JOURNAL oF BAcTERIoLoGY, Feb. 1977, p. 623-629Copyright 0 1977 American Society for Microbiology

Vol. 129, No. 2Printed in U.S.A.

Occurrence ofFacultative Anoxygenic Photosynthesis AmongFilamentous and Unicellular Cyanobacteria

S. GARLICK,* A. OREN, AND E. PADANDepartment of Microbiological Chemistry, The Hebrew University-Hadassah Medical School,

Jerusalem, Israel

Received for publication 28 September 1976

Eleven of 21 cyanobacteria strains examined are capable of facultative anoxy-genic photosynthesis, as shown by their ability to photoassimilate CO2 in thepresence of Na2S, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 703-nm light.These include different cyanobacterial types (filamentous and unicellular) ofdifferent growth histories (aerobic, anaerobic, and marine and freshwater).Oscillatoria limnetica, Aphanothece halophytica (7418), and Lyngbya (7104)have different optimal concentrations of Na2S permitting C02 photoassimila-tion, above which the rate decreases: 3.5, 0.7, and 0.1 mM, respectively. In A.halophytica, for each C02 molecule photoassimilated two sulfide molecules areoxidized to elemental sulfur, which is excreted from the cells. The ecological andevolutionary significance of anoxygenic photosynthesis in the cyanobacteria isdiscussed.

The occurrence ofcyanobacteria ("blue-greenalgae") in anaerobic environments rich in sul-fide has long been reported (3-6, 9, 10, 13, 18-20, 24, 25, 29, 31). However, only recently,Cohen et al. (8) demonstrated that sulfide canbe used as electron donor for the photoassimila-tion of CO2 in the cyanobacterium Oscillatorialimnetica. This cyanobacterium displays typi-cal oxygenic photosynthesis. Nevertheless,when its photosystem H is inhibited by thepresence of 3-(3,4-dichlorophenyl)-l,ldimethyl-urea (DCMU) or not excited by 700-nm light,the photoassimilation ofC02 is driven by photo-system I with Na2S as sole electron donor (7, 8).Two sulfide molecules are oxidized to elementalsulfur for each C02 molecule photoassimilated(7).Since 0. limnetica displays both oxygenic

(plant type) and anoxygenic (bacterial type)photosynthesis and can readily shift from one tothe other, its phototrophic metabolism may rep-resent an interdiate pattern linking the twoknown types of phototrophic metabolism. Thissuggestion is tempting since the cyanobacteriaare the most ancient plant-type phototrophs onearth (23) and show procaryotic cellular organi-zation (26). Nevertheless, it is possible that 0.limnetica represents secondary specializationtowards anoxygenic photosynthesis due to ad-aptation to its biotype of isolation. In this habi-tat, aerobic and anaerobic conditions alternatewithin a yearly cycle (Y. Cohen, Ph.D. thesis,The Hebrew University, Jerusalem, 1975).Hence, the question arises whether 0. limnetica

is an exception to the prevailing concept thatcyanobacteria are capable of only oxygenicphotosynthesis, or whether anoxygenic photo-synthesis is an ability common among thecyanobacteria, which should be of ecologicalsignificance in those species found in anaerobicecosystems.

In this study, a number of cyanobacteriawere screened for their ability to carry out an-oxygenic photosynthesis with H2S as electrondonor. The cyanobacteria tested included orga-nisms isolated from sulfide-rich habitats aswell as strains available from culture collec-tions.

MATERIALS AND METHODSCyanobacteria strains and culture conditions.

The strains tested for the ability to photoassimilateCO2 with H2S a electron donor are listed in Table 1together with their history and the specific incuba-tion conditions used. Growth medium was Chu 11(27) prepared in the respective suspension waters(Table 1). The strains were grown in 250-ml flaskscontaining 100 ml of medium at an incident lightintensity of 5.103 ergs/cm2 per s, provided by whitefluorescent lamps (4,300 K, 20 W). The specificgrowth incubation temperature is indicated in Table1.CO2 photoassimilation measurements. Screening

for photoassimilation of CO2 with H2S as electrondonor was conducted in 5-day-old cultures (logarith-mic growth phase of 0. limnetica and Aphanothecehalophytica). Cells were washed and resuspended(20 to 40 ,ug of cell protein per ml) in the experi-mental medium (prepared in the respective suspen-sion waters as indicated in Table 1) containing thefollowing major elements (grams per liter): KH2PO4,

