j. bacteriol.-1972-abeliovich-682-9

JOURNAL OF BACTERIOLOGY, Sept. 1972, p. 682-689Copyright 0 1972 American Society for Microbiology

Vol. 111, No. 3Printed in U.S.A.

Photooxidative Death in Blue-Green AlgaeA. ABELIOVICH AND M. SHILO

Department of Microbiological Chemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received for publication 15 May 1972

When incubated in the light under 100% oxygen, wild-type blue-green algae(Anacystis nidulans, Synechococcus cedrorum) die out rapidly at temperaturesof 4 to 15 C, and at 35 C (or at 26 C in the case of S. cedrorum) in the absenceof CO2. Photosynthesis is impaired in these cells long before they die. Blockingof photosystem II at high temperatures in the presence of CO2 sensitizes thealgae to photooxidative death. Photooxidative death and bleaching of photo-synthetic pigments are separable phenomena. Photooxidative conditions weredemonstrated in Israeli fish ponds using A. nidulans as the test organismduring dense summer blooms, when dissolved CO2 is low, and in winter, whenwater temperatures generally drop below 15 C. This finding suggests that pho-tooxidative death may be responsible for the sudden decomposition of blue-green blooms in summer, and may be a factor in the absence of blue-greenblooms in winter.

The term "photooxidative death" refers tothe lethal effect to cells exposed to light in thepresence of oxygen and sensitized by internalor external dyes. Carotenoids were found toprotect certain organisms from the photooxi-dative effects at physiological temperatures.Many organisms possessing carotenoids havebeen found to be sensitive to photooxidativereactions when deprived of this pigment (5-7,11, 15, 17). Wild-type organisms, containingcarotenoids, were sensitive to photooxidationwhen incubated at low temperatures (16, 17),indicating that photooxidation is prevented inthese organisms by an enzymatic mechanism.The frequent observations of sudden and

spontaneous disintegration of the blue-greenalgal blooms, which appear in summer in Is-raeli fish ponds, raised the question of whethera photooxidative effect is involved in this phe-nomenon, since conditions of oxygen supersat-uration and high light intensities prevail atthat time (1). A characteristic feature pre-ceding massive die-off of blue-green algalblooms is the low CO2 content (as expressed inelevated pH values during the light period) ofthe highly alkaline waters. Therefore, the ef-fect of CO2 on die-off was investigated.The investigations reported here show that

several blue-green algae are susceptible to pho-tooxidative effects when illuminated in anatmosphere of 100% oxygen in a CO2-free me-dium. Conditions for photooxidation of blue-green algae were found in nature, and the pos-sible ecological significance of this is dis-cussed.

MATERIALS AND METHODSCultivation and enumeration of algae. Axenic

Anacystis nidulans strain 6311 and Synechococcuscedrorum (from the collection of the Department ofBacteriology and Immunology, University of Cali-fornia at Berkeley) were used. Zehnder and Gorhammedium no. 11 (25) with NaNOs concentration mod-ified to 1.5 g/liter served as medium. Standardgrowth of algal cultures was under an atmosphere ofair at 35 C in a New Brunswick Psychrotherm in250-ml flasks containing 100 ml of medium. Illumi-nation was provided by 40-w white fluorescent lampsgiving an incident light intensity of 104 ergs percm per sec at the flasks. Unless stated otherwise, allexperiments were carried out with A. nidulans har-vested at day 5 of growth (end of logarithmic phase;protein concentration, about 300 mg/liter; chloro-phyll concentration, 3 mg/liter, and total carotenoidsconcentration, 1 mg/liter). Solid medium was pre-pared by mixing equal volumes of double-strengthmedium and 2% agar in distilled water, after theyhad been autoclaved separately. Viability countswere determined by conventional dilution andplating in soft agar technique. In certain cases, themost probable number of living cells was evaluatedon the basis of growth in series of highly diluted sus-pension.Photooxidation experiments. Photooxidation

experiments were carried out in an illuminated re-ciprocal shaking bath (150 strokes/min). The lightintensity at the flasks was 104 ergs per cm per sec(cool white fluorescent light). Cell suspensions werekept in complete medium or in Na2CO,-free me-dium under an atmosphere of 100% of 02 (or N2 incontrols) in glass-stoppered 250-ml Erlenmeyerflasks. To remove 2, the flasks were equippedwith a central well containing 0.5 ml of 50% KOHsoaked on filter paper. Photooxidation experiments in

