Figure 3 Phylogenetic tree showing the affiliations of bacterial 16S rRNA gene sequences detected from S2 to selected reference BYL719 sequences. Enrichment of ANME-2 and SRB CARD-FISH results showed that percentages of ANME-2 and SRB biovolume increased from 13.4 ± 4.2% and 22.7 ± 5.3% in S1 to 50.4 ± 15.9%

and 60.6 ± 5.5% in S2 (Table 2). By combining with the total biovolume data from DAPI staining (Figure 1B), the biovolume of ANME-2 in S1 was: (1.28*109 μm3/ml slurry) * 13.4% = 1.7*108 μm3/ml slurry The biovolume of ANME-2 in S2 was: (4.49*109 μm3/ml slurry) * 50.4% = 2.3*109 μm3/ml slurry Therefore after 286 days incubation, the ANME-2 population increased for 12.5 times. Following the same method of calculation, the SRB population increased for 8.4 times after 286 days incubation in this high-pressure bioreactor. The populations of ANME-2 and SRB both MM-102 nmr increased faster than the total biomass, which indicated that ANME-2 and SRB were selectively enriched in the system. This selective enrichment of ANME-2 and SRB was another proof that the incubation condition inside this high-pressure bioreactor was favourable for SR-AOM community. To our knowledge, this is the first report on the enrichment of SR-AOM community under high methane pressure, although

potential growth of ANME-1, ANME-2 and SRB has been reported in other engineered systems at ambient or low methane pressures (Table 3). The different inocula showed different

doubling times. When ANME-1 and ANME-2c were incubated in continuous flow bioreactors under ambient methane partial pressure, ANME-1 had doubling time of 1.1 months while ANME-2c had doubling time of 1.4 months [16]. High methane partial pressure appeared to have advantage on MK-0457 cost stimulating the growth of ANME. In the experiment of Krüger et al. [22], the methane-dependent uptake of 15N-NH4 by AOM community dominated by ANME-1 was higher at 1.5 MPa methane pressure than at ambient methane pressure. If we assume the ANME-2a cells in our system were following a logarithmic growth curve, a doubling time of 2.5 months can be estimated based Dolutegravir supplier on ANME-2 biovolume in S1 and S2, which is shorter than the result (3.8 months of doubling time of ANME-2a from an ambient pressure bioreactor) obtained by Meulepas et al. [10]. The increase of energy gained from SR-AOM process by increasing methane pressure may favour the biomass growth [8, 22]. Continuous flow also stimulated growth: ANME-2a/2c had longer doubling time in a fed-batch bioreactor (7.5 months) than in continuous flow bioreactors (1.4-3.8 months) (Table 3). Table 3 Comparison of doubling times of ANME in different enrichment systems Sediment origin ANME group Methane pressure Operational mode Doubling time (months) Reference Monterey Bay ANME-1 Ambient Continuous flow 1.1 [16] Gulf of Mexico ANME-1 1.5 MPa Batch 2-3.4 [22] Eckernforde Bay ANME-2a Ambient Continuous flow 3.