ALBERT

Notes: Abstract The interconversion of adenine nucleotides during acetate fermentation was investigated with concentrated cell suspensions of Methanothrix soehngenii. Starved cells contained high levels of AMP (2.2 nmol/mg protein), but had hardly any ADP or ATP. The energy charge of these cells was 0.1. Immediately after the addition of the substrate acetate, the level of ATP increased, reaching a maximum of 1.4 nmol/mg protein, corresponding to an energy charge of 0.7 when half of the acetate was consumed. Once the acetate was depleted, the ATP concentration decreased to its original level of 0.1 nmol/mg protein. As M. soehngenii contained relatively high amounts of AMP, the luciferase system for the determination of ATP gave not always satisfactory results. Therefore a reliable method based on the separation of adenine nucleotides by anion exchange HPLC was used.

Notes: Abstract The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to Mr 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has Km apparent values of 23 μM and 2 mM for N-7-mercaptoheptanoylthreonine phosphate (HS- HTP=componentB) and methyl-coenzyme M (CH3?CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein.

Notes: A newly isolated Pseudomonas species, strain P51, growing aerobically on all dichlorobenzene isomers and on 1,2,4-trichlorobenzene as sole carbon and energy sources was tested for its ability to mineralize these components also in a non-sterile soil environment. Untreated sand from the river Rhine in which none of the dichloro- and trichlorobenzenes were degraded was placed in a percolation column and inoculated with Pseudomonas sp. strain P51. The column was fed continuously with synthetic river water containing the chlorinated compounds at concentrations between 10 μg/1 and 1 mg/1. The inoculated microorganisms were able to degrade the chlorinated benzenes and survived for at least 60 days in the column. For each compound a specific threshold concentration was observed below which no further degradation took place, and which was independent of the initial concentration. These threshold were 6 ± 4 μg/1 for 1,2-dichlorobenzene, 20 ± 5 μg/1 for 1,2,4-trichlorobenzene and more than 20 μg/1 for the 1,3- and 1,4-isomers. Repeated inoculation of the column with strain P51 did not affect this minimal concentration. In noninoculated soil columns the native microbial population adapted to degrade 1,2-dichlorobenzene after a long lag phase, and reduced it from 25 μg/1 to a threshold concentration of 0.1 μg/1.

Notes: Abstract The minimum threshold concentrations of acetate utilization and the enzymes responsible for acetate activation of several methanogenic bacteria were investigated and compared with literature data. The minimum acetate concentrations reached by hydrogenotrophic methane bacteria, which require acetate as carbon source, were between 0.4 and 0.6 mM. The acetoclastic Methanosarcina achieves acetate concentrations between 0.2 and 1.2 mM and Methanothrix between 7 and 70 μM. For the activation of acetate most of the hydrogenotrophic methane bacteria investigated use an acetyl-CoA synthetase with a relatively low Km (40–90 μM) for acetate. although the affinity for acetate was high, the hydrogenotrophic methane bacteria were not able to remove acetate to lower concentrations than the acetoclastic methane bacteria, neither in pure cultures nor in anaerobic granular sludge samples. Based on these observations, it is not likely that hydrogenotrophic methanogens compete strongly for acetate with the acetoclastic methane bacteria.

