| Methanogenesis is one of the important contributors to greenhouse effect, performed by methanogenic archaea, a specialised group of microbes present in several anaerobic environments including the rumen. In the rumen, methanogens utilize predominantly H2 and CO2 as substrates to produce methane, filling an important functional niche in the ecosystem. However, in addition to methanogens, other microbes also have an influence on methane production, either because they are involved in hydrogen metabolism or because they affect the numbers of methanogens or other members of the microbiota. Although there are numbers of microbes in the rumen with complex ecosystem, all of the microbes could be divided into hydrogenogens and hydrogenotrophs. This study explores the relationship between some of these microbes and methanogenesis and highlights fumarate-reducing groups that could play a role in decreasing methane emissions in the rumen.Part 1 Effect of the addition of disodium fumarate on rumen fermentation and overall microbial communityThis part was divided into in vitro and in vivo experiments. In vitro experiments were conducted to investigate the effect of the combination of tea saponins and disodium fumarate (DF) addition on rumen fermentation. Tea saponins and disodium fumarate had positive interactions on rumen methane production, gas production, propionate concentration and acetate to propionate ratio. Futher in vivo experiments were carried out to investigate effects of DF on fermentation characteristics and overall microbial community change in the rumen of Hu-sheep fed on high-forage diets. The propionate concentration increased significantly at week 6 with DF addition. The populations of methanogens and R. flavefaciens decreased in the fluid samples (P<0.001), while addition of DF stimulated the population of solid-associated Fibrobacter succinogenes. Population of R. albus increased in weeks 2 to 4 in fluid associated samples and threefold higher in week 4 than week 0 in solid samples. Analysis of denaturing gradient gel electrophoresis (DGGE) fingerprints revealed that there were significant changes in rumen microbiota after adding DF. Thirteen of total 15 sequences from cut-out bands appeared only in week 4 were represented by Clostridium species (95-99% similarity). Ten of total 15 clone sequences from cut-out bands appeared in both week 2 and 4 were 94-100% similar to Prevotella-like bacteria, and four sequences showed 95-98% similarity to Selenomonas Dianae. In summary, the microorganisms positively responding to DF addition were cellulolytic bacteria, R. albus, F. succinogenes, and B. fibrisolvens as well as proteolytic bacteria, B. fibrisolvens, P. ruminicola and Clostridium sp.Part 2 Community Composition of ruminal bacterial in Hu-sheep added with fumarateTwo Libraries of rumen bacterial 16S rRNA gene sequences of hu-sheep were cloned and sequenced in the present work. One library was based on rumen samples without DF treatment (control), the other library was based on the sample with DF addition at week 6 (fumarate-treated group). In total, there were 155 clones in control and 231 clones in treatment. The majority of sequences retrieved from the two libraries were fell into the phyla of low G+C subdivision(LGCGPB) and Cytophaga-Flexibacter-Bacteroides (CFB). In control library,73% of total diversity belonged to CFB, and 24% diversity to LGCGPB. While the treatment library showed the opposite situation with 82% diversity close to LGCGPB and 10% diversity to CFB. In the treatment library, there appeared 4% of clones closely related to Proteobacteria, which might play a role in fumarate-reducing pathway. The remaining clones in two libraries belonged to the phyla of Proteobacter, high G+C gram positive bacteria (HGCGPB) and Spirochaetes. There were 75 OTUs present in control library and the number increased to 153 OTUs in fumarate-treated group. The coverage value for control and treatment was 79 and 51%, respectively. There were 12 clones closely related to Butyrivibrio-like bacteria. Among them, there were 3 clones belong to Butyrivibrio fibrisolvens with 93% identity,8 clones close to Butyrivibrio hungatei with 99% identity, and the remaining 9 clones showed 95% similarity to Butyrivibrio proteoclasticus. It is inferred that fumarate stimulate the growth of Butyrivibrio-like bacteria which is recognized as main butyrate-producing bacteria in rumen as well as fibrolytic bacteria. There were 163 Prevotella-like clones found in control library, with only 15 Prevotella-like clones in treatment. There were 27 Clostridium-like clones detected in fumarate-treated group with 86% identity, which corresponded to the DGGE results. Clostridium-like bacteria could be involved in fumarate-reducing pathway. From these results, it is indicated that DF addition increased the bacteria diversity in rumen, and changed the microbial structure at different taxonomy