Analysis of methanogens adsorption and biogas production characteristics from different coal surfaces.

The aim of this study was to examine the biogas production and the adsorption aspect of microorganism from different coals. Coal samples were obtained from Qianqiu mine and Guandi mine. Microbial populations were cultured from the coal mine drainage. After an anaerobic reaction period at about 35 °C, adsorption rate was determined by the spectrophotometer, while a scanning electron microscopy was used to observe the microorganisms on the coal and the headspace methane was analyzed using gas chromatography. Results show that the coal rank and particle size serve as important factors influencing the adsorption of microorganism and biogenic methane production. With decreasing particle size, the Qianqiu coal produced a considerable adsorption rate between 75 and 79%, while the adsorption rate of Guandi coal was between 52 and 74%. Meanwhile, the density of microorganisms from the Qianqiu coal surface demonstrated a higher level of adsorption than that of Guandi coal following the scanning electron microscopy images. Additionally, Qianqiu coal produced a higher level of biogas production (391.766-629.199 µmol/g) than that of Guandi coal (292.835-393.744 µmol/g) and the Qianqiu coal also generated a higher concentration of methane during the incubation. When the adsorption rate decreasing, the biogas production from various pulverized coals appeared to be decreased and demonstrated a positive correlation to the adsorption rate. The results of this study suggest that the adsorption behavior of microorganisms is closely related to the effect of coal biodegradation and contributes to the biogenic methane production potential.

Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides.

The methanogenic archaeon Methanomassiliicoccus luminyensis strain B10T was isolated from human feces just a few years ago. Due to its remarkable metabolic properties, particularly the degradation of trimethylamines, this strain was supposed to be used as "Archaebiotic" during metabolic disorders of the human intestine. However, there is still no data published regarding adaptations to the natural habitat of M. luminyensis as it has been shown for the other two reported mucosa-associated methanoarchaea. This study aimed at unraveling susceptibility of M. luminyensis to antimicrobial peptides as well as its immunogenicity. By using the established microtiter plate assay adapted to the anaerobic growth requirements of methanogenic archaea, we demonstrated that M. luminyensis is highly sensitive against LL32, a derivative of human cathelicidin (MIC = 2 µM). However, the strain was highly resistant against the porcine lysin NK-2 (MIC = 10 µM) and the synthetic antilipopolysaccharide peptide (Lpep) (MIC>10 µM) and overall differed from the two other methanoarchaea, Methanobrevibacter smithii and Methanosphaera stadtmanae in respect to AMP sensitivity. Moreover, only weak immunogenic potential of M. luminyensis was demonstrated using peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (moDCs) by determining release of pro-inflammatory cytokines. Overall, our findings clearly demonstrate that the archaeal gut inhabitant M. luminyensis is susceptible to the release of human-derived antimicrobial peptides and exhibits low immunogenicity towards human immune cells in vitro-revealing characteristics of a typical commensal gut microbe.

Ammonia effect on hydrogenotrophic methanogens and syntrophic acetate-oxidizing bacteria.

Ammonia-rich substrates can cause inhibition on anaerobic digestion process. Syntrophic acetate-oxidizing bacteria (SAOB) and hydrogenotrophic methanogens are important for the ammonia inhibitory mechanism on anaerobic digestion. The roles and interactions of SAOB and hydrogenotrophic methanogens to ammonia inhibition effect are still unclear. The aim of the current study was to determine the ammonia toxicity levels of various pure strains of SAOB and hydrogenotrophic methanogens. Moreover, ammonia toxicity on the syntrophic-cultivated strains of SAOB and hydrogenotrophic methanogens was tested. Thus, four hydrogenotrophic methanogens (i.e. Methanoculleus bourgensis, Methanobacterium congolense, Methanoculleu thermophilus and Methanothermobacter thermautotrophicus), two SAOB (i.e. Tepidanaerobacter acetatoxydans and Thermacetogenium phaeum) and their syntrophic cultivation were assessed under 0.26, 3, 5 and 7 g NH4 (+)-N L(-1). The results showed that some hydrogenotrophic methanogens were equally, or in some cases, more tolerant to high ammonia levels compared to SAOB. Furthermore, a mesophilic hydrogenotrophic methanogen was more sensitive to ammonia toxicity compared to thermophilic methanogens tested in the study, which is contradicting to the general belief that thermophilic methanogens are more vulnerable to high ammonia loads compared to mesophilic. This unexpected finding underlines the fact that the complete knowledge of ammonia inhibition effect on hydrogenotrophic methanogens is still absent.

