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The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species ( Methanobrevibacter ruminantium, Methanobrevibacter wolinii , Methanosphaera stadtmanae and Methanobrevibacter smithii ), with about 89% of the methanogens belonging to the genus Methanobrevibacter . The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher ( P <0.05) for gilts fed LFD (3.31×10 9 copies/g dry matter (DM)) than gilts fed HFD (1.02×10 9 copies/g DM). No difference ( P >0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than ( P <0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up in vitro fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the in vitro fermentation confirmed that the predominant methanogens belonged to the genus of Methanobrevibacter , and about 23% methanogens was found to be distantly related (90%) to Thermogymnomonas acidicola. In vitro fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of Methanobrevibacter species was positively correlated with CH 4 production in both in vivo ( P <0.01, r =0.737) and in vitro trials ( P <0.05, r =0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH 4 emission from gilts fed HFD.

Anaerobic digestion of agricultural wastes provides an opportunity for renewable energy production while reducing emissions of greenhouse gasses such as carbon dioxide and methane from crop and livestock production. While anaerobic digestion is possible under a wide range of temperatures and reactor configurations, it does require a stable methanogenic community composed of hydrolytic and fermentative bacteria and methanogenic archaea in order to maintain robust methane production. Research focused on characterizing and optimizing the microbial community during anaerobic digestion is increasingly exploiting DNA-based methods. In addition to providing an in-depth phylogenetic survey, these techniques permit examination of dynamic changes in α- and β-diversity during the digestion process and in response to perturbations in the system. This study used universal target amplification, next generation sequencing, and quantitative PCR to characterize the Bacteria and Archaea in digestate from thermophilic batch anaerobic digesters processing different combinations wheat ethanol stillage waste and cattle manure. The results indicated that the bacterial community was composed primarily of Firmicutes, with Proteobacteria and Bacteroidetes also numerically abundant. While less phylogenetically diverse, the archaeal community showed robust populations of both hydrogenotrophic and acetoclastic methanogens. A core microbiome present across all reactors was identified and differences in the relative abundances of the bacteria within the core community suggested significant niche overlap and metabolic redundancy in the reactors. A time-course study correlating the abundances of individual Bacteria and Archaea to methane production and volatile fatty acid catabolization identified several microorganisms hypothesized to be critical to both hydrogenotrophic and acetoclastic methanogenesis. Individual Bacteria most closely related to Clostridium spp. and Acetivibrio spp. were 10-1000-fold less abundant in reactors suffering from volatile fatty acid accumulation and inhibition of methanogenesis. Additionally, failing reactors were devoid of robust populations of acetoclastic methanogens. Microorganisms identified as critical during the time-course study were targeted for isolation in vitro and a robust methanogenic consortium consisting of at least 9 bacteria and both a hydrogenotrophic and an acetoclastic methanogen was stably propagated. Addition of this bioaugmentation consortium to digesters experiencing classic symptoms of acid crisis resulted in reduced acetate accumulation and initiation of methanogenesis. One acetoclastic methanogen, most likely a novel species from the genus Methanosarcina, showed particularly robust growth in the recovered bioaugmented reactors, increasing 100-fold in the first 7 days post-treatment. A combination of Illumina shotgun and Roche 454 paired-end sequencing chemistry was used to generate a high quality draft genome for this organism. Analysis of the annotated genome revealed diverse metabolic potential with a full complement of genes for acetoclastic, hydrogenotrophic and methylotrophic methanogenesis pathways represented. Taken as a whole, this thesis provides the foundation for using microbial community characterization to inform anaerobic digester design and operation. By identifying organisms of interest, correlating their abundance to specific biochemical functions and confirming their hypothesized functions in situ, microorganisms critical for robust methane production were acquired. The logical extension of this work is to establish monitoring tools for microorganisms identified as critical to specific performance parameters, to enumerate them in real-time, and to use that data to improve reactor operation.

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In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.

In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.

