Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.     

Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.     

Contribution of propionate to glucose synthesis in sheep

1. The production rate of propionate in the rumen and the entry rate of glucose into the body pool of glucose in sheep were measured by isotope-dilution methods. Propionate production rates were measured by using a continuous infusion of specifically labelled [(14)C]propionate. Glucose entry rates were estimated by using either a primed infusion or a continuous infusion of [U-(14)C]glucose. 2. The specific radioactivity of plasma glucose was constant between 4 and 9hr. after the commencement of intravenous infusion of [U-(14)C]glucose and between 1 and 3hr. when a primed infusion was used. 3. Infusion of [(14)C]propionate intraruminally resulted in a fairly constant specific radioactivity of rumen propionate between about 4 and 9hr. and of plasma glucose between 6 and 9hr. after the commencement of the infusion. Comparison of the mean specific radioactivities of glucose and propionate during these periods allowed estimates to be made of the contribution of propionate to glucose synthesis. 4. Comparisons of the specific radioactivities of plasma glucose and rumen propionate during intraruminal infusions of one of [1-(14)C]-, [2-(14)C]-, [3-(14)C]- and [U-(14)C]-propionate indicated considerable exchange of C-1 of propionate on conversion into glucose. The incorporation of C-2 and C-3 of propionate into glucose and lactate indicated that 54% of both the glucose and lactate synthesized arose from propionate carbon. 5. No differences were found for glucose entry rates measured either by a primed infusion or by a continuous infusion. The mean entry rate (+/-s.e.m.) of glucose estimated by using a continuous infusion into sheep was 0.33+/-0.03 (4) m-mole/min. and by using a primed infusion was 0.32+/-0.01 (4) m-mole/min. The mean propionate production rate was 1.24+/-0.03 (8) m-moles/min. The conversion of propionate into glucose was 0.36 m-mole/min., indicating that 32% of the propionate produced in the rumen is used for glucose synthesis. 6. It was indicated that a considerable amount of the propionate converted into glucose was first converted into lactate.     

Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose

Propionibacterium acidipropionici was grown in a fed-batch culture, fed with glucose or lactate, or mixtures of lactate and glucose. Lactate and glucose were always simultaneously consumed. As co-substrate, glucose modified the propionate:acetate molar ratio (P/A) and increased the fraction of carbon used for biomass production. A P/A of 7.63 was obtained with a lactate:glucose molar ratio of 4; a P/A value of 1.34 was obtained with lactate alone and 1.85 with glucose alone. The fraction of carbon recovered in biomass was 0.09 for glucose, 0.12 for lactate, and 0.21 for a lactate:glucose molar ratio of 4.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

Extracellular iron reduction is mediated in part by neutral red and hydrogenase in Escherichia coli

Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, by Escherichia coli, ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts of E. coli were used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. An E. coli hypE mutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.     

Regulation of alanine dehydrogenase in Bacillus (licheniformis)

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

固定化光合细菌利用有机物产氢的研究

应用固定化细胞技术包埋荚膜红假单胞菌(Rhodopseudomonas capsulata)菌株386．研究在光照下利用有机物产氢的特性。实验观察到,光照培养120小时,悬浮培养物的产氢量为68．2ml·比产氢速率为104．1ml H2/g(生物量)·h;用琼脂包埋后．其产氢能力得到改善,产氢量和比产氢速率分别达到128．4ml和l 9s．8mlH2/g·h。该菌株除可利用苹果酸外,还可利用葡萄糖、乳酸、丙酸等基质高效地产氢。基质浓度只有控制在适当水平时,才具有较高的基质转化产氢效率。此外．菌体生物量、菌龄、培养液pH、光照强度、光照／黑暗时间比以及温度对产氢过程均有不同程度的影响。     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.