0 o I 1 1 0

cd 1) to a co or o cc Fl co $4

a n 9 - " w c n .i n) t , (ö v

N ý C -9.4 0 r4 to ) o 0 r-i . rh

C) co Cd = "ý to 4i U 4ý H 0 R

m 4a) I 1 C 1 1 1 4 4 4 I o

a co , -i 0

ý ý r -I 3

N º

1 O " 1

41 ä

1 p

a I o ö ö J 1 CO +

rl 4.3

-H 0 c a "r1 G) 4) O +J I c0 0 O O C ( ý r to to m Q ) "r -1 e 0

i

4J H O c " t 0 -i 4 " (p £'

l

.3 1 (U 4 ß 1 44

4-) 49 49

"r CC 4. N. O O O 4 ti ý' 3 3 ý o Ö CO

c ä " ºý ö ö 1 O ö . O

° ' - Ä ý ý ý

N U,

.p .0 to N. N. T- T-

N tr

b 4) tJ r4

'CJ

W d

.ý V d

a CO a,

4-4 0

ti

4-4 0 CO 4.3

CO w 0 U d

e-i p4

9 CO

v

+

-177-

Nitr. oren . letabolism.

The activity of nitrogonase of algal material

collected from the mares, as determined by the reduction of

acetylene, was measured during the summer of 1976 and the

results are given in tables 35 to 37. In Kettle More

(Table 35) activities are generally higher than thorsco

measured in the other mares. Activity falls as the

population of any species a _-os, but this reduction in

obscured by the increased activity of other species. Rates

of activity of A. circinalis appear to be comparitively 0

high andýA. fl os-aoua low, although on surface bloom

formation the influence of bacteria may be significant.

Ethylene production by soil bacteria has been reported by

Primrose and Dilworth (1976) although in anaerobic

environments. Like A. circinalia, A. snir. oiden appcre to

exhibit a high activity. During late Juno, while the

population of rah. flog-a: iixae was increasing, nitr. ogenase

activity of this species was fairly low, despite a

relatively high heterocyot frequency (Table 40). Within a

week activities had increased to near those of the Ane. bnona

species, although the heterocyet frequency had deolined.

Activities of material from white Mere (Table 36) aro

lower than those recorded for material from Kettle More.

The dominant algal species throughout the period of

measurements was A. flos-aquae, and the effects of the

increasing age of this population can be clearly soon.

Despite the increasing heterocyyst frequency (Table 40) the

total activity (per rig dry weight) and also the activity

" expressed in terms of hoterocyst numbers declines. Although

heterocysts were observed to undergo morphological changes,

no akinetes were observed (except. 3/7i 76) . Fluctuations in heterocyst frequency of algal material

collected throughout the year from Kettle Mere and Villite

Mere are given in tables 38 (data for 1974) and 39 (data

for 1975).

Although ni. trogenase activities of material collected

from Newton Mere (Table 37) are generally lowor than thorax

of Kettle: ; Iere, a different succession of species was

recorded In this mere. Algal material did not become

-178-

d cU 1f a0

" Pl .1c( 1$ 11111000OOOOOO >a 0 3°"

ö -4 to i IA

41 Aao000000 Co rn lD T- r- (- CO

ä

a) o >Z $4 N0

C) C) a r-i

0 aý 111 11 111101oOOo

a) "r w

"OOOOOOOOOO c2 Prr

to Ö" 't2 "ri d +ý 3

'. +a a) Ea

cd (O

LQ` ý- 1 ý- s- 1I11111

(D 8 r-i 0 c2

4-4 pp 1 NOroOOOOOOOOO

E)

"r4 'LS "rl O

H Pf vä c6 Co o "1 OOo110001011I1

Co 00 m (Lm "r", a. H 0" (U o IA NgOo vr4" m rn ONoOo0 r4N

0 cQ

1~ 4. ) f4 0 C) :a 4- zp

E>N 'D LD N. N. (M 0O'.

O '. O I T- T- L` 0O M "rý Lf%ö IA . ýD Mr ON ý' O"N IA N CO NN M 9:

0-OONO""""M"" Qr

N"v %D "

Co "

O" """"

rý M it

Hm

$. yN ýM

Co : r- C\ Ö (X3 Ö

CO V% N- r

CO N.

EOON r- N %Z MN r(" OM1. N

ýp M 4AN-==NNNN fV t! 1 ýO

4J .rj. r-4 W o . a3 LO a)

F3 . 14

w 0

a) +J +. 3 co to "rl as U

U0 0o

w

. ai (0 o o ti w +ý apý ä 4J 4-1

to ", co ö0

o Lý 0 b aý 4) +) ýa a)

nS RS

0

0.4 43 11-4 ;40 C) 0

U +1 co 0 ýS

"r4 U '"-4 +3 0 00 $4

.n C7

Co ö 4+ v0 ao

.ýý ono

VU ri 4N n! C) C) "P+ aa

cJ F. .: 7 (0

* ö4 3Q

-179-

d Co $. o a

oÖ ý1

1' 0 1 O 1 0 1 1 1 1 1 1 1

Co o o w 4 o aý "

x o rn o o O o O o O o 1 a p leIll

CY% (1) «4 10 93

:, r sf b 0 C ä Z to i a 0 10 to Cd to 0 4) p4 1 1 1 1 1 1 1 1 O o O 1 1 a) ö r4

ö 9.1 o m e

c o PL 4. ) lu 13 0

" r. N -t A " 0 0 0 0 0 0 0 0 M O 1 91

ý w 4) o m 0 0 o N r t t

3 q l CO 9 N 94 ý f" cd 1 >b cö R cu 0 4)

<» 54 A to o N -1 r 1.4 Ici cr - 9.9 %- cö

? 1 0 0 0 0 0 0 0 0 0 O 1 b

ß 0 4.3

9: Im to ri H

19 Ei 4-4 a)

, M 8

N 8

n 8

0 g 8 ri 3

43 A. 0 CO r Q. c- CO t- 0 to CO

t, . 1

CO k 3

43 Ü 1 Q

O r ý. {

4- in tq

c6 J ".. Cd 10 U d v 4-1 0 - 'd ti 0.. 14 O 4)) U C i4 .e

; .. i

p.

. ri 0 (Z Al

o P4 ö a: " "" 4-) Cl)

ý N o C) 5 " . .. 3 + w w 0) fi 0) * " /o 0 ` ' j, - * W, ) ° 3 > . CO O 4. o r M O O O o 0 1 . ý CO ö ä to a 0 Ü 94 . r

N

W 5 Z 43 0 Z

I -0 0 E

0) 0 * >

" 11 S ýO N

CO 0 CO -

ON CO

M N

N O c- M

M K1 `

H c0 t

4 0 10 r N [ M L cC o 4) M -0 0

O v 1 "

N "

-- "

r- " "

e- " O " O " O " O "

O N1 " 1 o

0 ý

to 9 0 ge 0) $Z

10 C) 10 to H 0 >a " p, ß +> +3

try 9, y

H 'C1 r

4- -

%0 O% 00

0 N

M f

%. 0 KI

8 4 -

e- N. -

N1 .. C. -

+1 4+ o

tom) P, - I \ N e t c 4- ON T P $4 F+ x .N bO " " . " " " " " " " " " " cti r1 G) G) U v O r M O tf1 L` r O O O O O 93 0 a s . 0 4

w w 4-5 ýi

, ri "" (. ý {. 1 (Q Is

> "ri

+1 Qi

i-N 1A

\ \ '. Q

\ '0

\ \ . -.

t\

-"

ti

"

n

A

n "

43 Q)

c ä

U m Co L. N \ \ \ \ \ " A c- e- N N N N N . -r fV M UA ?C 9 -

-180-

43 Co U 0

"y

dr

P4

Ö $4

+) O 3 O"

43 "

1 2110 s' "rI O C)

"º4 3

U Ps

, goo

av f'f

r-I Pi V- -P N .b C

vd U

4. ) "rl 0

"r4 P

U C) Cd 0

C, O ri U Cd Iz $4 4> 9ý a, 0N

4D o zý

N K Co d Cd

H to to a, IL, P, x (1)

'Ta

"rI

U

c1 Si 41 0 Co a

`Ö ä 0 0 0 0 0 0 0 0 0 0 a' .o w

rn 0 0 0 CY% 0 0 0 Co O

0

cu $4 :1 0 Pt to O O O O O O O O O O

to a, 'O r 0 0 0 0 0 0 0 0 0

CY

00 I '- r O O N O O O O O

to O t0 H 43 tH m

N tll O O Kl O O O O O

"rl 0

r N

t+1 O r N r *

O

0 " O

8 o a -

+ N

- t(\ - a'. a

t11

to 44rI

.O- co e- p. L r\ t! ý 0

U\ -Y öN NN - r+ c0 öLrö

0 r- AOOO -- OoOOO 4

n ?a"

3 -yC

u1 ON N C\j

e- r- OwN ., t "0 n1 co "N. N. C" N 4-) LO C) 0 vENOMOOO

v

ANN tC\ r

fý( M

t\ W

0

4J Cd

0 43 to

"H

0

0 4)

-j ýs 0U i ý9

t) rj ,., "ä 4'

co 4.3 0 V

"° 0

0 4)

.N o0

"rl 0 4-3 4, p 4. ) 0 -rl (D

0 4-4 0or U

tao

cz 4-) 4-31 0Ö

týA 0) V

aw "rý-j

tÖ ±"1 y 4-3 0 a) P4 Co

1t1ýý

-181- -

0 4 cd y 1 1 1 I O O

In . - O E- r-i ON 41

" 1 I 1 1 1 1 1 1

In d

r 0 "r l N K1 CO ON r4

a) .1 I M N A I 1 O O 8 X. 4-1 4) "r1

rh 0 Ea

0 O O I 1 1 1 I 1 1

4.4 " U 4

U 4-3

N r-I N C` 141 CO " .) " am

. rj 3 I t(\

N r I 1 I 1 Ci

G 4-1 0 0 -r

"rI a 0

Cd 1 1 ö M

1 1 1 I 4 + 0 4) " U 0 +) o 93 Cl) U N O $4 CD ri " r4 " C) cd O ON O' C- -4' N U U P-4 O = " " " " " " rI O

r 1 N N N N 1 O O 4-4 W 4. ) CH ý Cl) 1 0

0 (A 4) U 0 t1

-I O "r4 +> 0 (D I 1 N I I I 1 1 4.3 4-4

4-) " 0 ýC .o w

b ri

10 0 "rl H 4- N1 N I! 1 LA M N f4

co ý, y i t! ý rýl rn týl 1ý1 O ý' (1) 4J SI

" .ý ¬3 0 U O

H " K" a ) C O

. + C ) Gi

1 O M 1 1 1 I V] "r1 C) fti " ýa U : c. N

0 44 U

Li H r1 4) G) O 4) +3

r. - a) 0 (D Q) N 00 CEO 0' d x H 9

cd 0 Kl N O Z0 0 A M r ciý N N r s 1

-182-

ur% C- rn

N a) a,

a)

6 0

a) ca rl Cd 4-4 0 aý 9b c)

a) U a) P4

4-3 ra U 0 a) m

rn ON

12 H

e- Il'

cý I I I 1 N v 1 1 1 1 I 1 1 O N O

H CH T; O O

" 1 O O 1 1 1 1 M N I 1 1 1 P

c r c 0

3 1 1 1 1 K\ 1 I 1 1 1 I 1 1

"rl 0

N L` M C` r N- O\ N

1 1 O 1 O O O M M M I 1 1 1

"ý :. I 1 1 1 N 1 1 M I I 1 1 1 1

O 1 1 Ö 1 c- I 1 1 1 1 r 1

ce N M O' e- M M U1 0 UN N " " " " " " " " " " " O O ý' N N N N M M r 1 1

1 N O

H CO C-

1 O O 1 1 1 1 1 1 1 1 1 1 1 "

in ,H _e N e- 00

ý cS 3 O 1 I N N r 1 1 I 1 1 1 " O H

U O

"r1 N N U 4

1 eý " 1 1 I 1 I I 1 I 1 1 1

N H A

- H v K1 ý0 [` CO CO o0 CO 0 r N 0 . M \

' N '. D CO M ýD \ Lý tt1 \ O

A N t1' O\ N L, N c" - N N '. 0 N T- Co C/2

-183-

N. rn

U)

0

0)

E 0 ti

N Cd ea H Cd 44 0 4) bu c)

w a N

4-31 U)

U 0 d a)

0 C) r4

H

a)

N 0

Q

cý,

"r4

O

N U

ro 0

d)

cd }

4

K% 00 u1 e- 11I1I111I11ttN c- -! 1-0

.o ao UN o a. 111111111111: NN4O

1111111111111II1I

"1 11111111111I11I1

QN N 111111111111". N1

Oý Qý Oý t1ý 1111II1111I+NMNrM

OOOt! 1 O a' NM 11s+1r1f- c- rNNM -ý- r1

O co 1111I111 cý I11I11N

" b

_r 0NI! \ N Ltd -4- r .ý -t .* hO O ý- rO c- r c- rriI1IiMMMH

to 0

W LL NON

1I11I11O1"1NNr11 org

H

cti NM .ý tf1 t! ý ýO ýD vý ý 00

O

N oO

NLN M- NWrNN t\ to

-184-

4) Ö

.O r to U1 0% r' Cl- -e N e- C, "

ý "

ýp o" ' "

K\ "

N I 1 1 "

- "

N "

-- 1 T: " "

- 4) O

Cd -1

4. ) O

O' N

O `- [

ýO O

a 04 " N O O O O O O O N 1 O O

b0 X4ý

0 "r4

4)

6

H j2

Ä

2 g e-

0 e-

g c-

p 0 q . 2) 94 N C' .* .

t` CO

rr % sz M

O l`

o M

4-4 1491

o 1 0

O H 0

cö

K7 Q) to O N

4)

4)

O Rý ö

U

to C)

"i4 0

p, 4

O

1 p O

r

"

1

d

R 1

r lO

"

Cý

RS 1 U) O

r-1 .4

"

O R

1

rl 4-i

"

to r

l

P ". a U

r1 O

"

i N ei

-i

q "rl U t+ U r1

1

"

O Cd U0 O

"

p2 r1 mi

rl U

"r1 U

"

O R

C 1

IO O

r-I a

"

N M

r-i

ri U

"rl U

"

N

(0 1

(A O

S"l1

"

t0 ti

U f,

"rý U

"

4)

1 O

f1 `4'i

" vý a a s a s a ¢ 'C -s a 'c a

to 0 Ö ö ö ö 0

0 0 o 0 8 " " " "

. ah Z r- c- r r r r r ". .. .. ". ." .. ." ." of ..

93 "0

cm r*I %D

CM ON

cm Co Co

Z

0 ö ö ö ö ö ö ö C'

S. to ." ." .. .. .. ..

C' N L1- '. O e- '. D T- _e

%Z 9 %D

CY% Co ;Z C' rdl%

\o s7 cad

*lO %iö %D %0

q v oO N N Pal Pn 'lD

d O

C) r-1 Q) O x _

"rI H 4) . Ci c, cn ca se :ý z

-1c5-

abundant until m-d June and the species which bloome-d wirst,

A. circinalis, remained so until mid July, in contrast to

its early dominance and equally early disappearance in

White I"Iere and its small populations in Kettle. Nero.

Activities of A. circinalin, although showing a decreasing

trend, are more variable than that observed for A. f). os-

aquae in White ! -ere. There is no significant chunge with

the onset of sporulation. Colonies of G. echinulatn, which

were rapidly separated from the rest of the phytoplankt-on

by filtering through gauge and by purification using the

technique of differential buoyancy, also showed a declinjnc,

activity.

The low activity on nitrogenase of White Iiera algae is

also reflected in the higher-carbon to nitrogen ratios (Table -4s). An increase in the carbon to nitrogen ratio (C: 11) is also apdarant in the

. kettle Mere po.; )ulation of

A. flus-%auae which had undergone extensive sporulation in

early June. This change suggests a relative decrease in the

amount of nitrogen fixed or a relative increase in the rate

of carbon fixation as akinetes mature. There is comparitively

little change in the C: 21 ratio in Newton Mere algae, which

was characterised by the continuous dominance of a oinglb

species.

Ox r en i1ralucticn.

complexity of considerations to that which

is involved in the interpretation of the nitrodenase assays

is appNrax.;; with data on the photo-production of oxygen in

natural populations of algae incubated in the laboratory

(Tables 42 to 44). In general the patterns are E, im-EUtr.,

with hier gross photosyxithetia ret( in Kettle Niere than

in the other raeres, and a general climinution of SIctivi. t. 7 Ha

the sum . er proce, do. Both gro; 3s and net rates of oxy; can

production: fall, the net values becoming negative :t the

end of ýw:. e tie respiratory uptake exceeds photo:; , nthetic

evolution of oxy �eil.

In the Kettle ; "iere i: laterial ('fable 42) ,; rosa rat..:. of

oxygen production are low durjne, early Jurte, whc n A.

