The effect of vinyl acetate in acetoclastic methanogenesis

The influence of vinyl acetate (VA) in the methanogenesis was evaluated, by using an upflow anaerobic sludge blanket reactor of 1.5L. The reactor was operated at 33.5 g/L volatile suspended solids to 30±2 °C, a hydraulic residence time of 1 day, an organic loading rate of 1 kgCOD/m3/d of two different mixtures of VA and glucose. The VA was methanized to 81% when its proportion was of 10% into reactor loading rate, when VA proportion increased to 25%, the methane production rate decreased to 62% and the acetate production rate increased almost 8 times. These results indicated that VA was only hydrolyzed and glucose was not used as a co-substrate. The effect of glucose on VA methanogenic degradation was evaluated through batch reactors of 60 mL, concluding that the glucose supported the methanogenesis without favoring the VA elimination. On the other hand, the results of the sludge from the reactor in the presence of VA demonstrated that VA caused an irreversibly inhibition of acetoclastic methanogenesis when the anaerobic sludge was exposed to this compound.     

Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: Involvement of an ancient horizontal gene transfer

The sulfate reducing bacteria Desulfovibrio vulgaris and the methanogenic archaea Methanosarcina barkeri can grow syntrophically on lactate. In this study, a set of three closely located genes, DVU2103, DVU2104, and DVU2108 of D. vulgaris, was found to be up-regulated 2- to 4-fold following the lifestyle shift from syntroph to sulfate reducer; moreover, none of the genes in this gene set were differentially regulated when comparing gene expression from various D. vulgaris pure culture experiments. Although exact function of this gene set is unknown, the results suggest that it may play roles related to the lifestyle change of D. vulgaris from syntroph to sulfate reducer. This hypothesis is further supported by phylogenomic analyses showing that homologies of this gene set were only narrowly present in several groups of bacteria, most of which are restricted to a syntrophic lifestyle, such as Pelobacter carbinolicus, Syntrophobacter fumaroxidans, Syntrophomonas wolfei, and Syntrophus aciditrophicus. Phylogenetic analysis showed that all three individual genes in the gene set tended to be clustered with their homologies from archaeal genera, and they were rooted on archaeal species in the phylogenetic trees, suggesting that they were horizontally transferred from archaeal methanogens. In addition, no significant bias in codon and amino acid usages was detected between these genes and the rest of the D. vulgaris genome, suggesting the gene transfer may have occurred early in the evolutionary history so that sufficient time has elapsed to allow an adaptation to the codon and amino acid usages of D. vulgaris. This report provides novel insights into the origin and evolution of bacterial genes linked to the lifestyle change of D. vulgaris from a syntrophic to a sulfate-reducing lifestyle.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

Hydrogen metabolism during methanogenesis from acetate by Methanosarcina barkeri

A tritium exchange assay and a sensitive gas chromatographic technique were used to demonstrate that hydrogenase was active and that hydrogen was produced by Methanosarcina barkeri strain MS grown on acetate. Both methane and hydrogen production rates were dependent on the concentration of acetate in the medium. H₂ was produced at 0.5–2% of the rate of CH₄ formation. Chloroform and potassium cyanide, inhibitors of methanogenesis from acetate, inhibited H₂ production but not hydrogenase activity. The addition of hydrogen gas to cell suspensions did not inhibit CH₄ or carbon dioxide production from the methyl group of acetate. H₂ production appears to be linked to several intracellular redox processes which follow the cleavage of acetate.     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.     

Involvement of a corrinoid enzyme in methanogenesis from acetate in Methanosarcina barkeri

Abstract Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H₂. Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate.
An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate.     

Evolution of gene fusions: horizontal transfer versus independent events

Background  

Gene fusions can be used as tools for functional prediction and also as evolutionary markers. Fused genes often show a scattered phyletic distribution, which suggests a role for processes other than vertical inheritance in their evolution.     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Effect of redox potential on methanogenesis by Methanosarcina barkeri

Concentrations of 0.5% O₂ immediately inhibited CH₄ production from methanol by Methanosarcina barkeri. Simultaneously, the redox potential of the medium increased to about +100 mV. However, the rates of CH₄ production were not significantly affected, when the redox potential of an anoxic medium was adjusted to values between -420 mV and +100 mV by addition of titanium (III) citrate, sodium dithionite, flavin adenine dinucleotide, or sodium ascorbate. When the redox potential was adjusted to values between -80 mV and +550 mV by means of mixtures of ferrocyanide and ferricyanide, CH₄ production was not inhibited until a redox potential of about +420 mV was reached. M. barkeri was able to reduce 0.5 mM ferricyanide solution at +430 mV within <30 min to a value of about +50 mV, and then to start CH₄ production. Higher ferricyanide concentrations were only partially reduced. The extent of reduction of ferricyanide was also dependent on the substrate concentration (methanol) and the density of the bacterial suspension. The results show that M. barkeri was able to generate to a certain extent by itself the redox environment which suited the production of CH₄. However, the bacteria probably have not enough reducing power to decrease the redox potential below the critical level of +50 mV, if O₂ is present at concentrations >0.005%.     

