30Y 97.7 LGM-AF13 1 1260 DQ985550 Methanobrevibactersp. Z8 97.4 A total of 66 clones were examined. Figure 2 Phylogenetic analysis of 13 phylotypes of methanogens from the 25th anaerobic fungal subculture. The sequences determined in this study are marked in bold type. Accession numbers are

given in parentheses. The root was determined by using Pyrolobus fumarius (× 9555) as outgroup. The topology of the tree was estimated by bootstraps, based on 1000 replications. Bootstrap values greater than 80% are shown on the internal nodes. Further, in order to understand the methanogens which survived in the long-term transferred fungal subcultures, the two strong bands from the 62nd subcultures were excised from the DGGE gel for further cloning. Five clones generated www.selleckchem.com/products/jnk-in-8.html from each band were sequenced and showed to be identical. AC220 cell line One band had its sequence (EF222222) 99% similar to LGM-AF04, and the other had its sequence (EF222223) 98% similar to Methanobrevibacter sp. Z8. Transfer frequency affects the abundance of the novel RCC species in the fungal subcultures To monitor the abundance of the novel RCC species, PCR specific primers (LGM178f/434r) to this novel RCC were

designed. BLAST searches of the primer sequences showed homology to sequences within the novel RCC species only. Their specificity was further confirmed by running PCR, and results showed that the primers only targeted the novel RCC species, and did not target other methanogen isolates or clones, or bacteria species tested in this study (Figure 3). Figure 3 Detection of the PCR specific primers for the novel RCC species.

M, DNA marker; LGM, the novel RCC clone; M4, Methanobacterium beijingense like strain; M6, Methanobacterium formicicum like strain; MEF2, filipin Methanobrevibacter smithii like strain; RPS4/RPS15, Methanoculleus sp. like strain; RPS13/RPS37, Methanosarcina mazei like strain; R24, Methanomicrobium mobile clone; Y76, Methanosphaera stadtmanii clone; K88, E. coli K88; RE, E. coli isolated from rumen digesta; C, PCR control. The effects of the transfer frequency on the abundance of the novel RCC species in the anaerobic fungal subculture were investigated using the specific primers. The results showed that, as the transfer proceeded, the16S rRNA gene copy numbers of the novel RCC species significantly increased in the mixed cultures with the five-day transfer frequency and the seven-day transfer frequency (P<0.05), while it decreased in the three-day subcultures (Figure 4). This finding suggested that low transfer frequency might benefit the enrichment of the novel RCC species in the mixed cultures. Figure 4 The relative abundance of the novel RCC species in the anaerobic fungal cultures transferred with three transfer frequencies. Fungal cultures were transferred every 3, 5, and 7 days, and the samples were collected at the 2nd, 4th, and 9th subcultures.