Study On The Screening Of Aerobic Denitrifiers From Source Water Reservoir And The Corresponding Denitrification Performance

In recent years, with the discovery of aerobic denitrifying bacteria, subverts the traditional opinion of biological denitrification process, l aerobic denitrifying also has been becoming a new research hotspot. Existing studies have shown that aerobic denitrifying bacteria widely exist in the natural environment. They have strong adaptability to oxygen and the carbon-source they can use is widely. So the aerobic denitrifying bacterium has great application potential in natural water bodies bioremediation. However, the application research on aerobic denitrifying bacteria are mainly concentrated in wastewater treatment system, few report on aerobic denitrifying bacteria in natural water bodies, especially the aerobic denitrification in drinking water reservoir is rarely reported.In this research, a multifunctional device for water lifting and aeration(WLA) was combined with in-situ biological purification technology(ISBP) for the denitrification and anaerobic-release endogenous pollution of drinking source water. Several aerobic denitrifying bacteria were screened from the sediment of Zhoucun reservoir. Carry out the study of denitrification characteristics, pathway and product. Denitrifying functional bacteria populations were constructed by self-adjustment and eco-recombination for the purpose of denitrification under oligotrophic and complex conditions in drinking-water reservoir. The ultimate aim of this research is to gain the mixed bacteria agent which has a good aerobic denitrification performance in the raw water of drinking water reservoir. The main results and conclusions of this study are as follows:(1) Domestication and isolation of oligotrophic aerobic denitrifiers. In this research, several kinds of enrichment and domestication methods were used to screening 693 strains from the sedmint of the reservoir. Thirty-three strains were choosed for their high efficiency of aerobic denitrifying ability. According to their physiological, biochemical methods and the sequence analysis of 16 S r DNA, these strains was identified as Pseudomonas sp., Acinetobacter sp., Sinorhizobium sp., Zoogloea sp., and Bacillus sp., Iron-reducing, Streptomyces, Ensifer. These strains were seldom reported for their discovery in drinking water reservoir, especially the aerobic denitrifying bacteria of Streptomyces, and Ensifer was first reported by this research.(2) Denitrification characteristics and mechanism analysis of the optimum strains. By the results of Periplasmic-bound nitrate reductase(nap A),eight strains named ZF10、ZF31、WP1、WP27、WP129、WP152、WP278、WP404 was selected for further research.A new calculation method for nitrogen balance of aerobic denitrifying process was introduced to this research work, and Response Surface Methodology(RSM) method was used to construct a mathematical model of nitrogen efficiency and environmental factors. Furthermore, nitrate reductase genes including nitrite reductase(NIR)、nitric oxide reductase(NOR)、and nitrousoxide reductase(NOS) were all detected to analysis denitrification mechanism, ways and products of these strains in oligotrophic environment. Characteristics of heterotrophic nitrification and the oxygen tolerance ability were all studied for their further applied in slightly polluted source water to provide theoretical guidance and basis.The experiment results show that, the efficiency of nitrogen removal of six superior strains named WP were still above 55% when the DO in the range of 1.6±0.5~7.8±0.5mg/L. The strains have powerful ability of oxygen tolerance. Strain WP1 、 WP27 、 WP278 has the capability of heterotrophic nitrification-aerobic denitrification.The PCR amplification results of ZF31 suggest the presence of four key enzymes––NAP, NIR, NOR, and NOS. The existence of these four enzymes provides additional evidence for aerobic denitrifying ability and identified the denitrifying pathway of strain ZF31 as NO3--N? NO2--N ? NO? N2O? N2.A comparison of the initial and final TN mass in this reaction system showed that 19.78±2.10% of the NO3--N was changed to biomass, 6.64±1.93% was converted to nitrification products(NO2--N, NH4+-N, and organic nitrogen), and approximately 75% of the initial nitrogen was lost, which was probably removed in the form of gas products.The optimum level of each variable in the denitrification efficiency of strain ZF31 was obtained by solving the regression equation build by RSM. The results indicate that the highest TN removal efficiency of 90.96% was reached when the C/N ratio was 6.68, temperature was 27.72°C, shaking speed was 54.15 rpm and initial p H was 8.23. Predicted value by the mathematic model showed ZF31 has high nitrogen removal efficiency when C/N ratio=3 or temperature was10°C. These results should be useful information for those operating an aerobic denitrification process in oligotrophic water bioremediation.(3) The construction of oligotrophic denitrifying functional bacteria groups. In response to the complex environment problems in slightly polluted water reservoir, 26 functional bacteria groups were constructed by eco-recombination using the selected eight strains from the sediment。The denitrifying effection of these functional bacteria groups were tested in the artificial water simulation of Zhoucun reservoir water quality. The test results showed that the nitrate removal efficiency of 67.3% and TIN removal efficiency of 59.6% was gained by group L2, and the nitrate removal efficiency of 70.2% and TIN removal efficiency of 60.3% was gained by group L19. So, functional bacteria groups L2 and L19 were selected for raw water denitrification experiments.(4) Denitrification efficiency of bacteria group in raw water and their ecological impact analysis. The results of nitrogen removal experiments conducted by adding bacteria groups L2 and L19 in raw water showed that, the economical and effective methods of nitrogen removal was adding bacteria groups L19 when the DO was 3-4mg/L. The concertration of TN remained in the range of 0.771~0.939mg/L, and the corresponding nitrogen removal efficiency can reached to 44.54%~54.46%. The nitrogen removal effects in steady running period could meet the requirements of class Ⅲ of surface water quality according to GB3838-2002.Therefore, the compound bacterium agent gained in this study can combined with oxygen aeration filling technology. The combined technology provided new techniques and approaches for in-situ biological denitrification in slightly polluted source water reservoir.It has important research significance and application value in the control of eutrophication and the endogenous pollution release.