| Nitrogen contamination which is responsible for eutrophication has been a serious environmental problem gradually noticed by the whole world, especially in the aquaculture process. Due to low conversion rate and high toxicity of nitrite nitrogen, lots of aquatic animals are died from oxygen shortage. In addition, nitrite combined with amine come into being nitrosamine of acute carcinogenic action. Pathogenic fish and shrimp are caused primarily by high nitrite concentration. According to conventional bioremoval of nitrogen theory, denitrification is completed in anoxic or anaerobic condition. However, aerobic denitrification is proposed lately and some aerobic denitrifiers are identified by foreigner scholars. Noting but aerobic denitrifiers with nitrite reductase are capable of completely denitrification. Study on screening and investigation of aerobic denitrifiers in domestic field still has a long way to go, few reports about the reduction of nitrite nitrogen accumulation in aerobic denitrification process were existed in the literature.The purpose of this study is expected to screen aerobic denitrifier, nitrogen removal of which is higher than that of original strains, with the method of UV mutagenesis coupled protoplast fusion. Besides, the research on reduction of nitrite accumulation is also investigated, looking forward to provide references in aquaculture wastewater treatment. Some aerobic denitrifiers have been isolated from activated sludge by SBR, an aerobic denitrifier of high nitrogen-removal, named R1, was screened through shaking flask. Several aspects regarding the morphological and colony characteristic, physiological characteristic and dentitrification characteristic of strain were explored. The results showed that, the color was straw yellow, the shape was round, the fringe was regular, the surface was glossy and viscid. The figure of R1 was bacilliform, G+. There are four phases in the growth process of R1: lag phase, logarithmic phase, stationary phase, decline phase. Aerobic denitrification by R1 occurred mainly in logarithmic phase, that is culture time of 6～24h. Generation period of R1 is 5 days. It was found that nitrate removal rate by R1 was 75.56%, but nitrogen removal rate was only 21.42% due to high nitrite nitrogen accumulation of 75.05mg·L-1. It was concluded that nitrite reductase activities of R1 was low in aerobic denitrification, which lead to nitrite nitrogen accumulation during conversion from NO2- to N2O and N2.It is generally believed that the method of UV mutagenesis is widely applied for its safety operation, simple equipment and high efficiency. Hence, strain R1 was induced by UV mutagenesis, aimed to increase nitrogen removal rate and decrease nitrite nitrogen accumulation of aerobic denitrifier. As a result, a mutant, named ZR43, was obtained. Several aspects regarding the morphological characteristic, growth condition, genetic stability and dentitrification characteristic of strain ZR43 were explored. The results showed that the method of UV mutagenesis was successfully employed to enhance nitrite reductase activities of aerobic denitrifier, nitrite nitrogen accumulation in the dentitrification process was significantly lessen, only 17.42 mg·L-1.Nitrogen removal rate was enhanced by 18.06%, compared with original strain R1. The results proved that, the figure of ZR43 was global, G+, the color was yellow, the fringe was regular and the surface was glossy.The effect of environmental factors on growth of strain ZR43 was observed by orthogonal experiment. The optimal temperature was 30℃, pH was 8, C/N ratio was 10 and carbon source was succinic acid. It was found that aerobic denitrification performance of the strain ZR43 was influenced mostly by C/N ratio. In the case when carbon source was insufficient, electron was not enough, provided by carbon source for growth of strain ZR43. In the case when carbon source was sufficient, the growth of strain ZR43 was not limited by carbon source, the growth and metabolize were in stable state. However, denitrification performance of the ZR43 was influenced less by temperature and pH, which proved that ZR43 adapted to large extent of temperature and pH. It would have big application value in waste water treament by ZR43. It was concluded that the degree of the important effect on growth of strain ZR43 from small to large was ordinal:C/N ratio, carbon source, pH, temperature.Serial subcultivation of ZR43 from passage 5 to passage 8 was investigated and denitrification performances of passages from 5 to 8 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 nitrogen removal. Thereby, genetic stability of ZR43 was great. Furthermore, the effects of biomass and C/N ratio on denitrification performance of ZR43 were discussed. Bioactivities and nitrogen removal rate of ZR43 in different biomass were all high, without obvious variation. The highest cell concentration was achieved, at 3.1g·L-1, nitrogen removal rate was 37% or so. Hence, aerobic denitrification performance of ZR43 was not much influenced in different biomass. Nevertheless, it was found that aerobic denitrification performance of ZR43 was influenced mostly by C/N ratio and nitrogen removal rate was above 32% with a C/N ratio of above 8. Nitrogen removal rate was hardly increased by more C/N ratio.In recent 20 years, protoplast fusion technique has been developed by investigators. The formation of protoplast can be produced by lysozyme, then protoplast fusion is accelerated through PEG. Therefore, the method of protoplast fusion was adopted to investigated, aimed to enhance nitrite reductase activities and nitrogen removal rate more. The formation conditions of strain were optimized with the orthogonal experiment, the optimal culture time was 16h, absorbency of cellular fluid was 0.23, enzyme concentration was 20mg·mL-1, enzymolysis temperature was 42℃, enzymolysis time was 1h, thus formation rate of 99.8% was obtained. As a result, protoplast fusion technique had been successfully employed to increase nitrite reductase activities and nitrogen removal rate, as well as a fusant that named F3 was screened, doubling the excellent characters of parent strains: ZR43 and ZR24. The nitrate nitrogen removal rate and TN removal rate of F3 was 99.86%, 73.21%. Correspondingly, the nitrite nitrogen accumulation was 36.95 mg·L-1.The physiological characteristic and dentitrification characteristic of F3 were discussed. The results showed that the color of F3 was yellow, the shape was round, the fringe was regular, the surface was glossy and viscid. Compared with strain ZR43 and R1, the growth of F3 was lentamente in logarithmic phase, 24h later. The highest cell concentration was achieved after 24h-culture. It was concluded that TN removal rate of fusant F3 was almost 52% higher than original strain, made by protoplast fusion technique, whenas the increase of TN removal rate by mutant was few, only 18%.In a word, the method of UV mutagenesis coupled protoplast fusion was successfully employed to enhance nitrite and nitrate reductase activitie of aerobic denitrifier demonstrated by this experiment, accordingly improve aerobic denitrification efficiency. It will have big engineering significance in aquaculture wastewater treatment in the future. |
PDF Full Text Request