`! _. (><; -t1C' 1'! t? (tu e doC1=211i21t : -&l(*a)

is 1L71Cýi? 2^t; n]_I1ý? F3ý7C)7Ll . tit'1U21.

-186-

ca

v C)

'. O 0

94 Q)

" P,

Cý C, m0

C) H

C) zs

" c- o to

vr 1

U it 'CS

U

C äJ C

U t\;

Nv

C M

tr) E-1 O

U)

S" iJ

Z7 C)

10 ýý ca o0o8000000 LN -t "a 1t\ N rn r.

to Q) O00000O0O C0 N0OOr

pr c- -rNrr

P. N

a

11------- . :; rZ 0 r+ U ri "ri

U y

1d Sý

O ct3

oovvvvv Li u0O0NNN co "o C0 U. Cý nl c- c-- N Kl r\ 'U 4i c- FS O"

"rj All 4-5 P

p "ri to

ýi "ri O VOOOOOOONO CO IA M lý %, 0 ON c- Nr Cý CT L(\ U -t _l tV m ýo vi

cS 0 LI Sti "rl C) r-! ýý c3

000000000n C1\ OC

v

it

r-A

U

ON CG CO N Cam- co r - O Cam- 11II11., `

Cr

OI111II" U" "Ia

O C: UO c0- t^

ttl ,t III

Raj v

0

4-3 0j \U te\ 0 C7 J U'\ C; ) G> "N c- N t. c1 ONG'C. UO\

K\ 0N \O (D L\ Cý CJý hlN c- CT C7 -1I cI '11

C7

O ý` UO LC\ O 'O 1\ C; ̀

(` tf \L7 }P\ ý_) iU 9ý 111111O C`ý O 47 ?

U 'D `- N\ C) -t C rN týl cJ` J0NN

O

tý Ö" [z) NMQ ir

O O' 0

C) -t

Cam`-- 0CJ C7 U-\.? O` ýý to N4rr'. O N^\ c- b.., N c- N c- c (`1 N

fl c- CU NNI\

ä

0

II 1 i

m

ý-1

it

"

o 0 N o 2ý

aJ JJ H

" ý7 C. ý

"r1

Gý " N r7 aý

+' 4 1 I 1 "rl ý:

4J "ri ra ý

Th 1 - t i1 C U

. )

1 1 1 "i

aý C) dJ

[- l"i ý: "1 tom{

r{ W

rH FI c.

4. ) C) o

-H N U1

1 V-, c- c- r ti ,.,

-1 t37-

c0 C)

-0 J O OOOOrWN Cý r Nl pr 4

O r v. +) (6 H

R OV

L7 w U' "r' I

10 a) Q0 00000o0 u) º+ý cý rý "" 0 ,

0 A.

in C) Q\ U aý N Ö

lý cC c F I 110 I 0 U "F-> O += u ýý a"**" 0H C"% 0 0 0 0 0 0 o O C) :; P, 4-

O r` O -P Ili Q) CYN Ls

r) EO r-:. . ý

C) .. G -

-H 4-1 Fl

t-4 I; C) "^ 0 Si OOOO O O -t O Cý O O O\ O

C H

O G C7 S+ O 4. ) i7 t) (l± "0

Cj 4-)

N C) ( V1 " C- O w

! G! 1 w O to CJ t: 3 "

CO 8 'iJ " 'T,

rye r'l i * ** L^ "i= (3y ýý

o 4 i O Id

1' "r ö

R+ N 1 H <tl

O y Ll 0 to , -I . PP-1, r-l

O S-, r-l N- rf\ N ý, O CJ O CO c- '. O FOB.

V

N ÖÜ! ! 11 C7 Ü r ("IN 0

U "r+ I I II " " " " 0

Fý7 tj 0 O O O

" " .3 4.3

0

; J. Zv 1 1 I 1 1 ! P O 43

O 1 º) Gi rq (a 0 1.14 14 EA 10 y 'b 1 l`J

N (C

F= OP\ C) " CO

C co UN -r

Cc 0 C

Ul 0 '

C`_ (

O 0 L(", r- c 4'

4.1 - "rl . t N Cam- ON o . C, c-, , 10 ,

f+ - CJ M G, t C) ci

tQ ý

c U

c 1 1

f -N x 1 1 1 1 , 'N } N H

". ) 0 to 0 4) C) rf i

0 -iý c- tý U1 C: 7 1ý1 C'- N h1 L^ Lý N

CV N I'0 (V Ifs Lý ý- "ri 4-). ( ! 11 " " "

O C O O " O " r

" h1

" N\ "

C'- " hl

N 17 0 (u

tC tý

ri N

-i S

-, t: P, w

U) O O" `(1

Cl -7'

OO ^\ C"

L` ' 0

C) C\

M U-\ r1 C` \ N" " . r '. 0 ' 10 rh 4- Hc

Fat " . - r- (\l G Cý ý. ýil U. r+ r

\ 41 \` \\ f - ý> N" ý r ý c r..

' C- V " ci hý

iý t ` tip (ý , .` \ i\I i+\

\ iIý \ ; t

ý ( v (sJ C\1 (V (\! Y1

. J- ! ;y C/ M t,

-188-

U) a) p

OOOOOO J4 Iý

C9 U

" e-1 Q

of cl

H0 t, J `li

VOC. rMNr ý' N r+l Cý Q rid +) HrlIn

0 4-+ c- 0 94 rHl C

" ¢ý '3 CJ ,i

CG4: HO . F)

'" >~ fti 1 ý110 cl- C11% 110 N-U C7 O\ '. 0 Wo CT CJ'. l0ý- N- '°' CJ tUv;

ctý H L) Vý ro

Z" cu "''1

"r 1~ 1 6) O ö) UO

", 4 eH "`ý r{ c- r r- N If\ C7 '. O -. I" rfl

0" Or '= Or CU N C' oo iOV -ý ttl rl ul NNr Ca N 03 O `ý F '-D OOOO r- 0" OOOA0 . 4y

C' 10

11I1 U "zý

p 1: 14

0 :j

tti .: J 0 P, " "''ý ti

P' a b3 C P+ L` NNr lý CO r cm T!

a4""" CO Cj 10 Q

" U\ H £11tI r- 0

a)

"rl C;

44 " C .ý0 4) H 'd $: H"

v0 , -i L- CO to - ßr1 N-\ N as '. 0 ä0 N- LO Cýn. i. n N

ný H U)

va 0 ice- LO C, 04""""Q: N f4 ýi '. Cý OOr0rr c-" ON

Lv 4)

0 co

Pl U9 n ''

+5 N

11 CO rl i==. L: 'U a""""

o tt lOC c- oO N--\ n h\ ,i I-

rr c- trr c:

Cr \, D '0 \, O N- N- N C' C-

ýa N ýt rJl o ri

-189-

Net photosynthesis is only slightly loiter thz-in gross ratter.,

indicating a low rate of respiration. With the decline of

the A. x'7.0. =, -,, ciu<<e j)O )Ula. tion and an ineref. se, of (rc*_-n algae (including Volvo, ) there is an increase of j_)hotosynjt}jetic

activity. The relationship between gross and net rates

appear to be cior. e norrial for the iiiorocy, itin and.

A. circinalis populations monitored in mid Juno. Activities

were high when compared with the A. f7. c. )s-ncWae population,

but A. circinalis appears to have undergone a slight

decline in activity towards the latter }calf of June.

From late June there was a marked fa"L. I., particularly

of the net photosynthetic rate. Although there is a

downward trend of gross rates of oxygen production, the

increase in respiratory activity appears to be greater.

This leads to a large difference between ßroc; s and net

rates of oxygen production. This period coincides with a

marked' increase in temperature of the surface waters (Fig.

10), which results not only in the increase of algFil

respiration, but also enhanced bacterial Growth. Accumulation

at the water surface of algal material during this calm

hot weather, and its subsequent breakdown, would represent

the source of nutrients required for rapid bacterial

growth. Bacterial respiration is probably an important

factor in the uptake of oxygen. Increasing numbers of

bacteria were found attached to the algal material during

June Hnd July (Table 45). This may explain the low rates of

net oxygen production (i. e. the high respircation rates)

observed on some occasions during early July'.

Distinct differences in the rate of oxygen evolution

also occur with the char_r; e of domi %ance from one species to

another. A high gros, uptake was observed for alf; al

material, principally Volvox and other crec; n algae, on the

27th and 30th of June. Activities in terms of cell numbers

were also high. On the 29th of June the incubation

eam1) eJ were composed almost totally of A01. floO-ricuae,

and it was observed that production in terms of cell

nunbers was much lower than in the incuibationt: of gree-ri

algae . This is probably due to the smaller volunin uf the

cu? J_:: Of : lIpl. r1_0 -U uno. When OX 7 (. n production its

-190-

expressed in terms of amount per mg dry weight the values

are similar. she respiration rate of the green algae

appears to be higher than that of Ah. i'l os--r IF , under the

conditions applied. in contrast to Arh, fý-orj-rac Llae, A. s; siroidee appears to be as active an the green algae

described above, but a high rate of oxygen uptake reduces

net production to a negative value. Further comparisons

between different species cannot be made during early July,

due to the mixture of a number of species in more or lens

equal proportions. %

Table 45. Bacterial numbers in algal samples, 1976.

Numbers x 10-3 mg-1 (dry weicht).

Dates, "Kettle ilere White Mere Newton More 19,16 iso Alga No

" Alga No Alga,

6/6 1105 A. floc-aauao ----

22/6 -- 84 A. flor-nguan -

3/7 6281 A. sniroides 4330 " of 1622 A. circa. nrQ ici .

5/7 5085 and 938 " 10676

6/7 ---- 193501 Mixture

27671 A. circinnlio

2625 G. echinulata

A similar response to changing conditions (for exarriple

temperature, Fig. 1 1) , to that found in Kettle More, was

also observed in White Mere (Table 43). Gross rates of

oxygen production appear to be fairly high throughout the

period of observation although net production declines.

Thus although photosZ; ynthesis remains relatively constant,

respiratory activity increases, producing negative values

for net oxygen production. Like Kettle More, bacterial

numbers in White Mere increased during, this period (Table

45). Both photosynthetic and respiratory ectivity appear to

be inhibited in material collected from a dense surface

scum on the 29th of June. It is probable that this is a

reý--u_lt of d1ýrnaý; o to the 'alga_l cells rather than nutrient

deppl. etion, nines both photo; s; jnthcisis und ror31Dirotiun were

affected. '11i material WELD also diluted with filt*. rt: d lnktý

-191-

water to a concentration oinilar to that which was u.., ually

used in these incubations.

tiiLi rates for both not and gross oxyj; en production,

calculated as a function of cell numbers, wat3 observed on

the 5th of July, when a mixture of A. oriroid¬'s and

colonial green algae (Scenedesmus and was

present. It is possible that the green pleite are re, 3pono blo

for the increa e, although when next day the o::, on

production of a population predoriinaintly of green algae war

measured, it was found to be much lower. Ae vir.. tller

population of A. spiroicles did not Show a siriiiar light

photosynthetic activity. However, this was with a DaMple

obtained from a dense scum and therefore probably coffered

considerable damage. Oxygen production exprosoed as a, function of dry weight had also increased, Wt to a emallor

extent.

Gross oxygen production was loss variable during the

period of sampling In late Juno and early Ju: Ly in Newton

I1ere. (Table 44) than in Kettle Mere and White Mere. Thin

may be a reflection of the stability of the population of A. circina? _is, which remained thn dominant alga throughout

the period of blooming in this mere. Rates calculated as an expression of dry weight were similar to those estimated in

the other rzeres, but when calculated in turm: -e of cell

numbers, lower rates were sometimes obtained. However, for

species of similar size (ouch as the A. floes-a uae_ in White

Mere) similar rates were recorded. The difference in rates

shown by algae of different size is clear when compared

with samples taken from Mettle Ner. e during the dominance of A. spiroidos, a large celled alga.

Although variable, rates of net oxygen production in

Newton Teere tended to be lower and neýEitlve values were

obtained on fewer occasions, than in Kettle Mere and White

Mere. Towards the end of the sampling period an increase in

respiratory activity was observed, which is probably

correlated with the increase in numbers of bacteria (Table

45). -

ý cý ýý .

D12cussIOll Phosphorus.

The abundance of orthophosphate, which prevails

throughout the thermally stratified periods in the meros,

is reflected in the high levels of intracellular phosphorus

of natural algal populations. Intracellular phosphorus was

found to be high in the early phäees of bloom formation of

Ah. flos-aciune (Haley and HHendoel, 1976) and of Perid3, nittm

cinctum (jerruya and Berman, 1975). In contrast to the

observations made on algae from the mores, the level of

intracellular phosphorus declined at the end of growth and

prior to the collapse of blooms in both the investi. gationt;

quoted. Gerloff and : 3koog (1954) found that the cellular

levels of phosphorus and nitrogen in natural. blooms of

M. aeruginosa are comparable to laboratory cultures grown

in an excess of these nutrients. Concentrations of nitrogen

and phosphorus in the lakes from which the materiel was

collected were not, however, stated. Healey and Hendzel

(1976) found changes of cell nitrogen and phosphorus much

larger than the change of concentration of nutrients in the

water and suggested that the former are more accurate

indications of the progress of a bloom than the latter.

The low levels of alkaline phoaphataso observed in

bloom populations of algae from the meres also indicate a

sufficiency of phosphorus. An increase of alkaline

phosphatase activity (Healey, 1973; Beath and Cooke, 1975)

and enzyme synthesis (Ih_lenfeldt and Gibson, 1975) have

been demonstrated under conditions of phosphorus depletion.

However, Healey and Hiendzel (1 976) found that alkaline

phosphatase activity was variable and could not be related

to changes in phosphate uptake and cell phosphorus. bone (1971) suggests that an inverse relationship between coil

phosphorus and alkaline phosphatese activity is exhibited

only in phosphorus starved cells rather than in actively

growing phosphorus limited cells.

The indications frort the results discussed in this

section are that there is a plentiful supply of biologics iiy

available phosphorus as orthophoe -, hate and that the algal

-19: 5-

material which accumulates as a surface scum does not

become phosphorus depleted.

Nitrogen And Carbon.

The nitrogenase enzyme has been the subject of much

study, and many factors have been investigated which affect

its synthesis and activity in a range of biological

systems. Cell-free extracts of the enzyme are highly

sensitive to oxygen (Pay and Cox, 1967), and in the non- heterocystous species enzyme activity is only detected in

micro-aerophilic conditions (Rippka et aJ., 1971; Kenyon

et al., 1972). An exception appears to be Glococapas app. (Wyatt and Silvey, 1969), and apparently the marine blue-

green alga Trichodeernium, which can reduce acetylene to

ethylene in both aerobic and "anaerobio conditions ('. I ay1er

et al., 1973). It has been suggested that the colonial

nature of the alga protects the inner cello from oxygen (Carpenter and Price, 1976).

Despite the conditions of a high oxygen partial

pressure-in the surface waters of the merea during the

summer, nitrogenase activity was detected in all the

samples which contained h©terocystous blue-green algae, indicating that heterocysts provide effective protection

against oxygen inactivation of the enzyme. It is possible

that the colonial organization of some of the species, such

as G. echinalata, may give additional protection to the

central heterocysts from the inhibitory effect of oxygen. However the nitrogenase activity of G. echinulata, expressed in terns of heterocyst efficiency, was not significantly

greater than that measured for A. circinnl: in collected from

the same site at the same time.

Nevertheless the observed activities of algae from the

meres may be below the pötential activity. Stewart and Pearson (1970) have shown that nitrogen fixation and growth

of A. floc--p. 'uae and N. muscorum in laboratory cultures is

greatly increased at partial pressures of oxygen below 0.2

atmospheres. Data presented by Stewart (1971) for natural

populations of Gi oeotricM. a and Rivu2,, aria demonstrated that

nitrogen fixation can proceed oven, at elevate. }d oxygen

-194-

partial pressures. "A doubling from 2U to 40 reduces the

the naximurn activity (at 2U; ß) by 5U, ß. Inhibition of

activity by oxygen is-rapidly reversible (Stewart and Pearson, 1970). This factor may be of importance in the

markedly stratified environment of the mores, whore oxygen is greatly depleted a metre or two below the surface.

As mentioned in the Discussion to Section 1, the

soluble inorganic nitrogen to phosphorus atomic ratio in

the mores investigated was about 5: 1. Although nitrogen

will therefore become limiting before phosphorus, the

conce ntratio no 'of nitrate in the surface samples eollooted during periods of bloom formation was not significantly

reduced. Its presence would, however, be expected to have

an effect on nitrogenase activity. Horns and Fogg (1970)

" observed that although there was no direct correlation

between the rate of nitrogen fixation and nitrate

concentration, fixation was generally limited to periods

when the amount of dissolved nitrate nitrogen was below

0.3 mg l- A similar limitation of nitrogen fixation to

periods of low nitrate concentration was also reported by

Dugdale and Dugdale (1962) and Horne at n : L. (19'j2).

