Plant Physiol. (1982) 69, 1268-12730032-0889/82/69/ 1268/06/$00.50/0

H2 and CO, Evolution by Anaerobically Adapted Chiamydomonasreinhardtii F-601

Received for publication September 15, 1981 and in revised form December 17, 1981

ELCHANAN S. BAMBERGER2, DAN KING3, DAVID L. ERBES4, AND MARTIN GIBBSInstitutefor Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT

Using manometric and enzymic techniques, H2 and CO2 evolution indarkness and light has been studied in the green alga Chianydomonasreinhardtii F-60. F-60 is a mutant strain characterized by an incompletephotosynthetic carbon reduction cycle but an intact electron transportchain.

In the dark, starch was broken down, and H2 and CO2 was released.The uncoupler, carbonyl cyanide m-fluorophenylhydrazone with an opti-mum concentration of 5 to 10 micromolar, increased the rate of CO2release and starch breakdown but depressed H2 formation. It was suggestedthat carbonyl cyanide m-fluorophenylhydrazone increased the rate ofstarchbreakdown by making the chloroplast membrane permeable to H+, remov-ing a rate-limiting step, and leading to an altered fermentative pattern.

Photoevolution of H2 and CO2, but not starch breakdown, was stimu-lated by acetate. Maximum stimulation occurred at concentrations from 1to 10 millimolar. Carbonyl cyanide m-fluorophenylhydrazone stimulatedstarch breakdown and CO2 and H2 release in the light, but not to theextent of acetate. Inasmuch as the uptake and subsequent metabolism ofacetate required ATP, it was suggested that acetate, like carbonyl cyanidem-fluorophenylhydrazone, stimulated H2 photoproduction by removingATP which limited the sequence of reactions. The contribution of photo-system II to the photoproduction of H2, as judged from the effect of 10micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, was at least 80%.CO2 photoevolution increased Unearly with time, but H2 photoevolution

occurred in two phases: a rapid initial phase folowed by a second slowerphase. The rate of H2 release increased hyperbolically with light intensity,but the rate of CO2 production tended to level off and decrease withincreasing light intensity, up to 145 watts per square meter. It was proposedthat a changing CO2 and H2 ratio is the result of interaction between thecarbon and hydrogen metabolism and the photosynthetic electron transportchain.

Studies with chlorophyllous algae have established that H2evolution in the light is the result of electron transport throughthe photosystems associated with an adaptable hydrogenase. Thesealgae also produce H2 in the dark but at a lower rate. Twomechanisms have been proposed to account for H2 release in thelight, while the pathway for the dark release of H2 has received

'Supported by Department of Energy (10-EY-76-5-02-3231) and Na-tional Science Foundation (PCM 79-22612).

2 Present address: School of Education, University of Haifa, Oranim,Israel.

3 Present address: Biology Department, Lycoming College, Williams-port, PA 17701.

4 Biochemicals Department, E. I. DuPont de Nemours and Company,Wilmington, DE 19898.

scant attention since Gaffron and Rubin (5) first described hydro-gen metabolism in algae in 1942. Of the two mechanisms describ-ing H2 photoproduction, one (4, 17, 19) involves H20 photolysiscoupled to electron transport through PSII and PSI. This meta-bolic route is characterized by the simultaneous formation of H2and 02, with a molar ratio of approximately 2, and inhibition byDCMU. The alternate pathway (5, 6, 10, 11) involves the couplingof oxidative carbon metabolism to PSI only and is characterizedby the release ofH2 and CO2 and insensitivity to DCMU. Whetherboth mechanisms function simultaneously in the adapted, illumi-nated alga remains unresolved.Although the DCMU-sensitive pathway is well documented,