Methanogenesis, a metabolic pathway unique to Archaea, is severely inhibited by the reduced form of arsenic (As). Despite this inhibition, methanogenic Archaea are present in some hydrothermal features at the El Tatio Geyser Field (ETGF), a high-arsenic site with 100+ hydrothermal features, including boiling pools, geyers, fumaroles, and springs. The ability of methanogenic Archaea and other microorganisms to withstand elevated arsenic concentrations, and a variety of other extreme environmental conditions at ETGF, may be due to unique adaptations or syntrophic relationships with other microorganisms. ETGF is situated in the Andes Mountains at an altitude of ~4300 meters. UV radiation is elevated in this region and air temperatures fluctuate widely. Most hydrothermal waters discharge at ~85˚C, the local boiling point, and rapidly evaporate due to the arid climate. This concentrates hydrothermal salts and metals, including arsenic (As) and antimony (Sb). Additionally, dissolved inorganic carbon (DIC) concentrations are extremely low in most features and may limit life. Water chemistry analyses done for this study show variability in dissolved constituents between features that are consistent over time. Variations may be due to the source or residence time of waters, and differences in chemistry could be responsible for the presence or absence of methanogenic Archaea at hydrothermal sites. The overlying control on microbial diversity and community composition may be water geochemistry, and potentially specific constituents. The goals of this study were to detect novel microbial taxa at ETGF, including novel methanogens, as well as to document microbial community composition at select hydrothermal features. The distribution and diversity of microorganisms at each feature was analyzed phylogenetically and within an ecological context in order to determine physicochemical and biological controls on community composition. Additionally, a model methanogen was used in laboratory analyses to determine how concentrations and oxidation states affected growth and methane production. This methanogen, Methanothermobacter thermautotrophicus, is found at ETGF, Yellowstone, and other hydrothermal fields, and thrives in high-temperature environments. MPN (most probable number) analyses show that culturable biomass from multiple sites contain metabolically active methanogens. These results support the biogenicity of dissolved methane detected in the field. 16S rRNA surveys of Archaea at four sites show that Archaea are diverse, and archaeal community composition varies across features. Phylogenetic tree construction indicates that Archaea from ETGF group together, suggesting that the isolation and broad environmental constrains on ETGF have some control on phylogenetic diversity. Laboratory analyses of As and Sb concentrations on M. thermautotrophicus suggest that Sb may decrease the inhibition of methanogenesis by As by preventing the formation of As(III) from As(V). Statistical analyses correlating microbial community composition and structure to physicochemical parameters show that archaeal and bacterial communities relate to different variables; with Bacteria correlating to water temperature, and Archaea correlating to dissolved constituents such as hydrogen gas and sulfate. / text

The archaeal branch of life represents some of the oldest life forms on Earth. Archaea are believed to have diverged from Prokaryotes roughly 3.5 billion years ago and it’s theorized that biological methane production started around this time as well. This would make methanogenesis one of the oldest metabolisms on our planet. Methanogenesis is a process that, so far, is known to be unique to archaea. Since their evolution, methanogens have had massive impacts on Earth’s climate and biology. Methane is an important part of the global carbon cycle, but is also a major greenhouse gas, making it a vital area of research. The methanogen studied in this thesis is referred to as the wild type (WT) and was isolated from an active serpentinization system at Chimaera seep in Çıralı, Antalya Gulf, Turkey. The Chimaera seep is a geological formation analogous to mid-ocean ridges, but exposed and above land. The research in this thesis focuses on the description of the WT, which is believed to be a new species. Describing the WT consisted of the characterization of extremes as well as optimal growth conditions. The WT was found to grow at initial pH levels of 9.0 and 9.5. It grew from 15 °C to 45 °C, but not at 47 °C and not at 12 °C, and had an optimum at 39 °C. The WT had measurable growth up to 40 g/L NaCl, and had its optimum at 0 g/L. The WT grew best with H2/CO2 substrate, but also grew well on formate.

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