Ferredoxin- and Nicotinamide Adenine Dinucleotide-Dependent H2 Production from Ethanol and Formate in Extracts of S Organism Isolated from “Methanobacillus omelianskii”

S organism ferments ethanol to acetate and H(2) but grows poorly on ethanol unless the partial pressure of H(2) is kept low, as when it is grown in combination with an H(2)-utilizing methanogenic bacterium. The present study shows that S organism contains an alcohol dehydrogenase and a formate dehydrogenase, both of which require nicotinamide adenine dinucleotide (NAD) for activity. Hydrogen is evolved from NADH generated by these activities via a ferredoxin-dependent oxidation of NADH to NAD and H(2). NADH:NADP oxido-reductase activity was also demonstrated. The relationship of these activities to the growth of S organism is discussed.     

Characteristics of S Organism Isolated from Methanobacillus omelianskii

Previous work showed that Methanobacillus omelianskii was a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H(2) produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H(2) gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO(2), in addition to a small amount of H(2). When combined with M. ruminantium, no H(2) and very little ethanol were produced and acetate production was increased. When M. ruminantium was present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H(2) and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 +/- 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH(4))(2)SO(4) as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Microbial Oxidation of Methane and Methanol: Isolation of Methane-Utilizing Bacteria and Characterization of a Facultative Methane-Utilizing Isolate

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.     

The inhibition of acetate oxidation by ethanol in Acetobacter aceti

Ethanol grown Acetobacter aceti differed from acetate grown. In ethanol grown cells, acetate uptake, caused by the oxidation of acetate, was completely inhibited by ethanol, in acetate grown cells only to 20%. This was correlated with a 65-fold higher specific activity of the membrane bound NAD(P)-independent alcohol dehydrogenase in ethanol grown than in acetate grown cells. In comparison with ethanol grown cells, acetate grown cells showed a 3-fold higher acetate respiration rate and 3-fold higher specific activities of some tricarboxylic acid cycle enzymes tested. Both adaptations were due to induction by the homologous and not to repression by the heterologous growth substrate. A. aceti showed a membrane bound NAD(P)-independent malate dehydrogenase and no activity of a soluble NAD(P)-dependent one, as was known before from A. xylinum. A hypothesis was proposed explaining the observed inhibition of malate dehydrogenase and of functioning of the tricarboxylic acid cycle in the presence of ethanol or butanol or glucose by a competition of two electron currents for a common link in the convergent electron transport chains. The electrons coming from the quinoproteins, alcohol dehydrogenase and glucose dehydrogenase on the one side and those coming from the flavoproteins, malate dehydrogenase and succinate dehydrogenase via ubiquinonecytochrome c reductase on the other side are meeting at cytochrome c. Here the quinoproteins may be favoured by higher affinity and so inhibit the flavoproteins. Inhibition could be alleviated in the cell free system by increasing the oxygen supply.Dedicated to Professor Carl Martius on the occasion of his 80th birthday, March 1st 1986     

Isolation and characterization of "Methanosphaera cuniculi" sp. nov

A nonmotile, gram-positive, spherical organism was isolated from the intestinal tracts of rabbits. Both hydrogen and methanol were required for growth. No methane was produced from hydrogen-carbon dioxide, formate, acetate, methylamines, ethanol, or isopropanol. The optimum pH was 6.8, and the optimum temperature was 35 to 40 degrees C. The DNA G+C content is 23 mol%. The pseudomurein cell wall contained serine. These characteristics and the immunological fingerprinting results are consistent with its placement in the genus Methanosphaera as a new species.     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Production and Utilization of Ethanol by the Homoacetogen Acetobacterium woodii

