Characteristics Of Denitrification Bacteria In Wastewater Treatment And Self-Immobilization

With the increasing severity of the eutrophication and discharge standard, the control of nitrogen pollution has become a focus problem in wastewater treatment field. The technique of biological nitrogen removal is a common and effective method in controlling nitrogen pollution at present. Because of the different requirement of the nitrifying and denitrifying bacteria for nourishment, the process of nitrogen removal is very complex and the application is limited. Recently, some new "players" in the nitrogen cycling process have been found, which provide new method for developing the application of the biological removal of nitrogen. In this experiment, a strain of bacterium with the capacity of heterotrophic nitrification and aerobic denitrification, a strain of traditional denitrifying bacterium were isolated from activated sludge. The nitrogen removal characteristics of two strains and an aerobic denitrifying bacterium were evaluated. The role of calcium in the formation of glucose-fed aerobic granular sludge in Sequencing Batch Reactor (SBR) was investigated. Meanwhile, the optimum conditions of aerobic granular sludge formation were obtained. On the basis of this analysis, nitrogen removal bacteria,for example,strains Al, AD6, and D6 were immobilized in aerobic granular sludge. The characteristics of immobilized aerobic granular sludge were studied using synthetic wastewater in two simulated sequencing batch reactors. The stability of functional bacteria, immobilized in granular sludge, was investigated in an open wastewater treatment system. SND process based on functional bacteria and microenvironment was constructed. The main results were presented as follows:A strain of heterotrophic nitrifying bacteria, Al, isolated from actived sludge reactor was studied for its characteristics of nitrification and nitrogen removal. Based on analysis of physiological and biochemical characters and sequence analysis of the 16S rDNA, strain Al was identified as Bacillus subtilis. Nitrifying and denitrifying performances of Al at various NH4+ concentrations were investigated through flask experiment. When the concentration of NH4+ increased from 105.58 to 257.23mg/L, the COD of culture solution arrived at 94.77-90.26mg/L and NH4+,TN removal efficiency was 36.32-19.53%, and 24.88-13.09%,respectively after 120h incubation. when the concentration of NH4+ increased to 536.21-1014.17mg/L, the COD of culture arrived at 56.80-33.56mg/L and NH4+,TN removal efficiency were 6.54-2.26% and 4.07-2.27%,respectively after 120h incubation. Denitrifying performances of Bacillus subtilis Al at various carbon sources and C/N ratios were evaluated through flask experiment. The variety of carbon sources including glucose,citrate, succinate, and acetate, as near as made no difference in nitrogen removal efficiency of strain A1.After 120h culture with acetate, the COD, NH4+,TN removal efficiency by strain Al were 71.08%,60.35% and 52.22%, respectively. When the C/N ratio was 6 and 12, the COD,TN, NH4+ removal efficiency by the strain Al was 79.35-71.08%,59.21-52.22%, and 67.07-60.35%, respectively. During the incubation process, there was almost no nitrite and low nitrate accumulation, DO concentration being up 4.01mg/L. Therefore, the heterotrophic nitrifying bacteria had aerobic denitrification ability.By checking the individual morphology, colony culture characteristics, DNA sequencing and 16S rDNA gene bank, AD6, an aerobic denitrifying bacterium was identified as Pseudomonas mendocina. Aerobic denitrifying performances of P. mendocina AD6 at various C/N ratios, dissolved oxygen (DO) concentrations, carbon sources including citrate, acetate, and glucose and nitrogen sources including ammonia, nitrate, and nitrite were evaluated through flask experiment. At an insufficient carbon concentration, namely C/N ration of 3, TN removal efficiency was 41.71% by aerobic denitrification. When the C/N ratio was increased to 8,15, and 23, the TN removal efficiency by AD6 quickly increased to 55.40%,70.41%, and 69.33%, in which 6.93%,20.09%, and 24.60% of TN losses could be contributed to the aerobic denitrification by AD6, respectively. Batch cultures were carried out in a series of 250mL conical flasks containing 100,50, and 25mL of the nitrate-supplemented basal medium with 15 of C/N ratio,7.15 mg/L-8.08 mg/L of DO and strain AD6. Flasks were shaken in a rotary shaker at 28℃and 180r/min to allow better gas exchange. DO concentration of culture medium declined rapidly to anoxia condition at the beginnings of reaction followed by an aerobic level with above 2.0 mg/L of DO after 24 h for reaction. Different carbon sources had strong influence on the aerobic denitrifying performance of strain AD6. The maximum consumption rates of citrate, acetate, and glucose by strain AD6 were 89.64%,92.11%, and 40.56%, respectively. In the medium spiked with acetate as carbon source, TN removal efficiency and N losses due to aerobic denitrification increased to 13.63% and 5.18%, respectively, with comparison to that spiked with citrate. Under the aerobic conditions, initial nitrogen sources including ammonia, nitrite, and nitrate, strain AD6 had good growth and nitrogen removal efficiency ability,16.57%,74.84%,and 74.61% of TN removal rates were obtained after 29-33 hours incubation respectively. The strain AD6 showed the weak ability of heterotrophic nitrifying when initial nitrogen sources of medium was ammonia.A strain of denitrifying bacteria, D6, isolated from actived