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624 GARLICK, OREN, AND PADAN

TABLE 1. Strain histories and specific incubation conditions of cyanobacteria tested for anoxygenicphotosynthesis with H,S as electron donor

Temp Suspen-Name (if any) History (C) of sion wa-growth ter of me-

and expt diaa

Oscillatoria type(6407) Culture collection 25 DW(6412) R. Y. Stanierb 25 DW(6506) R. Y. Stanierb 25 DW(6602) R. Y. Stanierb 25 DW

Lyngbya typeSchizothrix calcicola (7004) R. Y. Stanierb 25 DWLyngbya lagerheimii (7104) R. Y. Stanierb 25 DW

Microchaete typeNostoc (73102) R. Y. Stanierb 25 DW

Chlorogloeopsis type (6712) R. Y. Stanierb 25 DWAphanocapsa type (6714) R. Y. Stanierb 25 DWSynechococcus type (6311) R. Y. Stanierb 35 DWPseudoanabaena (7402) R. Y. Stanierb 25 DWPseudoanabaena (7403) R. Y. Stanierb 25 DW

Plectonema boryanum (594) Indiana Culture Collection (Bloomington) 25 DWGomot)

Oscillatoria limnetica Solar Lake isolatec 35 SLWOscillatoria salina Solar Lake isolatec"d 35 SLWMicrocoleus sp. Solar Lake isolate"d 35 SLWAphanothece halophytica (7418) Solar Lake isolatec.e 35 TISPhormidium-like Wadi Natrun salt marsh isolate (Egypt)d 35 SLWOscillatoria-like Bardawil salt marsh isolate (Northern Sinai)d 35 TISPhormidium-like I Baja salt marsh isolate (California)df 25 TISPhormidium-like II Baja salt marsh isolate (California)d"f 35 TIS

a DW, Distilled water; SLW, Solar Lake water with a salinity of 7.3% (determined by the method ofStrickland and Parsons [30]); TIS, Turks Island Salt solution (17), prepared in double strength.

b For details, see references 14 and 27.c Isolated by Y. Cohen, Microbiological Chemistry, Hadassah Medical School, Jerusalem, Israel; identi-

fied by I. Dor.d Not axenic.e Purified and numbered by R. Y. Stanier.f Isolated from mud samples obtained from J. William Schopf, Department of Geology, University of

California, Los Angeles.

0.33; NH4Cl, 0.33; MgCl2 6H20, 0.33; KCl, 0.33; vita-min B12, 10-5; Na2CO3, 1.5; as well as SL-4 trace ele-ments (21). The sulfide concentration of this mediumwas varied in the different experiments, as noted.The final pH was adjusted to 6.8 with HCI. The cellsuspensions were preincubated in the presence of 1.0mM Na2S in completely filled 150-ml glass-stopperedbottles for 18 h under the growth conditions de-scribed above. The cells were then resuspended (20to 40 ,ug of cell protein per ml) in 5 ml of freshexperimental medium containing 5 IAM DCMU andthe sulfide concentrations indicated in Table 2 incompletely filled, stoppered vials and then furtherpreincubated for 2 h in light provided by 60-W tung-sten lamps (incident light intensity of 2.104 ergs/cm2per s). Subsequently, 4 ml of the suspension wastransferred to stoppered cuvettes (4 ml) togetherwith NaH14CO3 (Amersham, England), yielding afinal specific activity of 1 ,uCi/,4mol. The cell sus-pensions were incubated (temperature indicated inTable 1) with stirring for 6 min under actinic lightprovided by a Prado Universal-Leitz projector, fil-

tered through a Corning HR 2-60 filter and a BairdAtomic sharp cut-off interference filter blocked toinfinity, peaking at 703 nm (17-nm half-bandwidth). The incident intensity of the actinic light,varied with the aid of a powerstat, was measured bya Yellow Springs Instrument radiometer, model 65.Cell suspensions were filtered through glass filterpaper (Whatman GF/C), and the collected cells werewashed with 40 ml of the respective (Table 1) cold(4°C) suspension water. Three drops of acetic acid(10%, vol/vol) was spread on the filters; cell radioac-tivity was counted with the aid ofa gas-flow counter(Nuclear-Chicago, model C-11OB) after the sampleshad been dried overnight at room temperature. Forprotein determination (16) filters were washed with5 ml of absolute ethanol to remove elemental sulfur.Na2S concentrations were determined by the meth-ylene blue photometric method (2). Experimentalsystems in the absence of Na2S were the same, butthe preincubations were carried out in partiallyfilled flasks, stoppered with cotton plugs.

