污泥浓缩消化一体化反应器微生物群落结构与功能研究
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摘要
由于三峡库区特殊的山地地形，雨水的冲刷极易导致污水厂剩余污泥对库区水体造成污染，因此库区的污泥处理处置问题就显得尤为重要。库区内已经建成运行的污水处理厂中，污泥减量和稳定处置方面都很欠缺，同时传统的污泥厌氧处理工艺存在的负荷低、消化时间长、投资费用高等问题，因此，开发新型高效污泥处理技术，特别是污泥减容和污泥稳定技术的研究就显得非常迫切。
本课题组在前期试验研究的基础上进一步优化设计了两相一体式浓缩消化（TISTD）反应器，实现污泥浓缩消化一体化并相互促进，反应器运行效能的小试试验表明其浓缩、消化效果优于普通浓缩池、消化池。TISTD反应器中处理效能与反应器中的微生物直接相关，TISTD反应器中有两种关系极为密切的功能菌群：产酸菌群和产甲烷菌群，产酸菌将有机物转化为挥发性有机酸，而产甲烷菌利用这些有机酸转化为甲烷、二氧化碳等气体从而实现污泥的减量化。虽然产酸相中主要有产酸发酵细菌存在，产甲烷相中有产甲烷细菌存在，但整个系统也包含多种其它菌群。反应器运行的稳定性和效率在很大程度上取决于其中微生物种群多样性及优势种群的活性。不同条件下反应器运行的稳定性及效率与系统群落结构的变迁有必然的联系。为了实现TISTD反应器在工程上的推广和应用，论文在课题组前期小试研究成果的基础上，以重庆鸡冠石污水处理厂二沉池污泥为处理对象，对TISTD反应器运行效能和微生物群落结构与功能进行了研究。研究内容包括：运用传统的分离培养方法及16S rDNA序列分析方法研究了TISTD反应器在最优工况下反应器内微生物的分布特性及优势菌属；并分别在反应器的启动期、投配率20%、30%、40%和50%下达到稳定运行时取样，提取污泥总DNA，使用PCR-TGGE指纹图谱技术研究分析了反应器各个阶段的群落结构和生态变化；并结合厌氧污泥脱氢酶活性测定，分析不同阶段下反应器系统中微生物群落代谢活性的动态变化规律，全面揭示TISTD反应器在中温运行条件下维持良好浓缩消化功能的微生物机理。取得主要研究成果有：
①试验考察了中温条件下投配率为10%、20%、30%、40%、50%时TISTD反应器的运行效能，结果表明反应器在中温条件下，运行状况良好，最优投配率为30%，相应的水力停留时间为3.33天。在最优投配率下，当进泥含水率为99.4~99.6%，排泥含水率在89.5%~93.5%之间；当进泥VS/TS为0.62~0.77，排泥VS/TS在0.21~0.28之间；排水SS在0.15g/L~0.6g/L之间；其浓缩和消化效果优于普通浓缩池和消化池。
②外反应室中有变形虫、豆形虫、鞭毛虫和纤毛虫等众多原生动物；电镜扫描可以看到内反应室菌群种类复杂，各种形态的细菌互营互生，菌丝交错相互结合形成了复杂的菌群结构，细胞形态包括球状、杆状等，有许多菌胶团存在；厌氧分离和培养得到的优势菌株的形态包括短杆状、丝状和球状。其中3株优势细菌经16S rDNA序列分析鉴定分别为芽孢杆菌属和产甲烷菌属，表明TISTD反应器有效的实现了污泥厌氧消化过程中的生物相分离，即外反应室的优势菌群是芽孢杆菌属，是污泥浓缩酸化主要场所，内反应室的优势菌群是古细菌和甲烷菌属，是污泥消化产甲烷的主要场所。
③TGGE图谱中显著条带数量较多，各个阶段条带的位置不尽相同，说明TISTD反应器在中温条件下内部微生物种群呈现较高的多样性分布，微生物优势种群呈现一定的差异，随着反应器负荷的改变，微生物的群落结构和种群数量也呈现出明显的演替过程，优势种群的功能地位处在动态变化中，表明反应器中污泥浓缩消化过程是由多种细菌种属共同协作完成的。典型条带割胶回收分析结果显示，与TISTD反应器系统厌氧污泥中的大部分优势功能菌群亲缘关系较近的菌属为未培养菌株。
④在TISTD反应器的不同处理阶段，结合厌氧污泥脱氢酶和辅酶活性的测定，较为全面的了解了TISTD反应器中微生物群落结构的演变过程中群落代谢活性是处于动态变化的，都是呈先增高后降低的趋势，其中内反应室污泥脱氢酶活性始终低于外反应室，根据脱氢酶活性的变化曲线确定TISTD反应器的最佳投配率为30%，此时内反应室脱氢酶活性为80.1μg·L~(-1)·h~(-1)，外反应室脱氢酶活性为97.7μg·L~(-1)·h~(-1)，微生物的种群数量和功能菌株代谢活性都处于很高的水平。
研究成果为TISTD反应器工程应用的设计、启动和运行提供了较系统的微生物理论和技术支撑，对推广TISTD反应器的工程应用具有实质的基础理论意义。
Due to the mountainous terrain of Three Gorges Reservoir Area, it risks the severepollution of the surplus sludge from the WWTPs, which is easily washed away into thesurrounding water body. Accordingly, sludge treatment and disposal in the reservoirarea is of particular importance. However, the WWTPs built in the Three Gorgesreservoir area lack of the technologies in the reduction and stabilization of the sludge;meanwhile, the traditional sludge anaerobic treatment processes are subjected tolow-load, long digestion duration, high costs, and etc. From a comprehensiveperspective, the development of a new efficient sludge treatment technology, especiallyfocusing on the volume reduction and stabilization of was very urgent.
