Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs

Energetic and reaction-rate interactions between hydrogenic (hydrogen-producing) and hydrogenotrophic (hydrogen-consuming) bacteria were investigated in five perturbation experiments performed on steady-state, mixed-culture methanogenic CSTRs receiving ethanol, propionate, or both hydrogenic substrates. When a large quantity of propionate was suddenly added to a propionatefed CSTR, P(H(2) ) increased to 10(-4) atm and propionate oxidation remained energetically favorable. When ethanol was added to a CSTR receiving ethanol, P(H(2) ) rose to 6.3 x 10(-3) atm within 5 h. In both perturbations, P(H(2) ) remained at levels such that oxidation of the hydrogenic substrate remained energetically favorable throughout the transient. Sudden increase in ethanol concentration in the ethanol- and propionate-fed CSTR resulted in an increase in P(H(2) ) such that propionate oxidation became energetically unfavorable and was blocked. Propionate utilization resumed when the added ethanol was depleted and P(H(2) ) returned to its previous steady-state levels. Ethanol perturbation of ethanol- and propionate-fed CSTRs led to the formation of reduced products, including n-propanol and four-through seven-carbon n-carboxylic acids, when P(H(2) ) was elevated; these products disappeared after P(H(2) ) returned to previous, steady-state levels. The transformations were consistent with reaction energetics. Reduced product formation may have been a sink for reducing equivalents, as an alternative to oxidation for propionate utilization, as indicated by an electron equivalents balance over the time course of experiments.     

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.     

Glucose and Pyruvate Metabolism of Spirochaeta litoralis, an Anaerobic Marine Spirochete

The pathways of glucose and pyruvate metabolism in Spirochaeta litoralis, a free-living, strictly anaerobic marine spirochete, were studied. Addition of 0.2 to 0.4 M NaCl (final concentration) to suspending buffers prevented cell lysis and was necessary for gas evolution from various substrates by cell suspensions. The organism fermented glucose mainly to ethanol, acetate, CO(2), and H(2). Determination of radioactivity in products formed from (14)C-labeled glucose and assays of enzymatic activities in cell extracts indicated that S. litoralis catabolized glucose via the Embden-Meyerhof pathway. A clostridial-type clastic reaction was utilized by the spirochete to degrade pyruvate to acetyl-coenzyme A, CO(2), and H(2). Formation of acetate from acetyl-coenzyme A was catalyzed by phosphotransacetylase and acetate kinase. Nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenases converted acetyl-coenzyme A to ethanol. A reversible hydrogenase activity was detected in cell extracts. S. litoralis cell extracts contained a rubredoxin similar in spectral properties to other bacterial rubredoxins.     

Propionate formation from alcohols or aldehydes by Desulfobulbus propionicus in the absence of sulfate

Fermentative degradation of alcohols and aldehydes in the absence of sulfate was investigated using a propionate-oxidizing, sulfate-reducing bacterium, Desulfobulbus propionicus strain MUD (DSM 6523). The organism converted ethanol plus CO₂ to acetate and propionate. The conversion was not affected by the presence of hydrogen. Strain MUD converted propanol plus acetate to propionate. Acetaldehyde and propionaldehyde were also converted with a dismutation reaction in the absence of sulfate. The products were propionate and acetate from acetaldehyde, and propionate from propionaldehyde plus acetate.     

Syntrophic association of a butyrate-degrading bacterium and methanosarcina enriched from bovine rumen fluid.

An anaerobic butyrate-degrading bacterium, morphologically similar to Syntrophomonas wolfei, was isolated in coculture with Desulfovibrio strain G11 from an enrichment of bovine rumen fluid. A Methanosarcina species was the major H2-using organism in the enrichment. The results are discussed in relationship to the absence of Methanospirillum hungatei, the H2-using methanogen usually found in association with S. wolfei, and the finding of Methanosarcina rather than Methanobrevibacter ruminantium as the major H2-using bacterium in the enrichments. The finding of butyrate degraders in the rumen suggests that, if the retention time of the rumen contents becomes more prolonged, butyrate and longer-chained fatty acids might be significantly degraded.     

Formation of an oxo-radical of peroxovanadate during reduction of diperoxovanadate with vanadyl sulfate or ferrous sulfate

Formation of oxygen radicals during reduction of H(2)O(2) or diperoxovanadate with vanadyl sulfate or ferrous sulfate was indicated by the 1:2:2:1 electron spin resonance (ESR) signals of the DMPO adduct typical of standard ()OH radical. Signals derived from diperoxovanadate remained unchanged in the presence of ethanol in contrast to those from H(2)O(2). This gave the clue that they represent a different radical, possibly (*)OV(O(2))(2+), formed on breaking a peroxo-bridge of diperoxovanadate complex. The above reaction mixtures evolved dioxygen or, when NADH was present, oxidized it rapidly which was accompanied by consumption of dioxygen. Operation of a cycle of peroxovanadates including this new radical is suggested to explain these redox activities both with vanadyl and ferrous sulfates. It can be triggered by ferrous ions released from cellular stores in the presence of catalytic amounts of peroxovanadates.     

The Products and Pathways of Glucose Catabolism in Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum

ABSTRACT. The products and pathways of glucose catabolism in the insect trypanosomatids Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum have been studied with the aim of elucidating how both organisms are able to proliferate well under aerobic and anaerobic conditions. When incubated in medium containing glucose as the only exogenous carbon source, catabolism was found to be fermentative in both cases. Acetate was a major product of both organisms while H. m. ingenoplastis produced more ethanol and propionate and less succinate than H. m. muscarum . Ethanol production by H. m. ingenoplastis decreased both under anaerobic conditions and in the presence of elevated CO₂ concentrations, whereas succinate and propionate release by this organism were greater in high CO₂ and anoxia, respectively. Succinate production by H. m. muscarum was greatest under anaerobic conditions in elevated CO₂ whereas propionate was only a minor product. The same four products were released during growth of the organisms in complex medium, but the relative proportions differed suggesting that other substrates were being used. Both organisms contained enzymes of the glycolytic and pentose phosphate pathways, but while all activities of the TCA cycle were present in H. m. muscarum . NAD-linked isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate CoA synthase and succinate dehydrogenase were not detected in H. m. ingenoplastis . Fumarate reductase activity was present in both organisms. The data presented suggest that CO₂-fixation and reverse flux through the TCA cycle may be important factors that enable the organisms to undergo anaerobiosis.     

Stereochemical studies of the pyruvate kinase reaction with (Z)- and (E)-phosphoenol-alpha-ketobutyrate

(Z)-and (E)-phosphoenol-2-ketobutyrate were synthesized. [3-2H]-2-Ketobutyrates were formed from both isomers in the pyruvate kinase reaction in 2H2O and were converted to chiral propionates. Authentic (2S)-[2-2H]propionic acid was also prepared, and the optical rotatory dispersion curves of the propionates were compared. The rotation compared with standard propionate at 240 nm of sodium (2R)-[2-2H]propionate from the Z isomer was 47% (i.e., 53% was RS), and of (2S)-[2-2H]propionate from the E isomer was 29% (i.e., 71% was RS). Protonation at C-3 of the 2 si, 3 re face of the pseudosubstrates would have yielded (2R)- and (2S)-[2-2H]propionates from the Z and E analogues, respectively. An explanation offered for the nonstereoselective protonation that occurred is dissociation of the enol from the enzyme and subsequent random protonation in solution.     

Fe(III) and S0 reduction by Pelobacter carbinolicus.