much less is known about the reactions of the oxidation of organiccompounds resulting in the generation of CO2 and the reductantfor the evolution of H2 in darkness and in light (3, 14). It is knownthat gas evolution is stimulated by added glucose (5). Data ob-tained with position-labeled glucose led Kaltwasser et al. (13) topropose that classical glycolysis in Scenedesmus obliquus is thepathway involved. From a stoichiometric analysis of the prod-ucts-which included ethanol, glycerol, and acetate, in additionto CO2 and H2-Klein and Betz (15) also proposed glycolysis forthe heterofermentative breakdown ofthe reserve substance, starch,in Chlamydomonas reinhardii. This suggestion was supported bya similar fermentation pattern in Chlorella vulgaris (22). On thebasis of a depression of H2 evolution in the dark by uncouplers ofphosphorylation, Gaffron and Rubin (5) suggested that the fer-mentation yielded not only CO2 but also ATP. ATP would berequired to raise the redox potential of the electrons from thereductant (NADH) to a higher one necessary for H2 production.In contrast to inhibiting H2 evolution in the dark, the uncouplerelevated the photorelease of H2 and CO2. Acetate has been re-ported to stimulate H2 evolution insensitive to DCMU, and Healy(10) suggested an anaerobically functioning citric acid cycle inChlamydomonas moewusii to explain this observation. In his for-mulation, NADH generated in the citric acid cycle would feedelectrons into PSI beyond the DCMU block, a reaction reportedin isolated chloroplast preparations of Chlamydomonas reinhardii(2).There have been attempts to determine the ratio in which H2

and CO2 were produced. While H2 evolution was stimulated bylight, CO2 production was found to be unchanged (15) or slightlystimulated (13). It was recognized that, in illuminated cells poi-soned with DCMU, a reutilization of gases could occur via pho-tosynthesis or by photoreduction, a serious drawback in determin-ing valid ratios (13, 21).

In the present study, we describe the relationship betweencarbon and hydrogen metabolism in anaerobically adapted Chla-mydomonas reinhardtii F-60. This mutant strain is characterizedby an incomplete photosynthetic carbon reduction pathway, butit does contain an intact photosynthetic electron transport chain(16). Whereas the adapted cells are able to carry out H2 and CO2production in darkness and light, they are incapable of photosyn-

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1269H2 AND CO2 EVOLUTION BY CHLAMYDOMONAS REINHARDTII

Table I. Effect of Growth Conditions, Starvation, and Acetate on Hydrogen PhotoevolutionCells were adapted under N2 for I h. The Chl concentration was 200 to 300 yg in a final volume of 3 ml

containing 10 mm K-phosphate (pH 7.4), 20 mm KCI, and 2.5 mM MgCI2. Algae were starved by suspendingfreshly harvested cells in a medium of 10 mm K-phosphate (pH 7.4), 20 mm KCI, and 2.5 mm MgCl2 and bubblingfiltered air through the darkened suspension. Light intensity was 100 w/m2.

Growth Hydrogen PhotoevolutionOrganism Conditions Control +Acetate (1 mM)

lumollmg Chl M-h

Chlamydomonas reinhardtii, wt Minimal 6 12TAP 15 15TAP, starved 1 5

Chlamydomonas reinhardtii F-60 TAP 18 35TAP, starved 3 8

thesis and photoreduction (reduction of CO2 by H2). We haveutilized the mutant to sort out the effects of uncouplers on gas

release in darkness and in light and have examined their effect onstarch degradation, the proposed substrate of CO2. Similar toChlamydomonas moewusii, acetate was found to stimulate, in thelight, H2 as well as CO2 evolution in F-60, and data are presentedto account for this finding.

Finally, we have taken advantage of the mutant's incompletephotosynthetic carbon reduction cycle to analyze the ratio inwhich H2 and CO2 are released in cells illuminated at high lightintensity. A preliminary accounting of these results has beenpublished (7).

