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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. SAOB were more sensitive to high ammonia compared to the hydrogenotrophic methanogens tested. Thus, hydrogenotrophic methanogens could be equally, if not more, tolerant to high ammonia levels compared to SAOB.

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.). 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)). Strain HZ254(T) could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.

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.

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.

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 (P L  = 0.0033) and Methanimicrococcus (P L  = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (P L  = 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 (P Q  = 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.

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 gvolatile solids−1 and at initial metabolite concentrations (∼ 0.2 gorganic 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. The study describes the inhibition of the anaerobic digestion process by the secondary plant metabolite coumarin. The anaerobic degradation of coumarin was accompanied by an adaptation of the bacterial community.

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.

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.

Three strictly anaerobic, thermophilic methanogens (ZC-2T, ZC-3 and ZC-6) were isolated from Shengli oil field, China. The 16S rRNA gene sequences of the three strains were nearly identical, possessing >99.8% sequence similarity. They also possessed high sequence similarity, 97.4%, to Methanoculleus palmolei strain INSLUZT (97.4% and 97.5%, respectively), indicating that they represented a novel species within the genus Methanoculleus. Cells of strain ZC-2T were nonmotile cocci, 0.8-1.7 μm in diameter, and always occurred singly or in pairs. The three strains used H₂/CO₂ or sodium formate as substrates for methanogenesis but not sodium acetate, trimethylamine, monomethylamine, ethanol, dimethyl sulfide, isopropanol, isobutanol, butan-2-ol or H₂/CO. Optimum growth of strain ZC-2T occurred in the presence of 0.2 M NaCl, pH 7.5-7.8 and temperature 50-55 °C with a specific growth rate of 0.084 h⁻¹. The mol% G+C content of the genomic DNA was 55.2 mol%. Based on these phenotypic and phylogenetic characteristics, strains ZC-2T, ZC-3 and ZC-6 are proposed to represent a novel species in the genus Methanoculleus and named Methanoculleus receptaculi sp. nov. The type strain is ZC-2T (CGMCC 1.5087T=DSM 18860T).

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.