Rates of CO2 photoassimilation ofA. halophytica

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ANOXYGENIC PHOTOSYNTHESIS AMONG CYANOBACTERIA 625

and Lyngbya (7104) were determined in the respec-tive experimental systems containing varying con-

centrations of Na2S by exposure to 703-nm light forvarious intervals. For comparison of the rate of CO2photoassimilation byA. halophytica at 580 rm (pref-erentially exciting photosystem HI) with that at 703nm (preferentially exciting photosystem I) underlimiting light intensity, the 580-nm light was ob-tained by filtering the actinic light through 1.5 cm ofa saturated solution of CuS04 and a Baird Atomicsharp cut-off interference filter, blocked to infinity,peaking at 580 nm (14.5-nm half-band width).

For simultaneous determination of sulfide uptakefrom the medium and CO2 photoassimilation by A.halophytica, its respective experimental system wasused containing 5 ,uM DCMU and 40 yg of cellprotein per ml. The reaction mixture in sealed flaskswas flushed with pure nitrogen for 20 min, afterwhich sulfide (1.25 mM) and NaH'4C03 were added.After different intervals of incubation at 3500 inlight provided by tungsten lamps (40 W; incidentlight intensity, 2.104 ergs/cm2 per s), sulfide concen-tration and photoassimilated CO2 were determined.

RESULTS

Table 2 summarizes the results of screening21 different strains of cyanobacteria for theability to use sulfide as electron donor for thephotoassimilation of CO2 driven by photosys-tem I. Data on the ability of several strains tocarry out facultative photoheterotrophy(growth in light on glucose [1%] in the presenceof DCMU [10 ,uM]), taken from Kenyon et al.(14), Rippka (22), and our preliminary results,are also shown.

All the strains carried out oxygenic photo-synthesis. They grew aerobically in light in amineral medium, and their C02 photoassimila-tion reaction, under these conditions, in thepresence ofDCMU or 703-nm light was negligi-ble. Eleven strains (including 0. limnetica)photoassimilated C02 in the presence of Na2Swhen only photosystem I functioned, i.e., in thepresence of DCMU and 703-nm light. Photoas-

TABLz 2. Results ofa screening test forphotosystem I-driven anoxygenicphotoassimilation ofCO2 with Na,Sas electron donor"

Maximal mea- Sulfide concn (mM) at which CO2sured rates of photoassimilation was: Aityof facul-

Strain Cmilationp not tative photo-of COJmg of Detected Maximal Tested heterotrophy'protein per h)

Oscillatoria limneticac 1,600 1-8.5 3.5Aphanothece halophytica (7418)Y 686 0.25-3.5 0.68Oscillatoria (6407) 12 0.77 0.77 +Lyngbya (7004) 3,750 0.05-4.1 0.28 +Lyngbya (7104) 751 0.08-0.4 0.12 +Microcoleus Sp.d 500 0.45-0.90 0.90Oscillatoria salinac.d 2,270 0.45-0.90 0.45Phormidium-like (Wadi Natrun)d 540 0.50-0.80 0.50Phormidium-like (Baja) Ic.d 158 0.35-1.7 0.35Phormidium-like (Baja) lId 1,040 0.68 0.68Oscillatoria-like (Bardawil)d 146 0.83 0.83

Oscillatoria (6412) 0 0.7-2.0 +Oscillatoria (6506) 0 0.67-2.0 +Oscillatoria (6602) 0 1.0-2.0 +Chlorogloeopsis (6712) 0 1.1-2.0 +Pseudoanabaena (7402) 0 1.3Pseudoanabaena (7403) 0 1.4-2.5Aphanocapsa (6714) 0 1.2-2.0 +Nostoc (73102) 0 1.5-2.7Synechococcus (6311) 0 0.55Plectonema boryanum (594) 0 0.66 +

a Photoassimilation of CO2 at 703-nm light with H2S as electron donor, determined as described inMaterials and Methods. The incident light intensity was 0.9 ,tE/cm2 per min.

b Facultative photoheterotrophy: growth in light on glucose (1%) in the presence ofDCMU (10 ,uM). Datataken from references 14 and 21 and from our preliminary results. +, Growth; -, no detectable growth.

c Extracellular granules resembling elemental sulfur granules were observed microscopically.d Experiments with nonaxenic cultures were carried out after having previously washed the cells with the

respective reaction media on Millipore filter membranes (8 ,um) until no bacteria could be detected bymicroscopic examination.