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PHOTOOXIDATIVE DEATH IN BLUE-GREEN ALGAE

presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea(DCMU) were carried out on cells grown in an at-mosphere of air + 5% CO2.Oxygen and pH measurements. Kinetic meas-

urements of oxygen evolution and consumption bycells suspended in complete medium and CO2-freemedium were performed in a thermostatically con-trolled 20-ml Perspex cell, illuminated by a 500-wphotoflood lamp (Tungsraphot-B, Tungsram, Ger-many). The light intensity was 1.5 x 101 ergs percm per sec at the surface of the cell. The cell wasequipped with a Clark-type oxygen electrode(YSI5331, Yellow Springs Instruments Co., YellowSprings, Ohio) connected to Gilson oxygraph modelKM (Gilson Medical Electronics, Middleton, Wisc.)and with a combined pH electrode (GK2320, Radi-ometer) connected to a Radiometer titrator (TTT2 +ABU 11, Radiometer, Copenhagen, Denmark).Measurement of photosynthetic pigments. A 10-

ml algal suspension in 0.1 M potassium phosphatebuffer (pH 7.0) was disintegrated at 4 C using 0.1 to0.11 mm 0 glass beads in a Braun homogenizer(Model MSK, Braun Apparatbau Melsungen, Ger-many) at 4,000 cycles/min for 30 sec. The resultingextract was centrifuged at 12,000 x g for 10 min,and the C-phycocyanin in the supernatant wasmeasured at 620 to 625 nm in a Perkin Elmer 137UV spectrophotometer (Perkin-Elmer Corp. Nor-walk, Conn.). Chlorophyll a and carotenoids weremeasured after acetone extraction as described byParsons and Strickland (18).

RESULTSPhotooxidative death of A. nidulans. Pho-

tooxidative death of A. nidulans occurs whencells are incubated at 35 C under a 100% 02atmosphere in media devoid of CO2, whereascells remained viable when incubated at thistemperature and oxygen concentration in aCO2-containing medium (Fig. 1). At this tem-perature, the death rate becomes quite rapidafter an initial lag period of 6 to 8 hr. On theother hand, photooxidative death at lowertemperatures (4 C, 10 C, 15 C) occurs in mediaboth containing and lacking CO2 (Fig. 1; Table1) with a considerably shortened lag period of2 to 4 hr. S. cedrorum was also killed after 16hr of incubation in photooxidative conditions(100% 02 atmosphere in Na2CO ,-free mediumor in distilled water) at 26 C.To determine whether oxygen partial pres-

sure plays a role in susceptibility to photooxi-dative death, A. nidulans cells were incubatedfor up to 20 hr in CO2-free medium under anatmosphere of air or under pure oxygen (Fig. 1).The algae remained viable in air, and weresensitive to photooxidation at 100% oxygenconcentrations. This is in agreement with thefindings of Franck and French (10), that pho-tooxidative processes in hydrangea leaf tissueare fully expressed only at partial pressures of60% 02 saturation or more, as well as those of

108 N

10-

610

E

105

2 4 6 8 10 12l 4.0 148T im e (hours)

FIG. 1. Viability of Anacystis nidulans aftertransfer from photosynthetic to photooxidative con-ditions. A. nidulans was grown in complete mediumin air for 5 days under continuous illumination (104ergs per cm2 per sec), and then was exposed to thefollowing environments: (A), complete medium andC02-free medium, 100%0 oxygen, light, 4 C; (0),C02-free medium, 100%o oxygen, light, 35 C; (0),complete medium and C02-free medium, 100% oxy-gen, dark, 4 C and 35 C; (0), complete medium,100%o oxygen, light, 35 C; (0), CO2-free medium,air or 100% N2, light and dark, 4 C and 35 C; (0)and (d), A. nidulans grown in air enriched with 5%CO2 and transferred to C02-free medium, 100% oxy-gen, 35 C in light (0) or dark (a).

