【作者】 杨新萍；

【导师】 周立祥；

【作者基本信息】 南京农业大学 ， 生态学， 2009， 博士

【摘要】 随着水体富营养化的日趋严重与污水排放标准、氮素限值的日趋严格,水中氮素的去除已成为当今水污染防治的热点问题之一。生物脱氮是从废水中去除氮素污染的最为经济有效的方法,可实现真正意义的氮去除。目前为止,尽管传统生物脱氮工艺是多数废水生物脱氮处理的主要承担者,但仍存在着硝化速率低、工艺复杂、脱氮效率低等缺点。而在氮素循环过程中发现的新型脱氮微生物,可以克服传统生物脱氮工艺固有的问题,对这些微生物的研究与新型脱氮工艺开发,是对传统生物脱氮理论与实践的丰富与突破。本文从活性污泥中分离出一株异养硝化/好氧反硝化细菌A1、一株高效缺氧反硝化细菌D6,研究了两株细菌以及已有的好氧反硝化细菌AD6脱氮过程的生态影响因子。同时考察了ca2+在好氧颗粒污泥形成中的作用,确定了SBR中快速培育好氧颗粒污泥的条件。在此基础上,将分离、鉴定的3株脱氮功能菌以好氧颗粒污泥形式固定化,研究了其在开放的污水处理系统中的作用与稳定性,构建了基于微生物及环境因子的同步硝化与反硝化工艺(Simultaneous Nitrification and Denitrification, SND)。本文主要研究结果为：从活性污泥中分离得到一株异养硝化细菌A1,通过形态学、生理生化特征并结合16S rDNA序列同源性分析,初步鉴定该菌为枯草芽孢杆菌(Bacillus subtilis).菌株A1可以自养生长。当培养液NH4+为105.58、257.23 mg/L时,菌株A1培养120h,培养液COD可达94.77、90.26mg/L,对NH4+去除率为36.32%、19.53%,对TN去除率为24.88、13.09%；当NH4+为536.21、1014.17mg/L时,菌株A1生长受到抑制,培养120h,培养液cOD分别为56.80、33.56mg/L,菌株A1对NH4+去除率仅有6.54%、2.26%,对TN去除率仅为4.07%、2.27%,基本没有脱氮效果。考察了碳源种类与数量对菌株A1脱氮特性的影响。葡萄糖、柠檬酸盐、丁二酸盐、乙酸盐4种碳源对菌株A1脱氮特性的影响没有明显差异,以乙酸盐为碳源时,最大COD、NH4+、TN去除效率分别为71.08%、60.35%、52.22%。培养液碳源为乙酸盐,菌株A1在c/N值为6、12时,对COD, TN, NH4+-去除效率较高,分别为79.35%、71.08%,59.21%、52.22%,67.07%、60.35%,培养过程中培养液DO始终大于4.01mg/L。由此,菌株A1具有异养硝化/好氧反硝化功能。通过菌株形态观察、生理生化特征及16S rDNA序列分析,鉴定一株可进行好氧反硝化作用的细菌AD6为假单胞菌属门多萨菌(Pseudomonas mendocina)。采用摇瓶试验,研究了C/N值,培养液DO及柠檬酸盐、乙酸盐、葡萄糖3种有机碳源,NH4+、NO2-和NO3-3种氮源对好氧反硝化细菌AD6脱氮特性的影响。C/N值不仅直接影响好氧反硝化过程中碳源数量,也显著影响培养液DO变化。起始c/N值为3时,因碳源数量不够而导致AD6好氧反硝化脱氮效率仅有41.71%；当起始C/N值为23、15、8时,培养液DO先快速下降至缺氧状态然后再升至好氧状态,反应末期TN损失率分别为69.33%、70.41%、55.40%,其中好氧阶段反硝化作用引起的TN损失约为6.93%、20.09%、24.60%。培养液起始DO为7.15～8.08 mg/L, C/N值为15及以下,摇瓶(250mL三角瓶)装液量为25～100mL,摇床转速为180r/min,培养液DO先下降至缺氧状态,但可在反硝化反应进行24h后恢复至好氧状态,减少摇瓶装液量的充氧措施提高培养液DO效果有限。柠檬酸盐与乙酸盐是AD6能够高效利用的碳源,利用率可达89.64%、92.110%,而同样条件下葡萄糖的利用率仅有40.56%。乙酸盐促进了AD6的好氧反硝化功能,TN去除率、好氧反硝化脱氮效率分别比以柠檬酸盐为碳源时高13.63%、5.18%。该菌株可分别以NH4+、NO2-和N03-为起始氮源进行生长与脱氮作用,培养29～33h达到最大氮去除效率,分别为16.57%、74.84%与74.61%,AD6表现出微弱的异养硝化／好氧反硝化功能。