MATERIALS AND METHODS

Cell Culture. Chlamydomonas reinhardtii Dangeard wt5 and F-60 mutant strain (obtained from Dr. R. K. Togasaki) were grown

photoheterotrophically at 25°C on a vigorously aerated, acetate-supplemented medium (TAP) under fluorescent light (9). Cellswere harvested in the logarithmic phase of growth by centrifuga-tion (about 100g for 5 min), washed twice, and resuspended in 10mm K-phosphate (pH 6.5), 20 mm KCI, and 2.5 mM MgC12.H2 and CO2 Production. In gas exchange assays, algal cells

containing 100 to 300 Mig Chl in 3 ml of a solution containing 10mm K-phosphate (pH 6.5), 20 mm KCI, and 2.5 mM MgCl2 were

placed into the main compartment of 15-ml Warburg flasks in a

Gilson differential respirometer bath maintained at 28°C. Toinitiate H2 metabolism, the cells were incubated for I to 3 h in thedark under an atmosphere of N2 (less than 5 Ml1/l 02). For gasevolution, a light intensity of 100 w/m2 was generally used. Lightwas provided by General Electric 75-w reflector flood bulbs in theGilson respirometer.When the simultaneous formation of H2 and CO2 was measured,

one set of flasks was prepared containing 0.2 ml KOH-pyrogallolor KOH in the center well to trap CO2. Another set of flaskscontained no trapping agent. The difference in micrometer read-ings between the two flasks was taken to represent CO2 evolution.An occasional flask contained glucose-glucose oxidase as an O2trap; no difference in gas evolution was observed by its absence.Gas evolution data are expressed as the total gas evolved/mg

Chl.4 h, since the rate of gas evolution in most experiments was

not linear with time, and expression as initial rates may beerroneous (see also Ref. 20 for a kinetic description of H2 evolu-tion). H2 uptake was less than 5% of H2 evolution.

In some experiments, after a period of gas exchange, KOH or

palladium-methylene blue was injected into side arms (through

r)Abbreviations: wt, wild type; FCCP, carbonyl cyanide m-fluorophenylhydrazone; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DHAP,dhydroxyacetone-P.

rubber septum stoppers sealing the side arm ports) to consumeCO2 and H2, respectively. These agents also served as means ofconfirming the likely nature of the evolved gases. When used,DCMU and FCCP were added in a final concentration of 1%ethanol. This concentration of ethanol did not affect the rates ofgas evolution.

Assays. Starch was determined as glucose after enzymic hy-drolysis, according to Klein and Betz (15). Two ml of the cellsuspension were centrifuged, and the precipitate was extractedtwice with 5 ml acetone to remove Chl. The residue was washedonce with 3 ml 20 mm K-phosphate (pH 6.9) and suspended in 2ml 100 mm Na-acetate (pH 4.5). The suspension was sonicated ina Branson sonifer 200 for 15 s at full power and subsequentlyheated in a boiling water bath for 5 min to solubilize the starch.Enzymic hydrolysis of starch was initiated by adding to the sample1.1 units of amyloglucosidase (gluco-amylase, 1,4-glucan gluco-hydrolase, EC 3.2.1.3), purchased from Sigma. The samples wereincubated in a shaking water bath at 55°C for 2 h, and thenglucose was determined in the supernatant fraction by an enzymic-colorimetric determination, using glucose oxidase and peroxidase(Sigma Kit No. 510).

Acetate, in the supernatant of cell suspensions, was assayedenzymically using a Test combination kit (Boehringer) containingmalate dehydrogenase, acetyl-CoA synthase, citrate synthase,ATP, CoA, and NAD. Chl was determined by the method ofArnon (1).

RESULTS

Effect of Growth Conditions, Starvation, and Acetate on H2Photoevolution. There is evidence of a stimulation of H2 photo-evolution by 1 mm acetate (10) and by the composition (hetero-trophic or autotrophic) of the growth medium (20) in strains ofChlamydomonas. In preliminary experiments, we compared theseeffects in wt and in the mutant. Photoheterotrophically growncultures of the wt yielded H2 at a higher rate than did those grownon minimal media (Table I). The presence of 1 mm acetatestimulated H2-photoevolution, except in the unstarved, TAP-grown Chlamydomonas reinhardtii wt. Similar results (data notshown) were found with S. obliquus.Even though the acetate stimulated the rates higher in the

starved cells, we preferred freshly harvested F-60, mainly becauseit exhibited not only higher, but also more consistent, time-courserates of gas evolution.H2 and CO2 Evolution, Starch, and FCCP. The dark rate of H2

and CO2 evolution in our cells was generally about I to 8 ,umol/mg Chl -4 h, and these values were 3 to 5 times higher in the light(Table II). Apparently, more starch was consumed in the darkthan in the light. FCCP at 5 ,UM greatly stimulated photoevolutionof both gases, but, in the dark, H2 release was depressed, and CO2