Stewart and Pearson (23), that growth of Phor-midium and Anabaena is inhibited at this par-tial oxygen pressure. Because the pH of theCO2-depleted medium rises to values of 10.5and higher within 1 hr in light at 35 C, westudied the effect of pH on the system. Lethalphotooxidation occurs as well at pH 7.0 (Fig.2) and 9.2 (Table 1). Therefore, cells are sensi-tive to high oxygen partial pressure at lowerpH values as long as CO2 is absent.The kinetics of photooxidative death de-

pends not only on experimental conditions,but also upon the preincubation history of thealgae. For example, A. nidulans cells grown inair enriched with 5% CO2 began to die only 48hr after transfer to photooxidative conditions(light, -CO2, + 100% 02) although the pHrose to 11.0 within 6 hr after transfer.

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684 ABELIOVIC

TABLE 1. Effect of light and 02 on viability of A.nidulans at different temperatures

Viability of A. nidulans

Temperature Illumination Sodium(C) Complete Distilled (001 M)medium' water (.1mbuffer

(pH 9.6)

4 Dark 100 100 1004 Light 0.1 1 110 Dark 10010 Light 0.115 Dark 10015 Light 0.135 Dark 100 100 10035 Light 100 1 1

a Initial concentration, 102 cells/mi; incubationtime, 12 hr under 100% oxygen.

b Final pH of the incubation suspensions at 35 Creached 10.5, whereas, at 4, 10, and 15 C, the pHremained 9.2.

10

< 10

CL

0 510

E

z

T i m e (hours)FIG. 2. Photooxidative death at pH 7.0. A. nidu-

lans cells were suspended in 0.05 M potassium phos-phate buffer (pH 7.0) and incubated in the light at35 C in an atmosphere of 99% 02 + 1% CO2 (0) orin 100% 02(0).

AND SHILO J. BACTERIOL.

Effect of DCMU on photooxidation. Wefound that 10-6 M DCMU, which inhibits ox-ygen evolution and CO2 assimilation (3), in-hibits within 10 sec both photoassimilation ofCO,2 and oxygen evolution of A. nidulans,when incubated in complete medium underatmosphere of air. Therefore, DCMU wasadded to A. nidulans cells incubated under100% oxygen in the light at 35 C (Table 2).The table shows that, in the presence ofDCMU, the cells are sensitized to photooxida-tive death even in complete medium.

Effect of photooxidative conditions onphotosynthetic oxygen evolution and darkrespiration. When photosynthesizing A. nidu-lans cells are transferred to an atmosphere andmedium devoid of CO2, both the photosyn-thetic capacity and dark respiration are inhib-ited (Fig. 3) long before the onset of photooxi-dative death (cf. Fig. 1). Results identical tothese were obtained when the cells were sus-pended in distilled water. Within 6 hr of incu-bation under photooxidative conditions, onlypart of the initial photosynthetic capacity (asmeasured by 02 evolution) of these cells wasrestored when CO2 (as sodium bicarbonate)was added to the medium. When the cells wereilluminated under 100% N2 in medium devoidof CO2, the photosynthetic capacity of thealgae remained stable at 20% of its initial rate(Fig. 4). Addition of the sodium bicarbonatehad no effect on oxygen evolution by thesecells, but restored the initial rate of oxygenevolution in cells incubated in distilled waterinstead of CO2-free medium. The depressingeffect on photosynthesis of incubation in CO2-free medium under 100% N2 is still not clear.In comparison, A. nidulans cells, which hadbeen incubated in the dark under either 100%N2 or 02 and without CO2, fully retained theirphotosynthetic capacities (Fig. 3, 4).

TABLE 2. Effect of DCMUa on photooxidative deathin Anacystis nidulans

Viability (%)bIllmiaton DCMUIllumination (10- M) Complete Na,CO,-free

medium medium

Light + 1 0.01_ 100 0.01

Dark + 100 100_ 100 100

aDCMU = 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea.

'Initial cell concentration, 106 cells/ml; incuba-tion time, 16 hr at 35 C, in 100% 02 atmosphere.

