| 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. The dynamic interaction between anaerobic fungi and methanogens was investigated in this study. Firstly, the DGGE method for molecular ecological analysis of ruminal archaea was established; the co-culture of anaerobic fungi and methanogens was obtained from rumen content and the fermentation and diversity of the co-culture was assessed; the impact of the viability of anaerobic fungi, the effect of transfer frequency and number of transfers on the establishment and stability of the co-cultures was assessed; relative anaerobic fungi and methanogens were isolated. The main results are shown as follows:1DGGE method for molecular ecological analysis of ruminal archaeaA denaturing gradient gel electrophoresis (DGGE) method for analyzing16S rDNA of methanogenic archaeal community in the rumen is presented. Ten primer sets for16S rDNA of methanogenic archaea, which were used for DGGE analysis of environmental Archaea were first evaluated by PCR amplification of DNAs of rumen digesta and pure bacterial culture. Six of the ten primers were used for direct PCR amplification and the other four for nested PCR. The DGGE analysis was possible with three primers (519f/915rGC,1106fGC/1378r and344fGC/915r) of the ten pairs tested. These three primer pairs were further evaluated for use in analysis of methanogenic archaeal community in the rumen. Good separation and quality of patterns were obtained in DGGE analysis with two primer pairs (519f/915rGC and1106fGC/1378r). Only one band existed in the DGGE profile with primer pair344fGC/915r. A total of40DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 519f/915rGC is suitable for investigating methanogenic archaeal community in the rumen.2Establishment and diversity analysis of a developing stable co-culture of ruminal anaerobic fungi and methanogenic archaeaRumen digesta from a goat fed a diet of hay was used to inoculate Orpin’s medium supplemented with1%(w/v) rice straw. Antibiotics were added (penicillin and streptomycin) to inhibit the growth of Eubacteria. Subculturing of the batch culture was then performed every three days, for a total of62transfers. Samples of the rumen digesta and co-culture (5th,15th,25th,35th,45th,55th, and62nd transfer) were also processed for DNA extraction. Molecular techniques were then used to analyse the diversity of anaerobic fungi (ARISA) and methanogens (DGGE) present. A methano gen16S rDNA clone library was also prepared from the25th subculture.ARISA and DGGE analysis demonstrated that the diversity of anaerobic fungi (13peaks) and methanogens (17bands) in the original rumen digesta was complex. The diversity of anaerobic fungi in the co-culture in contrast was simple (two peaks), and remained consistent throughout the series of transfers. Microscopic observations confirmed that single fungal moncentric morphology was evident. The diversity of methanogenic Archaea in the co-culture in contrast was more complex with variation occurring over the series of transfers, resulting in a final profile of two bands by the62nd transfer. From the methanogen clone library a total of thirteen different sequences were obtained, two of which represented the two bands in the62nd transfer:Methanobrevibacter sp. and a novel methanogen (<80%similarity to any known taxon).These results demonstrate that methanogens and fungi representative in rumen digesta do not grow equally well in co-culture and population composition changes considerable with time/transfer. Identification of the methanogens present also indicated that co-cultivation based isolation strategies may prove valuable in the future for isolation of novel methanogens.Primers for the novel uncultivated lineage of methanogens were designed. And the PCR assay for determining the novel methanogen was established.3In vitro enrichment, activity and composition of ruminal anaerobic fungal and methanogen populations grown together in consecutive batch culturesConsecutive batch culture (CBC) was used to investigate the ability to establish stable, methane generating co-cultures of anaerobic fungi and methanogens from rumen digesta. Antibiotics were used to remove Eubacteria and fungal enrichment was encouraged by growing cultures on particles of barley straw. Three CBC series, each involving nine serial transfers at3,5and7day intervals respectively, were established with the initiating cultures being inoculated with rumen digesta. Microbial diversity and liquid and gaseous fermentation end-products were determined on selected cultures and at appropriate intervals throughout each of the CBC time-courses of27,45and63days respectively. At each measurement interval, cultures were found to contain anaerobic fungi and methanogens but Eubacteria and protozoa were shown to be absent. Anaerobic fungal diversity in the co-cultures was found to decrease with number of transfers and appeared related to transfer interval, diversity in the5-and7-day CBC series differing from that in the3-day CBC series. In general, a diverse population of methanogens was maintained throughout transfers (although results varied by restriction enzymes; Taq I and Msp I) and clustering of diversity profiles by transfer interval or transfer number was not observed. It was noted however that a novel, previously uncultivated methanogen persisted only in the5-and7-day CBC series. Fermentation profiling revealed the accumulation of minimal quantities of the typical anaerobic fungal fermentation end-products, formate and D-lactate, and larger amounts of acetate. Production of other typical Eubacterial end-products, such as propionate and butyrate, was not detected. Fermentation gas, including methane, was detected in all CBC cultures with the amount generated per culture increasing as the3,5or7day transfer interval increased. However, while amounts increased with increasing transfer interval, the rate of gas and methane production was inversely related to transfer interval with the fastest rates being achieved in the3-day CBC series. Moreover, the amount of gas produced in co-culture was significantly greater than that produced in cultures where the methanogens were removed. In conclusion, we found it relatively easy to establish rapidly growing, methane producing co-cultures of anaerobic fungi and methanogens from an inoculum of rumen digesta and to maintain them by routine transfer over considerable time periods (from27-63days). The co-cultures appeared more resilient than their axenic counterparts and if harnessed, our novel observation could have considerable application as an industrial anaerobic digestion process. The detection of novel uncultivated methanogen revealed that it only persisted in5&7d co-cultures.4Isolation and characterization of ruminal methanogens and anaerobic fungiRuminal methangens and anaerobic fungi were isolated using the Hungate anaerobic techniques. The16S rRNA genes of the metanogens were sequencing and ARISA analysis was done with anaerobic fungal strains. Four methanogens were isolated from rumen digesta. The16S rRNA gene sequences showed that they were similar (>99%) to Methanobacterium beijingense, Methanobacterium formicicum, Methanoculleus sp. and Methanosarcina mazei respectively. This was the first time that Methanobacterium beijingense strain was isolated from the rumen. Six anaerobic fungi were isolated from the co-cultures. A comparative study was performed on their morphology and ITS1sequences. Results showed that strain YC301belonged to Caecomyces, the remaining five strains belonged to Neocallimastix. The ARISA profiles of the remaining five strains were similar to each other which indicated that they might be the same strain. |
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