Molecular Biology Identification Of Functional Bacteria In Single-stage Autotrophic Nitrogen Removal System And Gene Cloning And Expression Of Key Enzyme In Ammonia-oxidizing Reaction

Single-stage autotrophic nitrogen removal process is achieved in one single reactor, where ammonia is oxidized to nitrogen by autotrophic bacteria simultaneously. It is a new nitrogen removal process and has many advantages such as less gas and power supplying, no extra carbon source, fewer costs of processing etc, and it will have a better application prospect in the field of environmental protection. Until now the mechanism of nitrogen removal is not yet clear, which impedes its application. Research on the functional bacterial in the system has been in progress in order to explicate the mechanism of ammonia romoval. Here this study focused on phylogenetic analysis of two important groups in the single-stage autotrophic nitrogen removal system, anaerobic ammonium-oxidizing bacteria (ANAMMOX bacteria) and ammonium-oxidizing bacteria (AOB), and prokaryotic expression of functional gene amoA and hao in order to provide a theoretical foundation for the mechanism of nitrogen removal.In this study, using the cultivated sludge from the single-stage autotrophic nitrogen removal system with stable operation and 90% of ammonia conversion rate and 80% of total nitrogen removal rate, several aspects were investigated as follows.(1) With the total DNA extracted from cultivated sludge, partial 16S rDNA sequence of ANAMMOX bacteria was amplified by polymerase chain reaction (PCR) with a pair of specific primers Pla46rc/Amx820. Amplified product was cloned, sequenced and analyzed by BLAST. The result indicated that the sequence has 99% identities with ANAMMOX bacteria Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans. The phylogenetic analysis showed that the anaerobic ammonium-oxidizing bacteria in the single-stage autotrophic nitrogen removal system have closer relationship with Candidatus Kuenenia stuttgartiensis evolutionarily.(2) With the total DNA extracted from cultivated sludge through four times of enrichments, partial sequence of 16S rDNA, the full length sequences of ammonia monooxygenase (amoA) gene and hydroxylamine oxidoreductase (hao) gene of AOB were amplified by PCR with specific primers which were designed according to sequence published NCBI GenBank. Amplified product was cloned, sequenced and analyzed. The result showed that the sequences of 16S rDNA, amoA and hao have 99%, 99% and 100% identities with Nitrosomonas europaea ATCC 19178, Uncultured bacterium clone amoA_SBR_JJY clone, Nitrosomonas sp.ENI-11, respectively. The phylogenetic analysis revealed that the AOB in the single-stage autotrophic nitrogen removal system have closer relationship with"Nitrosomonas sp. DYS323","Nitrosomonas sp. DYS317","Uncultured bacterium clone amoA_SBR_JJY 61clone","Uncultured bacterium clone amoA_SBR_JJY 70clone"and"Nitrosomonas sp.ENI-11hao2". The results described above demonstrated that ANAMMOX bacteria and AOB exist in the system, and that the AOB belongs to different species of Nitrosomonas sp.(3) The full-length sequences of the amoA and hao were successfully amplified with the specific primers based on published amoA and hao sequences in NCBI. The amoA and hao had been cloned into the prokaryotic expression vector pET-32a, generating the recombinant plasmid pET-32a-amoA and pET-32a-hao that were then transformed into E. Coli BL21 (DE3). The best induction of recombinant AMO and HAO was obtained when 1.0 mmol/L IPTG was added into the E. Coli BL21 (DE3) incubated for 6 h at 37℃and 30℃, respectively. Western Blot detection showed that the fusion protein was correctly expressed in E. Coli BL21 (DE3). Detection of crude enzyme activity showed that the activity of AMO and HAO was stronger in vitro. This result established the theoretical basis for building engineering bacteria.