Methane-generating ammonia oxidizing nitrifiers within bio-filters in aquaculture tanks

Barry Kamira, Lei Lei Shi, Li Min Fan, Cong Zhang, Yao Zheng, Chao Song, Shun Long Meng, Geng Dong Hu, Xu Wen Bing, Zhang Jia Chen, Pao Xu

The discovery of aerobic and anammox bacteria capable of generating methane in bio-filters in freshwater aquaculture systems is generating interest in studies to understand the activity, diversity, distribution and roles of these environmental bacteria. In this study, we used microbial enrichment of bio-filters to assess their effect on water quality. Profiles of ammonia-oxidizing bacterial communities generated using nested PCR methods and DGGE were used to assess the expression of 16S rRNA genes using DNA sequencing. Five dominant ammonia-oxidizing bacterial strains-clones; KB.13, KB.15, KB.16, KB.17 and KB.18-were isolated and identified by phylogenetic analysis as environmental samples closely related to genera Methylobacillus, Stanieria, Nitrosomonas, and Heliorestis. The methyl ammonia-oxidizing microbes thereby found suggest a biochemical pathway involving electron donors and carbon sources, and all strains were functional in freshwater aquaculture systems. Environmental parameters including TN (2.69-20.43); COD (9.34-31.47); NH4+-N (0.44-11.78); NO2-N (0.00-3.67); NO3-N (0.05-1.82), mg/L and DO (1.47-10.31 µg/L) assessed varied in the ranges in the different tanks. Principal component analysis revealed that these water quality parameters significantly influenced the ammonia oxidizing microbial community composition. Temperature rises to about 40 °C significantly affected environmental characteristics-especially DO, TN and NH4+-N-and directly or indirectly affected the microbial communities. Although the nested PCR design was preferred due to its high sensitivity for amplifying specific DNA regions, a more concise method is recommended, as an equimolar mixture of degenerate PCR primer pairs, CTO189f-GC and CTO654r, never amplified only 16S rRNA of ammonia-oxidizing bacteria.