Carrier-mediated acetate transport in Acetobacterium woodii

Intracellular and extracellular acetate concentrations of Acetobacterium woodii DSM 1030 were determined during growth or incubation of resting cell suspensions. The internal concentrations during growth decreased from initially 350 mM to 145 mM at the end of the experiment. The intracellular pH was lowered from 7.5 to 6.6 and the pH was enlarged from 0.2 to 0.6 units. Both, growing and resting cells of A. woodii showed no equilibrium between internal and external acetate concentrations during glucose consumption; the internal concentrations were always higher than expected assuming equal concentrations of the free acid inside and outside the cells. From counterflow experiments it is suggested that acetate does not only leave A. woodii cells by passive diffusion but also by carrier-mediated transport.     

Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodii.

Growth of Acetobacterium woodii on fructose was stimulated by Na+; this stimulation was paralleled by a shift of the acetate-fructose ratio from 2.1 to 2.7. Growth on H2-CO2 or on methanol plus CO2 was strictly dependent on the presence of sodium ions in the medium. Acetate formation from formaldehyde plus H2-CO by resting cells required Na+, but from methanol plus H2-CO did not. This is analogous to H2-CO2 reduction to methane by Methanosarcina barkeri, which involves a sodium pump (V. Müller, C. Winner, and G. Gottschalk, Eur. J. Biochem. 178:519-525, 1988). This suggests that the reduction of methylenetetrahydrofolate to methyltetrahydrofolate is the Na+-requiring reaction. A sodium gradient (Na+ out/Na+ in = 32, delta pNa = -91 mV) was built up when resting cells of A. woodii were incubated under H2-CO2. Acetogenesis was inhibited when the delta pNa was dissipated by monensin.     

Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.     

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂‐fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch‐operated stirred‐tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g_acetate L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH₂ a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g_acetate g day⁻¹ under hydrogenotrophic conditions. Biotechnol. Bioeng. 2011;108: 470–474. © 2010 Wiley Periodicals, Inc.     

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

CO Metabolism in the Acetogen Acetobacterium woodii

The Wood-Ljungdahl pathway allows acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model acetogen Acetobacterium woodii. When cells grew on H₂-CO₂, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H₂ consumption. Experiments with resting cells revealed that the degree of inhibition of H₂ consumption was a function of the CO concentration. Since the hydrogen-dependent CO₂ reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.     

Catechol production by O-demethylation of 2-methoxyphenol using the obligate anaerobe, Acetobacterium woodii

Acetobacterium woodii produced catechol (up to 7.84 mM) by demethylating 2-methoxyphenol during growth in the presence or absence of fructose. The highest product concentrations were obtained when 2-methoxyphenol was the sole energy source but the highest substrate conversion (97%) was obtained in fructose-limited chemostat culture. Growing cells were the most suitable form of the biocatalyst since the catalytic activity was 5-fold higher than in harvested cells.     

Production and Utilization of Ethanol by the Homoacetogen Acetobacterium woodii

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

Nickel requirement of Acetobacterium woodii.

Growth of Acetobacterium woodii on H2 and CO2 rather than on fructose was dependent on nickel. Nickel-deprived cultures growing on fructose did not synthesize acetate from CO2; under these conditions hydrogen formation was used as the electron sink. The data indicate that nickel is involved in CO2 reduction to acetate in A. woodii.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 10³ transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 × 10^-6 to 7 × 10^-6 per donor or recipient.     

Tetrahydrofolate enzyme levels in Acetobacterium woodii and their implication in the synthesis of acetate from CO2

Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate.     

Transformation of Tetrachloromethane to Dichloromethane and Carbon Dioxide by Acetobacterium woodii

[This corrects the article on p. 2822 in vol. 54.].     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO₂ in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.     

Regulation of Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO₂, and once a culture was induced, caffeate and CO₂ were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H₂ plus CO₂ as the substrate, but the lag phase was much longer. Again, caffeate and CO₂ were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO₂ simultaneously with fructose or hydrogen as electron donors, but CO₂ was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.