Enrichment Of Nitrite-dependent Anaerobic Methane Oxidation Bacteria And Medium Optimization

Nitrite-dependent anaerobic methane oxidation (N-DAMO) is a novel bioprocess that couples anaerobic methane oxidation to nitrite reduction, which is mediated by Candidatus Methylomirabilis oxyfera (M. oxyfera) affiliated to NC10 phylum.Perivious research indicated that heterotrophic denitrifiers exist together with N-DAMO bacteria during the whole enrichment period and heterotrophic denitrifiers share complex relationships with N-DAMO bacteria, including competition and mutuality. However, hitherto there is no report on this. On the other hand, N-DAMO bacteria grow slowly, which limits the theoretical research and practical application of N-DAMO. To date, all of the N-DAMO biomass was enriched from fresh water ecosystems, although some NC10 16S rRNA and pmoA gene sequences were retrieved from some saline water ecosystems, suggesting the existence of N-DAMO bacteria and the possible occurrence of N-DAMO process in saline waters.In the present work, community successions of N-DAMO bacteria and heterotrophic denitrifiers were investigated to provide guidance for obtainment of N-DAMO enrichment harbouring highly purified and active N-DAMO bacteria. The mineral elements in the fresh water medium were optimized for N-DAMO bacteria growth to accelerate the enrichment of N-DAMO bacteria. Moreover, a novel way for pH control was proposed to maintain the pH near neutral. Finally, N-DAMO biomass was obtained from marine ecosystems to enrich the inoculum sources of N-DAMO culture. The main results were as follows:1. The community successions of N-DAMO bacteria and heterotrophic denitrifiers were revealed.① Fresh water N-DAMO biomass was successfully obtained with paddy soil as inoculum after 600 days’ enrichment. The course of the enrichment could be split into three phases, namely, cell lysis phase, activity lag phase and activity elevation phase. During activity lag and activity increase phases, N-DAMO was the dominant process. During activity increase phase, N-DAMO activity increased exponentially, while heterotrophic denitrifiers’activity inceresed slightly, even decereased a bit later. N-DAMO biomass was enriched after 600 days’incubation.② A new group of NC10 phylum was classified in the inoculum, named after group E, which is distantly relative to M. oxyfera with low sequence similarities (<90%) of 16S rRNA genes. However, the members of group E in the inoculum were eliminated after 6 months’ incubation, and only group A members were enriched.2. The mineral elements in enrichment medium and pH control for reactor operation were optimized.① Four mineral elements (CaCl2, MgSO4, KH2PO4 and KHCO3) in the fresh water medium were optimized with orthogonal tests and acid-base titration curves. The optimal mineral elements was 0.3 g L-1 CaCl2,0.2 g L-1 MgSO4,0.3 g L-1 KH2PO4 and 0.2 g L-1 KHCO3.② Addition of CO2 in the headspace of the bioreactor was an efficient way to control pH, and both theoretical estimation and practical experiment indicated that 1% CO2 could keep the pH near neutral in the optimal medium containing 0.2 g L-1 KHCO3.3. Marine N-DAMO biomass was enriched and its properties were investigated.① First marine N-DAMO enrichment culture was obtained from coastal sediments. During the course of enrichment, N-DAMO specific activity and cell numbers increased exponentially, and the doubling times were 33.6-38.4 days, which were slightly longer than those of fresh water N-DAMO biomass (14-25 days). The N-DAMO activity per cell of this marine culture was 0.15±0.07 frnol CH4 d-1 cell-1, close to those of fresh water N-DAMO culture (0.09-0.34 fmol CH4 d-1 cell-1).② Microbial community of the obtained marine N-DAMO culture was analyzed. Similar to the N-DAMO enrichment of fresh water in this work, group E of NC10 phylum was also detected in the inoculum but eliminated in the enrichment, and only group A members were enriched. Therefore, members of group A could mediate N-DAMO process.③ The effects of substrates (methane and nitrite) and salinity on the obtained marine N-DAMO culture were investigated. Based on the best fittings of the data, apparent substrate affinity coefficients of this N-DAMO culture were both lower than those of fresh water N-DAMO culture. On the other hand, salinity was also a potent impact factor, and the optimal value was 20.5‰. Moreover, low salinity destroyed the marine N-DAMO bacteria more seriously than high salinity.