Combining modern high-rate anaerobic reactor theory with gravity concentrationtank for the first time, the research group has successfully developed the Two-phaseIntegrated Sludge Thickening and Digestion reactor (TISTD) to achieve integration ofthickening and digestion and the enhancement of the functions. The laboratory testsshowed that the effectiveness of TISTD is way better than the conventionalconcentrated tank and digester. In essence, the TISTD reactor still follows thethree-stage theory of the anaerobic digestion process; therefore, its performance isclosely associated with the two functional microbial populations inside the reactor:acid-producing bacteria and the methanogens. The acid-producing bacteria are capableof degrading organics into volatile fatty acids, from which the methanogens utilize anddegrade into methane, carbon dioxide and other gases in order to achieve the reductionof sludge. Besides these two major bacterial populations, the system contained variousmicroorganisms. The stability and efficiency of the reactor depended largely on theactivity of microbial diversity and dominant species. The stability and efficiency of thereactor under different operation conditions has certain correlation with the change ofthe microbial structure in the system. In this study, the traditional isolation and culturemethods and16S rDNA sequence analysis were used to study the distribution ofmicroorganisms and the predominant species under the optimal operating conditions inthe TISTD reactor. The sludge samples were taken during the start-up period and thesteady state with sludge feeding rate of20%,30%,40%and50%. Then the wholegenome was analyzed by PCR-TGGE fingerprinting technology to acquire themicrobial structure and ecological changes in the reactor at various stages. Additionally, dehydrogenase activity of anaerobic sludge was measured to reveal the dynamicvariation of the metabolic processes in the reactor at different stages. All the resultscomprehensively unraveled the mechanisms of the microbiological reactions in theTISTD under moderate temperature and provided some insights for the furtherapplication of the reactor and process improvement. The main results are as follows:
①The efficacy under different sludge feeding rate (20%,30%,40%and50%)showed that the optimal sludge feeding rate was30%, and the hydraulic retention timewas3.33d. When the water content and the ratio of VS/TS were in the range of99.4~99.6%and0.62~0.77and the water content and the ratio of VS/TS of effluentsludge was in the range89.5%~93.5%and0.21~0.28respectively and the suspendedsolids in effluent water were in the range of0.15~0.6g/L, the thickening and digestionefficacy of the TISTD were way better than conventional gravitational thickening tankand anaerobic digester.