Additions of nitrate and ammonia were found to have an inconsistent effect on'fixation, sometimes even increasing

the rate (Goering and Neess, 1964). Although the oonoontrat- ion of nitrate in the mores occasionally exceeds 0.3 mg 1-1

it is poss. hl. e that the concentration within the algal

clump, and colonies is reduced. Since the requirement for

nitrogen is higher than that for phosphorus, depletion of nitrogen within the cell is nor© likely to occur than

depletion of phosphorus given similar external concentrations. The fixation of atmospheric nitrogen requires a supply

of energy and reductant. Energy is supplied, in the light,

through the photosynthetic production of ATP. Reductant,

in theory, may be supplied directly by photosynthetic

electron transport processes, that is, by a direct photo-

reduction of nitrogen (Fogg and Than Tun, 1953). Fiore

recent work, reviewed by Fogg It al. (1973), indicates that dark processes are the main source of roductant to

nitrogemnLc> in aerobic heterocyutoua all;.,; wherc ti. otcrocy:; ts

-195-

are the nitrogen-fixing sites.

Duggdal e and I)ugdnle (1 962) and Gooring and Neese (1964) have shown that nitrogen fixation in plankton

samples is mainly light dependent, although some fixation

may occur in the dark (Dugdale and Dugdale, 1962; Pay,

1976). The depth profile of enzyme activity in 6±indormere (the "other" Lake District) shows a similar pattern to the

primary productivity/depth curve (Horne and Fogg, 1970).

The incubations of natural populations performed during

the summer of 1976 were done at a constant light intensity

of 1000 lux. However, during the period of sampling, from

May to July, the activity recorded in blooming populations decreased. This was also matched by a fall in the rote of

photosynthesis, although gross rates were generally

maintained. Although the fluctuations in the rate of

photosynthetic oxygen production and of acetylene reduction

were considerable, a decreasing trend was observable for

each of the species of algae to form a surface bloom. Thiu

was particularly marked in Newton Mere in which A. circinuijo

was the dominant alga throughout the period of sampling, The reduction of nitrogenase activity may be the

result of the reduction of the photosynthetic rate, or both

may be the consequence of the reduction of growth and thud the increasing average ago of the algal cells in a surface bloom. Weare and Benemann (1973) observed an increasing

sensitivity of ni trogenase activity to oxygen as brrtoh

cultures of A. ctriindricn increased in age. Algal material

collected from surface scums in areas sheltered from wind

, and turbulence appears to be less active than that collected from the more exposed and thoroughly mixed areas of the

mere. This indicate: i that the high light intensity at the

surface of the water during the summer is the probable

primary cause.

This reduction of nitroeenase activity was observed despite the increasing, heterocyest frequency which generally

occur.: during the sunurer. Heterocyct frequency, considered

alune, is not an accurate indication of activity. The

frequency of heterocy: 3ts, however, in general roflc. ctn the cellular nitrogen status, (Iul_noouri. -a et n-I 197-2) . Pay

-196-

and Vasconcelos (1974) found that in cultured of j1. cylirtdri.. r

grown in the absence of vanadium and molybddnum hoterocyst

differentiation was induced although there wao no increase

of nitrogenase activity. Kale et al. (1973) obberved a reduction of heterocyst numbers in cultures of A. r1ribigun

grown without trace elements, although no measurements of

nitrogenase activity were made. Horne and Goldman (1974)

observed that trace additions of copper to natural bloom

populations produced a marked fall of nitrogenase activity

which was more marked than that of photosynthesis or

chlorophyll a levels.

Other factors may also have an' effect on hotorocy©t

differentiation and nitrogenase activity. Fogg (1949) found

that a source of readily assimilable combined nitrogen

. inhibits heterocyst formation'wjiile a source of organic

carbon increases heterocyst frequency. Although nitrate wan

present in appreciable quantities during the summer in the

meres, suppression of heterocyst differentiation by this

compound is incomplete. Ogawa and Carr (1969) found

heterocysts to be present in filaments of A. variabilin

grown in media containing 28 mg 1-1 nitrate. In cultured of N. muscorurn transferred to media containing 100 mg J, ̂ 1

nitrate, heterocyst frequency fell from 6` to 1iß after

which there was no further decline (Stewart et e., 1968).

Organic nitrogen compounds were: observed to completely inhibit the differentiation of heterocyste of Aph. floan-

aa uas (see Section 2).

A sufficiency of phosphorus is also required. Stewart

et a?. (1970) and Stewart and Alexander (1971) demonstrated

a stimulation of nitrogenase activity in phosphor. ue

starved cultures to which phosphate war-: added. The ineroa©o

of activity occurs within 15 min, although the increase in

size of the ATP pool proceeds less rapidly (Stewart and

Alexander, 1971). A further characteristic of phosphorus

deficiency of A. variabilis observed by hiecley (1973) is a

reduction of heterocyst frequency from 4; u to U. 5, o within 60 h. Readdition of phosphate produced an increase of heterocyst numbers to a normal level within a similar

period. It is considered that the supply o]' Eihot; phL, rue to

-1 97-

the u1igai cells under the conditions encountered in the

mares is cuf-'icient, and that other factors' riny be

nrimari1y rc:: ponc ble for the reduction of nitroeannse

activity'.

Heterocysts display a limited period of physio1. oCical

activity (Tischer, 1957) and appear to have a limited life (Pay, 1973). Senescent heterocysts become vacuolato and

appear devoid of cell contents. A decline of nitrogenaso

activity of U. muscorum after 4 days in batch culture was

observed by Stew, =art et al. (1968) and a reduction of heterocyst frequency was also found. However, when the

acetylene reducing efficiency of the hetoroeysts was

calculated, it was found to have fallen to a third of the

maximum value after a week in batch culture (Stewart eat al., 1968). '.

Probably the most significant factor affecting the

change of nitrogenase activity of algal material from the

Tieres is the high light intensity to which the bloom is

exposed. The formation of a surface bloom probably marks the termination of growth of the species concerned, and thus any further differentiation of physiologically active heterocysts may be reduced. The decline of net rates of

photosynthesis may also affect the rate of nitrogen fixation. The generally low-ratio of carbon to nitrogen may be a reflection of previous nitrogen fixing activity. (or a

result of photosynthesis being more sensitive to high light

intensity than nitrogen fixation), an abundr; nco of

assimilable combined nitrogen or of a diminished photo-

synthetic activity. An increase of heterocyst differantiat-

ion and nitrogenase activity in A. cylinclricF has been

shown by Uulasooriya et al. (1972) to occur only when the

cellular C: N ratio increases from 4.5: 1 to about 6: 1.

Values of about 4.5: 1 were found in material from the mares, even in heterocystous filaments. Changes in the C: I1 ratio

can be rapid (Kulasooriya et n., 1972) and thus the

heterocyc3ts, which will persist in unbroken filaments, may be a legacy of an earlier deficiency of nitrogen produced as the flan, is actively growi. nt;. .

-198-

Chant*e; s Of 1: (-. tabo3. isn And jporulation.

A consistent feature of the A-nnbaeria species oboorvod

in the meres is the association between the . )osition of

akinetes in the filament in relation to the hoter. oo, st. In

the species encountered the first akinete to be formed

differentiates from the third vegetative cell from the

heterocyst. Small variations in nunbers of undifferentiated

cells between the akinete and the heteroc; rnt were found to

be due to further cell divisions subsequent to akineto

formation. Further. differentiation of akinetes, producing a

short chain of two or three akinetes, proooede ountripetally,

away from the heterocyst.

A similar relationship between hat©rocynts and akinetas has been observed for other species of blue-green a. iiae.

The akinetes of A. cylindrica d4fferentiato from cells

adjacent to the intercalary heterocysts (dolle, 1965; 1966).

In OTlindro - permum and Glo"eotriohia also akinetee are

produced next to the terminal hetorocyst.

This relationchi) led to the suggestion, first made

by Carter (1856) that the h©terocysts exert some control

over the formation of akinetes (Fritsch, 1904; Bharadwaju,

1933; Singh, 1942b). Experimental evidence for this .

interaction was obtained by Volk (1966), who found that

heterocysts could inhibit as well as stimulate akinote

differentiation under some conditions.

Despite the apparent relationship between akinetos rind

the presence of heterocysts, some exceptions exist. in come

species, such as ! ý'odularia (Fritsch, 1951) and A. doliolttn

(Tyagi, 1974), akinete differentiation starts in the cells

equidistant between two heterocycts. burger (1974) observed

a considerable decree of variability in the position of the

akinetes in the filament in natural populations of A. als nhtonica, although 6O;, of the akineten wore found

four or less cells away from the heterocyst. äi^tultaneoue

development of all the veC, etative cells into a_tinetes,

without apptlrant influence. of heteroe;; Fata, Was observed in

A. doliolutn by 3iný; h acid Srivaotava (1968). In a: tudy of

mutant : strsins of t1u stime species $in(; li (and Dikr31hit

c011cJ. ude; that il eterC)Cyst and akineto differw: Jitiatiuit sr4ý

-199-

seperate processes regulated by different genes. Also

mutant strains of A. doliolum were produced which lacked

heterocti'sts but could sporulate (: iinha and Kumar, 19 7: %) . A natural example of akinete production in a non-

heterocystous filamentous blue-green alga is provided by

the 1. aphidopsis species (Singh, 1942a; Hill, 1972; Croribcrg,

1973).

According to an alternative concept the recent history

of the potential akinete-forming cell may be of importance.

Mitchison and Wilcox (1972) observed assymetric call

division in Anabaena catenula and the differentiation of the

smaller cells only into heterocysts. In Cylinc rosnermuui Eiji. both heterocysts and akinetes are differentiated from

recently divided sub-terminal calla (Clark and Jansen, 1969),

which indicates that the metabolism of the ochs must be in

a susceptible phase before differentiation can proceed.

Although the pattern of response of different taxe

varies, the cellular C: 11, ratio is probably an important

indicator of the factors governing akinet© as well an

heterocyst development. Fritsch (1951) suggested that

heterocysts secrete growth stimulating subotanons, the

nature of which changes in the later stages of development

which will result in the stimulation of akineto difforent-

iation. Alternatively he suggests that the reaction of the

vegetative cells to the excreted compounds may change. The

substances involved are probably nitrogen compounds. It

was suggested by Fogg O951) that heterocysts release

nitrogenous substances to adjacent cells when the nitrogen

in the rnediur. l becomes low. Large amounts of fixed nitrogon

compounds are released by growing cultures of blue-Lroon

algae (%`og , 1952. ).

An inverse relationship, that of heterocysts acting as

a sinkt for carbon compounds, was demonstrated by Uolk

(1968). From this it may be suggested that a reduction of

carbon compounds in the cells adjacent to the heturocyi-It

nay initiate alrinote development. '. folk (1965) indicates

that pho_. phorus starvation, nlthouz -li it induces kiporuý atiori,

: iW c}rily sezieý_ti:: c: the vc3�etri-tivO CU11. s to the iýýflu 'nc: c of

the hetc: rocyst; s. It is also j)os ibl. c that a reduction c): ' tliu

It

-200-

sugar phosphate levels in the vegetative cells, as a result

of phosphorus starvation and the transfer of carbon

compounds from the vej; etative cells to. the hetorocysto racy

be the stimulus to sporulation (i+olk, 1975). A concentration

gradient of the relevant compound along the algal filament,

and the requirement of a critical concentration may account

for the generally consistent position of the akinetes

within the filament. however, in thoso tpecios in which

akinetes are found adjacent to the heter. ocyst, the root of

the filament is then isolated from the influence of the

heterocyst due to the dense and intact akinete envelope. A

suitable physiological state may have already boon reached

by any further cells which undergo differentiation before

being completely cut off from the hetorocyst. This may

explain the occurrence in many species of only short chains

of akinetes. Alternatively, external factors may asour, ie

importance.

These factors may act directly on the vegetative cello,

without the intervention of the hetorooysts, or, au in the

case of phosphorus starvation, E3ensitice the cells to the

influence of further factors. It *is pouc2ble that the

factors which stimulate svvorulation operate throut; h the

mir. iicking of the interaction between the vegetative cell

which may differentiate into an akinete, and the lieterocyst.

Although extracellulur products, probably organic nitrogen

compounds, have been shown to stimulate oporulation of

C. lichenif orme (Fisher and 4aolk, 1976) nitrate was found

to be inhibitory to the sporulation of A. dolioJum (Singh

and. Srivastava, 1968; Tyyagi, 1974). Glucose was found to

stimulate sporulation and, moreover, the reducing activity

of heterocysts was reduced in sporulatin ; cultures (Tyaj,

1974). A more vigorous nitrogenase activity was observed in

non-oporulatir. C mutants of A. dolio1_1um than in the normal

wild-type or in akincte-forming mutant strains (:; inch and

Dikshit, 1976).

The factors involved are therefore very cormp>l. e. x, and

insufficient data are available to Lpoytulato anythine more ti: rýn a c; er: ral : _iec}innism of the induction of f>porn Fatjcon in

t:: c r< res .he cellular C: IJ 13ocil ratio in probably of

-201-

considerable importance in the control of differentiation

in the populations collectod from the n roc .A ratio of C: 11

of about 4.5: 1 was found in sporulating r: iater. ial of A. floc-aau e in 'Kettle ziere and i31aI e : zero and also in

aporulating A. cireinalis in I; ewton Mere. Although the nsarao

ratio was observed in material collected on 3U Juno 1976

fron Newton Piere in which no al: irietoo were found,

sporul ation was immenant and in fact akinetes war c observed in other samples collected from the same more at the same time.. In contrast the material collected from White More

during the same period had a higher ON ratio of about 5,5: 1 to 6: 1. T1o akinetes were observed in this material (A. flus-aquae) throughout Juno or July. Although

comparisons between material from different sitee, even

when of similar taxa, are not strictly legitimate, it would

appear that a greater diminution of photosynthetic activity

than nitr"ogenase activity is a prerequisite of sporulation.

To suggest which would be affected to the greater extent,

nitrogenase by the high levels of omygen present in the

surface waters or photosynthesis by the depletion of carbon

dioxide for example or by the intense solar radiation,

would require further investigation. The idea, developed in

the previous sections, that nutrient deficiency (particularly

phosphate) is not the main and direct factor governing the

sportlation of algae from the mores is supported by the

evidence discussed here.

GENERAL SUN NARY

GUNERAL 8U 12 :: CRY

From June 1974 to August 1976 a study was made of the

abundance and distribution within the water column of a

range of planktonic blue-green algal species. Special

emphasis was laid on the early growth of bloom populations

and the method of survival during adverse conditions,

particularly during the winter. The sites used for the

investigation were the meres around Ellesmere, north Salop,

England. Two, Kettle Mere and White Mere, were investigated

throughout the project, and two others, Blake Moro and

Newton Mere, were occasionally studied.

The meres are a series of water-filled kettlo holoo

and hollows in the mantle of glacial drift which covers the

region. The water supply is phreatie, and thus the mores

represent a relatively stable physical and chemical

environment, although changes in water level have been

produced in some mores as a result of drainage schemes. The

meres investigated are naturally eutrophie, and have

probably always been so. The regular occurrence of

surface blooms of blue-green algae ("the breaking of the

meres") is well established in the local folk-lore (Jackson, 1883; Phillips, 1884).

A typical succession of blue-green algae (Reynolds,

1971) was found in Kettle Mere and White More. A. ciroinalis

was generally the first to form surface blooms, followed

by A. floe-aquae. Other species, including A. aniroidos,

A. solitaries and Aph. floc-aquae bloomed later in the

summer. Microcystis generally did not become abundant

until the late summer or autumn. This pattern of succession

appears to bG consistant throughout the temperate lakes in

which blue-green algae occur. Apparent optimal temperatures

for the occurrence of planktonic algae in Lake Michigan (Stoermer and Ladewski, 1976) are similar to those observed

for the meres. Populations were generally smaller in Kettle

Mere than in White Mere. This may be the result of earlier

bloom formation due to the very sheltered position of Kettle Mere and its low surface area to depth ratio. Another factor may be the strong absorption of light by the

-204-

peat coloured water of Kettle t. ere, which may be of

critical importance in comparivons between the two mares. Kirk (1976) observed that dissolved organic compounds

greatly increased the attenuation of photosynthetically

active radiation when compared with that of other

wavelengths. Differonces in nutrient composition and

concentration were, by comparison, arall. Although

concentrated at the surface during stagnant periods, algril blooms did not become nutrient (nitrate and phosphate) depleted.