Biogas production from coumarin-rich plants--inhibition by coumarin and recovery by adaptation of the bacterial community.

Plants like sweet clover (Melilotus spp.) are not suitable as fodder for cattle because of harmful effects of the plant secondary metabolite coumarin. As an alternative usage, the applicability of coumarin-rich plants as substrates for biogas production was investigated. When coumarin was added to continuous fermentation processes codigesting grass silage and cow manure, it caused a strong inhibition noticeable as decrease of biogas production by 19% and increase of metabolite concentrations to an organic acids/alkalinity ratio higher than 0.3(gorganic acids) gCaCO3 (-1). Microbial communities of methanogenic archaea were dominated by the genera Methanosarcina (77%) and Methanoculleus (11%). This community composition was not influenced by coumarin addition. The bacterial community analysis unraveled a divergence caused by coumarin addition correlating with the anaerobic degradation of coumarin and the recovery of the biogas process. As a consequence, biogas production resumed similar to the coumarin-free control with a biogas yield of 0.34 LN g(volatile solids) (-1) and at initial metabolite concentrations (∼ 0.2 g(organic acids) gCaCO3 (-1)). Coumarin acts as inhibitor and as substrate during anaerobic digestion. Hence, coumarin-rich plants might be suitable for biogas production, but should only be used after adaptation of the microbial community to coumarin.

Physico-chemical characteristics and methanogen communities in swine and dairy manure storage tanks: spatio-temporal variations and impact on methanogenic activity.

Greenhouse gas emissions represent a major environmental problem associated with the management of manure from the livestock industry. Methane is the primary GHG emitted during manure outdoor storage. In this paper, the variability of two swine and two dairy manure storage tanks was surveyed, in terms of physico-chemical and microbiological parameters. The impact of the inter-tank and spatio-temporal variations of these parameters on the methanogenic activity of manure was ascertained. A Partial Least Square regression was carried out, which demonstrated that physico-chemical as well as microbiological parameters had a major influence on the methanogenic activity. Among the 19 parameters included in the regression, the concentrations of VFAs had the strongest negative influence on the methane emission rate of manure, resulting from their well-known inhibitory effect. The relative abundance of two amplicons in archaeal fingerprints was found to positively influence the methanogenic activity, suggesting that Methanoculleus spp. and possibly Methanosarcina spp. are major contributors to methanogenesis in storage tanks. This work gave insights into the mechanisms, which drive methanogenesis in swine and dairy manure storage tanks.

Pulp mill wastewater sediment reveals novel methanogenic and cellulolytic populations.

Pulp mill wastewater generated from wheat straw is characterized as high alkalinity and very high COD pollution load. A naturally developed microbial community in a pulp mill wastewater storage pool that had been disused were investigated in this study. Owing to natural evaporation and a huge amount of lignocellulose's deposition, the wastewater sediment contains high concentrations of organic matters and sodium ions, but low concentrations of chloride and carbonate. The microbiota inhabiting especially anaerobic community, including methanogenic arhcaea and cellulolytic species, was studied. All archaeal sequences fall into 2 clusters of family Halobacteriaceae and methanogenic archaeon in the phylum Euryarchaeota. In the methanogenic community, phylogenetic analysis of methyl coenzyme M reductase A (mcrA) genes targeted to novel species in genus Methanoculleus or novel genus of order Methanomicrobiales. The predominance of Methanomicrobiales suggests that methanogenesis in this system might be driven by the hydrogenotrophic pathway. As the important primary fermenter for methane production, the cellulolytic community of enzyme GHF48 was found to be dominated by narrower breadth of novel clostridial cellulase genes. Novel anoxic functional members in such extreme sediment provide the possibility of enhancing the efficiency of anoxic treatment of saline and alkaline wastewaters, as well as benefiting to the biomass transformation and biofuel production processes.

Methanocella conradii sp. nov., a thermophilic, obligate hydrogenotrophic methanogen, isolated from Chinese rice field soil.

