Study On UV Mutagenesis And Isolation Of Aerobic Denitrifiers For Treatment Of Nitrate Wastewater

With the rapid economic development, nitrogen contamination from industrial wastewater, domestic sewage, agricultural fertilizer, aquaculture wastewater, which results in eutrophication of rivers and lakes, has been constantly increasing. Eutrophication has become a serious environmental problem all over the world,and therefore the requirement of nitrogen-containing wastewater disposal is much higher than before. According to traditional theory of biological nitrogen removal, denitrification is completed only in anoxic or anaerobic conditions. However, aerobic denitrification is proposed in recent years and some aerobic denitrifiers are identified by foreign and domestic scholars. Compared with traditional nitrogen removal process, simultaneous nitrification and denitrification by aerobic denitrification has a lot of advantages and become a research hotspot at present. Yet denitrification can not be carried out completely by each species of aerobic denitrifiers, and nitrite accumulation is often produced as intermediate products during the course of denitrification. Therefore, this thesis will focus on how to select aerobic denitrifiers with good performance that can remove nitrate more efficiently without producing the accumulation of nitrite at the same time, and study the application of aerobic denitrifiers in the treatment of nitrate wastewater.A strain of aerobic denitrifier named ZR47 had been selected in our laboratory in the prior period. The strain ZR47 was cultured in shaking flask containing medium with KNO3 as the sole nitrogen source for 4 days. The initial concentration of nitrate was 138.61mg/L, initial pH value was 7.0, temperature was 30℃, and the rotational speed was 140 r/min. Under these conditions, it was found that nitrate removal rate by ZR47 was 80.56%, but total inorganic nitrogen(TIN)removal rate was only 59.26% due to high nitrite nitrogen accumulation of 29.53mg/L. It was concluded that nitrite reductase activities of ZR47 was lower than nitrate reductase activities in aerobic denitrification, which resulted in nitrite nitrogen accumulation during conversion from NO2- to N2O and N2.On this basis, strain ZR47 was induced by UV mutagenesis, aimed to increase TIN removal rate and decrease nitrite nitrogen accumulation of aerobic denitrifiers. As a result, through screening numbers of mutants with the methods of taking NO3--BTB chromogenic medium for pH indicator and shaking culture, a mutant named SZR29 was obtained. Several aspects regarding the morphological characteristic, dentitrification characteristic and genetic stability of strain SZR29 were studied. The results showed that the method of UV mutagenesis was successfully employed to enhance nitrite reductase activities of aerobic denitrifiers, nitrite nitrogen accumulation in the dentitrification process was significantly lessened, only 9.32 mg/L.TIN removal rate reached 76.89%, which was enhanced by 18.06% compared with original strain ZR47. The cell morphology of SZR29 was global, G+, the colony morphology was circular, yellow, protuberant with slippery surface and regular fringe, and the generation cycle was 2.36h. There are four phases in the growth process of SZR29: lag phase, logarithmic phase, stationary phase and decline phase. Aerobic denitrification by SZR29 occurred mainly in logarithmic phase and culture time was between 6h and 30h. The maximum of nitrate nitrogen removal rate reached 3.75mg/(L·h) in this stage. After serial passage of SZR29 on the inclined medium, the initial strain and the eighth passage of SZR29 were investigated and denitrification performances of them were observed. The outcome indicated that nitrite reductase activities of respective passages keep high levels steadily, which made no significant differences with nitrite nitrogen accumulation and TIN removal rate. Thereby, genetic stability of SZR29 was great.Secondly, based on characteristics of aerobic denitrifiers that can grow both in anoxic and aerobic conditions, activated sludge was domesticated with nitrite as substrate by intermittent aeration operation in sequence batch reactor, to enrich aerobic denitrifiers. By gradually increasing aeration time and the concentration of COD, NO2- -N in influent water, the environment was progressively suitable for aerobic denitrifiers to survival, and eventually aerobic denitrifiers became dominant bacteria in the system to achieve enrichment. After 71 days, the removal efficiency of NO2--N, TIN, COD respectively reached 99.99%, 74.56% and 91.51% in the system, and sludge morphology was transformed from yellow, dispersed sludge to filemot, flocculent aerobic sludge, which marked the enrichment of aerobic denitrifiers was completed.By using plate dilution method and plate streaking method, 30 strains of aerobic denitrifiers were separated and purified from activated sludge. Then screening these strains with shaking culture method to seek for a strain that can remove nitrite efficiently in aerobic condition, and finally, a strain named B5 was obtained. Moreover, the morphological characteristic and dentitrification characteristic of strain B5 were studied. The strain B5 was cultured in shaking flask containing medium with NaNO2 as the sole nitrogen source for 4 days. The initial concentration of nitrite nitrogen was 138.61mg/L, initial pH value was 7.0, temperature was 30℃, and the rotational speed was 140 r/min. Under these conditions, it was found that nitrite removal rate by B5 was 90.25%,and TIN removal rate reached 90.02%. The cell morphology of B5 was short rod, G+, the colony morphology was circular, white, dry with smooth surface and regular fringe, and the generation cycle was 3.05h. Aerobic denitrification by B5 occurred mainly in logarithmic phase as well as stationary phase, and the culture time was between 12h and 36h. The maximum of nitrite nitrogen removal rate reached 3.47mg/(L·h) during these stages.There are several benefits by using immobilized cell technology to remove pollutants in wastewater, such as high efficiency, resistance to shock loading, easy for solid-liquid separation, etc. In this paper, strains SZR29 and B5 were mixed-immobilized by choosing polyvinyl alcohol (PVA) as entrapping agents, which was mixed with sodium alginate and activated carbon. The optimal conditions were chosen in terms of the mechanical stability and denitrification performance of the cells. Moreover , the optimum particle-filled rate and the influence of different environmental factors on nitrogen removal effect of immobilized cells were also studied. The results showed that the optimum conditions of immobilization were as follows: PVA entrapping consist of 10% (W/V) as its concentration, 1:2 as the denitrification amount, and 32h as the coagulant duration. The denitrifying activity of immobilized cells can be improved by adding 1% sodium alginate and 2% activated carbon powder to the gel. The filling ratio suitable to the immobilized particle was 10%, and the optimum conditions for denitrification of the entrapped immobilized denitrifiers were pH of 7~8, temperature of 30℃, carbon-nitrogen ratio of 10. Compared with aerobic denitrifiers not imbedded, the entrapped immobilized denitrifiers showed stronger tolerance ability to the change of pH value and temperature, and the denitrifying efficiency was obviously higher than that of the mobilized under low temperature (15℃), strong acid or alkaline condition (pH<6.0 or pH>8.0).Immobilized cells were filled into fluidized bed reactor with the filling ratio of 10% to dispose nitrate wastewater. The results showed that under the conditions of NO3--N influent concentration of 48.62~80.11mg/L, carbon-nitrogen ratio of 10, temperature of 30℃, pH value of 7~7.5, DO of 4~5mg/L, hydraulic retention time of 24h, the system exhibited good running stability within continuous operation for 15~20 days. The highest removal rate of TIN reached 94.99%, the concentration of nitrate and nitrite in effluent water decreased to 3.48 mg/L and 0.069mg/L.In a word, the research on the treatment of nitrate wastewater with immobilized aerobic denitrifiers obtained very good effect. It provides theoretical basis for the application of aerobic denitrifiers in wastewater disposal and will have certain engineering significance in the future.