从活性污泥中分离筛选出一株高效缺氧反硝化细菌D6,通过形态学、生理生化特征并结合16S rDNA序列同源性分析,初步鉴定该茵为施氏假单胞菌(Pseudomonas stutzeri)。当C/N值为6、12时,菌株D6经过54h培养,COD、TN去除效率可分别达到90%、80%以上。柠檬酸盐、乙酸盐是菌株D6反硝化过程中能高效利用的碳源,当C/N值为6,培养54h后,可分别取得80.72%、74.75%的TN去除率以及90.53%、86.30%的COD去除率；同样培养条件下,菌株D6以葡萄糖为碳源,TN与COD去除率分别只有56.56%、74.64%。当C/N值为6,NO3-浓度为182.38～358.30 mg/L,菌株D6脱氮效率不受NO3-浓度影响,可达80.72～82.50%。采用摇床振荡培养与培养箱静置培养2种方式考察菌株D6的反硝化特性,发现当培养液DO大于1.0mg/L,菌株D6的反硝化特性基本被抑制。通过运行序批式生物反应器(Sequencing Batch Reactor, SBR)与摇瓶试验相结合方法,在进水中投加不同数量Ca2+(0～200mg/L),考察了Ca在好氧颗粒污泥形成中的作用。设计了进水Ca2+含量分别为30(Ⅰ)、100 mg/L(Ⅱ)的2组SBR试验,运行20d后,两反应器中均出现了好氧颗粒污泥。运行前50d, SBR I中颗粒污泥浓度(Mixed Liquor Suspended Solid, MLSS)与污泥沉降指数(Sludge Volume Index, SVI)指标明显好于SBRⅡ;运行后期,SBRⅠ、Ⅱ中MLSS、SVI差异很小。反应器运行与摇瓶试验表明,当进水中Ca2+为0～200mg/L,进水中ca2+含量的增加对污泥Zeta电位影响很小,没有引起好氧颗粒污泥Zeta电位的明显差异,电中和在颗粒污泥形成过程中并不起重要作用。随着进水Ca2+浓度增加,颗粒污泥中的微生物数量与种类都逐渐丰富,Ca2+有助于促进污泥中微生物多样性。接种污泥及SBR中颗粒污泥的元素含量分析表明,颗粒污泥中Ca、Mg、K、Na含量均有所减少,Fe含量分别增加了4.42%、7.82%。具有良好絮凝作用的金属离子与EPS间形成的高分子生物聚合体可能是促进好氧颗粒污泥形成的主要原因。采用2套相同的模拟SBR装置,配制典型的高浓度生活污水,在上述运行条件下均形成好氧颗粒污泥。其中1套模拟SBR装置在好氧颗粒污泥形成过程中,按7天1次的频率投加了3次A1、AD6、D6的混合菌液。60d运行试验结果表明,投加功能菌的SBR中形成了具有良好脱氮效果的好氧颗粒污泥,对COD、TN、NH4+的去除效率分别达到96.53%、85.59%、99.54%,比普通好氧颗粒污泥分别高2.02%、22.16%、5.31%,反应过程中NO3-、NO2-的积累量很少,出水中COD、TN、NH4+浓度可以稳定地达到城镇污水处理厂污染物排放标准(GB18918-2002)一级A标准,具有较好的同步脱氮除碳功能。对装置运行过程中颗粒污泥PCR-DGGE图谱的动态研究分析表明,接入的异养硝化/好氧反硝化细菌A1、好氧反硝化细菌AD6、缺氧反硝化细菌D6能够固定在颗粒污泥中,在SBR反应器中具有一定的数量优势,有助于功能菌长期稳定的发挥脱氮功能。全文研究表明,异养硝化菌株A1、好氧反硝化菌株AD6、缺氧反硝化菌株D6均具有氮脱除功能,但菌株类型不同,生态因子如有机物浓度、碳源种类、DO、氮源等对脱氮特性的影响存在明显差异。将3类脱氮功能菌组合,形成好氧颗粒污泥,能有效将功能菌固持在开放的污水处理系统中,形成具有同步脱氮除碳性能的SND工艺,稳定高效地去除污水中有机物与氮素。异养硝化细菌和好氧反硝化细菌能够在生物同步脱氮中起重要的作用,为微生物学理论解释SND现象提供了微生物学理论依据；同时,缺氧反硝化细菌D6在颗粒污泥中的固定,表明好氧颗粒污泥中存在发生SND的环境因素。研究结果为全面评价SND工艺提供了基础研究,以颗粒污泥形式固定功能菌株为新型脱氮微生物应用于污水处理实践提供了新的途径。更多还原

【Abstract】 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.更多还原