BACKGROUND: Methanocellales contributes significantly to anthropogenic methane emissions that cause global warming, but few pure cultures for Methanocellales are available to permit subsequent laboratory studies (physiology, biochemistry, etc.). METHODOLOGY/PRINCIPAL FINDINGS: By combining anaerobic culture and molecular techniques, a novel thermophilic methanogen, strain HZ254(T) was isolated from a Chinese rice field soil located in Hangzhou, China. The phylogenetic analyses of both the 16S rRNA gene and mcrA gene (encoding the α subunit of methyl-coenzyme M reductase) confirmed its affiliation with Methanocellales, and Methanocella paludicola SANAE(T) was the most closely related species. Cells were non-motile rods, albeit with a flagellum, 1.4-2.8 µm long and by 0.2-0.3 µm in width. They grew at 37-60 °C (optimally at 55 °C) and salinity of 0-5 g NaCl l(-1) (optimally at 0-1 g NaCl l(-1)). The pH range for growth was 6.4-7.2 (optimum 6.8). Under the optimum growth condition, the doubling time was 6.5-7.8 h, which is the shortest ever observed in Methanocellales. Strain HZ254(T) utilized H(2)/CO(2) but not formate for growth and methane production. The DNA G+C content of this organism was 52.7 mol%. The sequence identities of 16S rRNA gene and mcrA gene between strain HZ254(T) and SANAE(T) were 95.0 and 87.5% respectively, and the genome based Average Nucleotide Identity value between them was 74.8%. These two strains differed in phenotypic features with regard to substrate utilization, possession of a flagellum, doubling time (under optimal conditions), NaCl and temperature ranges. Taking account of the phenotypic and phylogenetic characteristics, we propose strain HZ254(T) as a representative of a novel species, Methanocella conradii sp. nov. The type strain is HZ254(T) ( = CGMCC 1.5162(T) = JCM 17849(T) = DSM 24694(T)). CONCLUSIONS/SIGNIFICANCE: Strain HZ254(T) could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.

Methanoculleus spp. as a biomarker of methanogenic activity in swine manure storage tanks.

Greenhouse gas emissions represent a major problem associated with manure management in the livestock industry. A prerequisite to mitigate methane emissions occurring during manure storage is a clearer understanding of how the microbial consortia involved in methanogenesis function. Here, we have examined manure stored in outdoor tanks from two different farms, at different locations and depths. Physico-chemical and microbiological characterization of these samples indicated differences between each tank, as well as differences within each tank dependent on the depth of sampling. The dynamics of both the bacterial and archaeal communities within these samples were monitored over a 150-day period of anaerobic incubation to identify and track emerging microorganisms, which may be temporally important in the methanogenesis process. Analyses based on DNA fingerprinting of microbial communities identified trends common among all samples as well as trends specific to certain samples. All archaeal communities became enriched with Methanoculleus spp. over time, indicating that the hydrogenotrophic pathway of methanogenesis predominated. Although the emerging species differed in samples obtained from shallow depths compared to deep samples, the temporal enrichment of Methanoculleus suggests that this genus may represent a relevant indicator of methanogenic activity in swine manure storage tanks.

Comparison of microbial community compositions of injection and production well samples in a long-term water-flooded petroleum reservoir.

Water flooding plays an important role in recovering oil from depleted petroleum reservoirs. Exactly how the microbial communities of production wells are affected by microorganisms introduced with injected water has previously not been adequately studied. Using denaturing gradient gel electrophoresis (DGGE) approach and 16S rRNA gene clone library analysis, the comparison of microbial communities is carried out between one injection water and two production waters collected from a working block of the water-flooded Gudao petroleum reservoir located in the Yellow River Delta. DGGE fingerprints showed that the similarities of the bacterial communities between the injection water and production waters were lower than between the two production waters. It was also observed that the archaeal composition among these three samples showed no significant difference. Analysis of the 16S rRNA gene clone libraries showed that the dominant groups within the injection water were Betaproteobacteria, Gammaproteobacteria and Methanomicrobia, while the dominant groups in the production waters were Gammaproteobacteria and Methanobacteria. Only 2 out of 54 bacterial operational taxonomic units (OTUs) and 5 out of 17 archaeal OTUs in the injection water were detected in the production waters, indicating that most of the microorganisms introduced by the injection water may not survive to be detected in the production waters. Additionally, there were 55.6% and 82.6% unique OTUs in the two production waters respectively, suggesting that each production well has its specific microbial composition, despite both wells being flooded with the same injection water.

Enrichment and characterization of a trichloroethene-dechlorinating consortium containing multiple "dehalococcoides" strains.