②Under an optical microscope, many protozoan amoeba, bean-shaped insect,flagellates and ciliates can be detected in the outer reaction chamber; Scanning electronmicroscope provided us with the complicated microbial community structure and cellmorphology. The predominant strains, enriched by anaerobic morphology isolation andculture, were shown to be short rod-shaped, filamentous and spherical. Threepredominant bacteria were identified as Bacillus and methanogenic genus by16S rDNAsequence analysis, indicating that TISTD realized biophase separation effectively duringthe anaerobic digestion in the reactor making the outer reaction chamber favorable forBacillus to thicken and acidify the sludge while the inner chamber suitable for archaeaand methanogens to digest the sludge and produce methane.
③TGGE profiles with many a significantly strips at different positions duringdifferent stages illustrated that the microbial populations inside the reactor were quitediverse under the moderate temperature condition and also the predominant populationsvaried. As the load of the reactor changed, the microbial community structure andpopulation showed a significant change dynamically, which indicated the significanceof the cooperation of all the microbial populations in the achievement of the efficiencyof TISTD. The analysis of the typical bands tapping recovery showed that, the strains inclose relationship with the predominant ones in anaerobic sludge TISTD reactor systemwere uncultured strains.
④The anaerobic sludge dehydrogenase activity was measured for acomprehensive understanding of the structural and functional diversity of the microbial community in the reactor system. Dehydrogenase activity showed the similar trend inthe outer and inner chamber of the reactor, which increased first and then decreased. Butthe dehydrogenase activity of the sludge from the inner reaction chamber was alwayslower than that from the outer reaction chamber. According to the dehydrogenaseactivity curve, the optimal sludge-feeding rate of TISTD reactor was30%, at whichpoint the dehydrogenase activity was80.1μg L~(-1) h~(-1)in the inner reaction chamber and97.7μg L~(-1) h~(-1)in the outer reaction chamber.
本课题组在前期试验研究的基础上进一步优化设计了两相一体式浓缩消化（TISTD）反应器，实现污泥浓缩消化一体化并相互促进，反应器运行效能的小试试验表明其浓缩、消化效果优于普通浓缩池、消化池。TISTD反应器中处理效能与反应器中的微生物直接相关，TISTD反应器中有两种关系极为密切的功能菌群：产酸菌群和产甲烷菌群，产酸菌将有机物转化为挥发性有机酸，而产甲烷菌利用这些有机酸转化为甲烷、二氧化碳等气体从而实现污泥的减量化。虽然产酸相中主要有产酸发酵细菌存在，产甲烷相中有产甲烷细菌存在，但整个系统也包含多种其它菌群。反应器运行的稳定性和效率在很大程度上取决于其中微生物种群多样性及优势种群的活性。不同条件下反应器运行的稳定性及效率与系统群落结构的变迁有必然的联系。为了实现TISTD反应器在工程上的推广和应用，论文在课题组前期小试研究成果的基础上，以重庆鸡冠石污水处理厂二沉池污泥为处理对象，对TISTD反应器运行效能和微生物群落结构与功能进行了研究。研究内容包括：运用传统的分离培养方法及16S rDNA序列分析方法研究了TISTD反应器在最优工况下反应器内微生物的分布特性及优势菌属；并分别在反应器的启动期、投配率20%、30%、40%和50%下达到稳定运行时取样，提取污泥总DNA，使用PCR-TGGE指纹图谱技术研究分析了反应器各个阶段的群落结构和生态变化；并结合厌氧污泥脱氢酶活性测定，分析不同阶段下反应器系统中微生物群落代谢活性的动态变化规律，全面揭示TISTD反应器在中温运行条件下维持良好浓缩消化功能的微生物机理。取得主要研究成果有：