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BAMBERGER ET AL. Plant Physiol. Vol. 69, 1982

Table II. Effect ofDCMU, FCCP, and Acetate on Light and Dark Starch Degradation and H2 and CO2Evolution in Anaerobically Adapted Chlamydomonas reinhardtii F-60

Cells were harvested at log phase, washed twice, and resuspended in 3 ml 10 mm K-phosphate (pH 7.4)containing 20 mM KCI and 2.5 mM MgCl2 in two sets of 15-ml Warburg flasks, one with 20%3o KOH in the centerwell and the other without KOH. The Chl concentration was 72 Ag/ml. For dark conditions, the flasks werewrapped with a double layer of aluminum foil. The light intensity was 120 w/m2. Acetate, FCCP, and DCMUwere added from the sidearm after the adaption period, 10 min before the light was turned on. After 4 h, the cellsuspensions were centrifuged, and acetate was assayed in the supernatant fluids; starch, as glucose, was determinedin the cells. The amount of starch degraded is the difference between starch determined in unadapted cells andthat determined in the adapted cells after 4 h in the dark or light.

Dark Light

H2 CO2 Glucose re- H2 CO2 Glucose re- Acetate inAdditions leased from leased from Medium

starch starchZero Aftertime 4 h

Amol/mg Chl .4 h ttmol

Control 8 0.4 8 27 4 6 0 0FCCP, 5,UM 3 13 12 44 16 15 0 0.5DCMU, 10AM 7 7 4 5 8 0 0Acetate, 3 mM 62 35 6 9.8 0.3Acetate, 3 mm plus FCCP, 5

JiM 43 24 15 9.8 13.2Acetate, 3 mm plus DCMU, 10

JiM 7 8 8 9.8 6.8

30

_ n

mn

0

m

0

C)

3_. , to

.n10 -3 _

J~o-6 10-5

FCCP (M) ACETATE (M)

FIG. 1. Effect of FCCP concentration of starch degradation and pho-toevolution of H2 and CO2 in anaerobically adapted Chlamydomonasreinhardtii F-60. FCCP was added from the side arm of the Warburg flask10 min before the lights were turned on. Chl concentration was 86.4 ,ug/ml. *, CO2 evolution; 0, H2 evolution; A, starch degradation.

was increased. FCCP caused an increase in polysaccharide deg-radation, and this effect was more striking in the light. Theoptimum concentration of uncoupler was 5 to 10 ,LM, but, beyond10 /M, FCCP inhibited gas production and starch dissimilation(Fig. 1).H2 and CO2 Evolution and Acetate. In the dark, acetate at 3

mM had no effect on gas production (Table II), and acetate uptakewas not detected (data not shown). On the contrary, there was

usually an increase of 1 to 3 ,umol acetate per 4 h in the reactionmedium. However, in the illuminated algae, all of the availableacetate in the medium was consumed, and H2 and CO2 releasewas increased 2- and 9-fold, respectively. On addition of 5 JiMFCCP to the reaction mixture containing 3 mm acetate, an increasein the level of acetate was found in the medium concomitant withgas release similar to that found with the uncoupler alone. As

FIG. 2. Acetate uptake and photoevolution ofH2 and CO2 as a functionof acetate concentration in anaerobically adapted Chlamydomonas rein-hardtii F-60. Cell suspensions (3 ml) were placed in Warburg flasks, andacetate was placed in the side arms and added to the cells after 2-hadaptation under N2 atmosphere and 10 min before the lights were turnedon. The suspension contained 20 mm K-phosphate (pH 6.5), 20 mM KCI,and 2.5 mM MgCl2. The Chl concentration was 60 ,ug/ml. At the end of 4h in light, the cell suspensions were centrifuged, and acetate was deter-mined in the supernatant fractions. 0, CO2 evolution; U, H2 evolution;A, acetate uptake.

shown in Figure 2, the optimum concentration of acetate was 1 to10 mm, and, at concentrations beyond 10 mm, acetate uptake andH2 and CO2 photoevolution were depressed.