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T im e of incubat 'on(hours)FIG. 3. Rate of 02 evolution and dark respiration

of A. nidulans (108 cells/mI) incubated in both thelight and dark under an 02 atmosphere in Na2CO.-free medium. Initial 02 evolution and dark respira-tion rates were 90 nmoles of 02 per min per ml and40 nmoles of 02 per min per ml, respectively. Ox-ygen evolution of cells incubated in photooxidativeconditions (0), and after addition of NaHCOs (up to0.05 M) (a); rate of dark respiration (0) and rate ofoxygen evolution of control cells incubated in thedark and transferred to light (A).

Light-dependent 02 consumption. Whentransferred to CO 2-free medium under anatmosphere of 100% oxygen at 35 C, A. nidu-tans cells developed a light-dependent oxygenconsumption (Fig. 5) after 4 hr, a time whenall the cells are still viable and capable of re-covering most of their initial rate of photosyn-thetic oxygen evolution (cf. Fig. 3). The light-dependent oxygen consumption proceeds at anincreasing rate as the cells die off (6 to 12 hr,cf. Fig. 1). A typical tracing of one such experi-ment is shown in Fig. 6. As seen in Fig. 5,light-dependent oxygen consumption does notoccur after the cells were incubated under a N2atmosphere in the CO2-free medium or in dis-tilled water, when the cells were incubatedunder 100% oxygen in distilled water, or whenincubated in CO2-free medium under an at-

mosphere of air. This light-dependent oxygenconsumption could be imitated by raising thepH of the medium, commencing within 5 min(Fig. 7) after the pH of the cells in standardgrowth medium was raised to 12.0. The light-induced oxygen consumption ceased after thepH of the medium was reduced to 9.5 (by bub-bling CO2 through the medium), and the orig-inal oxygen evolution rate was once again ob-tained. The photosynthetic capacity of thecells kept at pH 12 remained unimpaired forat least 25 min. The light-dependent oxygenconsumption was found not to be affected byKCN (10-2 M), dinitrophenol (4 x 106 M) orDCMU (5 x 10-6 M) under these conditions.

100 -----------

90

80

0> 700 6

0 650

E

C 4000

0)

2108 1a.)

o

T i me of incubation(hours)FIG. 4. Rate of 02 evolution and dark respiration

of A. nidulans under N2 atmosphere in Na2CO,-freemedium or distilled water. Initial 02 evolution anddark respiration rates were 115 nmoles 02 per minper ml and 40 nmoles per min per ml, respectively.Oxygen evolution of cells in medium and distilledwater (0); oxygen evolution after addition ofNaHCOs to cells incubated in Na2CO0-free medium(A), or in distilled water (A); rate of dark respiration(0); rate of 02 evolution of cells incubated inNa2CO2-free medium in the dark (-) upon illumina-tion.

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T ime of incubotion(hours)FIG. 5. Rate of light dependent oxygen consump-

tion in A. nidulans. Oxygen consumption in cellsincubated in 100%/o oxygen in Na2CO,-free mediumin the light (0); oxygen consumption of cells incu-bated in Na2COr-free medium in N2 or air, in com-plete medium under 02 or N2, or in distilled waterunder N2 or 02 (0) in light or dark.

Effect of pH and photooxidative death onthe pigmentation of the cells. Only those A.nidulans cells kept in the light under a pure02 atmosphere devoid of CO2 at high pH values(10 to 11) and at 35 C bleach rapidly and al-most totally (Table 3).Photooxidative death of A. nidulans in

fish ponds. We tried to determine whetherphotooxidative death is related to the suddendisintegration and decay of waterblooms ofblue-green algae observed in natural waterbodies and, in particular, in Israeli fishponds.Three neighboring fishponds having differentdiurnal pH curves were chosen to test this pos-sibility. In September 1971, the water temper-ature in the profile examined ranged from 28to 32 C. A. nidulans cells were suspended indialysis tubing at different depths. Algae incu-bated in pond 1 showed marked mortality inconditions on high illumination. Photooxida-tive death was also observed in pond 2,whereas no killing occurred in pond 3 (seeTable 4). A comparison of the pH fluctuations

J. BACTERIOL.

I

I III

FIG. 6. Oxygraph recording of light-dependentoxygen consumption in A. nidulans. Cells (109) wereincubated for 12 hr in Na2CO2-free medium at pH10.5 in oxygen.

ight Dark Li ht Dark Li ght

C4 ~ ~

T me (min)FIG. 7. Oxygraph recording of the light-dependent

oxygen consumption at elevated pH in A. nidulans.A. nidulans grown in standing conditions and thepH of the medium shifted to 12 (_) and to 9.5 (XO)by the addition of 0.01 N NaOH and by bubblingwith CO2, respectively.

in each pond to the respective mortalities indi-cated that viability depends on the presence ofavailable CO2, since pH fluctuations are pri-marily due to changes in CO2 concentration.

