H2 as an energy source for mixotrophic acetogenesis from the reduction of CO2 and syringate by Acetobacterium woodii and Eubacterium limosum

Acetobacterium woodii and Eubacterium limosum were grown in a defined medium or suspended in buffer containing syringate as the sole organic carbon source to test whether H₂ would support acetogenesis from a phenylmethylether and CO₂. When growing and resting cell preparations were incubated under various headspace gas compositions, consistent O-demethylation activity and growth were observed on syringate-CO₂ when H₂ served as the sole energy source. However, the dependency for H₂ was relieved by addition of yeast extract. A hypothetical scheme representing hydrogenolytic (reductive) O-demethylation and mixotrophic acetogenesis is proposed in light of these results and other literature observations.     

Anaerobic biodegradation of methyl esters byAcetobacterium woodii andEubacterium limosum

Summary The ability ofAcetobacterium woodii andEubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H₂ requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO₂ allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.     

A phylogenetic analysis ofAcetobacterium woodii,Clostridium barkeri,Clostridium butyricum,Clostridium lituseburense,Uubacterium limosum,andEubacterium tenue

Acetobacterium woodii, Butyribacterium rettgeri, Clostridium barkeri, Clostridium butyricum, Clostridium lituseburense, Eubacterium limosum, andEubacterium tenue have been characterized by oligonucleotide cataloging of their 16S ribosomal RNAs.A. woodii, C. barkeri, andE. limosum form a related group.C. lituseburense andE. tenue are highly related. As previously reported,E. limosum andB. rettgeri are synonymous. TheEubacterium species examined here are less related to each other than they are to different species ofClostridium. Mol% G+C of DNA and murein composition support the 16S ribisomal RNA data. These findings question the validity of current taxonomic distinctions for these Gram-positive anaerobes.     

Acetic acid from H2 and CO2

Cell extracts of a nonsporeforming strictly anaerobic bacterium, Acetobacterium woodii produced acetate in N-tris(Hydroxymethyl)methyl-2-aminoethane sulfonic acid or phosphate buffers from hydrogen and carbon dioxide. The formation of acetate was not dependent on the presence of ATP in the reaction mixture; ADP also did not influence the acetate production. Since acetic acid is the main fermentation product during growth of A. woodii with H₂ and CO₂, ATP must be synthesized in the course of acetate formation. The possible sites of ATP synthesis are discussed.     

Differential effects of sodium ions on motility in the homoacetogenic bacteriaAcetobacterium woodii andSporomusa sphaeroides

The strictly anaerobic homoacetogenic bacteria Acetobacterium woodii and Sporomusa sphaeroides differ with respect to their energy metabolism. Since growth as well as acetate and ATP formation of A. woodii is strictly dependent on Na⁺, but that of S. sphaeroides is not, the question arose whether these organisms also use different coupling ions for mechanical work, i.e. flagellar rotation. During growth on fructose in the presence of Na⁺ (50 mM), cells of A. woodii were vigorously motile, as judged by light microscopy. At low Na⁺ concentrations (0.3 mM), the growth rate decreased by only 15%, but the cells were completely non-motile. Addition of Na⁺ to such cultures restored motility instantaneously. Motility, as determined in swarm agar tubes, was strictly dependent on Na⁺; Li⁺, but not K⁺ partly substituted for Na⁺. Of the amilorides tested, phenamil proved to be a specific inhibitor of the flagellar motor of A. woodii. Growth and motility of S. sphaeroides was neither dependent on Na⁺ nor inhibited by amiloride derivatives. These results indicate that flagellar rotation is driven by Δμ_Na ⁺ in A. woodii, but by Δμ_H ⁺ in S. sphaeroides. Received: 30 May 1995 / Accepted: 31 August 1995     

Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

Transient Production of Formate During Chemolithotrophic Growth of Anaerobic Microorganisms on Hydrogen