Figure 3 depicts the kinetics of CO2 and H2 photoproductionand acetate uptake. H2 evolution in the control and acetate-treated, illuminated cells showed an initial burst of gas, followed

by a linear rate in the presence of acetate. CO2 photoevolutionwas not characterized by an initial burst, but CO2 release in the

control cells leveled off after 2 h, whereas, in the acetate-treatedcells, CO2 formation continued to increase with time. Acetate

1270

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H2 AND CO2 EVOLUTION BY CHLAMYDOMONAS REINHARDTII

D

i ,-40 m

--D

m

c

"Ir3tn

-20 :T

2.0TIME, (h)

10

FIG. 3. Kinetics of acetate uptake and photoevolution of H2 and CO2in anaerobically adapted Chlamydomonas reinhardtii F-60. Cell suspen-

sions (3 ml) were placed in Warburg flasks, and acetate was placed in theside arms. Adaptation was carried out for 2 h under N2. Acetate was addedto the cells 10 min before the lights were turned on; the final acetateconcentration was 8.6 mn. At each time interval, the contents of two flasks(one with KOH-pyrogallol and one without) were centrifuged, and acetatewas assayed in the supernatant fluid. Chl concentration was 71 ,ug/ml.*, Control H2 evolution; O-, control CO2 evolution; W-U,

+acetate, H2 evolution; E-, +acetate, CO2 evolution; A* A, acetateuptake.

taken up from the medium was relatively low during the first hbut gradually increased between the second and third h. In thefourth h, acetate uptake was more than double the uptake in theinitial 3 h.

Effect of DCMU. DCMU at 10 ,AM inhibited 80%o of the H2photoevolution in the control cells with little effect on CO2 pho-toproduction, dark H2 evolution, or starch degradation (Table II).In the presence of 3 mm acetate, DCMU inhibited 90%o of H2 and77% of CO2 photoevolution. Acetate uptake was depressed 66%by 10 iLM DCMU.Gas Release at Several Light Intensities. The effect of light

intensity on H2 and CO2 formation in the presence of 5 Am FCCPor 8.6 mm acetate was compared to control cells, and the resultsare presented in Figure 4. In all three conditions, the rate of H2release calculated for 4 h after exposure to light increased approx-imately linearly with increasing light intensity. In the presence ofacetate, H2 production was already light-saturated at 70 w/m2,while release of H2 in the control cells or in the presence ofuncoupler was evidently still limited by the 'light' reaction even at145 w/m2. The rate of CO2 release tended to reach saturation withrespect to light intensity, at relatively low intensities, but declinedat higher light intensities, and this pattern was observed under allthree conditions.

DISCUSSION

With respect to rates of endogenous and acetate-supportedrespiration, respiratory quotients endogenous and in the presence

of acetate, and H2 photoevolution response to DCMU, the F-60mutant reacted similarly to wt (data not shown), indicating that

14533 1>) j(f) 1,'CLIGHT INTI N¾'ITY (w lw")

FIG. 4. Photoevolution of H2 and CO2 as a function of light intensityin anaerobically adapted Chlamydomonas reinhardtii F-60. Gas exchangeswere measured in 15-ml Warburg flasks, as described in "Materials andMethods." Different light intensities were obtained by wrapping the flaskswith varying numbers of aluminum screens: 145 w/M2, 2 screens; 33 w/m2, 4 screens, 15 w/m2, 6 screens. For the dark samples, the flasks werewrapped with two layers of aluminum foil. A, H2 (0) and CO2 (A)evolution in the control cells; Chl concentration was 107 ag/flask. B, Inthe presence of 5 lsm FCCP; in this experiment, control H2 and CO2 rateswere 47 and 7 pmol/mg Chl.4 h, respectively, at 145 w/m2. Chl concen-

tration was 150 pug/flask. C, In the presence of 8.6 mm acetate; in thisexperiment, control H2 and CO2 rates were 46 and 12 pmol/mg Chl.4 hat 145 w/m2. Chl concentration was 140 pg/flask. Acetate and FCCP were

added from the sidearm 10 min before the lights were turned on.

