Rumen Cluster C:Distribution, Isolation And Hydrogenotrophy

Methane is one of the end products in the rumen which results in the energy loss for the ruminants as well as greenhouse gas for the environment. Therefore, it is important to understand the mechanism of methanogenesis and the ecology of methanogens in the rumen. Rumen Cluster C archaea (RCC) is possible to be a new order of rumen methanogens, however, for which, to date, there have been no direct evidences. In this study, work has been done to enrich RCC archaea from rumen fluid by the way of co-culturing them with anaerobic fungi. The characteristics of their hydrogenotrophy and enrichment have been investigated by in vitro co-culture model. Their abundance in the rumen of goat fed different diets has been investigated by quantitative PCR. The main results are shown as follows:1The abundance of RCC strain LGM in the anaerobic fungal culture and its distribution in the different fractions of rumenThis study established the specific quantitative PCR method for RCC strain LGM. The16S rRNA gene copy number of LGM and the mcrA gene copy number of total methanogens in the anaerobic fungal cultures with different transfer intervals were analyzed. The results showed that the percentage of LGM in total methanogens in the anaerobic fungal culture with five-day transfer interval was the highest (P<0.05). In contrast, LGM disappeared in the anaerobic fungal culture with three-day transfer interval. The percentage of LGM in total methanogens in the fraction of rumen liquid was the highest in the three rumen fractions (rumen epithelium, rumen solid and rumen liquid)(P<0.05). Conclusively, to enrich LGM in the anaerobic fungal culture, it is better to transfer the culture every five days and take rumen liquid as the inoculation.2The isolation of anaerobic fungus with methanogenIn the study, the method of enrichment and Hungate roll tube technique were combined in the isolation of anaerobic fungus with methanogen. The results showed that anaerobic fungal cultures containing methanogens were successfully isolated by short-time transfer enrichment, but no fungal cultures contained LGM. Morphology observation, nucleus staining, PCR-DGGE and16S rRNA gene sequencing were conducted to identify anaerobic fungi and methanogens in these cultures. The anaerobic fungi were identified as belonging to genera Piromyces based on their morphology and DAPI nuclear staining. PCR-DGGE analysis indicated that only one methanogen strain in each fungal culture, and all the methanogens were identified belonging to Methanobrevibacter sp. based on16S rRNA gene sequence. The fluorescence microscope photos and scan electron microscope photos showed that the lots of methanogens in the fungal culture attached to the fungal rhizoids. A fungal culture containing LGM was isolated by the method combining long-time transfer enrichment, Hungate roll tube technique and inhibitor of Methanobrevibacter sp. PCR and sequencing was performed to identify LGM. And the results showed that LGM contained the methanogen function gene coded methyl coenzyme M reductase subunit A for forming methane, which supported that LGM was a methanogenic species. Conclusively, RCC strain LGM is a methanogenic species, and co-culture is a feasible way to investigate uncultured rumen archaea.3Co-culturing strain MGC with hydrogen-producing bacteria provides insight into its hydrogenotrophyIn this study, the hydrogenotrophy of RCC strain MGC derived from chicken cecum was studied by in vitro co-culture model. The model was established by co-culturing R. albus with methanogens. Test for this model was carried out, and the result showed that methanogens did not affect the metabolism of R. albus, suggesting that R. albus in the model just acted as hydrogen source. When both methanol and trimethylamine were added to the model, strain MGC first consumed trimethylamine. M. smithii was inoculated into the model to compete hydrogen with strain MGC, the results showed that before the growth lag time of strain MGC ceased, most of the hydrogen produced by R. albus had been consumed by M. smithii. In the model with pH sharply decline, strain MGC was more resistant to low pH than M. smithii, and it converted most of the hydrogen to methane. When the hydrogen producing speed of R. albus was slowed down, strain MGC captured more hydrogen than M. smithii (67.8%). To further study the hydrogen competing ability of MGC, the hydrogen competing model was consecutively transferred six times. And the results showed that as the transfers increased, the methane production increased, and the16rRNA gene copy number of M. smithii was decreased in the model, suggesting that the growth of M. smithii was inhibited by strain MGC. Strain MGC was cultured with a mix culture directly enriched from rumen fluid. After120hours incubation, the16rRNA gene copy number of strain MGC increased almost100times, suggesting that strain MGC could survive under complex competing condition. Conclusively, strain MGC had strong ability to compete for hydrogen with other methanogens under low hydrogen partial pressure condition, and the in vitro co-culture model provides a way to study rumen methanogens.4The study of the enrichment method for RCCIn this study, RCC were tried to be enriched by adding methanol or trimethylamine in the in vitro co-culture model. Though a large amount of methane produced during the enrichment,16S rRNA gene copy number of RCC declined greatly in the culture. The growth of Methanobrevibacter sp. did not inhibited by adding Lumazine,16S rRNA gene copy number of Methanobrevibacter sp. maintained in high level. To inhibit the growth of Methanobrevibacter sp., BJ medium without carbon dioxide and sodium bicarbonate was used. The culture enriched by trimethylamine stopped producing methane, when inoculated into BJ medium. In cotrast, the culture enriched by methanol increased produced a large amount of methane after inoculated into BJ medium. After five transfers with treated by antibiotics, the culture was observed under fluorescence microscope. The result showed that all the microorganisms in the culture were globose, and substrate test experiment showed that these microorganisms exclusively reduced methanol by hydrogen to methane. Cloning and sequencing showed that the microorganisms belonged to Methanosphaera. Conclusively, RCC could not be enriched by just addition of methyl group chemicals in the in vitro co-culture model.5The effects of different ratio of concentrate in diets on the abundance of RCC in the goat rumenIn this study, the effects of different ratio of concentrate in diets on the abundance of RCC in the goat rumen were investigated by quantitative PCR. The results showed that the diets did not affect the abundance of rumen archaea in the rumen content, rumen epithelium mucosa, cecum content and cecum epithelium mucosa (P>0.05). The abundance of RCC in the rumen content and cecum content of goats fed high roughage was significantly higher than that of goats fed high concentrate (P<0.05). The percentage of the16S rRNA gene copy number of RCC in the16S rRNA gene copy number of rumen archaea in the rumen content of goats fed high roughage was8.1-28.3%, and the percentage was0.6-4.6%in the high concentrate group. The abundance of RCC in the rumen epithelium mucosa and cecum epithelium mucosa did not affected by diets (P>0.05), the percentages of the16S rRNA gene copy number of RCC in the16S rRNA gene copy number of rumen archaea in the two locations were3.4-4.3%and12.0-13.2%, respectively. Conclusively, high concentrate diet greatly declined the abundance of RCC in the goat rumen.