Isolation, Identification And Cultivation Of Two Strains Of Heterotrophic Nitrification And Aerobic Denitrification Bacteria And Integrated Denitrification Research

Bacterial strains YY24and HA2with simultaneous heterotrophic nitrifition and aerobicdenitrification abilities were isolated from a biofilter system in marine fish farm. Microbialsamples were identified by morphological analysis, physiological and biochemicalcharacteristic and16S rDNA. The heterotrophic nitrification and aerobic denitrificationperformance of every single strain were studied as follows:Samples were collected on August4th,2012and May12th,2013respectively.Degradingbacteria with high ammonia nitrogen degrading efficiency was screened byenrichment culturemethod. The bacteria was inoculated on the heterotrophic nitrification medium with the prop-ortion of1%.The bacteria was cultured for24h under the conditions （C/N10,23°C and120rpm） with ammonium chloride as the nitrogen source and sodium citrate as the carbon source.Two trains of degrading bacteria with high ammonia nitrogen degrading efficiency （YY24and HA2） were rescreened according to the removal rates of total ammonia nitrogen （TAN）and total nitrogen （TN）, of which YY24was isolated from samples collected on2012.8.4andHA2was isolated from samples collected on2013.5.12.According to the morphological analysis, physiological and biochemical characteristicand16S rDNA identification， the strain YY24showed high sequence similarity of99%toP.pseudoalcaligenes （NR037000.1） when identified by16S rDNA sequence analysis. Andthe strain HA2showed high sequence similarity of99%with Vibrio alginolyticus（NR₀44825.1）. The strain YY24was identified by microorganism identification instrumentto be Pseudomonas aeruginosa with a reliability of98.1%and the strain HA2was identifiedto be Vibrio alginolyticus with a reliability of96.1%.The heterotrophic nitrification and aerobic denitrification performance of YY24and HA2were analyzed by measuring its growth and nitrogen removal efficiency. Under the conditions （C/N10,23°C and120rpm） with ammonium chloride as the nitrogen source and sod-ium citrate as the carbon source,growth of YY24reached logarithmic phase after4h and ent-ered stationary phase after24h. The removal ratioof total ammonia （TAN） and total nitrogen（TN） were96.67%and95.36%respectively at24h. And HA2reached logarithmic phase after6h and entered stationary phase after27h. The removal ratios of total ammonia （TAN） andtotal nitrogen （TN） were97.24%and90.92%respectively at27h. During the incubition,there were almost no nitrite and nitrate accumulation. The independent effects of environmental factors on nitrogen removal were tested, andthe optimal conditions were as follows: pH ranged5.5-8.5, the C/N ratio was above10,temperature ranged from23to30°C and the most suitable carbon source was sodiumcitrate.The effects of environmental factors, including carbon source, pH, carbon to nitrogen（C/N） ratio, on growth and denitrification capability of the HA2were tested. Results showedthat the optimum growth conditions were as follows: carbon source sodium citrate,23℃, pH8.5, C/N ratio18, under which the ammonia nitrogen removal rate of the HA2was99.06%,nitrogen removal rate was97.27%.Based on productive practice, effects of YY24and HA2on the formation of biofilm andeffects on water purification under recirculating aquaculture system were studied. The aminonitrogen removal capacities of strain HA2under different organic loading were compared. Itshowed that the strains had a significant impact on the forming of biofilm under naturalconditions. The strain YY24was easier to form biofilm and the forming cycle to achieve theexpected effects, i.e. the maturation cycle of film-forming, was about14d. After theformation of biofilm, good effects of nitrification and denitrification were achieved: TANconcentration in the biological filter remained below0.8mg·L-1after19d; the concentrationof NO2--N remained below0.5mg·L-1after40d; the concentration of NO3--N remainedbelow1.5mg·L-1after38d. The biofilm culturing cycle of HA2was19d. After theformation of biofilm, TAN concentration in the biological filter remained below0.9mg·L-1after26d, the concentration of NO2--N remained below0.1mg·L-1after38d, and theconcentration of NO3--N remained below1.7mg·L-1after40d, which showed good removaleffects on TAN, NO2--N, NO3--N, except TN. The strains were easier to form biofilm underhigher organic loading, with19d; the forming cycle was21d under lower organic loading.