the mutant was not deficient in these reactions. Our observationson H2 and CO2 release and polysaccharide breakdown in the darkand light with uncouplers and acetate confirm and extend previousobservations on this and other strains of algae. However, ourobservations contrast with some previous reports on the effect ofDCMU and light intensity on these processes.To account for the in-depth observations of Gaffron and his

students on H2 and CO2 evolution in the dark and in illuminated,adapted algae and on the oxidative carbon metabolism coupled tothese processes, as documented by Frenkel (6), Healey (10, 11),Klein and Betz (15), and in this paper, Figure 5 is a representationof the overall reactions between the chloroplast, the site of poly-saccharide storage and the photosynthetic electron transport chainin the green algae, and the cytosol during the fermentative metab-olism of starch. We have assumed that the cellular location ofhydrogenese is the chloroplast. Relying upon results obtained withhigher plant chloroplasts (12), we have assumed that the principalmeans ofcommunication between chloroplast and cytosol in termsof glycolytic intermediates is the counterexchange of Pi for DHAPand glycerate 3-P catalyzed by the Pi translocator. Furthermore,

1271

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C'

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0

0

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Plant Physiol. Vol. 69, 1982

CHLOROPLAST

G6P- GIP GLUCOSEn+IPGA - - - --- --- ----- -

H2I= --- - F d"Ii- - - - -=X

DCMl

t--

-.A p 4C--I4A)F6P -

CATP)ADP

PS IT I1 tP PSI

H20

A2Z21+I\NADPH FdrY H2>1uiiii NADP XFdr 4 2H+

vFBP- G3P

-DHAP ---

- -PGA-

- pI

- -DHAP

2 PGA4

2 PYRUVATE

2C02 + 2CH3CH20H

FIG. 5. Proposed mechanism for C02 and H2 evolution and polysac-charide degradation in anaerobically adapted F-60. +-I-+ Electron flowduring dark release of H2; ---, photoevolution of H2 when H20 or

NADH is electron donor; - - -, influx of Pi and efflux of DHAP and PGAcatalyzed by the Pi translocator (shaded area in chloroplast envelope);

, oxidative carbon metabolism-linking polysaccharide (glucose) withCO2 in darkness and light. GIP, glucose I-P; G6P, glucose 6-P; F6P,fructose 6-P; FDP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone-P; G3P, glyceraldehyde 3-P; PGA, glycerate 3-P; Q and X, primary

electron acceptors of PSII and PSI, respectively; PQ, plastoquinone; Fd,ferredoxin; -P, energy-conserving site of photophosphorylation.

in this representation, we propose that 2 units of Pi are transportedinto the chloroplast and 2 units of triose (one each of DHAP andglycerate 3-P) are returned to the cytosol, where the glycolyticbreakdown of the glucosyl moiety derived phosphorolytically fromstarch is completed. One triose-P is oxidized to PGA, and ATPproduced by substrate level phosphorylation is consumed in thefructose 6-P kinase reaction, while NADP is regenerated byelectron flow through NADP-ferredoxin reductase coupled tohydrogenase resulting in the release of H2. This sequence isproposed as the principal means of H2 production from carbohy-drate in the dark but may also occur simultaneously with theDCMU-insensitive H2 photoevolution pathway.