sludge reactor was studied for its characteristics of denitrification. The result indicated that the isolated strain D6 was most similar to Pseudomonas stutzeri based on the analysis of morphologic characteristics, physiological and biochemical properties and phylogenic analysis of 16S rDNA sequence. Denitrifying performances of P. stutzeri D6 at various C/N ratios, carbon sources, nitrate concentrations and culture method were investigated through flask experiment. When the C/N ratio was range of 6,12, after 54h culture, the COD, TN removal efficiency by D6 reached 90%,80%, respectively. The results showed that denitrification activity was influenced by different carbon source. The nitrogen removal rate by using citrate and acetate was significantly higher than that by using glucose. However denitrification efficiency by using citrate was slightly higher than by using acetate, in which the TN, COD removal efficiency was 80.72%,74.75% and 90.53%,86.30% respectively with the C/N ratio of 6,54h culture period. Under the same conditions, the TN,COD removal efficiency was only 56.56% and 74.64% with glucose. In the range of C/N ratio of 6, when the nitrate concentration increased from 182.38 to 358.30 mg/L, the nitrogen removal efficiency of AD6 varied 80.72-82.50% Under two incubate conditions of flask experiment and chemostat, denitrifying performances of D6 was evaluated. It was proved that denitrification process of D6 was inhibited when DO of culture solution was higher than 1 mg/L. Strain D6 denitrifying ability was showed only under anoxic condition.Influence of calcium on glucose-fed aerobic granule was investigated through sequencing batch reactor (SBR) and shaking flask experiments. Granules of two SBRs spiked with 30 mg/L and 100 mg/L of Ca2+ concentration could be observed visually on day 20. In the first 50 days, the SBRⅡhad higher mixed liquor suspended solid (MLSS) and lower sludge volume index (SVI) than SBRⅠ. After 50 days, the difference of granule performance in two reactors gradually be reduced and little differences was found on day 80 or more, strain Alconcentration of influent had little influence on Zeta potential of sludge during granulation process. Microbiological observations confirmed some appreciable changes in microorganism population and diversities with the increase of Ca2+ concentration of influent. The role of Ca2+ in bioflocculation of granule can be mainly attributed to its cation bridging instead of charge neutralization. The elemental analysis of seed sludge and aerobic granule by x-ray fluorescence (XRF) showed that the content of Ca,Mg,K, and Na in granule were less than seed sludge expect for Fe, increasing 4.42%,7.82% respectively. Divalent metal ions such as Ca2+ and Fe2+ were probably constituent of biopolymer. It was possible that the binding of divalent metal ion with extracellular polymeric substances (EPS) enhanced the granulation of seed sludge. Consequently, the pollutant removal efficiency of SBRⅡwere higher than that of SBR I throughout the trial. Both NH4+-N and COD removal efficiencies reached 90%; TN and TP removal efficiency were both 65%-70% after days 70 in SBRs.With the same activated sludge inoculation and synthetic domestic sewage, granules of two simulated SBRs could be observed. One of SBRs was inoculated three strains of nitrogen removal bacteria with frequency of one time every 7 days. The results of 60 days operation revealed that the SBR inoculated nitrogen removal bacteria effluent concentration of COD,TN and NH4+-N were less than 50,15 and 5mg/L respectively, meeting the first level A criteria specified in the Discharge Standard of pollutants for municipal wastewater treatment plant (GB18918-2002). After 60 days culture, the COD, TN, N4+-N removal efficiency by the granule immobilized nitrogen removal strains were 96.53%,85.59%, and 99.54%, increasing 2.02%,22.16%, and 5.31% than common granule respectively. During the trial process, there was almost no nitrite and low nitrate accumulation in SBRs.Microbial communities in aerobic sludge shifted obviously with granulation of aerobic sludge. DGGE analysis illustrated that aerobic granules with more abundant DGGE bands and the community structures than activated sludge in steady state. Compared to common aerobic granules, the population diversity was higher in the nitrogen-removing aerobic granules. Results demonstrated that aerobic sludge granulation may play an important role in the enrichment and retaining of nitrogen-removing microorganisms include heterotrophic nitrifying strain Al, aerobic denitrifying strain AD6, and denitrifying strain D6.It was concluded that heterotrophic nitrifying strain Al, aerobic denitrifying strain AD6, and denitrifying strain D6 had good performance of nitrogen removal, but influencing factors such as carbon sources, DO, nitrogen sources were not the same. Three type functional bacteria were closely related to nitrogen-removing aerobic granules in SBR and showed ability of simultaneous nitrogen and carbon removal. Strains Al, AD6 play the important role in the SND, and it provides the microorganism influence factor for SND. There were strain D6 in aerobic granules, which indicated microenvironment evidence for SND. It was indicated that nitrogen-removing functional bacteria fixed with aerobic granules, as a potentially excellent immobilization technology, would play an important role in treatment of nitrogen-contaminated surface water. All these results may contribute to the establishment of new biology process to remove nitrogen from wastewater with high efficiency.