There is a close phylogenetic relationship between Pelobacter species and members of the genera Desulfuromonas and Geobacter, and yet there has been a perplexing lack of physiological similarities. Pelobacter species have been considered to have a fermentative metabolism. In contrast, Desulfuromonas and Geobacter species have a respiratory metabolism with Fe(III) serving as the common terminal electron acceptor in all species. However, the ability of Pelobacter species to reduce Fe(III) had not been previously evaluated. When a culture of Pelobacter carbinolicus that had grown by fermentation of 2,3-butanediol was inoculated into the same medium supplemented with Fe(III), the Fe(III) was reduced. There was less accumulation of ethanol and more production of acetate in the presence of Fe(III). P. carbinolicus grew with ethanol as the sole electron donor and Fe(III) as the sole electron acceptor. Ethanol was metabolized to acetate. Growth was also possible on Fe(III) with the oxidation of propanol to propionate or butanol to butyrate if acetate was provided as a carbon source. P. carbinolicus appears capable of conserving energy to support growth from Fe(III) respiration as it also grew with H2 or formate as the electron donor and Fe(III) as the electron acceptor. Once adapted to Fe(III) reduction, P. carbinolicus could also grow on ethanol or H2 with S0 as the electron acceptor. P. carbinolicus did not contain detectable concentrations of the c-type cytochromes that previous studies have suggested are involved in electron transport to Fe(III) in other organisms that conserve energy to support growth from Fe(III) reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spore-forming thermophilic sulfate-reducing bacteria isolated from north sea oil field waters

Thermophilic sulfate-reducing bacteria were isolated from oil field waters from oil production platforms in the Norwegian sector of the North Sea. Spore-forming rods dominated in the enrichments when lactate, propionate, butyrate, or a mixture of aliphatic fatty acids (C(4) through C(6)) was added as a carbon source and electron donor. Representative strains were isolated and characterized. The isolates grew autotrophically on H(2)-CO(2) and heterotrophically on fatty acids such as formate, propionate, butyrate, caproate, valerate, pyruvate, and lactate and on alcohols such as methanol, ethanol, and propanol. Sulfate, sulfite, and thiosulfate but not nitrate could be used as an electron acceptor. The temperature range for growth was 43 to 78 degrees C; the spores were extremely heat resistant and survived 131 degrees C for 20 min. The optimum pH was 7.0. The isolates grew well in salt concentrations ranging from 0 to 800 mmol of NaCl per liter. Sulfite reductase P582 was present, but cytochrome c and desulfoviridin were not found. Electron micrographs revealed a gram-positive cell organization. The isolates were classified as a Desulfotomaculum sp. on the basis of spore formation, general physiological characteristics, and submicroscopic organization. To detect thermophilic spore-forming sulfate-reducing bacteria in oil field water, polyvalent antisera raised against antigens from two isolates were used. These bacteria were shown to be widespread in oil field water from different platforms. The origin of thermophilic sulfate-reducing bacteria in the pore water of oil reservoirs is discussed.     

Response of a biohydrogen-producing reactor to the substrate shift from sucrose to lactose

The response of an upflow acidogenic granule-based reactor to the substrate shift from sucrose to lactose was investigated in this study. Experimental results show that it took 60h for the reactor to completely degrade the new substrate. Hydrogen production performance, in terms of H(2) partial pressure, H(2) production rate and H(2) yield, was affected. Acetate, propionate, butyrate, valerate, caporate, ethanol and propanol were present in the reactor effluent, and their distribution changed significantly after the substrate shift. As the substrate was changed, the caproate- and ethanol-type fermentation was weakened, while the propionate-type fermentation was strengthened. Throughout the experiment, the butyrate-type fermentation played an important role. The H(2) yield had a close correlation with both propionate and B/A (butyrate/acetate) in this substrate shift process.     

Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures

Saccharomyces uvarum, Geotrichum candidum, Endomycopsis burtonii and Hansemula canadensis have been isolated from deteriorated silages and/or spoiled lactic acid fermented foods. All yeasts could grow in relatively high (100 mmol/l) concentrations of acetate or lactate in Wickerham's assimilation broth but were inhibited by increasing concentrations of these acids. Propionate was much more inhibitory than acetate or lactate. Growth rate, but not final cell yield, was adversely affected by increasing concentrations of these acids. The three acids used in combination were synergistically inhibitory to H. canadensis and G. candidum. Saccharomyces uvarum was inhibited by synergistic mixtures of acetate/propionate. Endomycopsis burtonii and H. canadensis were inhibited by synergistic mixtures of acetate/lactate, acetate/propionate and lactate/propionate. Lactate and propionate had a synergistic inhibitory effect on G. candidum. The mechanism of inhibition of growth of S. uvarum by acetate and propionate was further investigated. Acetate and propionate each caused a reduction in cellular efficiency (Y, μg cells/μmole glucose consumed). Mixtures caused a drastic reduction in growth rate and Y. These results suggest that the cells may expend energy in reducing intracellular concentrations of acetate while propionate probably acts in a different manner.     

