Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.     

Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport

Polysulphide was formed according to reaction (1), when tetrathionate was (1) $${\text{S}}_4 {\text{O}}_6^{2 - } + {\text{HS}}^ - \to 2{\text{S}}_2 {\text{O}}_3^{2 - } + {\text{S(O)}} + {\text{H}}^ + $$ added to an anaerobic buffer (pH 8.5) containing excess sulphide. S(O) denotes the zero oxidation state sulphur in the polysulphide mixture S _infn ^sup2- . The addition of formate to the polysulphide solution in the presence of Wolinella succinogenes caused the reduction of polysulphide according to reaction (2). The bacteria grew in a medium containing formate and sulphide, (2) $${\text{HCO}}_2^ - + {\text{S(O)}} + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + {\text{HS}}^ - + {\text{H}}^ + $$ when tetrathionate was continuously added. The cell density increased proportional to reaction (3) which represents the sum of reactions (1) and (3) $${\text{HCO}}_2^ - + {\text{S}}_{\text{4}} {\text{O}}_6^{2 - } + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + 2{\text{S}}_{\text{2}} {\text{O}}_3^{2 - } + 2{\text{H}}^ + $$ (2). The cell yield per mol formate was nearly the same as during growth on formate and elemental sulphur, while the velocity of growth was greater. The specific activities of polysulphide reduction by formate measured with bacteria grown with tetrathionate or with elemental sulphur were consistent with the growth parameters. The results suggest that W. succinogenes grow at the expense of formate oxidation by polysulphide and that polysulphide is an intermediate during growth on formate and elemental sulphur.     

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants.

Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%).     

Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source

Saccharomyces cerevisiae is a widely used host organism for the production of heterologous proteins, often cultivated in glucose-based fed-batch processes. This production system however has many factors limiting the productivity, mainly towards the end of the fermentation. For the optimised production of a Camelid antibody fragment this process was evaluated. In shake flask cultivations, it was found that ethanol has a strong effect on productivity increase and therefore glucose and ethanol fed-batch fermentations were compared. It appeared that specific heterologous protein production was up to five times higher in the ethanol cultivation and could be further optimised. Then the key characteristics of ethanol fed-batch fermentations such as growth rate and specific production were determined under ethanol limitation and accumulation and growth limiting conditions in the final phase of the process. It appeared that an optimal production process should have an ethanol accumulation throughout the feed phase of approximately 1% v/v in the broth and that production remains very efficient even in the last phase of the process. This productivity increase on ethanol versus glucose was also proven for several other Camelid antibody fragments some of which were heavily impaired in secretion on glucose, but very well produced on ethanol. This leads to the suggestion that the ethanol effect on improved heterologous protein production is linked to a stress response and folding and secretion efficiency.     

Phosphorylative fumarate reduction in Vibrio succinogenes: Stoichiometry of ATP synthesis

1. Cells of Vibrio succinogenes, treated with EDTA at pH 8, catalyze the phosphorylation of their endogenous ADP and AMP as a function of the electron transport from formate to fumarate. The P/fumarate ratio obtained from the initial velocity of the phosphorylation on initiation of the electron transport and from the activity of fumarate reduction in the steady state was 0.90. The phosphorylation was prevented by 10μmol/g protein carbonylcyanide-3-chlorophenylhydrazone.
2. The esterification of external phosphate in the presence of ADP, hexokinase and glucose is catalysed by a membrane preparation of V. succinogenes in the steady state of fumarate reduction by H₂. The phosphorylation was fully abolished by either 5μmol/g protein carbonylcyanide-4-trifluoromethoxyphenylhydrazone or 30μmol/g protein carbonylcyanide-3-chlorphenylhydrazone. Phosphorylation was blocked also by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent membrane bound ATP synthase, and by low concentrations of the inhibitors of electron transport 2-(n-nonyl)-4-hydroxyquinoline-N-oxide or 4-chloromercuriphenylsulfonate.
3. The P/fumarate ratios, measured with the membrane preparation, were found to increase with progressive inhibition of the electron transport from hydrogen to fumarate by means of 4-chloromercuriphenylsulfonate. The extrapolated ratio at vanishing electron transport activity was 0.47.
4. About 50% of the membrane preparation was found to consist of inverted vesicles with the hydrogenase and formate dehydrogenase oriented to the inside. The residual part is considered as being incapable of performing energy transduction. The extrapolated P/fumarate ratio valid for the inverted vesicles was 0.94.