A microbial consortium that reductively dechlorinates trichloroethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene with methanogenesis was enriched from chloroethene-contaminated soil from Japan. Dechlorination activity was maintained for over 4 years. Using quantitative polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis targeting the "Dehalococcoides" 16S rRNA gene, four strains were detected. Their growth and dechlorination activities were classified into two types: one that grows by converting cis-DCE to ethene and the other that grows by converting cis-DCE to VC. Then, the vcrA and bvcA genes encoding cis-DCE/VC reductive dehalogenases were detected. Inhibitors of methanogenesis (2-bromoethanesulfonate) and sulfidogenesis (molybdate) led to accumulation of cis-DCE and of VC respectively. These results suggest that methanogens and sulfate-reducing bacteria can play a significant role in dechlorination by "Dehalococcoides."

[Acidification and its effect on the population distributions of microorganisms in an anaerobic baffled reactor].

The changes of pH, COD, volatile fatty acids (VFA) and microbial morphology of the acidification process in an anaerobic baffled reactor (ABR) were investigated. And the population succession process of the anaerobic microorganisms was quantitatively analyzed by using the Fluorescent In situ hybridization technology (FISH). The results show that the ABR reactor is acidified gradually from the front to the back. After the reactor is entirely acidified, the COD removal efficiency is only 30.9%, and the pH values are lowered by 1.0-2.2, while the VFA in effluent increases by 5.1 times. Additionally, the microbial morphology is significantly affected by the acidification process, in which not only the bacteria are deformed or died, but also the internal and external mass transfer of granular sludge becomes difficult. The quantitative analyses with FISH shows that in the acidification process the Archaea growth is inhibited but the Eubacteria growth is promoted, thus resulting in the sharp decrease of the three crucial microorganisms of the anaerobic digestion. The abundance of the butyrate-oxidizing acetogenic bacteria Syntrophomonas spp. reduces by 30.9%, the propionate-oxidizing acetogenic bacteria Syntrophobacter wolinii reduces by 85.5%, the homoacetogenic bacteria Acetobacterium species E. limosum reduces by 60.0%, and methanomicrobium Methanomicrobiales reduces by 54.3%. All these result in the upsetting of the mass transfer balances of different anaerobic microorganism populations.

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov.

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.

A comparative study of sulfidogenic and methanogenic activities during the treatment of landfill leachate: part I.

A comprehensive anaerobic leachate treatment in batch reactors was conducted for 9 months. Activities of two main bacterial groups, sulfidogens and methanogens were observed with monitoring parameters as chemical oxygen demand (COD), total gas production, pH, alkalinity, dissolved sulfide and volatile suspended solids/total suspended solids (VSS/TSS). Leachate contained high concentrations of volatile fatty acids. Some competition between microbial species was observed. COD was applied at a range of 1100-8200 mg/L at a slowly increasing rate over time. COD removal was mostly above 80%, dissolved sulfide was produced at a range of 100-450 mg/L in the first weeks, proving high sulfidogenic activity and making 40-50% of COD removal. Methanogenesis seemed under stress and improved after several weeks with increasing COD load whereas hydrogen sulfide (HS-) production leveled off around 100 mg/L, making 4-8% COD removal by sulfidogens. Then, 40-70% of overall COD removal was achieved by methanogens. Volatile fatty acids (VFA) except acetic acid (propionic, isobutyric, butyric, isovaleric, valeric, caproic and heptanoic acids) were readily degraded and found near detection limit on gas chromatographic runs, accordingly VFA-oxidizing bacteria were assumed to be highly active and tolerant to sulfide levels encountered in our study, also with hydrogenotrophic groups providing favorable conditions for the process. pH was the key parameter to determine the degree of inhibition from sulfurous species. Alkalinity was produced proportionally with COD applied as a result of VFA degradation. Overall system performance was good and optimum pH was 7.8-8.2 at which inhibition due to unionized VFA was eliminated.

Isolation and characterization of methylotrophic methanogens from anoxic marine sediments in Skan Bay, Alaska: description of Methanococcoides alaskense sp. nov., and emended description of Methanosarcina baltica.