Acetobacterium woodii formed ethanol as a fermentation product in addition to acetate when the phosphate concentration of the medium was between 0.2 and 8.4 mM. Considerable amounts of alanine were also found (2 to 11 mM). Supplementation with phosphate caused a shift to acetate as the only end product. Ethanol could also serve as a substrate for A. woodii. The fermentation yielded predominantly acetate and was strictly dependent on high bicarbonate concentrations. 1-Propanol, 1-butanol, and 1-pentanol were converted to the corresponding fatty acids but allowed only marginal growth. A. wieringae and A. carbinolicum grown under identical conditions were also able to form ethanol, and A. wieringae could use ethanol as a substrate, too. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were determined in A. woodii. Activity stains of polyacrylamide gels with crude extracts allowed the detection of acetaldehyde dehydrogenase but not of alcohol dehydrogenase. Trace amounts of methane were detected during growth of A. woodii on glucose and ethanol.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

Comparison of the Key Enzymes of Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

For Hyphomicrobium 53-49 capable of growing under various conditions, aerobic methanol, anaerobic methanol (with denitrification), autotrophic (H₂-O₂-CO₂), aerobic ethanol and aerobic acetate, investigation and comparison of the specific activities of the following enzymes were performed: alcohol dehydrogenase (NAD-ethanol linked and NAD-methanol linked), primary alcohol dehydrogenase, formaldehyde dehydrogenase (NAD-GSH linked and DCPIP linked), formate dehydrogenase, serine hydroxymethyl transferase, hydroxypyruvate reductase, isocitrate lyase (icl), malate lyase, malate dehydrogenase, ribulosebisphosphate (RuBP) carboxylase, phos-phoenolpyruvate (PEP) carboxykinase (ADP linked), PEP carboxylase (phosphorylating), pyruvate carboxylase (NADH linked and NADPH linked) and α-ketoglutarate carboxylase (NADH linked and NADPH linked). On the basis of the data obtained, it was concluded that during growth on methanol, aerobically and anaerobically, the icl+ serine pathway operated, while during autotrophic growth on H₂-O₂-CO₂, CO₂ was incorporated through the RuBP pathway and others, and during growth on ethanol or acetate, neither the serine pathway nor the RuBP pathway operated. The organism changed its metabolism through the regulation of the metabolic enzymes according to the growth conditions.     

Temperature effects on ethanol and isopropanol utilization by Candida krusei

Candida Krusei has a optimum growth temperature of 37°C on SASOL ethanol-isopropanol mixture. The organism was unable to grow on isopropanol, but oxidized it partially to acetone in the presence and absence of ethanol. Growth at 40°C in the alcohol mixture was slightly faster than at 30°C over an ethanol concentration range of 0.43 to 3.6% (v/v), although at both temperatures the growth rate declined continuously with increasing concentration. At an ethanol concentration greater than 3.6% (v/v), the mixture was much more inhibitory to growth at 40 and 30°C. The inhibitory effect was due to the ethanol rather than the isopropanol. Metabolites such as acetate, acetaldehyde, and ethyl acetate accumulated in the medium, but the degree of accumulation depended upon the temperature and alcohol mixture concentration. At 40°C, acetaldehyde and acetate accumulated to a greater extent than 30°C on a 4.0% (v/v) synthetic alcohol mixture and this may also cause the greater inhibition at this temperature. The alcohol mixture is unsuitable for single cell protein (SCP) production in batch culture because of the low cell densities observed at all alcohol concentrations.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Cellulolytic properties of an extremely thermophilic anaerobe

Summary An extremely thermophilic anaerobe was isolated from a New Zealand hot spring by incubating bacterial mat strands in a medium containing xylan. The Gramreaction-negative organism that was subsequently purified had a temperature optimum of 70° C and a pH optimum of 7.0. The isolate, designated strain H173, grew on a restricted range of carbon sources. In batch culture H173 could degrade Avicel completely when supplied at 5 or 10 g l^–1. There was an initial growth phase, during which a cellulase complex was produced and carbohydrates fermented to form acetic and lactic acids, followed by a phase where cells were not metabolising but the cellulase complex actively converted cellulose to glucose. When co-cultured with strain Rt8.B1, an ethanologenic extreme thermophile, glucose was fermented to ethanol and acetate, and no reducing sugars accumulated in the medium. In pH controlled batch culture H173 produced an increased amount of lactate and acetate but there was again a phase when reducing sugars accumulated in the medium, and these were converted to ethanol by co-culture with Rt8.B1.     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

