Isolation And Characteristic Of Three Heterotrophic Nitrifiers And Its Application In Aquaculture Wastewater Treatment

Aquatic productivity is usually based on the quantity and quality of nitrogen, which plays an important role in aquaculture system.As a serious threat to the aquaculture organisms,accumulated ammonia and nitrite in the aquaculture water should be highly concerned.Aimed at biological nitrogen removal in aquaculture system,the research is carried out through such aspects as isolation of heterotrophic nitrifiers,nitrogen removal characteristics of nitrifiers,cloning of nitrite reducatse encoding gene,construction and application of nitrite-removal engineered bacteria, etc.The main results obtained are as follows:(1) Isolation and ammonia removal capability of heterotrophic nitrifiersThree nitrifying strains were isolated from active sludge of Sibao sewage plant. Strain ZY-1,ZY-2 and ZY-3 were indentified as Bacillus flexus,Bacillus cereus and Pseudomonas aeruginosa,respectively.After cultured in ammonium medium,three strains all exhibited high ammonia and TN(total nitrogen) removal efficiency and low nitric oxide accumulation,which had further research value in nitrogen removal process.(2) Nitrogen removal characteristics of three heterotrophic nitrifiersUnder aerobic conditions,strain ZY-1,ZY-2 and ZY-3 showed high nitrite removal efficiency.65 mg/L nitrite in the medium was completely removed after 48 hours.TN removal rates of ZY-1,ZY-2 and ZY-3 could reach 84.83%,83.42%and 92.22%,respectively.Strong denitrification capabilities of three strains were detected.Nitrate removal rates of strain ZY-2,ZY-2 and ZY-3 in 72 hours reached 99.42%,99.31%and 96.26%,respectively.According to the nitrogen removal characteristics of three heterotrophic nitrifiers,nitrogen removal pathways of three strains were speculated as followed: NH₄⁺→NH₂OH→NO₂^-→NO₃^-→NO₂^-→NO→N₂O→N₂.Growth rates and ammonia removal capabilities of strain ZY-1 were significantly improved after optimization of culture conditions including initial medium pH, inoculating rate,shaking speed and incubation temperature. (3) Construction and application of engineered strain expressing nitrite reductaseNitrite reductase-encoding gene(nirS) was amplified from strain ZY-3 and transferred into E.coli BL21.The constructed engineered strain ZY-4 successfully removed accumulated nitrite both in raw and simulated aquaculture wastewater. Ammonium concentration in aquaculture wastewater decreased obviously.Moreover, mixed strains consisted of engineered bacterium and heterotrophic strain ZY-1 and ZY-2 showed great nitrogen removal efficiency in raw and simulated aquaculture wastewater treatment process.