Atypical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermoacetica strain

A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bioreactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the acetogenic bacterium Moorella thermoacetica DSM 521T (98.3% sequence similarity). DNA¿DNA hybridization showed 75.2 ± 4.7% similarity to M. thermoacetica DSM 521T, suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sustained in coculture with a hydrogen-consuming methanogen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulfide formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H2/CO2, unlike M. thermoacetica (DSM 521T). The lack of growth with H2/CO2 likely is due to the absence of cytochrome b in strain AM

Isolation and characterization of Alicycliphilus denitrificans strain BC, which grows on benzene with chlorate as the electron acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 microm wide and 1 to 2 microm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplie

Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts

A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic

Benzene degradation coupled with chlorate reduction in soil column study

Perchlorate and chlorate are electron acceptors that during reduction result in the formation of molecular oxygen. The produced oxygen can be used for activation of anaerobic persistent pollutants, like benzene. In this study chlorate was tested as potential electron acceptor to stimulate benzene degradation in anoxic polluted soil column. A chlorate amended benzene polluted soil column was operated over a period of 500 days. Benzene was immediately degraded in the column after start up, and benzene removal recovered completely after omission of chlorate or a too high influent chlorate concentration (22 mM). Mass balance calculations showed that per mole of benzene five mole of chlorate were reduced. At the end of the experiment higher loading rates were applied to measure the maximal benzene degradation rate in this system; a breakthrough of benzene was not observed. The average benzene degradation rate over this period was 31 ¿mol l¿1 h¿1 with a maximal of 78 ¿mol l¿1 h¿1. The high degradation rate and the necessity of chlorate indicate that oxygen produced during chlorate reduction indeed is used for the activation of benzene. This is the first column study where benzene biodegradation at a high rate coupled with anaerobic chlorate reduction is observe

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of -hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of -hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of -hexachlorocyclohexane within 4months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (-, -, -, and -), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30°C. Dechlorination of -hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10mM sulphate in the influent led to simultaneous dechlorination of -hexachlorocyclohexane and sulphate reduction. When the column was fed with 10mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of -HCH. The significance of these experiments for in situ bioremediation of polluted soils is discusse

Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen form an anaerobic reactor fed with methanol

A novel thermophilic, obligately methylotrophic, methanogenic archaeon, strain L2FAWT, was isolated from a thermophilic laboratory-scale upflow anaerobic sludge blanket reactor fed with methanol as the carbon and energy source. Cells of strain L2FAWT were non-motile, irregular cocci, 0·7¿1·5 µm in diameter and usually occurred singly (sometimes forming clusters of two or four cells). The cells stained Gram-negative and lysed immediately in 0·1 % (w/v) SDS. Growth was inhibited by chloramphenicol and tetracycline, but not by penicillin, bacitracin, spectinomycin, vancomycin or kanamycin. Methanol and mono-, di- and trimethylamine were used as substrates, but H2/CO2, formate, acetate, propanol, dimethyl sulfide and methanethiol were not. The temperature range for growth was 42¿58 °C, with an optimum at 50 °C. The fastest growth was observed at a salinity below 100 mM NaCl; no growth occurred above 300 mM NaCl. The optimal pH for growth was 6·5; growth was observed from pH 5 to pH 7·5. The G+C content of the genomic DNA was 37·6 mol%. Analysis of the 16S rRNA gene sequence and the partial methyl-CoM reductase gene sequence revealed that the organism was phylogenetically closely related to Methanomethylovorans hollandica DMS1T (98 % similarity for the 16S rRNA gene sequence and 91 % similarity for the methyl-CoM reductase gene sequence). The DNA¿DNA relatedness between L2FAWT and Methanomethylovorans hollandica DMS1T was 46 %. On the basis of these results, strain L2FAWT (=DSM 17232T=ATCC BAA-1173T) represents the type strain of a novel species, for which the name Methanomethylovorans thermophila sp. nov. is proposed

Reductive dechlorination of B-hexachlorocyclohexane (B-HCH) by a Dehalobacter species in coculture with a Sedimentibacter species

An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the ß-HCH isomer to benzene and chlorobenzene in a ratio of 0.5¿2 depending on the amount of ß-HCH degraded. The culture grows with H2 as electron donor and ß-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate ß-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of ß-HCH by a defined anaerobic bacterial culture