In the light of the experimental finding thatat low temperature photooxidative death oc-curs even in the presence of CO2, field obser-vations were made in winter during the monthof January in these ponds, at low temperatures(11 to 14 C), and at low pH values (8.1 to 8.4).When suspensions of A. nidulans were kept for8 hr in dialyzing tubing in the sunlight, sub-merged at a depth of 5 cm, more than 99% ofthe algae died. Suspensions kept in the sameconditions, but shaded from the light, re-mained fully viable.

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TABLE 3. Effect of photooxidative conditions onbleaching of photosynthetic pigments in Anacystis

nidulkns in the absence of CO2aPigment concentration (%)

Complete Me- PotassiumPhotsynhetc mdiu-Ce dium phosphatepigment medim-CO at (0.05 m, pHat32 4 CC 7.0), at 35 Cd

02 N2 O0 02 N2

C-phycocyanin 5 50 100 75 100Chlorophyll a 50 50 100 75 100a 8 x 108 cells/ml were incubated in the light for

12 hr before chlorophyll a and C-phycocyanin wereextracted and measured.

I Final pH, 10.5.c Final pH, 9.2.d Final pH, 7.0.

DISCUSSIONPhotooxidative damage has been observed in

many photosynthetic organisms including bac-teria, eukaryotic algae, and higher plants, afterinterference or suppression of carotenoid bio-synthesis (22), thus establishing the protectivefunction of carotenoid pigments in photosyn-thetic organisms kept under air. (However, ithas been recently shown that Micrococcus ro-seus and Acholeplasma laidlawii B are subjectto photooxidative killing although they arecarotenoid-containing organisms [Schwartzeland Cooney, Cooney and Krinsky, Abstr.Annu. Meet. Amer. Soc. Microbiol., p. 291-292, 1972].) The photooxidative death underatmosphere of pure oxygen in blue-green algaewhich is described in this paper can proceed incarotenoid-containing organisms (12) at physi-ological temperatures only in the absence ofCO2. It is possible that under these conditionscarotenoids do not afford sufficient protectionagainst photooxidation. Thus, the role of CO2in preventing photooxidative death in A. nidu-lans and S. cedrorum indicates that the pro-tective mechanism(s) is connected with thephotosynthetic activity of the cell. Indeed,addition of DCMU, which blocks photosystemII, leads to photooxidative death even in com-plete (CO2-containing) medium. It may bethat the photooxidation at physiological tem-peratures in the absence of CO2 involves aperoxide or a superoxide radical produced bydirect reduction of oxygen by some reducedelectron carrier that accumulates when photo-synthesis is inhibited by absence of CO2.

This hypothesis, however, fails to explainthe lethal photooxidation at low temperatures,

which probably proceeds by direct photosensi-tization of the cellular photosynthetic pig-ments. The death at low temperatures hasbeen observed (5, 6, 16) in many organismsstudied, including nonphotosynthetic microor-ganisms (such as Sarcina lutea, Mycobac-terium marinum, Halobacterium salinarium),as well as in several photosynthetic organisms(17). In the case of carotenoid-containing A.nidulans and S. cedrorum, photooxidativedeath occurred at a much more rapid rate atthe lower temperature. The considerable lag(ca. 8 hr) before the onset of mortality at 35 Cand the even longer lag (48 hr) when cells werepreincubated in an atmosphere of 5% CO2 in-dicate that the protective mechanism(s) maybe sustained by metabolic reserves possiblysupplying endogenous CO2 by respiration,even after photoassimilation of exogenous CO2ceases. Stewart and Pearson (23) have foundthat nitrogen fixation and photosyntheticcarbon fixation are inhibited by high 02 levels.We found that, under photooxidative condi-tions, A. nidulans cells show progressivedamage to their photosynthetic capacitiesduring the period preceding onset of death.Yet another indication of early photooxidativedamage to A. nidulans cells is the induction oflight-dependent oxygen consumption.The conclusion that oxygen uptake in the

light in photooxidatively damaged cells maybe a nonphysiological reaction is also sup-