A dual function of alcohol dehydrogenase in Drosophila

Alcohol dehydrogenase (ADH) of Drosophila not only catalyzes the oxidation of ethanol to acetaldehyde, but additionally catalyzes the conversion of this highly toxic product into acetate. This mechanism is demonstrated by using three different methods. After electrophoresis the oxidation of acetaldehyde is shown in an NAD-dependent reaction revealing bands coinciding with the bands likewise produced by a conventional ADH staining procedure. In spectrophotometric measurements acetaldehyde is oxidized in an NAD-dependent reaction. This activity is effectively inhibited by pyrazole, as specific inhibitor of ADH. By means of gas chromatographic analysis a quick generation of acetate from ethanol could be demonstrated. Our conclusion is further supported by experimental results obtained with either purified ADH^F enzyme or genotypes with or without ADH, aldehyde-oxidase, pyridoxal-oxidase and xanthine-dehydrogenase activity. These results are discussed in relation to ethanol tolerance in the living organism in particular with respect to differences found between ADH in Drosophila melanogaster and D. simulans, and in relation to the possible implications for the selective forces acting on ADH-polymorphism.     

Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase.

Mutants of Escherichia coli which overproduce alcohol dehydrogenase were obtained by selection for the ability to use ethanol as an acetate source in a strain auxotrophic for acetate. A mutant having a 20-fold overproduction of alcohol dehydrogenase was able to use ethanol only to fulfill its acetate requirement, whereas two mutants with a 60-fold overproduction were able to use ethanol as a sole carbon source. The latter two mutants produced only 25% of the wild-type level of nitrate reductase, when grown under anaerobic conditions. Alcohol dehydrogenase production was largely unaffected by catabolite repression but was repressed by nitrate under both aerobic and anaerobic conditions. The genetic locus responsible for alcohol dehydrogenase overproduction was located at min 27 on the E. coli genetic map; the gene order, as determined by transduction, was trp tonB adh chlC hemA. The possible relationship of alcohol dehydrogenase to anaerobic redox systems such as formate-nitrate reductase is discussed.     

Ethanol and amylase production by a newly isolated Clostridium sp.

A newly isolated, anaerobic, mesophillic bacterium, Clostridium sp. strain YK-3, ferments pentoses, hexoses, oligosaccharides and polysaccharides, such as soluble starch and glycogen, to ethanol and acetate. The potential of this strain for ethanol and amylase production has been examined. Ethanol was the major product and acetate a minor one. The organism could grow with soluble starch in the presence of 40 g ethanol/l. Extracellular -amylase activity was detected when the strain was cultivated with soluble starch, glycogen or dextrin. The optimum pH of this amylase was 5.5 to 7.5 with an optimum temperature of 50°C.The authors are with the Laboratory of Applied Microbiology, Faculty of Agriculture, Yamagata University, Tsuruoka 997, Japan.     

Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

Growth and fermentation of an anaerobic rumen fungus on various carbon sources and effect of temperature on development.

An anaerobic fungus (strain R1) resembling Neocallimastix spp. was isolated from sheep rumen. When grown on defined medium, the isolate utilized a wide range of polysaccharides and disaccharides, but of the eight monosaccharides tested only fructose, glucose, and xylose supported growth. The organism had doubling times of 5.56 h on glucose and 6.67 h on xylose, and in each case fermentation resulted in production of formate, acetate, lactate, and ethanol. During active growth, formate was a reliable indicator of fungal biomass. Growth on a medium containing glucose and xylose resulted in a doubling time of 8.70 h, but diauxic growth did not occur since both sugars were utilized simultaneously. The optimum temperature for zoospore and immature plant development was 39 degrees C, and no development occurred below 33 degrees C or above 41 degrees C.     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).