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626 GARLICK, OREN, AND PADAN

similation in the dark, both in the presence andabsence of Na2S, was negligible. It can there-fore be concluded that 11 of the 21 strains exam-ined are facultative anoxygenic phototrophs ca-pable of using Na2S as an electron donor forphotosystem I-driven photoassimilation of CO2.

It is also evident from Table 2 that both themaximal observed rates of the anoxygenic CO2photoassimilation and the respective sulfideconcentrations differed markedly among thestrains when measured under identical inci-dent light. These differences can be attributedto true variations in the cyanobacterial strains.Yet, nonoptimal experimental conditions suchas limiting light intensity, temperature, ornonlinear kinetics cannot be excluded for moststrains. As we observed that Lyngbya (7104)and A. halophytica are similar to 0. limnetica(7) in saturation intensities (0.9 uE/cm2 permin) of703-nm light and in their linear kineticsof CO2 photoassimilation, we compared the de-pendence of the CO2 photoassimilation rate onsulfide concentrations in the three strains (Fig.1). Each of the three strains has a differentoptimal concentration of Na2S for CO2 photoas-similation above which the rate decreases:Lyngbya (7104), 0.1 mM; A. halophytica, 0.7mM; and 0. limnetica, 3.5 mM.The unicellular strain A. halophytica was

further investigated for comparison with thefilamentous 0. limnetica (7, 8). Photoassimila-tion of CO2 was determined in A. halophyticacells at two wavelengths: 580-nm light excitingmostly phycocyanin, i.e., both photosystems;

1500

2,Cx

N 1000

0C

500

0

02a 01 2

o Sulfide co

703-nm light preferentially exciting photosys-tem I-localized chlorophyll. This experimentwas run at low cell densities (10 to 20 jig of cellprotein per ml) under limiting light intensity,so that the rate of CO2 photoassimilation wouldbe linearly related to the actinic light intensity.This permits a comparison of the rates of thephotoassimilation reactions at identical wave-lengths (Table 3). To compare the rates of CO2photoassimilation at wavelengths of 703 and580 nm, we determined the ratio of the ab-sorbed intensities of these wavelengths by thecells in an integrating sphere set-up (con-structed by S. Malkin, Department of Biochem-istry, Weizmann Institute of Science, Rehovot,Israel). The latter eliminates artefacts due tolight scattering. It was revealed that absorp-tion at 580 nm is eight times higher than at 703nm. This factor was then used for a comparisonof photoassimilation rates at the same absorbedquanta. Thus, rates of photoassimilation at 580nm corresponding to incident actinic intensityof 0.08 ,uE/cm2 per min were compared to ratesof incident actinic intensity of 8 x 0.08 AE/cm2per min at 703 nm (Table 3). Although thiscalculation does not show the absolute ab-sorbed intensity, it allows a comparison of therates at the two wavelengths.Table 3 shows that with water as electron

donor, the photoassimilation rate at 703-nmlight was very low in comparison to that at 580-nm light: 0.09. These results agree with the"red drop" phenomenon in the efficiency ofaerobic photosynthesis observed in cyanobac-

ncentrotion (mM)

FIG. 1. Photoassimilation rate of Lyngbya (7104) and A. halophytica (7418) as a function of the Na2Sconcentration. The rate of C02 photoassimilation at saturating intensity (0.9 pBE/cm2 per min) of 703-nmlight, in the presence of DCMU (5 ,uM), was measured at various NsyS concentrations as described inMaterials and Methods. Initial concentrations of Na2S indicated in the figure were determined by themethylene blue photometric method (2). Symbols: (a) Lyngbya (7104); (0) Aphanothece halophytica (7418);(0) 0. limnetica; values were taken from reference 7.