With the formation of a surface bloom during the

summer, many of the heterocystous species of blue-green

algae sporulated. This was observed to occur whether the

various species formed surface blooms in a distinct

succession, which occurred in Kettle Tiere, or when several

species bloomed simultaneously, as frequently happened in

White Mere. It has been suggested that sporulation in a

response to unfavourable nutrient conditions (Glade, 1914;

Wolk, 1965) or a result of the ageing of the culture and the concomitant increase in the concentration of extra-

cellular products (Fisher and Wolk, 1976). However, in the

meres studied, sporulation was not accompanied with any

significant change in the concentration of essential inorganic nutrients. A corroboratory observation wan made

on natural populations of G. echinu_lata, in which akineteo

were formed during active growth and in juvenile ooloinie© (Roelofs and Oglesby, 1970). It wee thorefore considered that the change of conditions encountered as the alga floats towards the surface of the water, rather than

direct nutrient deficiency, stimulates the differentiation

of akinetos. A factor of apparent iniportanee, which wan

consistent for all the instances of aporulation examined, is a high degree of insolation. This consideration is

supported by the observation that blooms of hetorocyatou©

algae which form during calm periods in the winter did

not epcrulate. However, no indication of the type of photo-inducible life cycle described by Lazaroff and Vishniac (1961,1962) for IT. muscorum was found in either natural or laboratory populations of the algal species

-205-

present in the rleres. This suggests that the factors

involved in the induction of sporulation act through

changes of physiological activity rather than controlling

directly the process of akinete differentiation.

During the period of bloom formation and sporulation,

the conditions at the water surface are optimal for

photo-oxidation (Abeliovich and $hilo, 1972) and may cause

the breakdown of surface blooms. The vegetative cells of

the filamentous species appear to be particularly sensitive

to such conditions. After the formation of akinetes the

mass of algal material descends through the water column

and settles over the surface sediments. Vegetative material

also sediments out, but little of this survives an

intensive surface bloom, particularly in Rettle Mere. The

process of sedimentation is essentially complete within a

few days following sporulation. Thus, in Kettle Mere, Fs

distinct succession of sedimenting algal material is

observed. While akinetes generally persist in the surface

sediments, vegetative material does not, particularly in

Kettle Mere, probably because of the anoxic conditions in

its hypolimnion.

In the period subsequent to sedimentation, during the

late summer and autumn, there is a marked decline in the

number of akinetee in the mud. This decline is probably not,

or scarcely due to ressuspension of sedimented material,

since the reduction in numbers sometimes preceeda the

breakdown of thermal stratification. Also very few akinetes

were found in the plankton after sedimentation, during the

winter period. It is noteworthy for comparison that

sedimented pollen grains are readily resuspended during

the autumn and winter in a lake of similar area and depth

as LJhite : ere (Davis, 1968). At this time, however, a

revival of the vegetative populations was observed. It is

suggested that marry of the akinetes germinate during late

summer and in the autumn and contribute to this population. That this immediate germination after sporulation occurs in

the natural environment is supported by the observation of large numbers of empty akinete envelopes in the plankton before sedimentation. Germination of akinetes, without a

-206-

resting period, in the original culture medium has been

observed in labcratory cultures (Fay, 1969a). The

contribution of germlings to the planktonic populations

is more important in Kettle Mere, where much less

vegetative material survives a surface bloom then in White

Mere. It is this vegetative population which ovorwiritoro in

the plankton. Extensive vernal germination of akinotea was

not observed, although numbers of akinetee in the sediments

gradually decreased throughout the winter and the following

spring. Previous investigations on the overwintering of

sporulating algae suggested that the nedimontod akinoton

remain in the mud during the winter and germinate in the

spring (Rose, 1934; Vladimirova, 1968; Wildman et n1., 1975).

The contrary observations reported here are in support of

the conclusions made by Reynolds (1972,1975a) in his

previous investigations on Crose Mere, in which he

suggested that akinete germination during the spring

probably did not contribute significantly to the population

maxima. Therefore it is considered that akinetes do not

represent a significant overwintering device but rather

serve to protect the population against the extreme

conditions prevalent during a surface bloom. Thus akinotes do act as a "buffer" (Lund, 1965) although not in the

manner which was suggested by Vladinirova (1968) to occur

in the deep Russian reservoirs, in which the ovorwintering

akinetes preserve the populations during the winter until

germination occurs under more favourable conditions. There

are indications that those filamentous heteroeystous

species which do not always produce akinetos (Aph fl, oo..

a uae. in Kettle Ziere), or those whose akinetea are prone

to attack by pathogens (A. solitaria and A. spiroiden) do

not develop such large populations end undergo greater

fluctuations in abundance from year to year than those

species which regularly produce large numbers of akinetee (A. circinalis and A. floe-aquae),

In contrast to the fragile filamentous species, the

coccoid colonial species, especially Mieroevs tin, survive

a surface bloom intact and are sedimented in an apparantly

-207-

healthy state. in a relatively non-turbulent lake the

colonies remain in the sediments during the winter, as was found in the sheltered Kettle Mere. During the summer the colonies migrate u; pwHrds through the water column to

form a surface bloom. This pattern of migration of i'icrocvatis is thus similar to that described for deeper

lakes (Sirenko et ei., 1969; Reynolds and Rogers, 1976). A low degree of turbulence appears to be critical for the

formation of clearly distinct migration patterns. Reynolds (1973a), in investigations on a relatively shallow lake (Crose Mere) found no such pattern. A similar masking of migrations by mixing was also observed in the larger of the two meres (White Mere) investigated here.

The impression gained from the field work, that

nutrient deficiency was not the stimulus for sporulation,

was confirmed by experiments performed under controlled

conditions using uni-algal isolates of the species

predonir_ant in the mores. It was found, however, that high

concentrations (above 10 rig 1-1) of orthophosphate could inhibit akinete formation. No inorganic nutrient (of those tested) acting singly, was found to consistently' induce

sporulation by its absence or presence. The converse relationship, that of cellular

differentiation being stimulated by extracellular compounds

accumulating in ageing cultures, such as was observed by

Fisher and Nolk (1976) in experiments with C. licheniforme,

was not found for any of the planktonic species grown in

the laboratory. However, some organic compounds, which

were supplied to the medium as buffering agents, were found to affect the morphology of the filament. The presence of the simple chi-peptide, N glycyl-glycine, totally inhibited

heterocyst formation. Cultures buffered with HHF. YES werd found to undergo earlier spcrulation than that observed in

control cultures. This behaviour was, however, only

observed for a single strain of Anh. floe-aquae. Otherwise HEPES did not affect the growth of the culture, except for an extension of the lag phase which was directly

proportional to the concentration of the compound in the growth medium.

-208-

$porulation in laboratory cultures of planl. tonio

isolates generally proceeds in the stationary phase of

the growth cycle. If the alga was prevented from rec+ohing

that stage, no akinetes would be produced. A delay of

sporulation may be achieved by limiting a critical

nutrient (ea. iron) required for growth, or by altering

another factor. Cultures exposed to very high or low

light intensity, or kept in the dark, grew very slowly,

if at all. In consequence no sporulation was observed

under these treatments. However, in studies on

heterotrophic cultures of Anabaenornin, Watanabe and

Yamamoto (1967) found that this alga will produce akinetes

in cultures grown in the dark with a supply of glucose.

A similar delay or absence of eporulation to that found in

cultures kept in the dark was observed when the cultures

were grown under u. v. light, or monochromatic light of a

wavelength which did not support growth. Also algae Crown

under continuous culture conditions did not sporulate. Thus

the formation of akinetes can equally proceed at the

expence of photosynthetic fixation of carbon dioxide or

through heterotrophic uptake of organic carbon compounds. In some species of algae, however, light seems to be a direct requirement for the induction of sporulation (3jazaroff and Vishriiac, 1962).

Sporulation marks a change in the physioloeic, Ll

e. ctivity of the algal population, a change from active logarithmic growth to a senescent phase in which overall

physiological activity declines.

Some support for these suggestions was obtained from

incubations of material collected from the field during

the course of surface blooms. Analysis of labile

orthophosphate and assays of alkaline phosphatase activity indicate that phosphorus does not become limiting during a

surface bloom. Nitrogenase activity and photosynthesis of

algal material incubated under controlled conditions were found to decline with increasing age of the bloom

population. In general material taken from an algal scum, previously exposed to high solar radiation, was loss

active than planktonic material which was disporaed in the

-209-

water or which occurred at the surface in shaded areaa, of

the mere. Values of the net rates of photosynthesis declined into negative figures, indicating increased rates

of oxygen uptake by respiration. Part of that respiration

may be bacterial, as the numbers of bacteria attached on

algal filaments, were found to increase during a bloom.

Blooms of a uingle species which survive for longer periods

undergo a less rapid decline of nitrogenace activity and

photosynthesis. This indicates a greater physiological

resistance of the alga to the conditions prevalent at the

surface during blooming, and thus a potential method of

assessing the state and possible future behaviour of the

bloom. In studies of blooms of Aph. fl_os-aquae, Healey

and Hendzel (1976) found changes of cellular nitrogen

and phosphorus composition comparable to those occurring

when batch cultures become nutrient deficient. Dissolved

nutrients in the weter, which were available to the natural

populations were, however, in abundant supply during a bloom (Healey and Hendzel, 1976). This abundance of

nutrients was reflected in this study, but signs of

nutrient deficiency were not observed.

Analysis of cellular carbon and nitrogen reveals that

sporulating populations have a low C: N ratio. Although not fully confirmed, it would appear that a greater reduction

of photosynthetic activity when compared to that of

nitrogen assimilation is a precondition of akinete

differentiation. Therefore, although the production of

akinetes may be indicative of a general decline of

physiological activity, the changes involved must follow

a controlled and pre-determined pattern.

The process of akinete maturation involves a

significant in rease of cell mass (growth) when conpared

with undifferentiated vegetative cells (Simon, 1977).

Sporulation thus represents a considerable investment in

terms of biosynthetic activity and is accompanied by

cellular re-organization, all of which cocur at the expense fand with the contribution of the other cells (vegetative

and hetorocysts) of the filamentous organism.

-210-

An important aspect of water quality control,

particularly with increasing outrophication and utilization

oý water resources, is the ability to predict likely,

population densities of algae under a given aeries of

conditions. Of equal importance, however, is the size of

algal populations in the period prior to. rapid growth and

the ability of these populations to undergo rapid

multiplication once conditions become favourable.

This "inoculu&" population, for the species of algae

and the environments investigated, probably bears some

relationship to the size of the surviving post-bloom

populations. The conditions at the water surface during the

summer have been shown to be unfavourable for the survival

of vegetative populations. Although there are differences

in the sines of the surviving and overwintering vegetative

populations between the two meres, the basic patterns of

akineie formation and germination, and vegetative growth,

are similar. The importance of akinete production for the

survival cf algal populations is also shown by both mores,

and there is some indication that those species which do

not produce abundant numbers of viable aknetes are

relatively limited in population development in future

years.

These findings are of critical importance in the

investigation of blue-green algal population changes in

response to eutrophication. That population levels of

future years may, in part, depend on the response of the

previous years population to adverse conditions suggests

that this approach could be used as an indicator of likely

maximal development of algal populations under postulated

conditions. However, before a quantitative relationship

can be established, it is essential that algal numbers,

their distribution and behaviour be monitored over an

extended period to test these assumptions.

Although oome factors, such as a high level of

inE: olation, were established as possible decisive factors

controlling differentiation, further work on these aspects is required to elucidate the mechanism involved. Of

particular importance is the monitoring of physiological

-21 1-

changes of algae in the period prior to blooms, and during

bloom formation and breakdown. Healey and Hendoel (1976)

found that changes of cellular phosphorus and nitrogen

content were Uretter than the changes of the same nutrients

in the water during the formation and collapse of a bloom.

They probably arise from a significant change of metabolism

during this period. A similar situation was observed in the

meres, where large changes of nitrogenase activity and

carbon fixation in the blooming algal population were

recorded against a background of a dramatically changing

physical and a relatively stable external chemical

environment. It thus appears that changes of physiological

activity are a' more accurate reflection of the state of the

algal population and more suitable indicators of its

likely future behaviour, including the induction of

sporulation.

0

REFERENCES

f

REFERENCES

Abeliovich, A. and Shilo, Pi. (1972) Photo-oxidative death

in blue-green bacteria. J. Bact. 111,682 - 689.

Allen, ii. 13. (1952) The cultivation of Iiyxophyceae. Arch.

Mikrobiol. 1,34 - 53.

and Arnon, D. I. (1955) Studies on nitrogen-fixi:: g

blue-green algae I. Growth and nitrogen fixation by

Anabaena cylindrica Lemm. Pl. Physiol.. Lancaster Q,

366 - 372.

Allen, M. Ii. and Stanier, R. Y. (1968) Selective isolation

of blue-green algae from water and soil. J. ren.

Microbiol. 51,203 - 209.

American Public Health Association (1971) Standard Methods

for the Examination of Water and Wastewater, 13 th ed.

Am. Publ. Health Assoc. Inc. New York.

Baker, A. L., Brook, A. J. and Ilemer, A. R. (1969) Some

photosynthetic characteristics of a naturally occurring

population of Oscillatoria atardhii Gomont. limnol.

Oceanogr. 14,327 - 333.

Batterton, J. C. and Van Baalen, C. (1968) Phosphorus

deficiency and phosphate uptake in the blue-Green alga Anacystie nidulans. Can. J. hiicrobiol. 1A, 341 - 348.

Bendschneider, X. and Robinson, R. J. (1952) A new opeotro-

photometric method for the determination of nitrite in

sea water. J. mar. Res. 11,87 - 96.

Berman, T. (1970) Alkaline phosphatases and phosphorus

availability in Lake Kinneret. Limnol. Oceanojr. J,

663 - 674.

Bharadwaja, Y. (1933) Contributions to our knowledge of the

lyxophyceae of India. Ann. Bot. A,, 117 - 143.

Bone, D. H. (1971) Relationship between phosphates and

alkaline phosphatase of Annbaena flos-a use in

continuous culture. Arch. Mikrobiol. 80,147 - 153.

Boulton, G. S. and Woreley, P. (1965) Late Weichselian

glaciation of the Cheshire - Shropshire basin. Nature,

Lond. '207,,

704 - 706. Boyd, C. E., Prather, E. E. and Parks, R. W. '(1975) Sudden

mortality of a massive phytoplankton bloom. Weed. Soi. 223,61 - 67.

-214-

Burt, J. S. (1961) Isolation of bacteria-free cultures- from horrnogone-producing blue-green algae. V'ature.

Lond. 192,1275 - 1276.

Burger, J. (1974) A study of two populations of Annbaenn

lnktonica Brunnth. (Cyanophyta) from Minnesota.

Phycolog; ia 11,125 - 129.

Canter, H. M. (1954) Fungal parasites of the phytoplankton

III. Studies on British Chytrids, XIII. Trans. Br.

mycol. Soc. 2Z, 111 - 133.

Carpenter, E. J. and Price, C. C. (1976) Marine Onoillr; toria (Trichodeemium): Explanation for aerobic nitrogen

fixation without heterocysts. Science. N. Y. 191.

1278 - 1280.

Carter, H. J. (1856) Notes on the freshwater infusoria of

the island of Bombay.. No. 1. Organisation. Ann. I-Inc.

nat. Hist. (Ser. II) 18,115 - 132 and 221 - 249.

Chernusova, V. ICI., Sirenko, C. A. and Arendarahuk, V. V. (1968) Localization and physiological condition of mass

species of blue-green algae during late autumn and

spring periods. In "Tsvetnie"Vody ýBloominr. " Water)

(ed. Topachevsky, A. V. ) pp 81 - 91. Izdat "Naukova"

Dumka, Kiev U. S. S. R. [English tranalationJ.

Chu, S. P. (1942) The influence of the mineral composition

of the medium on the growth of planktonic algae. Part

I. Methods and culture media. J. Ecol. 0,284 - 325.

Clark, R. L. and Jensen, T. E. (1969) Ultrastruoture of

akinete development in a blue-green alga, Cvindronpermum

ap. Cyto? oEia , 439 - 448.

Collingbourne, R. H. (1966) General principals of radiation

rneterology. In Light as an Boo? ogical Factor (ode:

Bainbridge, R., Evans, G. C. and Rackham, 0. ) pp 1- 15. .

Blackwell Scientific Publishers, Oxford and Edinburgh.

Costerton, J. W. F. (1973) Relationship of a wall

associated enzyme with specific layers of the cell wall

of a Gram-negative bacterium. J. Bact. 1-14,1281 - 1293.

Cronberg, G. (1973) Development and ecology of Rnrhidopnin

mediterranes Skuja in the Swedish Lake Trummen. Svens bot. Tidskr. Al, 59 - 64.

Daft, N. J., McCord, S. B. and Stewart, V1. D. P. (1975)

-215-

Ecological studies on algal-lysing bacteria in fresh-

waters. Freshwater Biol. a, 577 - 596.

Davis, M. B. (1968) Pollen grains in lake sediments:

redeposition caused by seasonal water circulation.

Science. N. Y. 162,796 - 799.

Demeter, C. (1956) Uber Ilodificationen bei Cyanophyoeon.

Arch. I'Iikrobiol. 24,105 - 133.

Dinsdale, N. T. and Walsby, A. E. (1972) The interrelations

of cell turgor pressure, gas-vacuolcition and buoyancy

in a blue-green alga. J. -exp. --Bot.

22,561 - 570.

Dokulil, M. (1971) Atmung und Anaerobioaerenteno von

Sußwasseralgen. Int. Revue tree. Hydrobioll.., 56,751 - 768.

Dugdale, V. A. and Dugdale, R. C. (1962) Nitrogen

metabolism in lakes: role of nitrogen fixation in

Sanctuary Lake, Pennsylvania. Limnol. Oceanojr.