H2, CO2 Evolution, and FCCP. In their original paper withanaerobically adapted S. obliquus, Gaffron and Rubin (5) ob-served that H2 evolution in darkness was depressed and H2 pho-toproduction was enhanced by the uncoupler 2,4-dinitrophenol,leading to the proposal that different pathways were involved inH2 release in the dark and light (Fig. 5). Later, Kaltwasser et al.( 13) repeated this observation with a second uncoupler, CCCP. Incontrast to the results with H2, CCCP increased CO2 release bothin the darkened and in the illuminated algae. Finally, theseauthors presented preliminary evidence that polysaccharide was

dissimilated more rapidly in the presence of the uncoupler.The response of these processes to uncoupler in S. obliquus and

in C. moewusii (10) was confirmed with the F-60 mutant. Inasmuchas CO2 fixation was not completely inhibited in S. obliquus and C.moewusii by the uncouplers, some residual photoreduction couldhave partially masked the gas evolution. What seemed to be a

stimulation of the rates of CO2 and H2 photoproduction may havebeen the result of an inhibition of gas uptake via photoreductionand, subsequently, an inhibition of starch synthesis which couldbe interpreted as an increase in the breakdown of the polysaccha-ride. Since F-60 is incapable ofCO2 assimilation, the data recordedin Table II indicate that the stimulation of gas evolution by theuncoupler is the result of a direct effect on the mechanismsyielding CO2 and H2. Clearly, gas release is independent ofphosphorylation, and, to the contrary, the energy-conserving siteof the photosynthetic electron transport chain impedes H2 evolu-tion (10, 21).Whereas polysaccharide is not synthesized from CO2 in the

mutant strain, the increased rate of polysaccharide degradation isthe result of an effect of the uncoupler on the glycolytic pathwayrather than on the phosphorylative step coupled to the fermenta-tion, because substrate level phosphorylation is not sensitive touncouplers. A reasonable explanation for these observations isthat the uncouplers-2,4-dinitrophenol, CCCP, and FCCP-havethe common property of being weak acids which are relativelylipid-soluble and dissolve in the chloroplast membrane, makingthe membrane permeable to H+, apparently lowering the pHwithin the chloroplast, and removing a rate-limiting step. Sup-portive of this speculation is the observation in this laboratory thathigh (100 mM) concentrations of weak acids, such as malic andacetic, can induce a rapid breakdown of polysaccharide in F-60and alter the fermentation pattern (R. Gfeller, unpublished).To account for the inhibitory effect of uncoupler on H2 release

in the dark, the proposal has been made that the formation of H2in darkness is dependent upon NAD(P)H and high-energy phos-phate (5, 10, 21). In our model (Fig. 5), we suggest a sequence ofNADPH-ferredoxin-*H2 when high energy phosphate is not a

requirement for gas release, and it follows that the uncoupleralters the fermentative metabolic pattern and electron flow, re-

sulting in the regeneration ofNADP coupled to the production ofglycerol (15), ethanol, and less H2. Using a reconstituted system,it has been shown in this laboratory that H2 release can be coupledto triose-P oxidation in a spinach chloroplast preparation fortifiedwith NADP, ferredoxin, and hydrogenase (unpublished).The chloroplastic uncoupling concentration of FCCP is I to 10

itM (8), but the phenylhydrazones, in addition to uncoupling, areknown to inhibit electron transport and cause membranal disrup-tion at concentrations beyond 10 tiM (23). It was, therefore, ex-

pected that the high levels of uncoupler caused inhibition of starchdegradation and gas evolution (Fig. 3).

H2, CO2 Evolution, and Acetate. Similar to Healey's (10) ex-

perience with C. moewusii, acetate stimulated the photoevolutionof H2 in unstarved cells of the wt and mutant cells of Chlamydo-monas reinhardtii (Table I; Fig. 3). We also observed a stimulationin starved cells. The degree of stimulation was always higher thanthat found with unstarved cells. This smaller stimulation byexogenous acetate on gas release by the unstarved cells may reflectstorage-acetate within the freshly harvested cells, which couldaccount for the comparable initial rates of H2 release in the controland acetate-treated cells, despite the lag in acetate uptake fromthe medium (Fig. 2, compare * with A). The lower basal rate ofgas release in starved cells may result from the absence of endog-enous acetate or of starch, which is known to yield acetate inChlamydomonas adapted to a H2 metabolism (15).