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TABLE 3. Comparison of the rates of CO2photoassimilation by A. halophytica supported by580- or 703-nm light and Na2S or water as electron

donora

C02 photoas-similated (nmol Propor-

Wavelength Io (0EI of COJImg of tion ofWavelngth cm2per protein per h) in rates(nm) min) the presence of (Na2S/electron donor water)

Water Na2S580 0.08 128 250 1.95703 0.64 12 259 21.58Proportion of rates 0.094 1.04

703/580a The experimental system was as described in Materials

and Methods. To compare the rates of CO2 photoassimila-tion at the different wavelengths, the experiments were runat a very low cell density (15 jAg of cell protein per h) withstirring and under limiting light intensity so that "self-shading" was avoided and the rate ofCO, photoassimilationwas linearly related to the actinic light intensity. The ratioof the absorbed intensity of 580- and 703-nm light by thecells in the respective reaction mixtures was determined inan integrating sphere set-up. The absorption at 580 nm iseight times higher than that at 703 nm. Thus, incident lightintensity (Io) of 0.08 AtE/cm' per min at 580 nm is equivalentto Io of 0.64 AE/cm2 per min at 703-nm light with respect tothe absorbed intensity. Where indicated, Na2S concentra-tion was 0.7 mM.

terial whole cells and thoroughly studied ingreen algae and chloroplasts (11). However,

i when Na2S served as electron donor for thephotoassimilation reaction, the rates at 703 nmwere drastically increased. In the presence ofNa2S, the rates at 580 and 703 nm were identi-cal and two times higher than those of theoxygenic photosynthesis (580 nm, water), indi-cating high efficiency of anoxygenic photosyn-thesis.Table 4 shows the relation between CO2 pho-

toassimilation and sulfide depletion in a systemcontaining DCMU (5 ,uM). As CO2 is photoas-similated, the sulfide concentration is reduced.The ratio between the two rates is 1.7-2.5. Sinceno other electron donor is available in the reac-tion system, the ratio of rates of C02 photoas-similation to H2S uptake suggests the followingequation: C02 + 2H2S -_ (HCHO) + 2S° +H20. Free elemental sulfur was observed in thereaction medium in the form of typical refrac-tile granules.

DISCUSSIONEleven of the 21 strains of cyanobacteria ex-

amined are capable of facultative anoxygenicphotosynthesis. This is shown by their abilityto photoassimilate C02 in the presence of Na2Sunder conditions in which photosystem II is notfunctioning and there is no electron flow from

water, i.e., in the presence of DCMU and 703-rum light. The facultative anoxygenic photosyn-thetic strains include axenic representatives ofdifferent typological groups of cyanobacteria,previously defined (14, 22, 27) on the basis ofmorphological, physiological, and biochemicalproperties: Oscillatoria type and Lyngbya typeof the filamentous and Aphanothece type of theunicellular cyanobacteria. Six nonaxenicstrains, washed free of bacteria, which includedone strain of Oscillatoria, one strain of Micro-coleus, and four as yet unidentified strains,showed anoxygenic photosynthesis. We thus in-fer that these strains are also facultative anox-ygenic phototrophs. The property ofanoxygenicphotosynthesis, therefore, is not confined to asingle cyanobacterial type. Moreover, it is notlimited to strains of specific growth history,ecosystems, and geographic location. Eightstrains were isolated from marine environ-ments (salt marshs and Solar Lake) rich in H2Sin the case of three strains (0. limnetica, 0.

salina, and Microcoleus sp.; Y. Cohen, Ph.D.thesis, 1975). The remainder of the positivestrains (Oscillatoria 6407 and Lyngbya 7004and 7104) are freshwater strains grown in cul-ture collections under aerobic conditions forseveral years and for which no indication oforigin in anaerobic environments has been re-ported (14).The observed differences in the maximal

rates of anoxygenic photoassimilation (Fig. 1)may be due to actual differences in the cyano-bacterial strains. However, it should be notedthat the pattern of sulfide concentration de-pendence of anoxygenic photoassimilation re-

veals inhibition by sulfide. Thus, the maximalrates observed are not the true rates. The toxic-ity of sulfide has been demonstrated in botheucaryotic (15) and procaryotic photosyntheticcells and is well known as a determinativefactor in the ecology of sulfur bacteria (3). Theobserved variability in the range of sulfide con-

centrations permitting anoxygenic photosyn-

TABLE 4. Relation between CO2 photoassimilationand sulfide consumption by A. halophyticaa

Length of CO2 photoas- Na2S consumedincubation similated (Ao fN2/N2/O

(h) (&mo°l of C02/ (mo of Na2S/Na)S/C02mg of protein) m fpoen

2 1.8 4.5 2.55 5.3 9.0 1.78 7.3 12.4 1.7

a Photoassimilation of CO2 was determined withNaH14CO3 and sulfide consumption was determinedby the photometric method, as described in Materi-als and Methods.