TABLE 4. Photooxidative death of A. nidulans infishpondsa

Depth of immersion Percent of viable cellsb(cm) Pond 1' Pond 2' Pond 3e

5 0.001 10 10015 1025 1040 10 100Dark control 100 100 100

(5 cm)a The experiments were carried out in three adja-

cent fishponds in Beit Shan valley in September1971 when water temperature ranged from 28 to 32C. A. nidulans were suspended in closed, clear di-alysis tubing (1-inch diameter) at different depths inthe ponds. Each bag initially contained 50 ml ofsuspension of the algae (107 cells/ml) in logarithmicgrowth phase, washed in distilled water. Sampleswere kept in the ponds from sunrise to sunset (0500-1800 hr).

b Initial concentration, 107 cells/ml.cpH shift (0500-1400 hr), 9.4 , 9.8.dpH shift (0500-1400 hr), 8.0 - 9.1.epH shift (0500-1400 hr), 8.0 , 8.6.

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ABELIOVICH AND SHILO

ported by observations of such oxygen uptakein boiled leaf tissues of hydrangea (11) and A.cylindrica heterocysts (Bradley and Carr, Proc.Soc. Gen. Microbiol., p. XII, 1971), and ontransfer of the A. nidulans cells to very highpH.The destructive nature of photooxidative

conditions is further emphasized by thebleaching of photosynthetic pigments observedin A. nidulans. However, this bleaching bearsno direct relation to the photooxidative deathof the algae, since unbleached cells die at 4 Cand at 35 C at low pH values, and A. nidulanshas been observed to bleach in the absence ofnitrogen source (nitrogen chlorosis) while re-maining viable (2). Bleaching and photooxida-tive death are thus clearly separable phe-nomena.The dependence of blue-green algae on CO2

for protection against photooxidative effects atphysiological temperature could partially ex-plain why planktonic blooms of blue-greenalgae in eutrophic waters are frequently denserand longer-lasting than in oligotrophic waters(8), although the algae are obligate photoauto-trophs. The preference of blue-green algae forpolluted waters could well be related to thehigh evolution of CO2 by bacteria in such con-ditions (13). This assumption is furtherstrengthened by the finding of Lange (14) thatcarbohydrates enhance growth of blue-greenalgae when associated with bacteria. He foundthat both carbohydrates and bacteria could bereplaced by CO2 added to the algal cultures.M. E. Meffert (Abstr. 13th Limnological Con-gress, Leningrad, p. 72, 1971) found that Oscil-latoria redekei lysed in axenic cultures whenCO2 was removed, but not in bacterized cul-tures.The requirement for CO2 as protection

against photooxidative death is indicated bythe finding that Anabaena variabilis and A.nidulans die even in the presence of acetatewhen deprived of CO2 (19).The broad sensitivity of blue-green algae to

photooxidation may also explain why thisgroup thrives in environments of reduced ox-ygen supply (23), and why certain blue-greenalgae, such as Gleocapsa, are killed at highlight intensities and can grow only in dim light(22). Furthermore, the low light penetrationand reducing conditions may be factors forenrichment of blue-green algae in eutrophicwaters, as stated by Bozniak and Kennedy (4).Massive water blooms of blue-green algae tendto disintegrate spontaneously and suddenlywithout preliminary visible symptom. Theconditions proven experimentally to be re-

quired for photooxidative death (presence oflight and oxygen, lack of CO 2) are not un-common in the upper layers of dense algalwater blooms in ponds and lakes. Oxygen con-centrations of 300 to 400% saturation and highpH values (pH 10) following CO2 depletionfrequently occur in freshwater ponds duringthe summer (1). A possible mechanism foravoiding photooxidative death of blue-greenalgae which contain gas vacuoles in oxygen-rich layers could be the change in buoyancy ofthe algae due to changes in gas volume, de-scribed for Aphanizomenon (20) and Anabaenaflos aqua (8, 9, 24), causing migration from suchlayers.

