Pyruvate fermentation in Rhodospirillum rubrum and after transfer from aerobic to anaerobic conditions in the dark

The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F₁, S₁) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO₂ and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F₁ and S₁ metabolized the formate further to H₂ and CO₂. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

H2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions

Rhodospirillum rubrum is able to produce H₂ during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H₂ production by the nitrogenase could be partially inhibited by N₂. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP chloramphenicol - DSM Deutsche Sammlung von Mikroorganismen, Göttingen - FHL formate hydrogen lyase - O.D optical density - PFL pyruvate formate lyase     

Bildung flüchtiger Säuren bei der Vergärung von Pyruvat und Fructose in anaerober Dunkelkultur von Rhodospirillum rubrum

Zusammenfassung R. rubrum bildet anaerob im Dunkeln aus exogenen und endogenen Substraten hauptsächlich Acetat und Propionat.In Kulturen mit Pyruvat als Substrat wurde in der Regel mehr Acetat als Propionat gebildet (4:1–1:1), mit Fructose dagegen weniger Acetat (1:2–1:3). Ruhende Zellen produzierten aus Pyruvat, im Vergleich zu Kulturen mit (NH₄)₂SO₄ im Medium, relativ mehr Propionat als Acetat.Beim Abbau von gespeicherter PHBS wurde relativ mehr Acetat und weniger Propionat als beim Abbau endogener Polysaccharide produziert.Eine Zugabe von Ascorbat (0,8 u. 1,6%) oder K₃[Fe(CN)₆] (0,08 u 0,32%) hatte nur einen geringen Effekt auf das Verhältnis von Acetat zu Propionat. Exogenes CO₂ war, besonders bei Zugabe von Fructose als Substrat, für die Synthese von Propionat notwendig. Die Wege der Acetat- und Propionatbildung unter anaeroben Bedingungen im Dunkeln werden diskutiert.

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The synthesis of volatile acids by fermentation of pyruvate and fructose in anaerobic dark cultures of Rhodospirillum rubrum

Summary Under anaerobic conditions in the dark R. rubrum produced mainly acetate and propionate from exogenous and endogenous substrates.With pyruvate as a substrate usually less propionate than acetate was synthesized, with fructose, however, more propionate than acetate was produced.In resting cells, compared with cultures having (NH₄)₂SO₄ in the medium, more propionate than acetate was synthesized from pyruvate.By degradation of stored poly--hydroxybutyric acid under anaerobic dark conditions relatively more acetate is produced than by degradation of endogenous polysaccharide.Addition of ascorbate (0.8 and 1.6%) or K₃[Fe(CN)₆] (0.08 and 0.32%) had little influence on the relative concentrations of acetate and propionate.Exogenous CO₂ was necessary for the synthesis of propionate, especially when fructose was the substrate. The pathways of acetate and propionate production anaerobically in the dark are discussed.

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Abkürzungen BChl Bacteriochlorophyll - PEP Phosphoenolpyruvat - PHBS Poly--hydroxybuttersäure - I.p.m. Impulse (¹⁴C) pro Minute     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata

Rhodospirillum rubrum and Rhodopseudomonas capsulata were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of R. rubrum fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide; R. capsulata produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide. R. rubrum and R. capsulata were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of R. rubrum and R. capsulata on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O₂ in conserving energy through electron transport-linked respiration.     

Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation

Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H₂ but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH₄Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H₂, and CO₂ increased with the addition of NH₄Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO₂ as its main products, with H₂, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H₂ evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.     

Fermentation in the unicellular cyanobacterium Microcystis PCC7806

The cyanobacterium Microcystis PCC7806 fermented endogenously stored glycogen to ethanol, acetate, CO₂, and H₂ when incubated anaerobically in the dark. The switch from photoautotrophic to fermentative metabolism did not require de novo protein synthesis, and fermentation started immediately after cells had been transferred to dark anoxic conditions. From the molar ratios of the products and from enzyme activities in cell-free extracts, it was concluded that glucose derived from glycogen was degraded via the Embden-Meyerhof-Parnas pathway. In addition, CoA-dependent pyruvate:ferredoxin oxidoreductase, alcohol dehydrogenase, acetate kinase, and hydrogenase were present. The specific activities of these enzymes were sufficiently high to account for the rates of product formation by cell suspensions.     

Metabolism of Propane, n-Propylamine, and Propionate by Hydrocarbon-Utilizing Bacteria

Studies were conducted on the oxidation and assimilation of various three-carbon compounds by a gram-positive rod isolated from soil and designated strain R-22. This organism can utilize propane, propionate, or n-propylamine as sole source of carbon and energy. Respiration rates, enzyme assays, and ¹⁴CO₂ incorporation experiments suggest that propane is metabolized via methyl ketone formation; propionate and n-propylamine are metabolized via the methylmalonyl-succinate pathway. Isocitrate lyase activity was found in cells grown on acetate and was not present in cells grown on propionate or n-propylamine. ¹⁴CO₂ was incorporated into pyruvate when propionate and n-propylamine were oxidized in the presence of NaAsO₂, but insignificant radioactivity was found in pyruvate produced during the oxidation of propane and acetone. The n-propylamine dissimilatory mechanism was inducible in strain R-22, and amine dehydrogenase activity was detected in cells grown on n-propylamine. Radiorespirometer and ¹⁴CO₂ incorporation studies with several propane-utilizing organisms indicate that the methylmalonyl-succinate pathway is the predominant one for the metabolism of propionate.     