170 - 177.

Eberly, V1. R. (1964) Preliminary results in the laboratory

culture of planktonic blue-green algae. Proo. Ind ans Acad. Sci. , 165 - 168.

(1966) rotes on some new and rare IIyxophyceao in

laboratory culture. Trans, Am. microsc. Boo.

130 - 133.

Eloff, J. N., Steinitz, Y. and Shilo, U. (1976) Photo-

oxidation of cyanobacteria in natural conditions. Appl,

Environ. r i. crobiol. 1,119 - 126.

Fay, P. (1969a) Metabolic activities of isolated spores of

Anabeena ovlindrica. J. exp. Bot. 20,100 - 109.

(1969b) Cell differentiation and pigment composition

in Anabaena cylindrica. Arch. ISikrobiol. 67,62 - 70.

(1973) The heterocyst. In The I3iolorv of the Bluo-

Green AlRao. (eds: Carr, N. G. and Whitton, B. A. ) Ch.

12, pp. 238 - 259. Blackwell Scientific Publications,

Oxford and Edinburgh.

(1976) Factors influencing dark nitrogen fixation in

a blue-green alga. Appl. Environ. Microbio.. J, 376 - 379.

and Cox, R. U. (1967) Oxygen inhibition of nitrogen fixation in cell-free preparations of blue-green algae.

loo

-216-

Biochem. bioph>is. Acta 142,562 - 569.

and Fogg, G. E. (1962) Studies on nitrogen fixation

by blue-green algae III. Growth and nitrogen fixation in

Chlorogloea fritschii Mitra. Arch. Mikrobiol. . 2,

310 - 321.

and Kulasooriya, S. A. (1973) A simple apparatus for

the continuous culture of photosynthetic micro-

organisms. Br. phycol J. 8,51 - 57.

, Stewart, W. D. P., Walsby, A. D. and Fogg, G. B.

(1966) Is the hoterocyst the site of nitrogen fixation

in blue-green algae? Nature, Lond. 220,810 - 812.

and Vasconcelos, L. (1974) Nitrogen metabolism and

ultrastructure in Anabaena cylindrica II. The effect of

molybdenum and vanadium. Arch. 14icr. obiol.. . 22,221 - 230.

Fisher, R. W. and Wolk, C. P. (1976) Substance stimulating

the differentiation of spores of the blue-green alga

C3, l indrospermum licheniforme. Nature. Lond. 259,

394 - 395" Fitzgerald, G. P. and Faust, S. L. (1967) Effects of water

sample preservation methods on the release of phosphorus from algae. Limnol. Oceanorr. 12,332 - 334"

and Nelson, T. C. (1966) Extractive and enzymatic

analyses for limiting or surplus phosphorus in algae. J. Phycol.

_29 32 - 37.

Fogg, G. E. (1949) Growth and heterocyst production in

Anabaena cylindrioa Lemm. II. In relation to carbon

and nitrogen metabolism. Ann. Bot.. N. S. 1J, 241 - 259-

(1951) Growth and heterocyst production in Antbaena

cylindrica Lenzen. III. The cytology of heteroeysts. Ann.

Bot., N. Ü. 11i 23 - 35.

(1952) The production of extracoellular nitrogenous

substances by a blue-green alga. Proo. R. Boo. B 139,

372 - 397. (1965) Algal Cultures and Phytopiankton Eeolorw.

University of Wisconcin Press, Madison and Milwaukee,

U. S. A.

(1969) The physiology of an algal nuisance. Proc. R. Soc. B jM, 175 - 189.

(1971) I'litrogen fixation in lakes. In Biolorioa1_

-217-

Nitroren Fixation in Natural and Af; riculteral Habitats

(eds: Lie, T. A. and Mulder, E. G. ) pp. 393 - 401. ELL

soil, Special volume.

and Stewart, N. D. P. (1965) Nitrogen fixation in

blue-green algae. Sci. Progr. ri, 191 - 201.

Pay, P. and 1? alsby, A. H. (1973) The Blue-

Green Algae. Academic Press, London and New York.

and Than Tun (1958) Photochemical reduction of

elementary nitrogen in the blue-green alga Anabeena

c. ylindrica. Biochem. _biophys.

Acta Q, 209 - 210.

and (1960) Interrelations of photosynthesis and

assimilation of elementary nitrogen in a blue-green

alga. Proc. R. Soc. B, 111 - 127.

Foy, R. H., Gibson, C. B. and Smith, R. V. (1976) The

influence of daylength, light intensity and temperature

on the growth rate of planktonic blue-Green algae. Br.

phycol J. 11,151 - 163.

Fritsch, F. E. (1904) Studies on Cyanophyceae III. Sore

points in the reproduction of Anabaena. New Phytol.

216 - 228. (1945) The Structure and Reproduction of the A1P, te

Vol. 2. University Press, Cambridge.

(1951) The heterocyst: a botanical enigma. Proc. Linn.

Soc. Lond. 162,194 - 211.

Fuhs, G. W., Demrierle, S. D. 9 Canelli, B. and Chen, 11. (1972) Characterization of phosphorus-limited plankton

algae (with reflections on the limiting-nutrient

concept). In Nutrients and Butrophieatinn (ed:

Likens, G. 'E. ) Am. Soc. Limnol. Oceanogr. Special

symposium 1. pp. 113 - 133.

Geitler, L. (1925) Cyanophyceae. Vol. 12 of Die Süsewasser-

flora Deutchlandn, 6sterreiche und der Schweiz (ad:

Pascher, A. ). Gustav Fischer, Jena.

Gentile, J. H. and Maloney, T. E. (1969) Toxicity and

environmental requirements of a strain of Anhanizomenon

floe-aquae (L. ) Ralfe. Can. J. Microbiol. j, 165 - 173. Gerloff, G. C., Fitzgerald, G. P. and Skoog, F. (1950) The

isolation, purification and culture of blue-croon algae. Am. J. Bot. 27,216 - 218.

-218-

and , _�_, (1952) The mineral nutrition of

r1icrocystis aeruFinosa. Am. J. Dot. ; LQ, 26 - 32.

and Skoog, F. (1954) Cell contents of nitrogen and

phosphorus as a measure of their availability for the

growth of Microcystis aeruginosa. Ecolofv ,,

348 - 353.

and (1957) Nitrogen as a limiting factor for

the growth of Micr. ocystis aeruginosa in Southern

Wisconcin lakes. Ecology M, 556 - 561.

Glade, R. (1914) Zur kenntnis der Gattung Clindrospermum.

Beitr. Biol. Pfl. 12,295 - 344.

Goering, J. J. and Neess, J. C. (1964) Nitrogen fixation

in two Wisconcin lakes. Limnol. Ocennogr. y, 535 - 539.

Goldman, C. R. (1965) Micronutrient limiting factors and

their detection in natural phytoplankton populations.

Mern. Ist. Ital. Idrobiol. 18 (Suppl. ), 121 - 135.

Goldman, J. C. (1976) Identification of nitrogen as a

growth limiting nutrient in wastewaters and coastal

marine waters through continuous culture algal assays.

Wat. Res. 10,97'- 104.

Golterman, H. L. (1971) Methods for Chemical Analysis of

Fresh Waters. International Biological Programme

Handbook No. 8. Blackwell Scientific Publications,

Oxford, London and Edinburgh.

Good, N. E. and Izawa, S. (1972) Hydrogen ion buffers. In

Methods in Enzymology Vol. 24 B (ed: San Pietro, A. )

PP- 53 - 68. Academic Press, New York and London.

, Winget, G. D., Winter, W., Connolly, T. N., Izawa, S.

and Singh, R. N. M. (1966) Hydrogen ion buffers for

biological research. Biochem. N. Y. !,, 467 - 477.

Gorham, E. (1957) The chemical composition of some waters

from lowland lakes in Shropshire, England. Tel us S,

174 - 179.

Gorham, P. R., McLachlan, J. S., Hammer, U. T. and Kim, W. K. (1964) Isolation and culture of toxic strains of Anrtbaena flos-aquae (Lyngb. ) de Breb. Veh_int. Verein. theor. anaew Limno . ,

1-ý, 796 - 004.

Griffiths, B. M. (1925) Studies in the phytoplankton of the lowland waters of Great Britain ICI. The phytoplankton

of Shropshire, Cheshire and Staffordshire. J. Linn.

-219-

. Soc. (IZot. ) 4,75 - 98 (1938) Early references to waterbloouis in British

lakes. Proc. Linn. Soc. fond. 151,12 - 19.

Grimshaw, H. 14. and Hudson, M. J. (1970) Some mineral

nutrient studies of a lowland more in Chenhiro,

England. FIydrobioloi 36,329 - 341.

Hammer, U. T. (1964) The succea:; ion of "bloom" species of

blue-green algae and some causal factors. Verh. int.

Verein. theor. sn, ew. Limnol. 829 - 836.

(1969) Blue-green algal blooms in Saskatchewan lakes.

Verh. int. Verein. theor. anRew. Limno_. j, 116 - 125.

Harder, R. (1917a) Uber die Beziehung der Keimung von

Cyanophyceensporen zum Licht. Ber. dt. bot. Gen. 2.5

58 - 64. (1917b) Uber die Bezeihung de© Lichtes our Keimung

von Cyanophyceensporen. Jb. Wise. Bot. 58,237 - 294.

Hardy, E. F. (1939) Studies on the poet glacial history of

the British vegetation V. The Shropshire and Flint

t1aelor mouses. New Phytol., 364 - 396.

Hardy, R. W. F., Holeten, R. D., Jackson, B. K. and

Burns, It. C. (1969) The acetylene-ethylene assay for

112 fixation: laboratory and field evaluation.

Physiol., Lancaster . 42,1 185 - 1207.

Harris, G. P. and Lott, J. N. A. (1973) Lieht intensity and

photosynthetic rates in phytoplankton. J. Fish. Res.

Bd Can. 0,1771 - 1778.

Hart, I. C. (1967) Nomograms to calculate dissolved-oxygen

contents and exchange (mass-transfer) ooeffioiontß. Wat. Res. 1,391 - 395.

Haynes, R. C. (1975) Some eoologioal effects of artificial

circulation on a small eutrophic lake with particular

emphasis on phytoplankton II. Kezar Lake experiment,

1969. H , drobiolo, iia -4-6,141 - 170.

Healey, P. P. (1973) Characteristics of pho©phorue

deficiency in Anabaena. J. Phvcol. , U, 383 - 394.

and Hendzel, L. L. (1976) Phyeioloßical changes during the course of blooms of A, Lhanizornenon floe-aquae.

J. Fish. RAs. Bd Can. 22,36 - 41.

Heaney, Be 1. (1973) Problems related to the growth of

-220-

blue-green algal populations. Ph. L. Thesis, New

University, Ulster, Coleraine.

Heath, R. T. and Cooke, G. D. (1975) The aignifieanoe of

alkaline phosphataee in a eutrophic lake. Verh, int.

Verein. theor. angew. Lirmnol. 19,959 - 965.

Herbes, S. E., Allen, H. E. and Mancy, X. H. (1975)

Enzymatic characterization of soluble organic phosphorus

in lake water. Science, N. Y. 18' , 432 - 434.

Heron, J. (1962) Determination of phosphate in water after

storage in polyethylene. Limnol. Ooeanoj3r. 3,, 316 - 321.

Hill, H. (1972) A new Rrnphidonsis species (Cyanophyta,

Rivulariaceae) from Ninnesota lakes. Phyaoloaia j1,

213 - 215.

Hobro, R. and YJillen, E. (1975) Phytoplankton counting and

volume calculations for the Baltic -a Method

comparison. Vatten j, 317 - 326.

Holm-Hansen, O., 'Gerloff, G. C. and Skoog, F. (1954) Cobalt

as an essential element for blue-green algae.

Physiologia Pl. 3,665 - 675.

Horne, A. J., Dillard, J. E., Fujita, D. X. and Goldman, C. R.

(1972) Nitrogen fixation in Clear Lake, California II.

Synoptic studies on the autumn Anabaenn bloom. Limno . Oceanogr.

_ 11,693 - 703.

and Fogg, G. B. (1970) Nitrogen fixation in some English lakes. Proo. R. Soc. B J, 351 - 366.

and Goldman, C. R. (1974) Suppression of nitrogen

fixation by blue-green algae in a eutrophic lake with

trace additions of copper. Science. N. Y. 1,409 - 410.

and Viner, A. B. (1971) Nitrogen fixation and its

significance in tropical Lake George, Uganda. Nature.

Lond_ 22,417 - 418.

Huntsman, S. A. and Barber, R. T. (1975) Modification of

phytoplankton growth by excreted compounds in low

_ density populations. J. Phvcol. 11,10 - 13.

Ihlenfeldt, M. J. A. and Gibson, J. (1975) Phosphate

utilisation and alkaline phosphataoe activity in

Anacystis nidulans (Synechocoocus). Arch. I"iicrobiol.

102,23 - 28.

-221-

Iverson, R. L. , Curl, H. C. , O' Cozinoro, 11. B. , , Kirk, D. - and

Zakar, K. (1974) Summer ph. 'toplankton blooms in Auke

Bay, Alaska, driven by wind mixing of the water column.

Lirno'_. Oceanopr. _

12,271 - 278.

Jackson, G. F. (1883) Shron5hire Folk-Lore (ed: Burne, C. S.,

from the collection of G. F. Jackson). Trubner, London.

Kale, S. R., Balial, M. and Talpai3ayi, E. R. U. (1970)

Heterocyst differentiation in Anabaena nnbirua Rao I.

Growth of the alga. Indian Biologist 2,30 - 40.

and -

(1973) Heterocyst development in

. Anabaena ambigua Rao IV. Effects of some mineral salts.

Phycos 12,11 - 17.

Kaushik, M. and Kumar, H. D. (1970) The effect of licht on

growth and development of two nitrogen-fixing blue-

green algae. Arch. Mikrobiol. , 52 - 57.

Kawana, K. (1975) Spectral distribution of underwater

irradiance. Bull. Fac. Fish. Hokkaido Univ. 26,

235 - 248.

Keating, K. I. (1975) Algal allelopathic involvement in

bloom sequence in freshwater. J. Phycol. 11, (Suppl. )

12 - 13. (Abotr. ).

Kenyon, C. IJ., Rippka, R. and Stanier, H. Y. (1972) Fatty n

acid composition and physiolog cal proportiea of some

filamentous blue-green algae. Arch. Mikr. ohio P2,

216 - 236.

Ketchum, B. H. (1939) The ievelopment and restoration of

deficiencies in the phosphorus and nitrogen composition

of unicellular plants. J. cell. oomp. Phvºsiol. 1,

373 - 381. Khan, K. R. (1974) Overwintering mechanisma in Gloeotrichirt

echinulata (J. M. Smith) Richt. J. Phycol. 10, (Surpl. )

4 only. (Abstr. ).

King, D. L. (1970) The role of carbon in eutrophication.

1 J. Wat. Pollut. Control Fed. 42,2035 - 2051.

Kirchner, W. B. (1975) An evaluation of sediment trap

methodology. Limnol. Oceano, r. 20,657 - 660.

Kirk, J. T. 0. (1976) Yellow substance (Gelbstoff) and its

contribution to the attenuation of photosynthetically active radiation in some inland and coastal south-

-222-

eastern Australian waters. Aust. J. mar. Freohwat. Res.

21,61 - 71.

Klebahn, H. (1895) Gasvakuolen, ein Be©tandteil der Zellen

der wasserblütenbildenden Phycochromaoeen. Flore. Jena

80,241 - 282.

I: noechel, I. and Kalff, J. (1975) Algal sedimentation: the

cause of a diatom - blue-green succession. Verh. int.

Verein. theor. angew. Limnol. 12-, 745 - 754.

Komarek, J. (1958) Die taxonomische Revision der Plankton-

ischen Blaualgen der Techechlowakei. In Algoliecho

Studien (Kou. irek, J. and Ettl, H. ) pp. 10 - 286. Czech

Academy of Sciences, Prague.

Kostlan, N. V. and Necterova, D. U. (1975) Change in

content of phosphorus compounds in Aphinizomenon floo-

ac uaG (L. ) Ralfs during vegetation. Ukr. Bot. Zh. ý2,

355 - 358- [In Ru©uifui, Lngliuh abotrcuot,.

Kuentzel, L. E. (1969) Bacteria, carbon dioxide and algal

blooms. J. Wat. Pollut. Control Fed. 'Al, 1737 - 1747.

Kulasooriya, S. A., Lang, N. J. and-Pay, P. (1972) The

heterocysts of blue-green algae Ill. Differentiation

and nitrogenase activity. Proc. R. Boo. B 181,199 - 209.

Lange, W. (1976) Speculations on a possable essential

function of the gelatinous sheath of blue-green algae.

Can. J. Mi_crobio_l_ 22,1181 - 1185.

Lazaroff, N. (1966) Photoinduotion and photoreversal of the

Nostooacean developmental cycle. J. Phyool. ,? � 7- 17.

and Schiff, J. (1962) Action spectrum for developmental

photo-induction of the blue-green alga Nostoo muncorum.

