Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids.

Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. The respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on each of the monochlorinated phenol and monochlorinated benzoic acid isomers, using sediment from the upper (freshwater) and lower (estuarine) Hudson River and the East River (estuarine) as source materials. Utilization of each chlorophenol and chlorobenzoate isomer was observed under at least one reducing condition; however, no single reducing condition permitted the metabolism of all six compounds tested. The anaerobic biodegradation of the chlorophenols and chlorobenzoates depended on the electron acceptor available and on the position of the chlorine substituent. In general, similar activities were observed under the different reducing conditions in both the freshwater and estuarine sediments. Under denitrifying conditions, degradation of 3- and 4-chlorobenzoate was accompanied by nitrate loss corresponding reasonably to the stoichiometric values expected for complete oxidation of the chlorobenzoate to CO2. Under sulfidogenic conditions, 3- and 4-chlorobenzoate, but not 2-chlorobenzoate, and all three monochlorophenol isomers were utilized, while under methanogenic conditions all compounds except 4-chlorobenzoate were metabolized. Given that the pattern of activity appears different for these chlorinated compounds under each reducing condition, their biodegradability appears to be more a function of the presence of competent microbial populations than one of inherent molecular structure.     

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.     

H2/CO2 metabolism in acetogenic bacteria isolated from the human colon

The present work reports on autotrophic metabolism in four H2/CO2-utilizing acetogenic bacteria isolated from the human colon (two Clostridium species, one Streptococcus species, and Ruminococcus hydrogenotrophicus). H2/CO2-utilization by these human acetogenic strains occurred during both exponential and stationary phases of growth. Acetate was the major metabolite produced by all isolates following the stoichiometric equation of reductive acetogenesis. Furthermore, the ability of these acetogenic bacteria to incorporate 13CO2 into acetate in the presence of H2 in the gas phase demonstrated the utilization of the reductive pathway of acetate formation from a one-carbon compound. Energy conservation during the autotrophic metabolism in colonic acetogens might involve sodium- or proton-chemiosmotic mechanisms. A sodium-dependent ATP generation was only demonstrated in one Clostridium species, whereas sodium could be replaced by potassium in other strains. The minimal thresholds of hydrogen uptake were determined and varied from 1100 to 3680 ppm depending on the acetogenic strain. These values appeared higher than those measured for the colonic methanogen,Methanobrevibacter smithii.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum.

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

UV—B辐射对小麦叶片H2O2代谢的影响

研究了温室种植的小麦在０（ＣＫ）、８．８２ｋＪ／ｍ＾２（Ｔ１）和１２．６ｋＪ／ｍ＾２（Ｔ２）三种剂量的紫外线Ｂ（ＵＶ－Ｂ）辐射下Ｈ２Ｏ２含量的变化及其机理。ＵＶ－Ｂ辐射下Ｈ２Ｏ２、还原型抗坏血酸（ＡｓＡ）和还原型谷胱甘肽（ＧＳＨ）含量增加,抗坏血酸过氧化物酶（ＡＰｘ）和谷胱甘肽不原酶（ＧＲ）活性升高,脂肪酸不饱和度指数（ＩＵＦＡ）降低。ＳＤＳ－ＰＡＧＥ谱图没有质上的差异,但凝胶着色深浅有变化。分析     

Metal centers in the anaerobic microbial metabolism of CO and CO2

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO(2), for harnessing 'green' energy and producing biofuels. One strategy is to convert CO(2) into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO(2) and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO(2), we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO(2) and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe(4)S(4) clusters, catalyzes the addition and elimination of CO(2) during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron-sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B(12) and a Fe(4)S(4) cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co(3+) intermediate. Studies of CO and CO(2) enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C-C and C-S bond formations.     

Electrometrical study of electron transfer from the terminal FA/FB iron-sulfur clusters to external acceptors in photosystem I

An electrometrical technique was used to investigate electron transfer between the terminal iron-sulfur centers F(A)/F(B) and external electron acceptors in photosystem I (PS I) complexes from the cyanobacterium Synechococcus sp. PCC 6301 and from spinach. The increase of the relative contribution of the slow components of the membrane potential decay kinetics in the presence of both native (ferredoxin, flavodoxin) and artificial (methyl viologen) electron acceptors indicate the effective interaction between the terminal 14Fe-4S] cluster and acceptors. The finding that FA fails to donate electrons to flavodoxin in F(B)-less (HgCl2-treated) PS I complexes suggests that F(B) is the direct electron donor to flavodoxin. The lack of additional electrogenicity under conditions of effective electron transfer from the F(B) redox center to soluble acceptors indicates that this reaction is electrically silent.     

Effect of physiological electron donors and acceptors on F1-ATPase

The hydrolytic activity of F1-ATPase isolated from rat liver was enhanced in the presence of NADH, FADH2, QH2 or reduced cyt c. The extent of this activation depended largely on substrate concentration. F1-ATPase sensitivity to bicarbonate or dinitrophenol activators decreased in the presence of any of those electron donors, which originated as well a slight sensitivity to oligomycin and a sensitivity increase to the inhibitory anion OCN-. In the presence of oxidized carriers the sensitivity to bicarbonate, dinitrophenol, or OCN- was not modified, and the enzyme remained oligomycin insensitive.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

Electron bifurcation involved in the energy metabolism of the acetogenic bacterium Moorella thermoacetica growing on glucose or H2 plus CO2

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO(2) molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd(red)(2-)) molecules. In the reductive part, 2 CO(2) molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO(2) are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO(2) reduction to acetic acid are generated by the reduction of 2 NADP(+) molecules with 1 NADH and 1 Fd(red)(2-) catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD(+) and Fd with 2 H(2) molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H(2)-CO(2)-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H(2)-CO(2)-grown cells additionally contain high specific activities of an NADP(+)-dependent hydrogenase that catalyzes the reduction of NADP(+) with H(2). This activity is hardly detectable in glucose-grown cells.