The anaerobic starch breakdown into end-products in the green alg Chiamydomonas reinhardtii F-60 has been investigted in the dark and in the light. The effects of 343,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigted.Anaerobic starch breakdown rnte (13.1 ± 3.5 micromoles C per milligram chlorophyll per hour) is increased 2-fold by FCCP in the dark. Light (100 watts per square meter) decreases up to 4-fold the dark rate, an inhibition reversed by FCCP. Stimulation of starch breakdown by the proton ionophore FCCP points to a pH-controlled rate-limiting step in the dark, while inhibition by light, and its reversal by FCCP, indicates a control by energy charge in the light.In the dark, formate, acetate, and ethanol are formed in the ratios of 2.07:1.07:0.91, and account for roughly 100% of the C from the starch.H2 production is OA3 mole per mole glucose in the starch. Glycerol >-lactate, and CO2 have been detected in minor amounts.In the light, with DCMU and FCCP present, acetate is produced in a 1:1 ratio to formate, and H2 evolution is 2.13 moles per mole glucose. When FCCP only is present, acetate production is lower, and CO2 and H2 evolution is 1.60 and 4.73 moles per mole glucose, respectively.When DCMU alone is present, CO2 and H2 photoevolution is higher than in the dark. Without DCMU, CO2 and H2 evolution is about 100% higher than in its presence. In both conditions, acetate is not formed. In all conditions in the light, ethanol is a minor product. Formate production is least affected by light.The stoichiometry in the dark indicates that starch is degraded via the glycolytic pathway, and pyruvate is broken down into acetyl- (3,19,20,28).Classical glycolysis followed by subsequent metabolism of the pyruvate to the various end-products has been proposed to account for the anaerobic catabolism of starch or glucose in the green algae (17,19,27). Except for H2 evolution, the effect of light on fermentative carbon flow has received little attention. This is principally due to the problems involved with photosynthetic fixation of the CO2 that might be evolved fermentatively. The first to attempt to resolve this question were Klein and Betz (20) who reported that light had no effect on the rate of starch breakdown or the pattern of fermentation in Chlamydomonas moewusii. But they used extremely low levels of light (160 lux (2) made use of C. reinhardtii F-60, a mutant characterized by an incomplete photosynthetic carbon reduction pathway but an intact photosynthetic electron transport chain, to monitor 'true' CO2 and H2 evolution. To account for their results, they proposed an involvement between anaerobic carbohydrate metabolism and the photosynthetic electron transport chain, implying that carbohydrate degradation is entirely or partially localized in the chloroplast. The purpose of our study was to establish for the first time a complete fermentative balance in C. reinhardtii F-60 between...

The anaerobic photodissimilation of acetate by Chlamydomonas reinhardii F-60 adapted to a hydrogen metabolism was studied utilizing manometric and isotopic techniques. The rate of photoanaerobic (N2) acetate uptake was approximately 20 moles per milligram chlorophyll per hour or one-half that of the photoaerobic (air) rate. Under N2, cells produced 1.7 moles H2 and 0.8 mole CO2 per mole of acetate consumed. Gas production and acetate uptake were inhibited by monofluoroacetic acid (MFA), 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and by H2. Acetate uptake was inhibited about 50% by 5% H2 (95% N2 (3) suggested that acetate increased gas production by consuming ATP which regulated the unspecified sequence of reactions giving rise to CO2 and H2. Healey (18) modifying a mechanism put forward by Jones and Myers (20) to explain the Kok effect in blue-green algae, proposed a flow of electrons from acetate via the citric acid cycle into PSI resulting in the photoevolution of H2 from reduced Fd. The operation of an anaerobic and light-dependent citric acid cycle which affects the stoichiometric conversion of acetate to CO2 and H2 had been documented in the photosynthetic purple bacteria ( 13).The present communication summarizes the results of a detailed investigation of the anaerobic photometabolism of acetate by C. reinhardii F-60, with reference to stoichiometry of gas (CO2 and H2) production, incorporation into cellular components, and sensitivity of the process to a variety of inhibitors. The stoichiometric relationships observed, together with the isotopic distribution following assimilation of ["4C]acetate, constitute strong evidence for the conclusion that anaerobic carbon oxidation occurs in part through the reactions of the glyoxylate and citric acid cycles. MATERIALS AND METHODSAlgal Growth Conditions. Chlamydomonas reinhardii (Dangeard) F-60, a mutant strain with an incomplete photosynthetic carbon reduction cycle but with an intact photosynthetic electron transport chain, was obtained from R. K. Togasaki, Indiana University. Cells were grown in batch cultures on an acetatesupplemented medium (14)

ABSTRACIEvidence is presented to substantiate a chloroplastic respiratory pathway in the green alga, Chlamydomonas reinhardtii, whereby reducing equivalents generated during the degradation of starch enter the thylakoidal chain at the plastoquinone site catalyzed by NADH-plastoquinone reductase. In this formulation, the reduced plastoquinone is oxidized either by the photoevolution (photosystem I) of H2 under anaerobic conditions or by 02 during dark respiration.In the preceding paper (7), we reported on the effect of light on the fermentative mode of starch breakdown in a strain of Chlamydomonas reinhardtii adapted to a hydrogen metabolism.When compared to cells kept in the dark, starch breakdown in the illuminated cells was decreased and the fermentative pattern was modified. Inasmuch as the rate of starch breakdown was restored on addition of the uncoupler, FCCP3, the light-induced inhibition was attributed to the role of the energy charge in regulating the glycolytic flux, in a way similar to that proposed to account for the Pasteur effect in this alga (15). The major modification of the fermentative pattern which was not reversed by FCCP or DCMU was a drop in ethanol yield relative to starch consumed. While, in the dark, ethanol formation accounted for the oxidation of the bulk of the reduced pyridine nucleotides generated in the glycolytic pathway, ethanol production in the light was a minor sink for reducing equivalents. To account for this difference, we (7) proposed that, in illuminated cells, most of the reducing equivalents formed during the oxidation of glyceraldehyde 3-P to glycerate 3-P were diverted to the thylakoidal chain at the plastoquinone site (1) with this transfer being catalyzed by NADH-plastoquinone oxido-reductase (9), thus halting the reduction of acetyl-CoA to acetaldehyde and subsequently to ethanol. PSI would catalyze the reoxidation of the reduced plastoquinone and the electrons would bleed off as H2.Making use of DBMIB, a plastoquinone antagonist (3) which is known to prevent not only reduced plastoquinone reoxidation but also noncyclic and cyclic photosynthetic electron transport, we report in this brief communication data substantiating the 'Supported by Department of Energy DE-AC02-76-ER03231 and National Science Foundation PCM 83-04147.