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H₂ and CO₂ as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H₂ and CO₂ using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H₂ was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H₂ partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H₂. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H₂. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H₂. Received: 24 November 1998 / Accepted: 30 December 1998     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Anaerobic degradation of tetrachloromethane by Acetobacterium woodii: separation of dechlorinative activities in cell extracts and roles for vitamin B12 and other factors

The transformations of ¹⁴CCl₄ by whole cells of Acetobacterium woodii suspended in phosphate buffer containing reducing agents, and by the cobalt corrinoid aquocobalamin in the same solution, were compared. Each catalyst transformed ¹⁴CCl₄ not only to reduced products (CHCl₃ and CH₂Cl₂) but also to CO and CO₂ as well as non-volatile products. The mass balance for radioactive carbon was complete in each case. Thus, the reactions of the pure cobalt corrinoid resemble the reactions in vivo. The proton in CHCl₃, formed from CCl₄ by A. woodii, was derived from water. Extracts of A. woodii were fractionated into large and small molecules, and each of the two fractions was separated chromatographically. Fractions of proteins demonstrated poor correlation between content of the corrinoid vitamin B₁₂ and rates of transformation of CCl₄. The correlation was somewhat improved if the fractions were autoclaved, but dechlorination in the absence of vitamin B₁₂ was observed. Separation of the small molecules yielded only one fraction containing vitamin B₁₂, and this fraction catalyzed dechlorination, whereas several other fractions were able to dechlorinate CCl₄ in the absence of vitamin B₁₂. We presume there to be unrecognized dechlorinative factors in anaerobic bacteria.Abbreviations GC gas chromatograph(y) - GC-MS (or-TCD or-FID) GC coupled to a mass spectrometer (or a thermal conductivity detector or a flame ionization detector) - HPLC high pressure liquid chromatograph(y) - FPLC high pressure protein chromatograph(y)     

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.     

Anaerobic fermentation of fructose in a mixed culture of fermentative and methanogenic bacteria

Summary Two newly isolated strains of Methanosarcina, strains JKAD and DALS, were grown in monoculture and in mixed culture in combination with Acetobacterium woodii WB1. Methanosarcina strains convert acetate into methane and carbon dioxide while Acetobacterium woodii grows on fructose, producing acetate via homoacetate fermentation. Monocultures of A. woodii in continuous culture consumed up to 6 mmoles g^-1 dry weight (dw) h^-1 of fructose and produced up to 12.9 mmoles g^-1 dw h^-1 of acetate at a dilution rate (D) of 0.13 h^-1. In batch growth the methanogenic bacteria produced up to 12.1 mmoles g^-1 dw h^-1 of CH₄ at a specific growth rate of 0.043 h^-1. In continuous cultivation the specific growth rate and the specific methane production of Methanosarcina were lower than in batch cultures, with values of 0.031 h^-1 and 3.1 mmoles g^-1 dw h^-1 of methane, respectively. In combination, A. woodii and Methanosarcina strain DALS in batch cultures completely converted fructose to methane and carbon dioxide with a maximum specific methane production rate of 1.9 mmoles g^-1 dw h^-1 of methane. In continuous cultivation these mixed cultures produced between 1.2 and 2 mmoles g^-1 dw h^-1 of CH₄ at a dilution rate of up to 0.043 h^-1. The methanogens were washed out at D values higher than 0.043 h^-1 for A. woodii and Methanosarcina strain JKAD, and higher than 0.05 h^-1 for A. woodii and Methanosarcina strain DALS. Data obtained from defined mixed cultures allow one to follow interactions in a mixed population of two species with different growth constants.     