Metabolic pathway to propionate of Pectinatus frisingensis,a strictly anaerobic beer-spoilage bacterium

      Pectinatus frisingensis, a recently described species of anaerobic mesophilic beer-spoilage bacteria, grows by fermenting various organic compounds, and produces mainly propionate, acetate, and succinate. Although acrylate and succinate were both dismutated by dense resting-cell suspensions, propionate production proceeded through the succinate pathway: [3-¹³C]pyruvate consumption led to equal ¹³C-labeling of propionate on methyl and methylene groups. Growth on glucose or glycerol led to a similar propionate to acetate ratio, suggesting dihydroxyacetone phosphate as being a common metabolic intermediate. Diacetyl, 1,3-propanediol, and 2,3-butanediol were not growth substrates or fermentation products, but they were all dismutated by dense resting-cell suspensions to acetate and propionate. Acetoin was a minor fermentation product. The consumption of [2-¹³C] or [3-¹³C]pyruvate by dense resting-cell suspensions demonstrated the involvement of two equivalent pyruvate molecules during acetoin production. Key enzymes involved in this metabolism were measured in anoxic cell-free extracts. A tentative metabolic pathway to the main fermentation products was proposed from the above results. Received: 17 February 1994 / Accepted: 30 August 1994     

Pathways of Anaerobic Acetate Utilization in Escherichia coli and Aerobacter cloacae

Acetate-1-¹⁴C was added to anaerobic glucose-fermenting cultures of Escherichia coli and Aerobacter cloacae. In the E. coli culture, lactate formation occurred late in the fermentation, when the rate of production of formate and acetate had decreased. The occurrence of acetate label in the lactate indicated formation of pyruvate from acetyl-coenzyme A (CoA) and formate. In the A. cloacae cultures, substantial amounts of acetate label were found in the 2,3-butanediol formed. Evidence is presented that the label could have entered the diol only by conversion of formate and acetyl-CoA into pyruvate. The observed levels of radioactivity in the diol indicated that during diol formation the reaction yielding formate and acetyl-CoA from pyruvate CoA was operating close to equilibrium. The shift in metabolism from formation of acetate, ethyl alcohol, and formate to the formation of butanediol or lactate appears to be due basically to an approach to equilibrium of the pyruvate-splitting reaction, whatever the induction mechanism by which the shift is implemented.     

Changes of Fermentation Pathways of Fecal Microbial Communities Associated with a Drug Treatment That Increases Dietary Starch in the Human Colon

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-¹³C]glucose and ¹²CO₂ or with unlabeled glucose and ¹³CO₂, the distribution of ¹³C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO₂. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO₂ reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.     

Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi

This work is concerned with the metabolism of Caldithrix abyssi—an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO₂ occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate: ferredoxin oxidoreductase (2.6 μmol/(min mg protein)), phosphate acetyltransferase (0.46 μmol/(min mg protein)), and acetate kinase (0.3 μmol/(min mg protein)). The activity of fumarate reductase (0.14 μmol/(min mg protein)), malate dehydrogenase (0.17 μmol/(min mg protein)), and fumarate hydratase (1.2 μmol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.     

Coenzyme specificity of dehydrogenases and fermentation of pyruvate by clostridia

Summary Four clostridial species (C. pasteurianum, C. butylicum, C. butyricum and C. tetanomorphum) grow on pyruvate. Two other species (C. roseum and C. rubrum) only ferment this compound; this is probably due to their inability to synthesize hexose phosphates from pyruvate (fructose-1,6-diphosphatase and pyruvate carboxylase are absent).The fermentation of pyruvate by the above clostridia yields acetate, carbon dioxide, hydrogen and small amounts of compounds more reduced than acetate. Hydrogen pressure increases the amount of ethanol, butanol and butyrate formed during the fermentation of pyruvate. Since C. roseum and C. rubrum contain a ferredoxin: NADP reductase it seems likely that NADPH₂ is the coenzyme involved in ethanol formation. In accordance with this acetaldehyde and alcohol dehydrogenases exhibit activity with NADPH₂.The glyceraldehyde-3-phosphate dehydrogenase of the clostridia under investigation is NAD specific and so is the -hydroxy-butyryl-CoA dehydrogenase with the exception of C. kluyveri.The specific activity of hydrogenase and the coenzyme specificity of NAD(P) reductase vary among the clostridial species.     

Effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in isolated rat hepatocytes

α-Ketobutyrate, an intermediate in the catabolism of threonine and methionine, is metabolized to CO₂ and propionyl-CoA. Recent studies have suggested that propionyl-CoA may interfere with normal hepatic oxidative metabolism. Based on these observations, the present study examined the effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in hepatocytes isolated from fed rats. α-Ketobutyrate (10 mM) inhibited the oxidation of palmitic acid by 34%. In the presence of 10 mM carnitine, the inhibition of palmitic acid oxidation by α-ketobutyrate was reduced to 21%. These observations are similar to those previously reported using propionate as an inhibitor of fatty acid oxidation, suggesting that propionyl-CoA may be responsible for the inhibition. α-Ketobutyrate (10 mM) inhibited ¹⁴CO₂ generation from [¹⁴C]pyruvate by more than 75%. This inhibition was quantitatively larger than seen with equal concentrations of propionate. Carnitine (10 mM) had no effect on the inhibition of pyruvate oxidation by α-ketobutyrate despite the generation of large amounts of propionylcarnitine during the incubation. α-Ketobutyate inhibited [¹⁴C]glucose formation from [¹⁴C]pyruvate by more than 60%. This contrasted to a 30% inhibition caused by propionate. These results suggest that α-ketobutyrate inhibits hepatic pyruvate metabolism by a mechanism independent of propionyl-CoA formation. The present study demonstrates that tissue accumulation of α-ketobutyrate may lead to disruption of normal cellular metabolism. Additionally, the production of propionyl-CoA from α-ketobutyrate is associated with increased generation of propionylcarnitine. These observations provide further evidence that organic acid accumulation associated with a number of disease states may result in interference with normal hepatic metabolism and increased carnitine requirements.     

Hydrogen metabolism by filamentous cyanobacteria

Apparent discrepancies in the literature concerning the amounts of H₂ produced by strains of Anabaena cylindrica are explained. These are not due to differences in strains used by different workers nor to differences in growth conditions, but rather appear to be due to the fact that cultures show an increasing dependence with age on CO₂ for sustained H₂ production. Two distinct hydrogenase activities were measured and characterized, both in vivo and in vitro in A. cylindrica B629; these were H₂ uptake activity and H₂ evolution from reduced methyl viologen. Gentle cell disruption techniques were used to gain further evidence that the latter activity was soluble. H₂ uptake was strongly inhibited by acetylene in vivo in the light or in the dark with phenazine methosulfate added, but only after a prolonged lag period. In extracts this lag did not occur. A detailed study of the nitrogenase and hydrogen uptake activities and their interrelationship both in the light and in the dark in A. cylindrica B629 showed that only in the dark in the presence of O₂ did H₂ uptake support C₂H₂ reduction significantly. Under several conditions in which nitrogenase activity was inhibited H₂ uptake was unaffected. H₂ metabolism was tested in three nonheterocystous filamentous cyanobacteria under different growth and incubation conditions. These were Plectonema boryanum, Schizothrix calcicola, and Oscillatoria brevis. Myxosarcina chroococcoides and Fischerella muscicola were also investigated. Cyanobacterial species vary markedly in their hydrogen metabolism and in the composition of the three H₂ metabolizing enzymes.     

Pyruvate — a novel substrate for growth and methane formation in Methanosarcina barkeri

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed.

1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO₂ and CH₄ were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH₄ and CO₂ according to following equation: Pyruvate^-+H⁺+0.5 H₂O » 1.25 CH₄+1.75 CO₂. The molar growth yield (Ych ₄) was about 14 g dry weight cells/mol CH₄. In contrast the growth yield (Ych ₄) of M. barkeri during growth on acctate (Acetate^-+H⁺ » CH₄+CO₂) was about 3 g/mol CH₄.
2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH₄, CO₂ and H₂ (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂. At higher cell concentration less H₂ and CO₂ and more CH₄ were formed due to CH₄ formation from H₂/CO₂. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂ at similar rates and with similar affinity for pyruvate as pyruvate-grown cells.
3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F₄₂₀ was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Pyruvate formate lyase inRhodospirillum rubrum Ha adapted to anaerobic dark conditions

The fermatation metabolism ofRhodospirillum rubrum Ha was studied after adaptation of both light-anaerobic and dark aerobic to dark anaerobic conditions.Pyruvate was metabolized to acetate, formate, CO₂ and propionate by suspensions of cells adapted to anaerobiosis. Pyruvate cleavage to formate accounted for about two-thirds of the pyruvate decomposed. This process was catalyzed by a coenzyme A dependent pyruvate formate lyase. In carboxylate- and nucleotide-free extracts, the substrate concentrations for half-maximal velocity [S]_0.5V were found to be 1.5 mM for pyruvate and 75 M for coenzyme A.Pyruvate formate lyase could practically not be demonstrated in light-anaerobic photosynthesizing cells. Lyase activity was low at a basic level in darkaerobic respiring cells. After adaptation of both types of cells under growth conditions to dark anaerobiosis lyase activity increased about 10-fold. Highest levels could be observed in cells grown aerobically in the dark on pyruvate after transition to dark anaerobic conditions. It is concluded that pyruvate formate lyase is the characteristic key enzyme of the dark-anaerobic fermentative metabolism ofR. rubrum Ha.     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.