ranks-domain, phylum, order, class, family, genus and species.Part 3 Molecular Diversity of Methanogens in the rumen of Hu-sheep added with fumarateIn this part, the molecular diversity of rumen methanogens in Hu-sheep rumen in control and fumarate-treated animals were investigated using mcrA and 16S rRNA gene libraries. Both 16S rRNA (340 clones) and mcrA gene libraries revealed the same three species, Methanobrevibacter spp., Methanosphaera stadtmanae and an uncultivated archaeon. For the 16S rRNA libraries, there were 158 clones in control and 182 clones in fumarate-treated group. All the clones from Hu-sheep fell into Euryarchaeota phylum. A total of 340 clones were examined, with Methanobrevibacter spp. accounting for 97.5% of the clones identified. From these 340 clones,21 different 16S rRNA phylotypes were identified. There were 17 phylotypes in control library, while only 6 phylotypes were found in fumarate-treated library. There were four abundant OTUs in control, which were Methanobrevibacter olleyae (46 clones,98% similarity), Mbb. millerae (37 clones,97% similarity) and Mbb. Thaueri (26 clones,97% similarity). There were only 2 abundant phylotypes in treatment which were Mbb. millerae, with 124 members (98% identity) and Mbb. Olleyae with 51 members (98% identity). Fumarate addition in Hu-sheep decreased the number of phylotypes in total but increased the members in the abundant phylotypes. Therefore, the abundant species become more abundant with fuamrate addition.Part 4 Diversity of Fumarate Reducing Proteobacteria in the rumen of Hu-sheep added with fumarateThe fumarate-reducing Proteobacterial-community in the Hu-sheep rumen was investigated using animals added with DF. Community structure of these bacteria was determined by phylogenetic analysis of the fumarate reductase gene (frdA) sub-unit that affiliates with the Proteobacteria. Two clone libraries based on frdA were constructed from pooled rumen samples (six animals) at the end of the adaptation period and following the fumarate addition. The frdA clone libraries were also prepared from enrichment cultures (fumarate-formate+H2 vs fumarate-H2 as substrates) of fumarate-reducing bacteria that were inoculated with rumen liquor from the fumarate added animals. In the animals, production of propionate increased (P=0.015) and diversity of fumarate reducing Proteobacteria changed significantly (P=0.0015; LIBSHUFF analysis) in response to DF addition. Phylogenetic analysis indicated that the majority of clones in the controls were affiliated with Proteus mirabilis (67%) and 18% showed homology to Vibrio furnissii, while in DF addition treatment the dominant clone was most similar to Vibrio furnissii (50%) and Proteus mirabilis (31%) was the minor population. In the enrichment cultures, sequences affiliating with Proteus mirabilis (70.6%) and E.coli (91.8%) frdA genes were predominant in the fumarate-formate+H2 and fumarate-H2 amended cultures respectively. Deduced amino acid sequences of cloned frdA amplicons from animal libraries were more distantly related to known sequences (75 to 89% identity) than enrichment libraries (83 to 100% identity). This suggests that uncultured fumarate reducing Proteobacteria exist in the rumen and that relatively easy-to-culture fumarate-reducing Proteobacteria tend to be dominant in enrichment cultures. Two phylogenetic trees were constructed which revealed the diverse relationships of fumarate-reducing bacteria and showed only one sequence (EH-OTU3) from enrichment trial was clustered into the Hu-sheep rumen cluster which from animal source; the other sequences from enrichment trial were distantly related to sequences from animal sources (Bovine rumen or Hu-sheep rumen). Vibrio-related species were found in both libraries in Hu-sheep rumen.The main functioning group of fumarate-reducing bacteria fell into Proteus mirabilis cluster in normal Hu-Sheep rumen (Lib. Control). After adding fumarate for 6 weeks, Vibrio furnissii became abundant Proteobacterial fumarate-reducing bacteria whose popularity was increased from 18% in 0 w to 50% in 6 w, and new species Shewanella sp. appeared in 6 w; the popularity of Proteus mirabilis decreased with the addition of fumarate from 67% in 0 w to 31% in week 6; The percentage of clones close to Pasteurella multocida declined from 15% in 0 w to 9%in 6 w, and one clone of sdhA sequence appeared in the sixth week in fumarate treated sheep.In summary, DF addition was assosciated with favorable changes in ruminal fermentation in vitro as well as in vivo. Bacterial structure in Hu-sheep rumen was significantly changed with fumarate addition at all taxonomic ranks, and molecular diversity of methanogens was also changed, but only at species level. Fumarate addition in vivo and enrichments with different hydrogen sources had significant effects on the community structure of fumarate reducing proteobacteria in Hu-sheep rumen.
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