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1.
Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.  相似文献   

2.
Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.  相似文献   

3.
Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.  相似文献   

4.
In free-living Rhizobium japonicum cultures, the stimulatory effect of CO2 on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO2 fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO2-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.  相似文献   

5.
The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH2) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997  相似文献   

6.
Electron mediators and electron supply through a cathode were examined to enhance the reducing power for butyrate production by an acidogenic clostridium strain, Clostridium tyrobutyricum BAS 7. Among the tested electron mediators, methyl viologen (MV)‐amended cultures showed an increase of butyrate productivity (1.3 times), final concentration (1.4 times), and yield (1.3 times). The electron flow altered by MV addition from the ferredoxin pool to the NADH pool was shown by one electron model, implying that more available NADH increased butyrate production. In the cathode compartment poised at ?400 mV versus the Ag/AgCl electrode, the neutral red (NR)‐amended cultures of Clostridium tyrobutyricum BAS 7 increased butyrate concentration (from 5 to 8.8 g/L) and yield (from 0.33 up to 0.44 g/g) with no acetate production at all. Given that electrically reduced NR (NRred, yellow) by the cathode was re‐oxidized (NRox, red) in the cells on the basis of color change, electron flow from NRred to NAD+ (i.e., NADH generation) induced an increase in butyrate production. This is the first report to show the increase of butyric acid production by electrically driven acidogenesis. These results show that the electron flow altered NADH formation by electron mediators and by the cathodic electron donor, increasing the yield and selectivity of reduced end‐products like butyrate. Biotechnol. Bioeng. 2012; 109: 2494–2502. © 2012 Wiley Periodicals, Inc.  相似文献   

7.
8.
Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD+ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD+, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.  相似文献   

9.
Pure cultures of H2/CO2- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H2/CO2 at H2 partial pressure in the gas phase above 200 kPa. At decreasing H2 partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (–180 to –200 mV), formate-utilizing methanogens produced high amounts of formate from H2/CO2. No formate was excreted by the species, which could only utilize H2/CO2 for methanogenesis. In contrast, H2 formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H2/CO2 and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H2, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H2 transfer. Correspondence to: J. Winter  相似文献   

10.
Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H+/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H+/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone  相似文献   

11.
The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP+-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.  相似文献   

12.
Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.  相似文献   

13.
The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.  相似文献   

14.
The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.  相似文献   

15.
A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [13C]formate, as well as 14C-labeling experiments, demonstrated production of labeled CO2 in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO2 formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.  相似文献   

16.
An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)3), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.  相似文献   

17.
  1. With fumarate as the terminal electron acceptor and either H2 or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium.
  2. Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate.
  3. Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis.
  4. In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present.
  5. During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H2 or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO2.
  6. The α-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H2 or formate were not detected in the bacterium.
  相似文献   

18.
Wild-type Anabaena cycadeae with normal glutamine synthetase (GS) activity utilized arginine as sole N source whereas a mutant strain lacking GS activity did not. Nitrate reductase (NR) activity, higher in the mutant strain than the wild-type strain, was inhibited by arginine though arginine-dependent NH 4 + generation was higher in the mutant strain than in the wild-type. This suggests that (1) NR activity is NO inf3 sup- -inducible and arginine-repressible; and (2) while GS activity is required for the assimilation of arginine as sole N-source, it is not required for arginine inhibition of NR activity.S. Singh was with the Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India, and is now with P.S. Bisen at the Department of Microbiology, Barkatullah University, Bhopal-462026, India  相似文献   

19.
A Δsud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The Δpsr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity. Received: 31 August 1995 / Accepted: 25 October 1995  相似文献   

20.
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