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628 GARLICK, OREN, AND PADAN

thesis in the various cyanobacterial strains istherefore attributed to differences in the appar-ent sulfide affinities of the sulfide-utilizing sys-tems as well as in the sensitivities to extracel-lular sulfide concentrations. This variabilitywould lead to different distribution patterns ofthe various cyanobacterial strains in the sulfideconcentration gradients prevailing in their an-aerobic habitats (Y. Cohen, Ph.D. thesis, 1975).In view of this variability the testing of newstrains for anoxygenic photosynthesis should becarried out in the presence of a wide range ofsulfide concentrations.As anoxygenic photosynthesis seems to be a

common feature among the cyanobacteria, itmay prove to be a useful taxonomic tool. Inview ofthe ease with which this property can bedemonstrated by the technique here described,it is now possible to routinely test for this fea-ture in the characterization of axenic strains ofcyanobacteria.

Similar to anoxygenic photosynthesis, photo-heterotrophic cyanobacterial growth (in thepresence of 1% glucose and 10 ,M DCMU) issupported by photosystem I independently ofphotosystem II. Table 2 shows that there is nodirect correlation between the ability of faculta-tive photoheterotrophic growth and facultativeanoxygenic photosynthesis.The pattern of H2S utilization as electron

donor in 0. limnetica (7, 8) and A. halophyticais thermodynamically inefficient in comparisonto its utilization by many photosynthetic bacte-ria. Whereas these two cyanobacterial strainsoxidize sulfide to elemental sulfur only, sulfurphotosynthetic bacteria can oxidize it to sul-fate. Oxidation of sulfide to elemental sulfurhas been suggested as a side chain of the mainpathway of sulfide oxidation in purple bacteria(H. G. Tuper, Abstr. Symposium on Prokar-yotic Photosynthetic Organisms, Freiburg,1973, p. 160-166). This inefficiency of cyanobac-teria may have ecological implications in anecosystem containing sulfide concentrationssuitable for both cyanobacteria and photosyn-thetic bacteria.The significance of anoxygenic photosyn-

thesis in the ecology ofcyanobacteria gains sup-port from accumulating reports on the world-wide and vast dimensions of H2S-rich biotypescontaining an abundance of cyanobacteria. Insalt marsh regions, cyanobacteria are ubiqui-tous and withstand extremes of desiccation, sa-linity, and low redox values (10). Recently,Fenchel and Riedl described (9) a complex an-aerobic, H2S-rich ecosystem ("thiobios") whichunderlies the oxidized surface layer of all ma-rine sandy bottoms and is rich in proliferatingcyanobacteria. This "thiobios" is suggested to

play a major role in the energy cycles of themarine ecosystems and to represent a possiblefirst stage in petroleum production (9). Amongfreshwater ecosystems, hot springs containingsulfide are densely inhabited by cyanobacteria,which had been suggested to utilize H2S inanoxygenic photosynthesis (6). The correlationbetween reducing conditions and developmentof cyanobacteria in benthic mats of freshwaterbodies and terrestrial habitats has been empha-sized (10).The capability of cyanobacterial cells to func-

tion under anaerobic or microaerophilic condi-tions is not necessarily related to the propertyof H2S utilization. Furthermore, microaero-philic conditions seem to favor several cyano-bacterial metabolic functions: photosynthesisin Phormidium species (31) and nitrogen fixa-tion in vegetative cells (28). Stimulation ofgrowth yield and rate have been observed incyanobacteria under microaerophilic conditions(1, 10, 12, 27, 29).

It is suggested that the ability of anoxygenicphotosynthesis in the presence of H2S and thepreference for microaerophilic conditions ofmany cyanobacterial cells are both metabolicrelicts of their evolutionary history. The twoproperties are common to different cyanobac-terial groups and not related to specific cellgrowth histories. In this respect, it is interest-ing to note that two of the H2S-rich ecosystemsdensely inhabited by cyanobacteria, the marine"thiobios" (9) and the sulfide hot springs (6),may represent old ecosystems, perhaps preced-ing the oxidized biosphere in age.

ACKNOWLEDGMENTSWe thank M. Shilo, Hebrew University, for extensive

and stimulating discussions, N. G. Carr, University of Liv-erpool, for critically reading the manuscript, S. Malkin,Weizmann Institute of Science, for the use of the integrat-ing sphere constructed by him, and Alexandra Mahler forhelp in the preparation of the manuscript.

This study was supported by a grant from the DeutscheForschungsgemeinschaft.

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