Science, N. Y. IM, 603 - 604.

and Vishniae, W. (1961) The effect of light on the

developmental cycle of Ilostoc muscortxm, a filamentous

blue-green alga. J. non. Nicrobiol. 25,365 - 374.

and (1962) The participation of filament

anastomosis in the developmental cycle of Nontoe

mus, a blue-green alga. J. non. TSiorobiol. 20,

203 - 210.

Lemmermann, E. (1900) Beiträge zur Kenntnis der Planktonalgen VII. Des Yhytoplankton den Zwiohenahner

-223-

I""Ieeres. Der. dt. bot. Ges. 18,135 - 143.

Lin, C. K. (1972) Phytoplankton succestiion in a eutrophic

lake with special reference to blue-green algal blooms.

Hydrobiologin 29,321 - 334.

Lind, H. M. (1944) The phytoplankton of some Cheshire mares.

Diem. Proc. Pianchr lit. phil. Soc. 86,83 - 105-

Lorenzen, M. and Mitchell, R. (1973) Theoretical offoote of

artificial destratification on algal production in

impoundments. Envir. Sei. Technol. Z, 939 - 944.

Lund, J. W. G. (1965) The ecology of the freshwater phyto-

plankton. Biol. Rev. 4,0,231 - 293. (1966) The importance of turbulence in the periodicity

of certain freshwater species of the genus Me os i_}r . Bot. Zhurn.

. 51 ,1 76 - 187. [In Rizssian] .,

, Kipling, C. and Le Cron, E. D. (1958) The inverted

microscope method of estimating algal numbers and the

statistical basis of estinations by counting.

Iiydrobiologia 11,143 - 170.

Lyon, A. J. (1970) Dealing with Data. Pergamon, Oxford.

McLachlan, J. and Gorham, P. R. (1961) Growth of Miorocyntip

aerum inosa I: utz. in a precipitate-free medium buffered

with TRIS. Can. J. Microbiol. . 7.869 - 882.

McNabb, C. D. (1960) Enumeration of freshwater plankton

concentrated on the membrane filter. Limnol.. Oonanor! r, E, 57 - 61.

Meffert, N. B. (1971) Cultivation and growth of two

planktonic Osci1_latorin species. Mitt, int. Vc, rein,

theor. angew. Limnol. 1, j, 189 - 205.

Diitchison, G. J. and Wilcox, M. (1972) Rule governing cell

division in Anabaena. iTature, Lond. ga, 110 - 111.

Mortimer, C. H. (1942) The exchange of din solved substanooo between mud and water in lakes III and IV. Summary and

references. J. Ecol. 0,147 - 201.

Morton, S. D. and Lee, T. H. (1974) Algal blooms - possible effects of iron. Envir. Sei. Technol. A, 673 674"

Moss, B. (1969) Vertical heterogeneity in the water column of Abbots Pond II. The influence of physical and chemical conditions on the spatial and temporal distribution of the phytop? ankton and of a coiamitnity of

4

-224-

epipelic algae. J. Ecol. . jZ, 397 - 414.

Ogawa, R. L. and Carr, J. F. (1969) The influence of

nitrogen on heterocyst production in blue-green algae. L mnol. 0ceanoir. 14,342 - 351.

Pearsall, U. 11. (1923) The phytoplankton of Rostherne More.

lem. Proc. Nanchr lit. Phil. Soc. U, 45 - 55.

Pennington, W. (1974) Seston and sediment formation in five

Lake District lakes. J. Boni. 62,215 - 251.

Phillips, W. (1384) The breaking of the Shropshire Lorca.

Trans. Shrops. Ar. chaeol. nat. Hießt. Soc. Z, 277 - 300. (1893) The breaking of the Shropshire Nerea. Mid j.

Nat. 16,1 - 15.

Primrose, S. B. and Dilworth, N. J. (1976) Ethylene

production by bacteria. J. Pen. T'linrohiol. , 177 -

182.

Reddy, P. N., Rao, P. S. N. and Talpasayi, E. R. 2. (1975)

Effect of red and far-red illuminations on the

germination of spores of two blue-croon algae. Cures

Sci. 44,678 - 679.

Reichardt, U. (1971) Catalytic mobilisation of phosphate in lake water and by Cyanophyta. Hydrobiolof; in 377 - 394.

Reif, C. B. and Fine, D. L. (1967) Two winter blue-green

algal populations. Ph'colotlia 6,105 -'106. Reynolds, C. S. (1971) The ecology of the planktonic blue-

green algae in the North Shrop; hire mores, Lngland. Fld

Stud. 2,409 - 432. (1972) Growth, gas vacuolation and buoyancy in a

natural population of a planktonic blue-croon alga. Freshwat. Biol. 2, E37 - 106.

(1973a) Growth and buoyancy of Ilicrocyatia aorupi. noaa Kütz. emend Elenkin in a shallow eutrophic lake. 'roc,

R. Soc. B 18A, 29 - 50. (1973b) Ph.,, ytoplankton periodicity of some ;: orth

Shropshire meres. Br. phN�ycol J. 8,301 - 32U. (1973o) The phytoplankton of Croce More, Shropshire.

Br. phycol J. - 8,153 - 162. (1975a) Interrelations of photosynthetic behaviour

and buoyancy regulation in a natural population of a

-225- blue-green alga. Freshwat. Biol. ý, 323 - 338.

(1975b) Temperature and nutrient concentration in the

characterization of the water supply to a small

kataglacial lake basin. Freshwat. Biol. ,.,

339 - 356"

and Rogers, D. A. (1976) Seasonal variations in the

vertical distribution and buoyancy of Microcyptin

aeruginosa K1tz. emend. Elenkin in Rostherne Mere,

'E'ngland. Hydrobiologin A. 8-, 17 - 23.

Rhee, G-Y. (1972) Competition between an alga and an

aquatic bacterium for phosphate. JLimnol. Ooenno, r. , l,

505 - 514.

Rippka, R., Nielson, A., Kunisawa, R. and Cohen-Bazire, G.

(1971) Nitrogen fixation by unicellular blue-green

algae. Arch. Mikrobiol_ 2-6,341 - 348.

Robinson, B. L. and Miller, J. H. (1970) Photomorphogenesia

in the blue-green alga Nostoo commune 584. Physio1oRin,

P1., 461 - 472.

Roelofs, T. D. and Oglesby, R. T. (1970) Ecological

observations on the planktonic cyanophyto Gloeotrichi3

echinulata. Limnol. Oceanofr_ j5,224 - 229.

Rose, E. T. (1934) Notes on the life history of A�3hanizomonon

flos-aquae. Univ. ' Iowa Utud. Nat. Hist. 16,129 - 141.

Schell, P. M. and Alexander, V. (1970) Improved incubation

and gas sampling techniques for nitrogen fixation

studies. Limnol. Oceanoir. , 961 -'962.

Schindler, D. W., Brunskill, G. J., Emerson, 2.,

Broecker, W. S. and Pang, T. -H. (1972) Atmospheric

carbon dioxide; its role in maintaining phytoplankton

standing crops. Science. N. Y. U, 1192 - 1194.

Schwabe, G. H. and Strange-Bursche, E. (1964) Ubor

A hanizomenon gracile Lemm. (Blaualgen und Lebensraum,

IX). Arch. Hydrobiol. 60,249 - 277.

Serruya, C. and Berman, T. (1975) Phosphorus, nitrogen and the growth of algae in Lake Kinneret. J. Phycol. 11,

155 - 162. Shapiro, J. (1973) Blue-green algae: why they become

dominant. Science. N. Y. 1 9,382 - 384.

and Glass, G. L. (1975) Synergistio effects of phosphate and manganese on growth of Lake Superior

-226-

al, gae. Verh. int. Verein. theor, anj ew. Limnol. jj, 395 - 404.

Shilo, M. (1975) Factors involved in dynamics of algal blooms in nature. In Unifvinn. ConeexAn of Eoolorv,

Proc. First Internat. Conf. Lcol. pp. 127 - 132. The

Hague.

Shubert, L. E. (1974) Utility of dilute algal media. J.

Phvco_l. 10, (Suppl. ) p21 only (Abstr. ).

and Trainor, F. R. (1974) Scenedesmue morphogenesis:

control of the unicell stage with phosphorus. Bre

phyycol J. 2,1 - 7.

Sicko-Goad, L. 111., Crang, R. E. and Jensen, T. E. (1975)

Phosphate metabolism in blue-green algae IV. In situ

analysis of polyphosphate bodies by x-ray energy

dispersive analysis. Cytobiol. 11,430 - 437.

and Jensen, T. E. (1974) X-ray energy dispersive

analysis of polyphosphate bodies in P. ec; tonerarz borg*anur.

In 32nd Ann. Proe. rleotr. MicroE3oop. Soo. Am©r,. (ad:

Arceneaux, C. J. ). St Louis, 11issouri.

_ and (1976) Phosphate metabolism in blue-green

algae II. Change in phosphate distribution during

starvation and the "polyphosphate overplue" phenomenon in Plectonema bor anum. Am. J. Bot. , 183 - 188.

Sigma. (1974) Phosphatase: acid, alkaline and proatatio. Sigma Tech. Bull. No. 104.

Simon, R. D. (1977) Sporulation in the filamentoun

cyanobacterium Anabaena oylindricn: the course of spore formation. Arch. Miorobiol. 111,283 - 288.

Singh, H. 11. (1967) Genetic control of aporuletion in the

blue-green alga Anabaena doliolum Bharadwaja. Plantr,

ZU, 33 - 38.

and Srivastava, B. 3. (1968) Studies on morphogenesi© in a blue-green alga 1, Effect of inorganic nitrogen sources on developmental morphology of Anabaen doliolum. Can. J. Microbiol. j, 1341 - 1346.

and Sunita, K. 1.1. (1974) A biochemical study of spore germination in the blue-green alga Anebanna doliolum. J. exp. Bot. 837 - 845.

Singh, R. N. (1942a) A new species of the genus Ranhidop sie

-227-

Fritsch and Rich (Raphidonsis indica sp. nov. )

exhibiting morphological variants. J. Indian bot. Soo.

21,239 - 243.

(1942b) Wollea bharadwa s. ep. nov., and its autecology.

Ann. Bot. N. S. 6,593 - 606.

and Dikshit, R. F. (1976). Mutaeenesia of the blue-

green alga, Anabaena doliolur Bharadwaja. Mutation it ea. 65 - 78.

Singh, H. N. and Sinha, H. (1967) Genetic control of

cellular differentiation in blue-green algae. Bull.

Nat. Inst. Sei., India No. 34,131 - 145. (Pron. Symp.

Newer Trends in Taxonomy, New Delhi).

Sinha, B. D. and Kumar, H. D. (1973) A mutational study of

the nitrogen-fixing blue-green alga Anabanna doliolum.

Ann. Bot. 2Z, 673 - 679.

Sinker, C. A. (1962) The North Shropshire mores and mosses:

a background for ecologists. Fld Stud. 1,101 - 138.

Sirenko, L. A., Chernousova, V. M., Arendarchuk, V. V. and

Kozitskaya, V. N. (1969) Factors of mass development of

blue-green algae. Hydrobiol J. ! j, 1-8.

Smith, R. V. (1973) Evidence for the obligate dependanoe of

a Lough Neagh strain of Oscillatorin redekii Van Goor

on the presence of bacteria for growth. Symp.

Prokaryotic Photoeynthetio Or tniems, Freiburg.

pp. 89 - 90 (Abstr. ).

and Foy, R. H. (1974) Improved hydrogen ion buffering

of media for the culture of fresh-water algae. Br.

phycol J. , 2,239 - 245.

Spratt, E. R. (1911) Some observations on the life history

of Anabaena cycadaceae. Ann. Bot. 25,369 - 380.

Stewart, W. D. P. (1971) Physiological studies on nitrogen-

fixing blue-green algae. In Biolorioel Nitro, on Fixation

in Natural and Aitriculteral Habitat rý (ede: Lie, T. A.

and I": ulder, B. G. ) pp. 377 - 391. Pl. Soil, special

volume. (1972) Algal metabolism and water pollution in the

Tay Region. Proc. R. Soo. Bdinb. 71,209 - 224. and Alexander, G. (1971) Phosphorus availability and

nitrogenase activity in aquatic blue-greon algae.

-228-

Freshwater Biol. 1,389 - 4U4.

, Fitzgerald, G. P. and Burris, R. H. (1967) In situ

studies on N2 fixation using the acetylene reduction technique. Proc. natn. Acad. Sci. U. S. A. 58,2071 - 2078.

md (1968) Acetylene reduction by

nitrogen fi:: ing blue-green algae. Arch. Tlikrobiol. 629

336 - 348.

and (1970) Acetylene reduction assay for determining phosphorus availability in 'Iisconcin

lakes. Proc. natn. Acad. Sci., U. S. A. 66,1104 - 1111.

and Pearson, H. W. (1970) Effects of aerobic and

anaerobic conditions on growth and metabolism on blue-

green algae. Yroc. R. Soc. B 1l, 293 - 311.

Stoermer, E. F. and Ladewski, T. B. (1976) Apparent optimal

temperatures for the occurrence of some common

phytoplankton species in southern Lake Michigan. Great

Lakes Res. Div., Publication 18. Univ. of Michigan,

Ann Arbor.

Sud'ina, O. G., 5hnyukova, Ye. 1., Kostlan, N. V.,

Mushak, Y. 0. and Tupyk, 14. D. (1973) Biochemical

features of blue-green algae which cause water-bloom. Ukr. Bot. Zh. 30,497 - 504. [Exiglish trunalation].

Sverdrup, H. U. (1953) On conditions for the vernal blooming of phytoplankton. J. Cons. perm. int. Exnlor.

Iter. 18,287 - 295.

Telling, J. F. (1971) The underwater light climate no a

controlling factor in the production ecology of fresh-

water phytoplankton. I'iitt. int Verein. theor. anaew. Limnol. 19,214 - 243.

(1976) The depletion of carbon dioxide from lake

water by phytoplankton. J. Lcol. 64,79 - 121.

Taylor, B. F., lee, C. C. and Bunt, J. S. (1973) Nitrogen

fixation associated with the marine blue-Green alga, Trichodesmium, as measured by the acetylene reduction technique. Arch. Mikrobiol. 88,205 - 212.

Taylor, F. J. (1975) Contamination of water samples during

collection (Note). II. Z. J. Mar. Freshwat. Res. 119. Telitchenko, M. I";., Teytsarian, G. V. and Shirkova, Ye. L.

-229-

(1970) Trace eler. ýents and algal "bloon". Hydrobiol+J.

6,1 - 6.

Thompson, L. M. N. and Macleod, R. A. (1974) Biochemical

localisation of alkaline phosphatase in the cell wall

of a marine pseudorioz. ad. J. Bract. 117,819 - 826.

Tischer, J. (1957) Untersuchungen i1ber die granulgren

Einschlüsse und das Reductions - Oxidationavormogen der

Cyanophyceen. Arch. Mikrobiol. al, 400 - 428.

Topachevskiy, A. V., Braginskiy, L. P. and Sirenko, L. A.

(1969) Massive development of blue-green algae as a

product of the ecosystem of a reservoir. Hydrobiol. J.

5,1 - 10. [English tranalutioni.

Trainor, F. R. and Shubert, L. E. (1974) Scenedesmus,

morphogeneois: colony control in dilute media. J. rhveo_

10,28 - 30.

Tyagi, V. V. S. (1974) Some observations on the pattern of

sporulation in a blue-green alga, Anabaena doliol. um.

Ann. Bot. M, 1107 - 1111.

Vance, B. D. (1965) Composition and succession of

cyanophyceaii water blooms. J. Yh col. 1,81 - 86.

Vasil'eva, V. E. and Levitin, M. G. (1974) Effect of carbon

dioxide starvation on certain blue-green algae. Finiol.

Rast. Most. )_ 21,1207 - 1211.

Vladimirova, K. S. (1968) The correlation between the

phytoplankton and the phytomicrobenthos of reservoirs.

In "Tsvetnie" Vody ("Blooming, Water) (ad: Topuohuvokky,

A. V. ) pp. 67 - 81. Izdat "Naukova" Durnka, Kiev,

U. S. S. R. [English translationJ.

Volk, S. L. and Phinney, H. K. (1968) Mineral requirements

for the growth of Anabaena spiroides in vitro. Can. J.

Bot. 4-6,619 - 630.

Vollenweider, R. A. (1974) A Manual on Methods for

Measuring, Primary Production in Aquatic Environments.

International Biological Programme Handbook No. 12.

Blackwell ; scientific Publications, Oxford, London and Edinburgh.

Walsby, A. E. (1971) The pressure relationships of Can

vacuoles. Proc. R. Soc. B J, 301 - 326.

and Booker, N. J. (1976) The physiology of water

-23C-

bloom formation by planktonic blue-green algae. Br. -

phycol J. 11,200 only (Abstr. ).

and Bichelberger, 11.11. (1968) The fine structure of

gas vacuoles released from the cells of the blue-green

alga Anabaena flos-acguse. Arch. Ilikrobiol. 60,76 - 83.

and Klever, A. R. (1974) The role of gas vacuoles in

the aicrostratification of a population of Oscillatoria

aagardhii var. isothrix in Deming Lake, Ilinnesota. Arch.