Notes: Abstract Acetate is the precursor of approximately two-thirds of the methane produced in anaerobic bioreactors. Only two genera of methanogenic archae are known to use acetate as sole energy source: Methanosarcina and Methanothrix. Methanosarcina appears to be a generalist with a high growth rate, but low affinity for acetate. Methanothrix is a specialist having a high affinity for acetate, but low growth rate. Methanothrix shows a much lower minimum threshold for acetate utilization (7–70 μM) than Methanosarcina (0.2–1.2 mM). This is consistent with the evidence that Methanothrix is found in environments with low acetate concentrations.The acetate degradation by acetotrophic methanogens starts with an activation of acetate to acetyl-coenzyme A. In Methanosarcina spp. this activation is catalysed by an acetate kinase/phosphotransacetylase system at the expense of one ATP. Acetyl-coenzyme A synthetase activates acetate in Methanothrix, with concomitant hydrolysis of one ATP to AMP and PPi. Both enzyme systems have been purified and comparison of the kinetic properties confirmed the hypothesis that low acetate concentrations favour Methanothrix. The gene encoding for acetyl-CoA synthetase of Methanothrix was isolated from a genomic library and actively expressed in Escherichia coli. The deduced amino acid sequence showed homology to proteins with similar function and contained two putative ATP binding sites.The most characteristic and complex enzyme involved in the acetate degradation by acetotrophic methanogens is carbon monoxide dehydrogenase. The enzyme has been purified from both Methanothrix and Methanosarcina, and represents 5–10% of the soluble protein of these microorganisms. CO dehydrogenase is proposed to catalyse both the cleavage of acetyl-CoA in a methyl-, carbonyl- and CoA-moiety, and the oxidation of the carbonyl group to CO2. This multifunctional redox enzyme contains several iron, acid-labile sulfur and nickel atoms. These atoms are arranged into several paramagnetic complexes, which have been studied by EPR spectroscopy. The low spin recovery of the different paramagnetic centers makes statements about structure and functions difficult. There are good spectroscopic and genetic indications that the CO dehydrogenase of Methanothrix contains at least one ferrodoxin-like [4Fe-4S] cluster, which could play a role in the electron transfer of the CO oxidation. Further, in EPR spectra of concentrated samples of CO dehydrogenase from Methanothrix a very unusual signal was observed, which showed great similarity to putative [6Fe-6S] prismane clusters.The final step in the methanogenesis from acetate, the reduction of methyl-coenzyme M, is catalysed by methyl-coenzyme M methylreductase. The enzyme purified from Methanothrix and Methanosarcina showed great homology with the methyl-CoM methylreductase of other methanogenic archae, although the specific activity was rather low (60–125 nmol min−1 mg−1).The reduction of the heterodisulfide between coenzyme M and component B is proposed to be the common site for energy conservation in all methanogens. Acetoclastic methanogens, however, need additional sites of energy conservation to compensate for their high energy input in acetate activation. The oxidation of CO to CO2 could form one possible site. The partially membrane-associated pyrophosphatase of Methanothrix could form another site of energy conservation.

Notes: Abstract All three isomers of trichlorobenzene were reductively dechlorinated to monochlorobenzene via dichlorobenzenes in anaerobic sediment columns. The dechlorination was specific: 1,2,3- and 1,3,5-trichlorobenzene were solely transformed to 1,3-dichlorobenzene, while 1,4-dichlorobenzene was the only product of 1,2,4-trichlorobenzene transformation. Microorganisms were responsible for the observed transformations. Since monochlorobenzene and dichlorobenzene are mineralized by bacteria in the presence of oxygen, the process of reductive dechlorination may be an important initial step to obtain complete mineralization of otherwise recalcitrant trichlorobenzenes. This is especially true for the 1,3,5-isomer, which seems to resist biodegradation in oxic environments.

Notes: Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min−1 mg−1 protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min−1 mg−1 protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F420 showed only low activity as electron acceptor.

Notes: Toluene degradation occurred in anaerobic flow-through sediment columns filled with contaminated sediment and sludge to which either amorphous or highly crystalline manganese oxide was added. An enrichment culture from these sediment columns was able to grow on toluene under strictly anaerobic conditions in the presence of manganese oxide. The oxidation of toluene was coupled to the production of CO2 and to the reduction of Mn(IV). Of the different manganese oxides tested, the rate was slowest with crystalline manganese oxide. After several transfers of the enrichment culture, its ability to degrade toluene became less and it was ultimately lost, unless sterilized Rhine river sediment was present in the medium. Direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could be further increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid.

Notes: A bacterial culture (LET-13) was enriched, which uses toluene as sole carbon and energy source, and manganese oxide as terminal electron acceptor. The culture is able to degrade a variety of substituted monoaromatic compounds like p-hydroxy-benzylalcohol, p-hydroxy-benzaldehyde, p-hydroxy-benzoate, phenol and the three isomers of cresol. Benzene, ethylbenzene, all xylenes and naphthalene were not degraded under the experimental conditions used. Based on the results of growth experiments and the detection of intermediates, it is concluded that toluene is degraded via a methyl hydroxylation. A possible side reaction can lead to the formation of cresol. The organisms in the culture look similar; motile rods, which are Gram-negative, oxidase-negative and catalase-negative. The culture was partly identified by phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria shows 70–80% similarity (based on 16S rRNA gene sequence data) with the Bacteroides-Cytophaga group, and one group consists of members of the β-subclass of the Proteobacteria.

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