A kinetic model for methanogenesis from whey permeate in a packed bed immobilized cell bioreactor

The fermentation kinetics of methane production from whey permeate in a packed bed immobilized cell bioreactor at mesophilic temperatures and pHs around neutral was studied. Propionate and acetate were the only two major organic intermediates found in the methanogenic fermentation of lactose. Based on this finding, a three-step reaction mechanism was proposed: lactose was first degraded to propionate, acetate, CO(2), and H(2) by fermentative bacteria; propionate was then converted to acetate by propionate-degrading bacteria; and finally, CH(4) and CO(2) were produced from acetate, H(2), and CO(2) by methanogenic bacteria. The second reaction step was found to be the rate-limiting step in the overall methanogenic fermentation of lactose. Monod-type mathematical equations were used to model these three step reactions. The kinetic constants in the models were sequentially determined by fitting the mathematical equations with the experimental data on acetate, propionate, and lactose concentrations. A mixed-culture fermentation model was also developed. This model simulates the methanogenic fermentation of whey permeate very well.     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

The characteristics of the `peroxidatic'' reaction of catalase in ethanol oxidation

Ethanol oxidation by rat liver catalase (the ;peroxidatic' reaction) was studied quantitatively with respect to the rate of H(2)O(2) generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase-H(2)O(2) intermediate (Compound I). At a low ratio of H(2)O(2)-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase-cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H(2)O(2) generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H(2)O(2)-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ;catalatic' and ;peroxidatic' reactions in the overall H(2)O(2)-decomposition reaction. There was a correlation between the proportion of the ;peroxidatic' reaction in the overall catalase reaction and the steady-state concentration of the catalase-H(2)O(2) intermediate. Regardless of the concentration of ethanol and the rate of H(2)O(2) generation, a half-saturation of the steady state of the catalase-H(2)O(2) intermediate indicated that about 45% of the H(2)O(2) was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ;microsomal ethanol-oxidation system' may be reinterpreted and the catalase ;peroxidatic' reaction provides a quantitative explanation for the activity hitherto attributed to the ;microsomal ethanol-oxidation system'.     

Mechanistic study of the urocanase reaction using deuterated substrates and 1H-NMR spectroscopy

1. Samples of (alpha-2H1, 5-2H1) and (alpha-2H1, beta-2H1) urocanic acid were prepared by a combination of chemical and enzymic methods. 2. The enzymic conversion of unlabelled urocanate was followed by 1H-NMR spectroscopy at 500 MHz in deuterium oxide. It was found (a) that urocanase promotes the exchange of the 5-hydrogen atom of the substrate faster than it catalyses the overall reaction, (b) that the product is an equilibrium mixture of racemic beta-(5-oxoimidazol-4-yl)propionate and beta-(5-hydroxyimidazol-4-yl)propionate and (c) that beta-(5-oxoimidazol-4-yl)-propionate is spontaneously hydrolysed under physiological conditions to N-formylisoglutamine. The rate of this hydrolysis is considerably diminished at +8 degrees C. 3. It was shown by ultraviolet and 1H-NMR spectroscopic measurements that beta-(5-hydroxyimidazol-4-yl)-propionate (gamma max approximately equal to 234 nm) exists in protonated from at low pH (less than 1) whereas pH (approximately equal to 7.5) it exists in equilibrium with beta-(5-oxoimidazol-4-yl)propionate (gamma max approximately equal to 269 nm). 4. (alpha-2H1, beta-2H1)Urocanate was reacted with urocanase in deuterium oxide. 1H-NMR spectroscopy at 500 MHz showed a slight incorporation of protium into the side-chain of the product. The incorporated protium corresponded roughly to the protium contamination of the solvent and was equally distributed between the alpha and beta positions. No transfer of the 5-hydrogen atom to the side-chain was detected. 5. Kinetic deuterium isotope effects of between 2 and 3 were measured when the urocanase reaction was conducted in deuterium oxide at different p2H values. 6. Implications of these findings for the mechanism of action of urocanase are discussed.