Understorey gaps influence regeneration dynamics in subtropical coastal dune forest

Dominant understorey species influence forest dynamics by preventing tree regeneration at the seedling stage. We examined factors driving the spatial distribution of the monocarpic species Isoglossa woodii, a dominant understorey herb in coastal dune forests, and the effect that its cover has on forest regeneration. We used line transects to quantify the area of the forest understorey with I. woodii cover and with gaps in the cover. Paired experimental plots were established in semi-permanent understorey gaps with I. woodii naturally absent and in adjacent areas with I. woodii present to compare plant community composition, soil, and light availability between the two habitats. Isoglossa woodii was widespread, covering 65–95% of the understorey, while gaps covered the remaining 5–35% of the area. The spatial distribution of this species was strongly related to tree canopy structure, with I.␣woodii excluded from sites with dense tree cover. Seedling establishment was inhibited by low light availability (<1% of PAR) beneath I.␣woodii. When present, I. woodii reduced the density and species richness of tree seedlings. The tree seedling community beneath I. woodii represented a subset of the seedling community in gaps. Some species that were found in gaps did not occur beneath I. woodii at all. There were no significant differences between the sapling and canopy tree communities in areas with I. woodii gaps and cover. In the coastal dune forest system, seedling survival under I. woodii is dependent on a species’ shade tolerance, its ability to grow quickly during I. woodii dieback, and/or the capacity to regenerate by re-sprouting and multi-stemming. We propose a general conceptual model of forest regeneration dynamics in which the abundant understorey species, I. woodii, limits local tree seedling establishment and survival but gaps in the understorey maintain tree species diversity on a landscape scale.     

Catalase and Superoxide Dismutase in the Cells of Strictly Anaerobic Microorganisms

Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity. All of the investigated anaerobes possessed SOD activity, necessary for protection against toxic products of oxygen reduction. High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae. Most of the investigated clostridia and acetogens were catalase-negative. A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens. Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin. Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms. In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.     

A phylogenetic analysis of anaerobic eubacteria capable of synthesizing acetate from carbon dioxide

Acetobacterium woodii, Acetogenium kivui, Clostridium aceticum, C. acidiurici, C. cylindrosporum, C. formicoaceticum, C. thermoaceticum, Eubacterium limosum, andPeptococcus glycinophilus were characterized by oligonucleotide cataloging of their 16S ribosomal RNA to determine whether the ability to synthesize acetate from CO₂ is a phylogenetic trait. The ability to synthesize acetate from CO₂ apparently is not a valid phylogenetic marker. TheEubacterium andPeptococcus species examined here are less related to other species in their genera than they are to different species ofClostridium. TheEubacterium species examined here show little relatedness to the genusPropionibacterium. The acetogenic eubacteria belong to the phylogenetic group defined basically by the Gram-positive sporeforming anaerobes.     

Adh4, an alcohol dehydrogenase controls alcohol formation within bacterial microcompartments in the acetogenic bacterium Acetobacterium woodii

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.     

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years.Clostridium botulinum C2 toxin,Clostridium perfringens iota toxin andClostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization.Clostridium botulium exoenzyme C3 andClostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.     

Utilization of cathodic hydrogen by hydrogen-oxidizing bacteria

Summary Hydrogen is consumed by methanogenic, sulphate-reducing, and homoacetogenic bacteria and members of these bacterial groups are able to grow chemolithotrophically with hydrogen as sole energy source. Cathodic hydrogen consumption by sulphate-reducing bacteria has been proposed as one of the factors in the anaerobic corrosion of metals. Desulfovibrio spp. were able to utilize cathodic hydrogen from mild steel as the only source of energy for growth with sulphate or nitrate as terminal electron acceptor. Other hydrogen-oxidizing bacteria such as Methanospirillum hungatei, Acetobacterium woodii and Wolinella succinogenes were also able to utilize cathodic hydrogen from mild steel for energy generation and growth. Weight loss studies of mild steel coupons under different growth conditions of Desulfovibrio spp. indicated that hydrogen removal alone is not the cause of corrosion and the depolarization phenomenon probably plays a role only in the initiation of the anaerobic microbial corrosion process.     

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.