Three novel strains of methylotrophic methanogens were isolated from Skan Bay, Alaska, by using anaerobic cultivation techniques. The water was 65 m deep at the sampling site. Strains AK-4 (=OCM 774), AK-5T (=OCM 775T=DSM 17273T) and AK-9 (=OCM 793) were isolated from the sulfate-reducing zone of the sediments. Each of the strains was a non-motile coccus and occurred singly. Cells grew with trimethylamine as a catabolic substrate and strain AK-4 could also catabolize methanol. Yeast extract and trypticase peptones were not required for growth, but their addition to the culture medium slightly stimulated growth. Each of the strains grew at temperatures of 5-28 degrees C; they were slight halophiles and grew fastest in the neutral pH range. Analysis of the 16S rRNA gene sequences indicated that strain AK-4 was most closely related to Methanosarcina baltica. DNA-DNA hybridization studies showed 88 % relatedness, suggesting that strain AK-4 represents a novel strain within this species. Strains AK-5T and AK-9 had identical 16S rRNA gene sequences that were most closely related to the sequence of Methanococcoides burtonii (99.8 % sequence similarity). DNA-DNA hybridization studies showed that strains AK-5T and AK-9 are members of the same species (88 % relatedness value), but strain AK-5T had a DNA-DNA relatedness value of only 55 % to Methanococcoides burtonii. This indicates that strains AK-5T and AK-9 should be considered as members of a novel species in the genus Methanococcoides. We propose the name Methanococcoides alaskense sp. nov., with strain AK-5T (=OCM 775T=DSM 17273T) as the type strain.

[Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups].

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.

Methanofollis formosanus sp. nov., isolated from a fish pond.

A mesophilic, hydrogenotrophic methanogen, strain ML15(T), was isolated from an aquaculture fish pond near Wang-gong, Taiwan. The cells were irregular cocci, non-motile, 1.5-2.0 microm in diameter and Gram-negative. Cells of strain ML15(T) lysed easily in the presence of SDS (0.1 g l(-1)) and the S-layer protein had an M(r) of 138 800. The catabolic substrates utilized by this strain included formate and H(2)/CO(2), but not acetate, methanol, trimethylamine or secondary alcohols. Growth did not occur in minimal medium, but was observed when yeast extract and tryptone were added. Strain ML15(T) grew fastest at 37 degrees C, pH 6.6-7.0 and with 3 % NaCl. Acetate was not required for cell growth. Trace amounts of tungstate promoted cell growth. The G+C contents of DNA of Methanofollis aquaemaris N2F9704(T) and strain ML15(T) were 59.1 and 58.4 mol%, respectively. Sequence analysis of the 16S rRNA genes of strain ML15(T) and selected Methanofollis species revealed similarities of 95-97 %. Based on the data presented here, it is proposed that strain ML15(T) (=OCM 789(T)=DSM 15483(T)) represents a novel species, Methanofollis formosanus sp. nov.

Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases.

This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell suspensions was investigated using batch activity tests and by operating a completely stirred tank reactor (CSTR). The maximum specific hydrogenotrophic sulfite/sulfate reduction rate increased with 10% and 300%, respectively, by crushing granular inoculum sludge and by cultivation of this sludge as cell suspension in a CSTR. Operation of a sulfite fed CSTR (hydraulic retention time 4 days; pH 7.0; sulfite loading rate 0.5-1.5 g SO3(2-) l(-1) d(-1)) with hydrogen as electron donor showed that high (up to 1.6 g l(-1)) H2S concentrations can be obtained within 10 days of operation. H2S inhibition, however, limited the sulfite reducing capacity of the CSTR. Methane production by the cell suspension disappeared within 20 days reactor operation. The outcompetition of methanogens in excess of H2 can be attributed to CO2 limitation and/or to sulfite or sulfide toxicity. The use of cell suspensions opens perspectives for monolith or packed bed reactor configurations, which have a much lower pressure drop compared to air lift reactors, to supply H2 to sulfite/sulfate reducing bioreactors.

Isolation of a methanogen from deep marine sediments that contain methane hydrates, and description of Methanoculleus submarinus sp. nov.

We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 micro m) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45 degrees C (no growth at /=55 degrees C). Cells grew with a maximum specific growth rate of 2.43 day(-1) at 45 degrees C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na(+). The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay, Alaska, and kinetics of H2 utilization.

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 microm), occurring singly. Cells grew by reducing CO2 with H2 or formate as electron donor. Growth on formate was much slower than that on H2. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B12, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 degrees C (range 5 degrees C to 25 degrees C), at a pH of 6.0-6.6 (growth range, pH 5.5-7.5), and at a salinity of 0.25-1.25 M Na+. Cells of this and other H2-using methanogens from saline environments metabolized H2 to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogeniumfrigidum Ace-2. DNA-DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain.