Anaerobic Biodegradation of Eleven Aromatic Compounds to Methane

A range of 11 simple aromatic lignin derivatives are biodegradable to methane and carbon dioxide under strict anaerobic conditions. A serum-bottle modification of the Hungate technique for growing anaerobes was used for methanogenic enrichments on vanillin, vanillic acid, ferulic acid, cinnamic acid, benzoic acid, catechol, protocatechuic acid, phenol, p-hydroxybenzoic acid, syringic acid, and syringaldehyde. Microbial populations acclimated to a particular aromatic substrate can be simultaneously acclimated to other selected aromatic substrates. Carbon balance measurements made on vanillic and ferulic acids indicate that the aromatic ring was cleaved and that the amount of methane produced from these substrates closely agrees with calculated stoichiometric values. These data suggest that more than half of the organic carbon of these aromatic compounds potentially can be converted to methane gas and that this type of methanogenic conversion of simple aromatics may not be uncommon.     

Bacterial Transformations of Naphthothiophenes

Naphthothiophenes are minor components of fossil fuels, and they can enter the environment from oil spills. Naphtho[2,1-b]thiophene, naphtho[2,3-b]thiophene, and 1-methylnaphtho[2,1-b]thiophene were synthesized and used in biodegradation studies with 1-methylnaphthalene (1-MN)-degrading Pseudomonas strains W1, F, and BT1. Cultures were incubated with one of the naphthothiophenes with or without 1-MN, acidified, and extracted with CH(inf2)Cl(inf2). The extracts were analyzed by gas chromatography with flame photometric and mass detectors to characterize sulfur-containing metabolites and with an atomic emission detector for quantification. Only strain W1 was able to grow on naphtho[2,1-b]thiophene, but strains F and BT1 cometabolized this compound if 1-MN was present. 1-MN was required by all three strains to metabolize naphtho[2,3-b]thiophene, which was more resistant to biodegradation than the [2,1-b] isomer. Two metabolites of naphtho [2,1-b]thiophene were purified, analyzed by (sup1)H nuclear magnetic resonance spectroscopy, and found to be 4-hydroxybenzothiophene-5-carboxylic acid (metabolite I) and 5-hydroxybenzothiophene-4-carboxylic acid (metabolite II). In cultures of strain W1 grown for 7 days on 52 (mu)mol of naphtho[2,1-b]thiophene, >84% of the substrate was degraded and metabolites I and II accounted for 19 and 9%, respectively, of the original amount of naphtho[2,1-b]thiophene. When 1-MN was present, strain W1 degraded >97% of the naphtho[2,1-b]thiophene and similar amounts of metabolite II were produced, but metabolite I did not accumulate. 1-MN was shown to promote the further degradation of metabolite I, but not of metabolite II, by strain W1. Thus, 1-MN enhanced the biodegradation of naphtho[2,1-b]thiophene. Approximately 70% of the 1-methylnaphtho [2,1-b]thiophene added to cultures of strain W1 with 1-MN was recovered as 4-hydroxy-3-methylbenzothiophene-5-carboxylic acid, the 3-methyl analog of metabolite I. The methyl substitution hindered further metabolism of 3-methyl-metabolite I even in the presence of 1-MN. Cometabolism of naphtho[2,3-b]thiophene yielded two products that were tentatively identified as 5-hydroxybenzothiophene-6-carboxylic and 6-hydroxybenzothiophene-5-carboxylic acids.     

Biogenesis of methane in primate dental plaque

Dental plaque samples collected from monkeys (Macaca mulatta) were found to contain a large amount of dissolved methane gas (0.6 nmol CH4/mg wet wt plaque). Enrichment cultures inoculated with dental plaque obtained from Macaca fascicularis produced methane when the medium contained ethanol, methanol, lactate, acetate or a hydrogen + CO2 atmosphere. Methane formation in the enrichments was inhibited by oxidation of the culture medium, autoclaving or the addition of 2-bromoethane sulfonic acid (BES). The methane producing enrichments were observed to contain fluorescent cocci occurring singly and in short chains. It was concluded that methane formation in the monkey dental plaque was the result of the presence of methanogenic bacteria.     

Transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans.

Cunninghamella elegans metabolized 1- and 2-methylnaphthalene primarily at the methyl group to form 1- and 2-hydroxymethylnaphthalene, respectively. Other compounds isolated and identified were 1- and 2-naphthoic acids, 5-hydroxy-1-naphthoic acid, 5-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, and phenolic derivatives of 1- and 2-methylnaphthalene. The metabolites were isolated by thin-layer and reverse-phase high-pressure liquid chromatography and characterized by the application of UV-visible absorption, 1H nuclear magnetic resonance, and mass spectral techniques. Experiments with [8-14C]2-methylnaphthalene indicated that over a 72-h period, 9.8% of 2-methylnaphthalene was oxidized to metabolic products. The ratio of organic-soluble in water-soluble metabolites at 2 h was 92:8, and at 72 h it was 41:59. Enzymatic treatment of the 48-h aqueous phase with either beta-glucuronidase or arylsulfatase released 60% of the metabolites of 2-methylnaphthalene that were extractable with ethyl acetate. In both cases, the major conjugates released were 5-hydroxy-2-naphthoic acid and 6-hydroxy-2-naphthoic acid. The ratio of the water-soluble glucuronide conjugates to sulfate conjugates was 1:1. Incubation of C. elegans with 2-methylnaphthalene under an 18O2 atmosphere and subsequent mass spectral analysis of 2-hydroxymethylnaphthalene indicated that hydroxylation of the methyl group is catalyzed by a monooxygenase.     

Methanogenesis,sulfate reduction and crude oil biodegradation in hot Alaskan oilfields

Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6–807 µmol S l^?1 day^?1), but of lesser consequence in Field B (0.1–10 µmol S l^?1 day^?1). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular‐based microbial community analysis of the methanogenic oil‐utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H₂‐using family Methanobacteriaceae. The findings demonstrate that oilfield‐associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.     

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 ± 0.003 nmol min⁻¹ mg of protein⁻¹ only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat

The emission of methane (1.3 mmol of CH(4) m(-2) day(-1)), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH(4)-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.     

Evidence of Active Methanogen Communities in Shallow Sediments of the Sonora Margin Cold Seeps

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.     

Analysis of the cytogenetic effect in human lymphocytes induced by metabolically activated 1- and 2-methylnaphthalene

Chromosome analyses were carried out in human lymphocytes treated in vitro with 1- and 2-methylnaphthalene (1-MN, 2-MN) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix there was no indication of induction of any significant cytogenetic effect by either compound. With S9 mix a weak clastogenic effect was apparent at 4 mM 2-MN only and sister-chromatid exchange frequencies were significantly increased at each dose of 1- and 2-MN, yet always less than twice the control level. The present observations do not indicate that 1- and 2-MN must be classified as potential genotoxic substances.     

Methanogenic Conversion of 3-S-Methylmercaptopropionate to 3-Mercaptopropionate

Anaerobic metabolism of dimethylsulfoniopropionate, an osmolyte of marine algae, in anoxic intertidal sediments involves either cleavage to dimethylsulfide or demethylation to 3-S-methylmercaptopropionate (MMPA) and subsequently to 3-mercaptopropionate. The methanogenic archaea Methanosarcina sp. strain MTP4 (DSM 6636), Methanosarcina acetivorans DSM 2834, and Methanosarcina (Methanolobus) siciliae DSM 3028 were found to use MMPA as a growth substrate and to convert it stoichiometrically to 3-mercaptopropionate. Approximately 0.75 mol of methane was formed per mol of MMPA degraded; methanethiol was not detected as an intermediate. Eight other methanogenic strains did not carry out this conversion. We also studied the conversion of MMPA in anoxic marine sediment slurries. Addition of MMPA (500 (mu)M) resulted in the production of methanethiol which was subsequently converted to methane (417 (mu)M). In the presence of the antibiotics ampicillin, vancomycin, and kanamycin (20 (mu)g/ml each), 275 (mu)M methane was formed from 380 (mu)M MMPA; no methanethiol was formed during these incubations. Only methanethiol was formed from MMPA when 2-bromoethanesulfonate (25 mM) was added to a sediment suspension. These results indicate that in natural environments MMPA could be directly or indirectly a substrate for methanogenic archaea.     

Methane-producing microbial community in a coal bed of the Illinois basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments

The potential for ferric iron reduction with fermentable substrates, fermentation products, and complex organic matter as electron donors was investigated with sediments from freshwater and brackish water sites in the Potomac River Estuary. In enrichments with glucose and hematite, iron reduction was a minor pathway for electron flow, and fermentation products accumulated. The substitution of amorphous ferric oxyhydroxide for hematite in glucose enrichments increased iron reduction 50-fold because the fermentation products could also be metabolized with concomitant iron reduction. Acetate, hydrogen, propionate, butyrate, ethanol, methanol, and trimethylamine stimulated the reduction of amorphous ferric oxyhydroxide in enrichments inoculated with sediments but not in uninoculated or heat-killed controls. The addition of ferric iron inhibited methane production in sediments. The degree of inhibition of methane production by various forms of ferric iron was related to the effectiveness of these ferric compounds as electron acceptors for the metabolism of acetate. The addition of acetate or hydrogen relieved the inhibition of methane production by ferric iron. The decrease of electron equivalents proceeding to methane in sediments supplemented with amorphous ferric oxyhydroxides was compensated for by a corresponding increase of electron equivalents in ferrous iron. These results indicate that iron reduction can outcompete methanogenic food chains for sediment organic matter. Thus, when amorphous ferric oxyhydroxides are available in anaerobic sediments, the transfer of electrons from organic matter to ferric iron can be a major pathway for organic matter decomposition.     

Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China

To identify the methanogenic pathways present in a deep coal bed methane (CBM) reservoir associated with Eastern Ordos Basin in China, a series of geochemical and microbiological studies was performed using gas and water samples produced from the Liulin CBM reservoir. The composition and stable isotopic ratios of CBM implied a mixed biogenic and thermogenic origin of the methane. Archaeal 16S rRNA gene analysis revealed the dominance of the methylotrophic methanogen Methanolobus in the water produced. The high potential of methane production by methylotrophic methanogens was found in the enrichments using the water samples amended with methanol and incubated at 25 and 35?°C. Methylotrophic methanogens were the dominant archaea in both enrichments as shown by polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE). Bacterial 16S rRNA gene analysis revealed that fermentative, sulfate-reducing, and nitrate-reducing bacteria inhabiting the water produced were a factor in coal biodegradation to fuel methanogens. These results suggested that past and ongoing biodegradation of coal by methylotrophic methanogens and syntrophic bacteria, as well as thermogenic CBM production, contributed to the Liulin CBM reserves associated with the Eastern Ordos Basin.     

Polycyclic Aromatic Hydrocarbon Degradation by a New Marine Bacterium, Neptunomonas naphthovorans gen. nov., sp. nov.

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans. The type strain of N. naphthovorans is strain NAG-2N-126.