Acetate consistently stimulated H2 and CO2 photoevolution toa greater extent than FCCP. Presumably, acetate is converted to

acetyl-CoA, a reaction which involves a pyrophosphoryl split ofATP, which is coupled to the acetylation of CoA by free acetate.Therefore, one may consider acetate equivalent to an 'uncoupler'in the sense that it reacts with available ATP and that its uptakedepends on a supply of ATP, since, in the presence of FCCP, alleffects of acetate were neutralized (Table II).

Acetate stimulated not only H2 photoevolution but also the

1272 BAMBERGER ET AL.

Il

-I

i - -Pj

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H2 AND C02 EVOLUTION BY CHLAMYDOMONAS REINHARDTII

release of C02, with a maximum stimulation between I and 10mm (Fig. 2). The gases were produced in a molar ratio of CO2 toH2 at 0.15 in the control cells and 0.56 on addition of 3 mMacetate. Inasmuch as CO2 released in the presence of [1 -4Clacetatewas essentially unlabeled (data not shown), the additional gas wasapparently derived from an endogenous source.The difference in the amount of H2 and CO2 photoproduction

by acetate and FCCP-treated cells remains unexplained. Thereduction of acetyl-CoA to ethanol, an end-product of Chlamy-domonas fermentation, would consume reducing equivalents aswould the conversion of acetyl-CoA to saturated fatty acids.Clearly, the role of acetate and its metabolism in the adapted,illuminated cells is more complex than previously thought (10).

Light Intensity and H2 and CO2 Evolution. Only a few studies(10, 13, 21) have been reported on the effect of light intensity onH2 and CO2 release from anaerobically adapted algae. Essentiallyall of these studies were done with poisoned or partially poisonedcells or at very low light intensities to maintain 02 evolution at arate sufficiently low in order not to inactivate the hydrogenasereaction. Furthermore, these algae retained the capacity to carryout photoreduction consuming CO2 and H2, thus complicatinginterpretation of a stoichiometric analysis. While the evolution ofH2 in light, was about twice as much as that in the dark, Kleinand Betz ( 15) reported no significant change in the release of CO2at a light intensity of 160 lux (- 1.2 w/m2). Taking advantage ofthe inability of F-60 to evolve O2 coupled to CO2 reduction, wewere able to determine the molar ratio of gas release up to 145 w/m (Fig. 4A) and in the presence of 5 AM FCCP (Fig. 4B) and 8.6mm acetate (Fig. 4C).

Clearly, the rate of H2 release responded hyperbolically to lightintensity, but the evolution of CO2 in the light seemed to be morecomplicated. The rate of CO2 evolution approached saturation,with respect to light intensity, at relatively low intensities. Athigher light intensities, CO2 evolution declined. Assuming that thesource ofCO2 is starch and that an anaerobic glycolytic breakdownoperates in darkness or light, then CO2 evolved should correspondclosely with the decarboxylation of pyruvate to yield the twocarbon units, ethanol and acetate. Furthermore, at high lightintensities where CO2 evolution is diminished, acetate and ethanollevels should be depressed, and glycerol and perhaps lactateshould accordingly increase. Clearly, starch breakdown in thesecells is a mixed fermentative process, and, if the early events arelocalized within the chloroplast (Fig. 5), then interaction betweenthe oxidative step of glycolysis (glyceraldehyde-3-P dehydrogen-ase) and the light process is to be expected, resulting in variationsin the ratio of end-products dependent upon light intensity.PSII Contribution. Earlier work with autotrophically grown

Chlamydomonas reinhardtii and C. moewusii indicated that H2photorelease was unimpaired by 10 zlM DCMU (10, 11). Thecontribution of PSII to the photoproduction of H2 by the F-60mutant strain, as judged from the effect of 10 /LM DCMU, was atleast 80% (Table II). This difference between our results and those

in the literature may be due to a number of reasons. The responseto inhibitors may vary with the age and growth conditions of theculture, and Chlamydomonas, in contrast to many other algaetested, appears to be very sensitive to inhibitors (21). Also, severalalgae, including Chlamydomonas, show different enzymic activi-ties when grown on acetate or CO2 (18).

Thus, the variable responses to DCMU may be related tochanges in the metabolic pathways of the cells.

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