The effect of detergent on methanogenesis by Methanosarcina barkeri

Abstract both growth and methanogenesis of Methanosarcina barkeri are completely inhibited by sodium dodecylbenzene sulphonate at between 15 and 20 mg·1⁻¹. At lower concentrations growth of cultures was delayed, but no uncoupling of methanogenesis from growth was observed. Higher concentrations of detergent (50 mg·1⁻¹) produced marked alterations in the surface structures of organisms observed in scanning electron micrographs. Thus levels of a detergent common in anaerobic sewage treatment plants can inhibit methanogenesis, the terminal stage in the anaerobic digestion process.     

The effect of aromatic structure on the inhibition of acetoclastic methanogenesis in granular sludge

Summary Benzene derivatives are important constituents of certain effluents discharged by pulp and paper, petrochemical and chemical industries. The anaerobic treatment of these waste-waters can be limited due to methanogenic inhibition exerted by aromatic compounds. The objective of this study was to evaluate the effect of aromatic structure on acetoclastic methanogenic inhibition. The toxicity to acetoclastic methanogens was assayed in serum flasks utilizing granular sludge as inoculum. Among the monosubstituted benzenes, chlorobenzene, methoxybenzene and benzaldehyde were the most toxic with 50% inhibition occurring at concentrations of 3.4, 4.2 and 5.2 mm, respectively. In contrast, benzoate was the least inhibitory: concentrations up to 57.3 mm were non-toxic. In general, the toxicity of aromatic compounds increased with increasing length of aliphatic side-chains, increasing the number of alkyl or chlorine groups. The logarithm of the partition coefficient octanol/water (log P), an indicator of hydrophobicity, was observed to be positively correlated with the methanogenic inhibition. The results indicate that hydrophobicity is an important factor contributing to the high toxicity of the most inhibitory aromatic compounds.     

Two stage methanogenesis of glucose byAcetogenium kivui and acetoclastic methanogenic Sp.

Summary A continuous two stage anaerobic digestion process was established using a homoacetogen,Acetogenium kivui, as the acidogenic organism and an acetoclastic culture for the methanogenic stage. In continuous culture,A.kivui fermented 83% of a glucose carbon source to acetate at a critical dilution rate of 0.13/h. The effluent acetate from this culture was readily utilised by an acetoclastic methanogenic culture enriched from sewage sludge. The long term stability of this system was demonstrated under a range of conditions, and the potential process advantages discussed.     

Identification of methyl coenzyme M as an intermediate in methanogenesis from acetate in Methanosarcina spp

The transfer of the methyl group of acetate to coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) during the metabolism of acetate to methane was investigated in cultures of Methanosarcina strain TM-1. The organism metabolized CD3COO- to 83% CD3H and 17% CD2H2 and produced no CDH3 or CH4. The isotopic composition of coenzyme M in cells grown on CD3COO- was analyzed with a novel gas chromatography-mass spectrometry technique. The cells contained CD3-D-CoM and CD2H-S-CoM) in a proportion similar to that of CD3H to CD2H2. These results, in conjunction with a report (J.K. Nelson and J.G. Ferry, J. Bacteriol. 160:526-532, 1984) that extracts of acetate-grown strain TM-1 contain high levels of CH3-S-CoM methylreductase, indicate that CH3-S-CoM is an intermediate in the metabolism of acetate to methane in this organism.     

Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp.

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.     

Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer

The isopenicillin N synthase genes from three fungal species, three Gram-positive species, and one Gram-negative bacterial species share an unusually high sequence similarity. A phylogenetic analysis was carried out to determine which type of evolutionary scenario best accounts for this similarity. The most plausible scenario is one in which a horizontal gene-transfer event, from the prokaryotes to the eukaryotes, occurred at a time close to the divergence between the Gram-positive and the Gram-negative bacteria.