     

Geobacter sulfurreducens can grow with oxygen as a terminal electron acceptor

Geobacter sulfurreducens, previously classified as a strict anaerobe, tolerated exposure to atmospheric oxygen for at least 24 h and grew with oxygen as the sole electron acceptor at concentrations of 10% or less in the headspace. These results help explain how Geobacter species may survive in oxic subsurface environments, being poised to rapidly take advantage of the development of anoxic conditions.     

Fe(III) reduction activity and cytochrome content of Shewanella putrefaciens grown on ten compounds as sole terminal electron acceptor

Shewanella putrefaciens was grown on a series of ten alternate compounds as sole terminal electron acceptor. Each cell type was analyzed for Fe(III) reduction activity, absorbance maxima in reduced-minus-oxidized difference spectra and heme-containing protein content. High-rate Fe(III) reduction activity, pronounced difference maxima at 521 and 551 nm and a predominant 29.3 kDa heme-containing protein expressed by cells grown on Fe(III), Mn(IV), U(VI), SO3(2-) and S2O3(2-), but not by cells grown on O2, NO3, NO2-, TMAO or fumarate. These results suggest that microbial Fe(III) reduction activity is enhanced by anaerobic growth on metals and sulfur compounds, yet is limited under all other terminal electron-accepting conditions.     

Vectorial electron transport in Degulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source in a medium containing excess iron. The topography of electron transport components was investigated. The bacterium contained per mg cells (dry weight) 30U hydrogenase (1U=1 mol/min), 35 g desulfoviridin (= bisulfite reductase), 0.6 U adenosine phosphosulfate reductase, 30 mU thiosulfate reductase, 0.3 nmol cytochrome c ₃ (M _r=13,000), 0.04 nmol cytochrome b, 0.85 nmol menaquinone, and 0.4 nmol ferredoxin. Hydrogenase (>95%) and cytochrome c ₃ (82%) were localized on the periplasmic side and desulfoviridin (95%), adenosine phosphosulfate reductase (87%), thiosulfate reductase (74%), and ferredoxin (71%) on the cytoplasmic side of the cytoplasmic membrane; menaquinone and cytochrome b were exlusively found in the membrane fraction. The location of the oxidoreductases indicate that in D. vulgaris (Marburg) H₂ oxidation and sulfate reduction take place on opposite sides of the cytoplasmic membrane rather than on the same side, as has recently been proposed.     

Respiration and growth of Shewanella decolorationis S12 with an Azo compound as the sole electron acceptor

The ability of Shewanella decolorationis S12 to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory azoreduction was investigated. This microorganism can reduce a variety of azo dyes by use of formate, lactate, pyruvate, or H(2) as the electron donor. Furthermore, strain S12 grew to a maximal density of 3.0 x 10(7) cells per ml after compete reduction of 2.0 mM amaranth in a defined medium. This was accompanied by a stoichiometric consumption of 4.0 mM formate over time when amaranth and formate were supplied as the sole electron acceptor and donor, respectively, suggesting that microbial azoreduction is an electron transport process and that this electron transport can yield energy to support growth. Purified membranous, periplasmic, and cytoplasmic fractions from S12 were analyzed, but only the membranous fraction was capable of reducing azo dyes with formate, lactate, pyruvate, or H(2) as the electron donor. The presence of 5 microM Cu(2+) ions, 200 microM dicumarol, 100 microM stigmatellin, and 100 microM metyrapone inhibited anaerobic azoreduction activity by both whole cells and the purified membrane fraction, showing that dehydrogenases, cytochromes, and menaquinone are essential electron transfer components for azoreduction. These results provide evidence that the microbial anaerobic azoreduction is linked to the electron transport chain and suggest that the dissimilatory azoreduction is a form of microbial anaerobic respiration. These findings not only expand the number of potential electron acceptors known for microbial energy conservation but also elucidate the mechanisms of microbial anaerobic azoreduction.     

Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Microbial arsenite oxidation with oxygen,nitrate, or an electrode as the sole electron acceptor

The purpose of this study was to identify bacteria that can perform As(III) oxidation for environmental bioremediation. Two bacterial strains, named JHS3 and JHW3, which can autotrophically oxidize As(III)–As(V) with oxygen as an electron acceptor, were isolated from soil and water samples collected in the vicinity of an arsenic-contaminated site. According to 16S ribosomal RNA sequence analysis, both strains belong to the ?-Proteobacteria class and share 99% sequence identity with previously described strains. JHS3 appears to be a new strain of the Acinetobacter genus, whereas JHW3 is likely to be a novel strain of the Klebsiella genus. Both strains possess the aioA gene encoding an arsenite oxidase and are capable of chemolithoautotrophic growth in the presence of As(III) up to 10 mM as a primary electron donor. Cell growth and As(III) oxidation rate of both strains were significantly enhanced during cultivation under heterotrophic conditions. Under anaerobic conditions, only strain JHW3 oxidized As(III) using nitrate or a solid-state electrode of a bioelectrochemical system as a terminal electron acceptor. Kinetic studies of As(III) oxidation under aerobic condition demonstrated a higher V _max and K _m from strain JHW3 than strain JHS3. This study indicated the potential application of strain JHW3 for remediation of subsurface environments contaminated with arsenic.     

Amperometric method for monitoring anaerobic respiration with nitrous oxide as the terminal electron acceptor

A commercially available Clark-type electrode with a platinum cathode, commonly used for monitoring oxygen uptake, may also be used to monitor respiration with N₂O. The disadvantages of this system include a high sensitivity to O₂, which may be overcome by excluding O₂, and sensitivity to acetylene. The advantage of the method is that it may be used to monitor directly the reduction of N₂O by respiring cells.     

Respiration and growth of Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor

Shewanella oneidensis MR-1 is a free-living gram-negative gamma-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex.     

Comparative analysis of membranous proteomics of Shewanella decolorationis S12 grown with azo compound or Fe (III) citrate as sole terminal electron acceptor

Shewanella decolorationis S12 is capable of carrying out anaerobic respiration using azo dyes and Fe (III) citrate as electron acceptors. In the present study, proteomic techniques including two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry were used to analyze the similarity and the dissimilarity of the membrane proteins isolated from strain S12 cells grown in amaranth or Fe (III) citrate with defined inorganic salt medium. The cells of strain S12 grown under a saturated dissolved oxygen condition served as controls. This is the first work that made the comparative analysis of cell membranous proteomics of strain S12 grown with azo compound or Fe (III) citrate as a sole terminal electron acceptor. The results showed that most of the membrane proteins of strain S12 under azo respiration are similar to those under Fe (III) respiration, but dissimilar from those of oxygen-grown cells. FdnH and FrdB were expressed specifically in azo respiration. NqrA-2, DctP, and hypothetical protein SO_4719 showed relative overexpression in azo respiration compared with Fe (III) respiration. OmpA family protein SO_3545 was detected to be specific to Fe (III) respiration. Furthermore, ArgF, SdhA, and HoxK were expressed markedly in both amaranth- and Fe (III) citrate-grown cultures compared with oxygen-grown cultures.     

Growth yield of denitrifiers using nitrous oxide as a terminal electron acceptor

The molar yields (g cell/mol) forAlcaligenes faecalis, Pseudomonas stutzeri, Paracoccus denitrificans andPseudomonas perfectomarinus batch cultures, under nitrous oxide (N₂O) as the electron acceptor, were 11.2, 8.2, 6.1 and 4.4, respectively.Paracoccus denitrificans andPseudomonas perfectomarinus, which had the slowest growth rates, gave the lowest yields. Large maintenance energy costs may be partially responsible for this. The growth efficiencies ofA. faecalis andPs. perfectomarinus on N₂O indicate that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N₂O reduction are about 49% and 39% of those in the electron transport system associated with O₂ respiration, respectively.     

Dibenzothiophene sulfur can serve as the sole electron acceptor during growth by sulfate-reducing bacteria

Summary Three species of Sulfate-Reducing Bacteria (SRB) were able to grow using dibenzothiophene (DBT) as their sole source of sulfur and sole electron acceptor. Desulfotomaculum orientis and Desulfovibrio desulfuricans were grown at 30°C while Thermodesulfobacterium commune was grown at 60°C, in media containing lactate and citrate. Hydrogen sulfide was the product of dissimilatory sulfur reduction.Research supported by the Office of Oil and Gas Processing of Fossil Energy, U.S. Department of Energy under contract DE-AC05-84OR21400 with Martin Marietta Energy Systems Inc.