| As the main form of surface water, reservoirs(lakes) have become the main drinking water of a modern city. With the problem of water quality pollution, eutrophication and algal blooms occurring frequently, the safety of water quality has aroused increased attention in recent years. In this research the high efficient heterotrophic simultaneous nitrification and denitrification bacteria and aerobic denitrifying bacteria are isolated and domesticated from the sediments of the Heihe reservoir and the species identification of the bacteria are carried out as well as the analysis of biological denitrification. In addition to, this study utilizes response surface methodology of the Design-Expert software to optimize the biological denitrification rate under many conditions. Aiming at the problem of the low nutrient of the reservoir, microbial agents are made from the oligotrophic denitrifier groups to form the in-situ biological denitrification technology which can effectively remove the nitrogen pollutants of the oligotrophic reservoir. The research also investigates the effect the carbon source has on the oligotrophic denitrifier groups and reveals the characteristics of the combined technology applied to the denitrification of the micro-polluted water body.(1) Domestication, isolation and denitrification properties of oligotrophic heterotrophic simultaneo us nitrification and denitrification bacteria. Two new strains with simultaneous nitrification and denitrification, named ZK-1 and J*26, are isolated from the sediment of the oligotrophic Heihe reservoir. Based on the sequence analysis of the 16 Sr RNA, strain ZK-1 was identified as a species of genus Acinetobacter with the similarity of 99%. Based on the sequence analysis of the 16 Sr RNA, strain J*26 was also identified as a species of genus Acinetobacter with the similarity of 98%. The genes encoding perip lasmic nitrate reductase(napA) and heme nitrite reductase(nirS) of the two strains,which are responsible for the aerobic reduction of nitrate and nitrite, respectively, were successfully amplified. The two strain ZK-1 and J*26 had a ammonium degradation rate of 94.87% and 82.77%, respectively, in the oligotrophic medium, but the research focused on the denitrification properties of the strain ZK-1. The growth curve of strain ZK-1 demonstrated that the growth rate was slow and the denitrification process mainly took place in the first three days. Various forms of nitrogen could be independently removed by the strain ZK-1. The most effective nitrogen removal rates were 97.45%?86.65%? 94.74%, respectively, with ammonia nitrogen, nitrite and nitrate as the sole nitrogen source in the rich nutrient medium. The nitrification rates was approximately 0.064 mg NH4+-N/L/h and the maximum nitrogen removal rates of ammonia nitrogen, nitrite and nitrate were 94.87%, 95.69% and 95.48%, respectively. The ammonium removal pathway for the strain ZK-1 was proposed to be via nitrite as the metabolic intermediate, that is NH4+?NH2OH?NO2-?NO3-, then NO3-?NO2-?N2. The factors including ammonia concentration, C/N ratio, initial pH and temperature significantly affected the degradation of ammonium by the strain ZK-1.Response surface methodology analysis demonstrated that the maximum ammonium removal of 94.98% occurred under the following conditions: NH4+-N concentration of 22.05 mg/L, C /N ratio of 4.31, initial pH of 7.78 and temperature of 29.73°C, where the initial p H and temperature had the largest influence on the ammonium removal.(2) Domestication, isolation and denitrification properties of oligotrophic aerobic denitrifiers. Three novel aerobic denitrifiers, named F4?DA15 and HF6, were isolated from the sediments of the Heihe reservoir. Based on their phenotypic and phylogenetic characteristics and the sequence analysis of 16 Sr RNA, strain F4, DA15 and HF6 were identified as Bacillus sp., Acinetobacter sp. and Bacillus sp., respectively, corresponding to the similarities of 100%, 98% and 99%. The gene encoding periplasmic nitrate reductase(napA) of three strains were successfully amplified and the heme nitrite reductase encoded the gene nirs about strain F4 was detectable. The three strains nitrate removal reached the rates of 100%, 91.13% and 91.74%, respectively, in the rich nutrient medium and the rates of 94.3%?77.59%? 75.94%, respectively, in the poor nutrient medium, but the research focused on the denitrification properties of the strain F4. The growth curve of strain F4 demonstrated that the growth rate was slightly slow and the denitrification process mainly took place between 1 and 3 days. The maximum nitrogen removal rates were94.3% and 97.01%, respectively, with nitrate and nitrite as the sole nitrogen source and the maximum nitrate degradation rate was 0.108 mg NO3--N/L/h. The important parameters which significantly influenced nitrate degradation rate were inoculum size(%, V/V), initial p H, C/N ratio and temperature. Response surface methodology analysis demonstrated the optimum inoculum size, initial p H, C/N ratio and temperature were found to be 18 %V/V, 8.31, 8.48 and 26.52°C, respectively. In the conditions, nitrate degradation rate was predicted to be 0.145mg/L/h and the C/N ratio and temperature produced the largest effect on the nitrate degradation rate. Besides, the isolated aerobic denitrifier, named ZK-4, had a function of flocculation with the flocculating rate of 87.34%.(3) Denitrification properties and effents of in-situ biological inoculation technology in the micro-polluted raw water. The combination of O ligotrophic denitrifier populations of K1( F4+ZK-2+J×26+ZK-1+ZK-4) and K2, K3, K4(the inoculum size of 1‰V/V ? 0.1‰V/V ? 0.01‰V/V, respectively, with the combination of W7×12+FJ-NO2(7-2)+DA15+HF6) were used for the microbial agents which could be added to the slightly polluted water. The group combination K1 reached the maximum removal of total nitrogen and nitrate of 44.13% and 46.77%, respectively, under the inoculum size of 0.1%. In the group of K2, K3 and K4, the K3 with the inoculum size of 0.1‰V/V had the optimum removal of total nitrogen and nitrate, with the maximum value of 43.05% and 45.07%, respectively. The carbon source was added to the tested water in the progress of the trial in order to investigate how the ratio of carbon and nitrogen affected the denitrification effect of oligotrophic in-situ biological inoculation technology. It was shown that the group M1(W7×12+FJ-NO2(7-2)+DA15+HF6) achieved the maximum removal of total nitrogen and nitrate of 47.22% and 49.69%, respectively, and the other M2(J×26+HF6+DA15+F4+ZK-1+ZK-4) also had the largest removal of total nitrogen and nitrate with the value of 48.96% and 51.37%, respectively. |
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