H_ý! drobiol . 74,375 - 392.

Wardle, T. (1897) The breaking of Copmere. Trans. North

Staffs. Field Club 1897 volume, 110 - 123.

Watanabe, A. and Yamamoto, Y. (1967) Heterotrophic nitrogen fixation by the blue-green alga Anabaenopsis circularie.

" Nature, Lond. 21A, 738.

Mosre, 11. M. and Benenann, J. R. (1973) Nitrogen fixation by

Anabaena cylindrica II. Nitrogenase activity during

induction and ageing of batch cultures. Arch. Mikrobiol.

22,101 - 112.

Westlake, D. F. (1966) The light climate for plants in

rivers. In Light as an Ecological Factor (eds:

Bainbridge, R., Evans, G. C. and Rackham, 0. ) pp. 99 - 119. Blackwell Scientific Publishers, Oxford and Edinburgh.

White, 11. S. and Wetzel, R. G. (1975) Nitrogen, phosphorus,

particulate and colloidal carbon content of aedimenting

sexton of a hard-water lake. Verh. int. Verein. theor.

annew. L mnol. 12,330 - 339. Whitton, B. A., Kirkby, S. M., Peat, A. and Sinclair, C-

(1973) Environmental effects on the morphology of flivulariaceae trichomes. Srmn. Prokaryotic Photosynthetic Organisms, Preiburg. pp. 180 - 181 (Abstr. ).

Wieringa, K. T. (1968) A new method for obtaining bacteria- free cultures of blue-green algae. Antonie van Leeuwenhoek , 54 - 56.

Wildman, R. B., Loescher, J. H. and Winger, C. L. (1975)

Development and germination of akinetes of Anhanizomenon flos-aquae. J. Phtºcol. 11,96 - 104.

Woelkerling, W. J., Kowal, R. R. and Gough, S. B. (1976)

Sedgwick - Rafter cell counts: a procedural analysis.

-231-

ý11- drobiolotIia 48,95 - 107.

''loll;, C. P. (1965) Control of cporulation in blue-green

algae. Devl Biol. 12,15 - 35.

(1956) Evidence of a role of heterocysts in the

sporulation of a blue-green alga. Am. J. Bot. ,

260 - 262.

(1968) Movement of carbon from vegetative cello to

heterocysts in Anabaena cylindrica. J. Bact. 269

2138 - 2143.

(1975) Differentiation and pattern formation in

fL lamentous blue-green algae. In Spores, Vol. VI.

PP. 85 - 96. Am. Soc. Microbiol.

Wood, E. D., Armstrong, F. A. J. and Rioharde, F. A. (1967)

Determination of nitrate in sea water by cadmium-copper

4, reduction to nitrite. J. mar. biol. Aso. U. R..

23 - 31. Wyatt, J. T. and Silvey, J. X. G. (1969) Nitrogen fixation

by Gloeoc_ansa. Science. N. Y. 165,908 - 909.

Proc. R. Soc. Lond. B. 196,317-332 (1977)

Printed in Great Britain

Sporulation and the development of planktonic blue-green

algae in two Salopian meres

BY J. A. ROTHER AND P. FAY

Department of Botany and Biochemistry, Westfield College, University of London

(Communicated by G. E. Fogg, F. R. S. - Received 23 June 1976)

Factors affecting akineto formation and the survival of planktonic blue- green algal populations after the formation of `water blooms' were investigated in two eutrophic kataglacial lakes. It is considered that the induction of sporulation may be due to extreme conditions at the water surface during the summer to which the algal bloom is exposed, rather than to nutrient, especially orthophosphate, deficiency. It has not been shown conclusively that akinetes constitute an overwintering stage of the planktonic algal life cycle, or that akinetes germinate in the spring to produce the inoculum for subsequent vegetative growth. Comparisons of planktonic, sedimenting and benthic algal material indicate that germination shortly after maturation may provide the greater part of the overwintering planktonic vegetative populations. Population sizes and life cycles appear to be influenced by the physical factors which control the stability of the water column.

INTRODUCTION

`Water bloom' formation has been shown by Reynolds (1971,1972,1973a) to result from the redistribution of buoyant planktonic blue-green algae. Buoyancy is imparted by gas vacuoles, and the rate of surface accumulation is dependent on the amount of gas vacuoles within the algal cell, the degree of turbulence within the water body and the size of the alga. The larger colonial species have a greater potential for rapid movement through the water column, both in terms of buoyancy and sedimentation (Reynolds & Walsby 1975). The conditions at the surface are highly unfavourable to the survival of vegetative algal material, and algae frequently undergo photo-oxidative death after bloom formation (Abeliovich & Shilo 1972; Boyd, Prather & Parks 1975). Some colony forming genera (Micro-

cystis and Coelosphaerium) are more resistant to damage than the filamentous forms discussed in this paper (Reynolds 1967). Since much of the total algal bio-

mass may be concentrated within a few centimetres at the surface during a bloom, the survival of some of this material will be critical for the development of future

populations. An important adaptation of many heterocystous blue-green algae in conditions

unfavourable for vegetative growth appears to be the formation of akinetes, [ 317 1

318 J. A. Rother and P. Fay

resistant cells well protected by an elaborate envelope (Fogg, Stewart, Fay & Walsby 1973) each of which differentiates from a single vegetative cell. Earlier

studies indicate that akinetes but not vegetative filaments survive in the surface mud sediments during the winter and germinate in the spring (Rose 1934; Roelofs & Oglesby 1970; Wildman, Loescher & Winger 1975). Germination of akinetes at a time when the lakes are still unstratified, apparently occurred in response to more favourable conditions of light and temperature (Reynolds 1971,1972). The

significance of this process is doubtful as the number of akinetes is never very large

and hence germination of akinetes may not contribute significantly to the population maximum (Reynolds 1972,1975). Growth can mostly be accounted for by

vegetative reproduction of a small overwintering stock of planktonic material. Non-sporulating species, especially Microcystis, have been shown to remain viable during the winter in the mud sediments of the deeper lakes, giving rise to planktonic populations the following spring (Sirenko, Chernousova, Arendarchuk & Kozitskaya 1966; Reynolds & Rogers 1976).

In laboratory experiments on akinete formation, it has been shown that a reduction in concentration of a critical nutrient such as orthophosphate (Wolk 1965) or the increase of extracellular products (Fisher & Wolk 1976) induces akinete development, that is, the phenomenon is characteristic of stationary phase cultures. Field studies on factors affecting akinete production are relatively sparse. Roelofs & Oglesby (1970) conclude from field and laboratory observations on Gloeotrichia ec1 inulata that akinete formation is not a result of unfavourable conditions, as akinetes were seen during the logarithmic phase of growth.

An attempt was made in this investigation to assess the importance of akinetes in the formation of planktonic algal propulations of a number of species, in two contrasted lacustrine environments, and to determine which factors controlled the various stages of the algal life cycle. The early stages of development may shed light on the mechanism of the annual succession of the heterocystous blue-green algal types and give an indication of the possible size of the population maximum.

THE SITES

Two meres, Kettle Mere and White Mere, of the Ellesmere group were investigated (figures la-c). Kettle Mere (National Grid reference: SJ 418 341) one of the smallest of the Salopian (Shropshire) meres, has a surface area of 2 ha and a maximum depth of 7 m. It is well sheltered from the wind, being at the bottom of a deep conical depression and fringed with woodland. White Mere (National Grid reference: SJ 415 330) with a surface area of 26 ha and a maximum depth of 15 m (in average between 4 and 8 m) is more exposed. Neither of the meres have significant inflows or outflows and rely on phreatic and surface drainage for water supply. Both meres are eutrophic and support large populations of blue-green algae. More detailed descriptions of the Meres are available in articles by Griffiths 1925), Gorham (1957), Sinker (1962) and Reynolds (1973b).

Sporulation of planktonic blue-green algae 319

METHODS

Sampling

Plankton samples from the sites indicated on figures 1b and 1c were collected using a Ruttner sampler, were preserved in the field in Lugol's iodine and enume- rated using the iodine sedimentation and inverted microscope technique of Lund, Kipling & Le Cren (1958). More rapid sedimentation which is less prone to resuspension, particularly of the larger colonial gas vacuolate species, was achieved by collapsing the gas vacuoles using the `hammer, cork and bottle' technique of Klebahn (1895). Estimates of heterocyst and akinete frequency were made by

counting a minimum of 1000 cells along randomly chosen filaments. Total suspended solids and algal dry mass were determined by weighing the amount of particulate material after filtration onto Whatman GF/C glass fibre filters and drying at 110 °C under atmospheric pressure for 24 h.

During 1975 a Jenkin surface mud sampler (Mortimer 1942) was used to collect mud samples at the sediment-water interface, at other times the Ruttner sampler was used. The top 5-10 cm section of the core was used to enumerate algal filaments, akinetes and empty akinete envelopes. Due to the loose and flocculent

FzoU I. The Ellesmere group of meres. (a) Location of meres. (b) Kettle Mere, indicat. ing position of sediment traps (0) and sampling point (0). (c) White Mere, symbols as in (b).


nature of the surface mud deposits it was not possible to section the cores and therefore the data are expressed in terms of number per unit volume. The mud samples were preserved in 4% formaldehyde. In order to facilitate identification of the algal material against the background of detritus, a Zeiss Standard 18 Micro- scope with Epi-fluorescence condenser IV/F was used, utilizing the natural fluorescence of chlorophyll a. This was supplemented by observations using normal transmission light microscopy.

Sedimentation of algal material through the water column was followed by using sediment traps. Each trap consisted of 14 or 16 plastic tubes 1.5 cm in diameter, held in a plastic container and suspended 2m above the surface mud sediments, below the summer thermocline. The amount of material collected in this type of trap has been shown by Kirchner (1975) not to vary on a unit area basis for traps of different sizes. Surface wave action was minimized by using submerged floats attached 2m above the trap itself. The trap was located by a small surface buoy attached to the submerged float by a loose line. No growth of epiphytes occurred on traps positioned 4m below the water surface, and any accumulation of bacterial material on the tubes was minimized by replacing them with a new set of clean tubes when samples were collected. These requirements prescribed that the traps were positioned in water which was not less than 6m deep. Total sedimentation rates were assessed in terms of dry mass of material sedimented per square metre per day. Quantitative and qualitative estimations of the sedimentation of algal material were also made.

Analysi8 Profiles of oxygen saturation (using an Electronic Instruments Ltd oxygen

probe), temperature, and light penetration (using a Griffin Environmental Comparator) were obtained.

Analyses of orthophosphate were carried out on sterile filtered samples using the molybdenum blue method as described by Golterman (1971) or on site using a Hach Direct Reading Engineers' Laboratory (model DR-EL/2). The analysis procedure used in the latter is derived from a method described in the American Public Health Association Standard Methods (1971) and is similar in principle to the method used in the laboratory. The results obtained are comparable.

Analyses of labile cellular orthophosphate were made on washed filtered algal material following the method of Fitzgerald & Nelson (1966), and assays of alkaline phosphatase activity were performed using the p-nitrophenol phosphate technique as described in Sigma Technical Bulletin No. 104 (1974). Algal material for these analyses was concentrated by allowing the buoyant material to float to the surface of the vessel, and then pipetting off a known amount of the dense suspension. Aliquots of the concentrate were used to determine dry mass. The results are expressed as milligrams of orthophosphate or enzyme activity per milligram of dry alga.


TABLE 1. AMOUNT OF ALGAL MATERIAL AT THE WATER SURFACE : FILAMENTS

COLONIES OF MICROCYSTIS) PER MILLILITRE, KETTLE MERE

date A. circi- A. foa. A. apiro- A. aoli- Aph. foa- Micro. (1974) nalia aquae idea taria aquae cyatis 10/6 277 0 0 0 3363 0 16/6 500 0 0 0 700 0

2/7 0 0 40 0 100 0 9/7 25 0 40 0 100 2

18/7 31 0 8 0 35 0 30/7 0 0 1 0 21 4 13/8 0.2 0.4 4.4 0 0 49 29/8 1.6 0 9 0 0 98

9/9 0 0 7 0 0 80 27/9 10 29 5 0 0 61 20/11 0.1 0.5 0 0 0.2 2.5 16/12 0.03 0.01 0 0 0 1.9

(1975) 14/3 0 000 0 1.1

5/5 104 51 00 1 0.6 9/6 22 68 7 14 28 13

23/6 0 0 0.9 1.3 6.3 10 7/7 0 2.7 3.3 4.7 0 2.7

22/7 0 001.4 0 56 16/8 0 05 225 0 44 18/8 0.2 00 120 0 74 23/8 2 06 255 24 33 26/8 0 0 0.3 251 28 28 6/10 0 0 1.5 0 0 0

G. echinulata was not observed.

RESULTS

Among the most important factors which control the annual cycle of growth and reproduction of algae are light and temperature. The differing responses of the

various algal species result in the typical succession of the bloom forming species (tables 1 and 2) in the meres investigated. Generally the filamentous species are the first to bloom, especially Anabaena circinalis (Kütz. ) Hansgirg and Aplaani-

zomenon floc-aquae (L. ) Ralfs., which are followed by Anabaena flos-aquae (Lyng. ) Breb., Anabaena solitaria Kleb., Anabaena spiroides Kleb. and Gloeotrichia echinulata (Smith) Richt. Microcystis aeruginosa Kütz. typically blooms in the autumn. The differences between the two meres are probably the result of differences in the physical factors. White Mere is considerably more exposed to wind action than Kettle Mere and consequently does not show thermal stratification until July (figure 2c) whereas Kettle Mere is stably stratified from June to September (figure 2a). Changes of dissolved oxygen (figure 2b, d) also reflect the stability of the water column. Below the relatively shallow epilimnion of Kettle Mere the

water becomes almost totally deoxygenated. This is less marked in White Mere


TABLE 2. AMOUNT OF ALGAL MATERIAL AT THE WATER SURFACE: FILAMENTS

COLONIES OF MICROCYSTIS) PER MILLILITRE, WHITE MERE

date A. circi- A. fos- A. Spiro- A. soli- Aph. fos- Micro. (1974) nalis aquae ides taria aquae cystic

10/6 0 0 0 0 0 0 16/6 40 0 0 0 0 0 2/7 235 0 0 0 0 86 9/7 366 17 0 0 0 4

18/7 603 301 247 20 0 219 30/7 3189 2035 6 7 0 0.4 13/8 490 0 18 8 0 11 29/8 20 21 7 5 0 12

9/9 106 50 1 1 8 36 27/9 109 5 0.2 0.2 15 33 20/11 18 71 0 0.6 3.3 0.7 16/12 4.5 24 0 1.3 0.2 0.2

(1975) 24/3 181 177 0 0 0 0

5/5 0 22 0 0 1 0 9/6 0 6 0 0 0 0

23/6 7 8 0 0 0 0 7/7 557 391 7 6 2.2 0.7

22/7 17 29 1.4 55 14 0 16/8 0 1741 0.4 4 0 0 18/8 0 965 0.2 4 2 3.4 23/8 0 680 0 0.6 0 7 26/8 0 741 0 1.4 0 8 6/10 0 0.04 0 0 0 0.6

Colonies of 0. echinulata were observed in the plankton in July and August 1974 and 1975.

which is stable for a shorter period of time. In both meres there are periods of supersaturation of dissolved oxygen in the surface layers during calm weather. The earlier stratification of Kettle Mere produces an earlier and more intense blooming of algae. As a consequence of the earlier stratification, of the shallow epilimnion and of the considerable absorbance (table 3) of the peaty water, population levels are generally lower in Kettle Mere than in White Mere. Populations also undergo a more dramatic decline which could be as a result of the greater surface concentration of the algae in Kettle Mere.

During this time of calm weather and intense solar radiation, and with the greater part of the algal population floating within a few millimetres of the surface of the water, a sudden change in the physiological state of the algae takes place, which manifests itself in extensive akinete formation. This process is particularly rapid in Kettle Mere and this rapidity accounts for the lack of detailed records in table 4. The situation is similar in White Mere, though modified by physical factors (table 4). The onset of akinete formation coincides with the stabilization of the water column and the formation of surface scums of the species concerned.


z

O b

db

4)

10 b

tl ö

q ti oý L4 äý v

1

äo

ao$ pD

ti

b

dý f7

GH

rü

(uz) ugdep


TABLE 3. DEPTH OF 10 % OF SURFACE RADIATION

depth (m) depth (m)

date Kettle White date Kettle White (1974) Mere Mere (1975) Mere Mere

10/0 1.5 4.5 24/3 1.6 2.0 18/8 1.0 3.0 5/5 2.0 4.1 2/7 1.0 2.5 2/6 2.0 3.7 9/7 1.0 1.7 2 310 1.4 4.7

18/7 1.0 1.7 7/7 1.4 3.0 30/7 1.0 1.0 22/7 0.9 1.9 13/8 1.5 1.0 18/8 1.0 1.5 29/8 1.0 1.7 18/8 1.0 1.0

9/9 1.5 1.6 23/8 1.0 1.3 20/10 0.9 2.0 26/8 1.0 1.0 20/11 1.0 - 6/10 2.5 1.0 16/12 1.0 1.5 27/10 1.0 5.0

18/11 1.0 2.0

TABLE 4. AKINETE PERCENTAGE OF ALGAE FROM THE MERES

Aphani.

