Adaptation of methane recovery, sludge characteristics and evolution of microbial community response to elevated nitrate under the methanogenic condition

Coupling of methanogenesis and denitrification with elevated nitrate under the methanogenic condition shows potential advantages for (N-) heterocyclic compounds removal and methane recovery simultaneously. However, methane production, sludge characteristics and microbial evolution response to elevated nitrate were seldom reported. Here, an experiment lasing about 300 days under the methanogenic condition with sequential influent nitrate addition as COD/NO₃⁻-N ratio from 486.03 to 8.00 was carried out. More than 50% decline of removal rate of COD was observed when the COD/NO₃⁻-N ratio varied from 19.97 to 8.00 (232 d). At last, the removal rate of COD recovered to around 94% at the COD/NO₃⁻-N ratio of 8.00. About 90% of nitrate was removed through denitrification, and a lower content of methane (40–45%) and effluent acetate and propionate (<10 mg/L) were observed with elevated nitrate. Damage of the granule-shaped and multi-layer structure of the sludge with the lift of nitrate addition was studied. Results of high-throughput sequencing hinted that bacteria accounted for about 70% of total microbes and Proteobacteria, Bacteroidetes, Firmicute and Chloroflexi were the dominant phyla shared with sludge at different COD/NO₃⁻-N ratio. Pseudomonas genus and Enterobacteriaceae family (Gammaproteobacteria class, Proteobacteria phylum), Brachymonas genus and Rhodocyclaceae family (Betaproteobacteria class, Proteobacteria phylum) might be vital for the denitrification process under the methanogenic condition. An increase of Smithella and Syntrophobacterale genus with elevated nitrate could be partly contributed to denitrification from the transformation of intermediates as propionate and butyrate. Methanosaeta and Methanobacterium genus were two major methanogens, and Methanosaeta seemed to be less adaptable for elevated nitrate than Methanobacterium. A suitable nitrate addition as influent COD/NO₃⁻-N ratio ranged from 121.43 to 15.18 was recommended for the coupling of methanogenesis and denitrification. The results of this work would enhance the understanding of the effects and adaptation with elevated nitrate under the methanogenic condition and instruct engineering practice for low COD/NO₃⁻-N wastewater treatment and methane recovery.