Since light intensity is high the year-roundin Israel, photooxidative conditions prevail inwinter when water temperatures generallyremain below 15 C, and in summer whenheavy algal blooms cause CO2 depletion.

Therefore, photooxidative conditions in na-ture might be tested in terms of sensitivity ofA. nidulans. A test system using this organismsuspended in dialysis tubing, as described inthe experimental part, which allows for rapidequilibration with external conditions, canserve for this purpose.The sudden and sporadic die-off of summer

blooms of blue-green algae, often with cata-strophic consequences to fish populations, ismost probably due to photooxidation whichoccurs when CO2 deficiency, caused by algaephotosynthesis, becomes critical.

In addition, the high sensitivity of blue-green algae, such as A. nidulans, to photooxi-dation at the low temperatures at which pro-tective or repair mechanisms are nonfunctionalis most probably a factor in the absence ofblue-green algal blooms observed in winter inIsraeli fish ponds.

ACKNOWLEDGMENTSOur thanks are due to A. M. Mayer and J. S. Holt for

reading the manuscript and for their helpful suggestions,and to Bina Golek for her aid in preparing the manuscript.

LITERATURE CITED1. Abeliovich, A. 1969. Water blooms of blue-green algae

and oxygen regime in fish ponds. Verh. Int. Verein.Limnol. 17:594-601.

2. Allen, M. M., and A. J. Smith. 1969. Nitrogen clorosisin blue-green algae. Arch. Mikrobiol. 69:114-120.

3. Bishop, M. I. 1958. The influence of the herbicide,DCMU, on the oxygen evolving system of photosyn-thesis. Biochim. Biophys. Acta 27:205-206.

4. Bozniak, E. G., and L. L. Kennedy. 1968. Periodicityand ecology of the phytoplankton in an oligotrophicand autrophic lake. Can. J. Bot. 46:1259-1271.

5. Burchard, R. P., and M. Dworkin. 1966. Light-inducedlysis and carotenogenesis in Myxococcus xanthus. J.Bacteriol. 91:535-545.

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8. Fogg, G. E. 1969. The physiology of an algal nuisance.Proc. Roy. Soc. Ser. B. Biol. Sci. 173:175-189.

9. Fogg, G. E., and A. E. Walsby. 1971. Buoyancy regula-tion and the growth of planktonic blue-green algae.Mitt. Int. Verein. Limnol. 19:182-188.

10. Franck, J., and C. S. French. 1941. Photooxidation proc-esses in plants. J. Gen. Physiol. 25:309-324.

11. Griffiths, M., W. R. Sistrom, G. Cohen-Bazire, and R.Y. Stanier. 1955. Function of carotenoides in photo-synthesis. Nature (London) 176:1211-1214.

12. Halfen, L. N., and G. W. Francis. 1972. The influence ofculture temperature on the carotenoid composition ofthe blue-green alga, Anacystis nidulans. Arch. Mikro-biol. 81:25-35.

13. Kuentzel, L. E. 1969. Bacteria, carbon dioxide and algalblooms. J. Water Pollut. Contr. Fed. 41:1737-1747.

14. Lange, W. 1967. Effect of carbohydrates on the sym-biotic growth of planktonic blue-green algae with bac-teria. Nature (London) 215:1277-1278.

15. Mathews, M. M. 1963. Studies on the localization, func-tion, and formation of the carotenoid pigments of astrain of Micobacterium marinum. Photochem. Photo-biol. 2:1-8.

16. Mathews, M. M. 1964. The effect of low temperature onthe protection by carotenoids against photosensitiz-ation in Sarcina lutea. Photochem. Photobiol. 3:75-77.

68917. Mathews, M. M., and W. R. Sistrom. 1960. The function

of the carotenoid pigments of Sarcina lutea. Arch.Mikrobiol. 35:139-146.

18. Parsons, T. R., and J. D. H. Strickland. 1963. Discussionof spectrophotometric determination of marine plantpigments, with revised equations for ascertainingchlorophylls and carotenoids. J. Mar. Res. 21:155-163.

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