A. circi- A. fl o8- A. epiro- A. Soli- zomenon nalis aquae ide8 taria

, los-aquae

date r--ýý----, ý-ý--, ý--ý-ý ý-ýý (1974) KM WM KM WM KM WM KM WM KM WM

18/7 t--000---- 30/7 - 3.1 - 0.2 - 3.0 - 1.1 -- 13/8 -02.0 1.0 - 4.1 - 1.5 -- 29/8 0 0.1 - 1.2 0.5 4.6 - 2.5 --

9/9 ---------- 27/9 ttttt-----

(1975)

24/3 -0-0-0-0-- 5/5 0-000----- 9/6 0$ -000-0-0-

23/6 -t-------- 7/7 -t--0.2 -0---

22/7 -0-0--00.3 -0 16/8 -0-0.2 --0.1 --- 18/8 ---0.1 --0-0- 23/8 ---1.0 --2.1 -0- 26/8 ---0------

6/10 ---0-----0

t Akinetes observed but insufficient for quantitative estimation. $ In Kettle Mere A. circinalis had undergone very extensive sporulation by early June;

on 9 June clumps of akinetes were found in the sheltered areas of the mere, but little remained in the plankton at the sampling site.

-, insufficient material of the species concerned. 0, no akinetes. KM, Kettle Mere. WM, White Mere.

Sporulation of planktonic blue-green algae 325 In contrast to the well-marked succession of species in Kettle Mere, many of the filamentous species in White Mere form surface blooms and produce akinetes more or less simultaneously.

The process of sporulation effectively removes the alga concerned from the plankton. The transformation of a gas vacuolate vegetative cell into an akinote lacking gas vesicles and accumulating large cyanophycin bodies leads to a considerable increase in the specific gravity of the cell. This could be the reason for the

rapid sedimentation of akinetes. Sedimentation is enhanced by the aggregation of akinetes into large clumps and by the breakdown of filamentous material. While in general clumps of detached akinetes sediment more rapidly than the surviving vegetative material, the greater turbulence in the surface layers of White More during July-August 1975, breaks up the clumps so that filaments and akinetes sediment at an equal pace. Rates of sedimentation of the total particulate matter are given in figure 3a. Maximum rates of sedimentation of particulate material generally correspond with high rates of sedimentation of algal material, particularly of Mierocystis in Kettle Mere during the autumn, and of Asterionella in White Mere in late spring. The smaller peaks of sedimentation in Kettle More are also reflections of the algal contribution (figure 3b). The pattern of sedimentation in White Mere (figure 3c) is disrupted by the earlier breakdown of stratification in August resulting in the resuspension of bottom deposits. Although the main algal component in the sedimented matter in Kettle Mere is A. circinalis, and the data in figure 3b are the total of all the Anabaena species, the clear succession of species results in distinct periods of sedimentation for each species. The minor increase during the second half of August 1975 in Kettle Mere is due to the sporulation and sedimentation of A. solitaria.

There is little survival during the winter of vegetative filaments in the mud of Kettle Mere. The only period during which material was observed in the mud was immediately after sedimentation. Filaments appeared to persist longer at the mud surface in White Mere (figure 4a) but much of this is due to an interchange

of material between the plankton and the benthos when stratification breaks down,

rather than indicating long survival of sedimented vegetative material. In both mares there is a considerable and rapid input of akinetes to the surface mud layers during the period immediately after a bloom (figure 4 b, c) but there is also an equally rapid decline in numbers in both meres. (Note that the data on akinete which are expressed in terms of numbers per millilitre cannot be quantitatively compared with data on rates of sedimentation expressed as numbers per square metre per day. )

Orthophosphate concentrations are given in table 5. In both mores the concentration is high throughout the year with only a slight decrease in the surface waters during stratification. Analyses of intracellular labile orthophosphate, an indication of surplus phosphate within the cell, show relatively high values of 0.01-0.05 mg orthophosphate per milligramme algal dry mass, particularly when compared with stationary phase batch cultures. The high levels of available phosphate are also reflected by the low levels of alkaline phosphatase activity (Fitzgerald &


" o

1

I. N O N O N O

1-'ABP '-tu 2 (e-01 x99 9Ibo. capJt) T-'c'ep : -w 1_ &gp '-ui xaquinu x 9-01

aequznu x 9-01

z O_

öw

mmý A mýý

ý' ao

vi n

z mý o

o hm

n +ý

Sporulation of planktonic blue-green algae

tl. "O cý o eý o 0 0

N cV

Gldtuvs T_jui aegmnu x a-Ol

zm m

ob

ým

d° 'O ä¢ý

ti IH 0

q

os

aa. q

Ö

.r

zö

mý n

C7 rM1"1

327


Nelson 1966). Algal material collected from surface blooms had alkaline phosphatase activities in the range of 1.0 to 2.0 Sigma units per milligramme dry mass of alga. (1 Sigma unit is equivalent to the liberation of 1 µM p-nitrophenol per h. ) This is comparable with activities recorded in logarithmically growing laboratory

cultures of algal species isolated from the meres. Old cultures in the post-logarithmic phase of growth had activities of 3-6 Sigma units with low levels of orthophosphate remaining in the medium at this stage of growth.

TABLE 5. ORTHOPHOSPHATE CONCENTRATIONS IN THE MERES

(mg P04 P PER LITRE), 1975

site Kettle Mere White Mere depth of

sample Om 3m 6m Om Mt B$

515 1.70 1.80 1.20 0.67 0.70 0.80 9/6 1.30 0.75 2.30 0.80 0.23 0.75

23/6 0.34 0.69 1.18 0.32 0.50 0.97 7/7 0.35 0.69 1.70 0.33 0.85 1.31

22/7 0.40 0.74 1.78 0.60 0.58 1.08 16/8 0.98 1.30 2.50 0.83 0.98 2.30 18/8 0.74 1.61 3.20 1.31 0.66 2.70 23/8 1.66 1.70 4.60 1.52 2.60 4.70 26/8 1.50 1.07 0.92 1.65 0.66 2.95 29/9 - - - 1.01 1.01 -

6/10 0.45 0.56 1.24 1.03 1.03 1.04 27/10 0.56 0.56 1.26 1.65 1.80 1.81 15/11 0.77 0.75 0.75 0.82 0.82 0.86

j Middle of water column (3-4 m). $1m above bottom (6-9 m).

DISCUSSION

The coincidence of akinete production with dense surface accumulations of algal material suggests that light may play a critical role, rather than nutrient deficiency,

especially of orthophosphate in the induction of sporulation. Despite the dense

scum of algae at the water surface, the individual cells do not appear to be deficient in phosphorus. Blue-green algae are capable of storing through luxury uptake, sufficient phosphorus reserves, in the form of polyphosphate bodies, to support the growth of two further generations when transferred to a medium deficient in phosphorus (Batterton & Van Baalen 1968). Thus, even if diffusion of phosphates through the stagnant surface layers of water were the only means of transport to the algal cell, there would be sufficient reserves for a limited period of active growth. The analyses of labile orthophosphate and also alkaline phosphat-

e activity of algae collected during a bloom indicate that either the supply from the water to the algal cells is sufficient to maintain a high level within the cell or that whatever reserves were accumulated previously were not utilized.

High light intensity together with supersaturated oxygen conditions results in the photo-oxidative death and bacterial breakdown of algal material (Abeliovich


& Shilo 1972). The production and release of organic compounds may initiate

sporulation in the manner suggested by Fisher & Wolk (1976), the light intensity

and quality (particularly in the u. v. region) may have a direct photoinductive effect of the type described by Lazaroff (1966) and Lazaroff & Schiff (1962), or akinete production may be the result of a physiological imbalance between

nitrogen fixation and photosynthesis while the latter is inhibited by the high light intensity. Tyagi (i974) observed that akinete production in Anabaena doliolum $haradw. is inhibited by inorganic nitrogen compounds but is stimulated by

glucose. In this species, akinete differentiation begins in cells which are equidistant between two heterocysts, and sporulation proceeds centrifugally. However, in all the planktonic species examined in the meres, the first akinete is developed from the third vegetative cell from the heterocyst and sporulation proceeds centri- petally to produce a short chain of two or three akinetes. Any small variation in the number of cells between the first akinete and the heterocyst may be due to cell division subsequent to the formation of the akinete. Large variations may be due to filament breakage resulting in the separation of closely associated heterocysta

and akinetes. This seems to be a consistent feature of the planktonic Anabaena species, and a similar relationship has also been observed in Aph. floc-aquae. A considerable amount of variability in the number of cells between heterocyst and akinete was observed in Anabaena planktonica Brunth. by Burger (1974)

although 60 % of akinetes were found four or less cells away from the heterocyst. From the consistent relationship between heterocysta and akinetea in the species

examined, it would seem to be that heterocysts exert some degree of control over akinete production (Wolk 1966). However, it seems that some external factor is

required to trigger off the process. During the calm weather of early December 1975, a dense surface scum of A. circinalis accumulated at the lee shore of White Mere. The filaments appeared intact and healthy, and they incorporated hetero-

cysts but no akinetes. This supports the idea that a high level of solar radiation is

required for sporulation. The increase of cytoplasmic density results in the sedimentation of akinetes to

the mud surface. Although there is a rapid increase in the number of akinetes in the surface deposits, there is also an equally rapid decline in numbers soon after in both meres. This could be, but is probably not, due to lifting of material by turbulence, since Kettle Mere was still stratified in early October of both years 1974 and 1975, as was White Mere at the end of July. Very few akinetes were observed in the plankton after sedimentation had occurred, certainly not enough to account for the fall in numbers in the mud even if it is considered that the material was evenly distributed through the water column. The decline is probably due to akinete germination soon after maturation, which may account for the

small increase of the filamentous population observed about a month after the

occurrence of surface blooming. From the point of view of future development of the algae this would be more important in Kettle Mere, where algal numbers were reduced to undetectable levels, than in White Mere where a considerable proportion

12 Vol. 196. B.


of the population had survived blooming. Although only a few instances of akinete germination have been observed in plankton samples, up to 60 % of the akinetes examined were empty while still within the plankton. Germination shortly after formation has been observed in laboratory cultures of isolates of algae from the meres and also in cultures of Anabaena cylindrica Lemm. (Fay 1969). The generally large overwintering planktonic populations of vegetative filaments, especially in White Mere, indicate that akinete germination is not an important source of algal material during the spring as most of the growth can be accounted for by the material already within the plankton. There was no marked decrease of akinetes in the spring, or any signs of extensive germination. From the observation that akinetes germinate soon after their maturation, their function is seen more in terms of survival of algal populations during the period of blooming, rather than promoting their survival during the winter. In Kettle Mere, where the overwintering planktonic populations are much lower, akinete germination during the spring may be more important to the early development of algal populations.

Griffiths (1925) observed that Aph. flos-aquae did not produce akinetes in Kettle Mere, but did so in a nearby mere, The Mere, Ellesmere. During the period of this investigation no akinetes were observed in the Kettle Mere strain, but the White Mere strain, although a minor component of the phytoplankton, produced akinetes. This is probably a phenotypic rather than a genotypic difference, as akinetes have been produced in the Kettle Mere strain which has been isolated and grown in the laboratory. However, any extrapolations to the field from experiments done in the laboratory using isolates grown from natural water bodies must be viewed with caution due to the genetic plasticity of blue-green algae. During 1974 a dense surface bloom of Apl&. flos-aquae was observed in Kettle Mere, which rapidly decreased in numbers. During the rest of the year, no material was found, either in the plankton or in the sediments. In 1975 the size of the population of the species was less than 1% of that in the previous year. This supports the idea that akinetes are important for the survival of a sufficient stock of algal material to produce a large population in future years.

We are indebted to the Trustees of the Grosvenor Estate for permission to work at White Mere, to Mr R. K. Mainwaring and Major K. W. Pettergree for permission to work at Kettle Mere, to Mr L. R. Jebb for allowing access to the shore of White Mere and for storage facilities, and to the Freshwater Biological Association for the use of the Meres Laboratory at Preston Montford Field Centre. We are grateful to Dr C. S. Reynolds for helpful comments on the manuscript of this paper. This work has been carried out with the support of a grant from the Natural Environ- ment Research Council.


REFERENCES

Abeliovich, A. & Shilo, M. 1972 Photo-oxidative death in blue-green bacteria. J. Bact. 111, 682-689.

American Public Health Association. 1971 Standard methods for the examination of water and wastewater, 13th ed. New York: Am. Publ. Health Assoc., Inc.

Batterton, J. C. & Van Baalen, C. 1968 Phosphorus deficiency and phosphate uptake in the blue-green alga Anacystis nidulans. Can. J. Microbiol. 14,341-348.

Boyd, C. E., Prather, E. E. & Parks, R. W. 975 Sudden mortality of a massive phyto. plankton bloom. Weed Sei. 23,61-67.

Burger, J. 1974 A study of two populations of Anabaena planktonica (Cyanophyta) from Minnesota. Phycologia 13,125-129.

Fay, P. 1969 Metabolic activities of isolated spores of Anabaena cylindrica. J. exp. Bot. 20, 100-109.

Fisher, R. W. & Volk, C. P. 1976 Substance stimulating the differentiation of spores of the blue-green alga Cylindrospermum lich. eniforme. Nature, Lend. 259,394-395.

Fitzgerald, G. P. & Nelson, T. C. 1966 Extractive and enzymatic analyses for limiting or surplus phosphorus in algae. J. Phycol. 2,32-37.

Fogg, G. E., Stewart, W. D. P., Fay, P. & Walsby, A. E. 1973 The blue-green algae. London and New York: Academic Press.

Golterman, H. L. 1971 Methods for chemical analysis of fresh waters. I. B. P. Handbook no. 8. Oxford, London and Edinburgh: Blackwell Scientific Publications.

Gorham, E. 1957 The chemical composition of some waters from lowland lakes in Shropshire, England. Tellus 9,174-179.

Griffiths, B. Al. 1925 Studies in the phytoplankton of the lowland waters of Great Britain. III. The phytoplankton of Shropshire, Cheshire and Staffordshire. J. Linn. Soc. (Bot. ) 47,75-98.

Kirchner, W. B. 1975 An evaluation of sediment trap methodology. Limnol. Oceanogr. 20, 657-660.

Klebahn, H. 1895 Gasvakuolen, ein Bestandteil der Zellen der wasserblütebildenden Phyco- chromaceen. Flora, Jena 80,241-282.

Lazaroff, N. 1966 Photoinduction and photoreversal of the Nostocacean developmental cycle. J. Phycol. 2,7-17.

Lazaroff, N. & Schiff, J. 1962 Action spectrum for developmental photo-induction of the blue-green alga Nostoc muscorum. Science, N. Y. 137,603-604.

Lund, J. W. G., Kipling, C. & Le Cren, E. D. 1958 The inverted microscope method of estimating algal numbers and the statistical basis of estimating by counting. Hydro- biologia 11,143-170.

Mortimer, C. H. 1942 The exchange of dissolved substances between mud and water in lakes. J. Ecol. 30,147-201.

Reynolds, C. S. 1967 The breaking of the Shropshire Mares: Some recent investigations. Bull. Shrops. Conserv. Trust. 10,9-14.

Reynolds, C. S. 1971 The ecology of the planktonic blue-green algae in the North Shropshire mares, England. Fld Stud. 3,409-432.

Reynolds, C. S. 1972 Growth, gas vacuolation and buoyancy in a natural population of a blue-green alga. Freshwat. Biol. 2,87-106.

Reynolds, C. S. 1973 a Growth and buoyancy of Microcystis aeruginosa Kütz. emend. Elenkin in a shallow eutrophic lake. Proc. R. Soc. Lond. B 184,29-50.

Reynolds, C. S. 1973b Phytoplankton periodicity of some North Shropshire meres. Br.

phycol. J. 8,301-320. Reynolds, C. S. 1975 Interrelations of photosynthetic behaviour and buoyancy regulation

in a natural population of a blue-green alga. Freshwat. Biol. 5,323-338. Reynolds, C. S. & Rogers, D. A. 1976 Seasonal variations in the vertical distribution and

buoyancy of 1llicrocystis aeruginosa Kütz. emend. Elenkin in Rostherne Mere, England. Hydrobiologia 48,17-23.

Reynolds, C. S. & Walsby, A. E. 1975 Water-blooms. Biol. Rev. 50,437-481. I2-2

ýýýý .ý ,-ýi ý: - QMRO Home

Documents

Transcript of ýýýý .ý ,-ýi ý: - QMRO Home