①试验考察了中温条件下投配率为10%、20%、30%、40%、50%时TISTD反应器的运行效能，结果表明反应器在中温条件下，运行状况良好，最优投配率为30%，相应的水力停留时间为3.33天。在最优投配率下，当进泥含水率为99.4~99.6%，排泥含水率在89.5%~93.5%之间；当进泥VS/TS为0.62~0.77，排泥VS/TS在0.21~0.28之间；排水SS在0.15g/L~0.6g/L之间；其浓缩和消化效果优于普通浓缩池和消化池。
②外反应室中有变形虫、豆形虫、鞭毛虫和纤毛虫等众多原生动物；电镜扫描可以看到内反应室菌群种类复杂，各种形态的细菌互营互生，菌丝交错相互结合形成了复杂的菌群结构，细胞形态包括球状、杆状等，有许多菌胶团存在；厌氧分离和培养得到的优势菌株的形态包括短杆状、丝状和球状。其中3株优势细菌经16S rDNA序列分析鉴定分别为芽孢杆菌属和产甲烷菌属，表明TISTD反应器有效的实现了污泥厌氧消化过程中的生物相分离，即外反应室的优势菌群是芽孢杆菌属，是污泥浓缩酸化主要场所，内反应室的优势菌群是古细菌和甲烷菌属，是污泥消化产甲烷的主要场所。
③TGGE图谱中显著条带数量较多，各个阶段条带的位置不尽相同，说明TISTD反应器在中温条件下内部微生物种群呈现较高的多样性分布，微生物优势种群呈现一定的差异，随着反应器负荷的改变，微生物的群落结构和种群数量也呈现出明显的演替过程，优势种群的功能地位处在动态变化中，表明反应器中污泥浓缩消化过程是由多种细菌种属共同协作完成的。典型条带割胶回收分析结果显示，与TISTD反应器系统厌氧污泥中的大部分优势功能菌群亲缘关系较近的菌属为未培养菌株。
④在TISTD反应器的不同处理阶段，结合厌氧污泥脱氢酶和辅酶活性的测定，较为全面的了解了TISTD反应器中微生物群落结构的演变过程中群落代谢活性是处于动态变化的，都是呈先增高后降低的趋势，其中内反应室污泥脱氢酶活性始终低于外反应室，根据脱氢酶活性的变化曲线确定TISTD反应器的最佳投配率为30%，此时内反应室脱氢酶活性为80.1μg·L~(-1)·h~(-1)，外反应室脱氢酶活性为97.7μg·L~(-1)·h~(-1)，微生物的种群数量和功能菌株代谢活性都处于很高的水平。
研究成果为TISTD反应器工程应用的设计、启动和运行提供了较系统的微生物理论和技术支撑，对推广TISTD反应器的工程应用具有实质的基础理论意义。
Due to the mountainous terrain of Three Gorges Reservoir Area, it risks the severepollution of the surplus sludge from the WWTPs, which is easily washed away into thesurrounding water body. Accordingly, sludge treatment and disposal in the reservoirarea is of particular importance. However, the WWTPs built in the Three Gorgesreservoir area lack of the technologies in the reduction and stabilization of the sludge;meanwhile, the traditional sludge anaerobic treatment processes are subjected tolow-load, long digestion duration, high costs, and etc. From a comprehensiveperspective, the development of a new efficient sludge treatment technology, especiallyfocusing on the volume reduction and stabilization of was very urgent.
Combining modern high-rate anaerobic reactor theory with gravity concentrationtank for the first time, the research group has successfully developed the Two-phaseIntegrated Sludge Thickening and Digestion reactor (TISTD) to achieve integration ofthickening and digestion and the enhancement of the functions. The laboratory testsshowed that the effectiveness of TISTD is way better than the conventionalconcentrated tank and digester. In essence, the TISTD reactor still follows thethree-stage theory of the anaerobic digestion process; therefore, its performance isclosely associated with the two functional microbial populations inside the reactor:acid-producing bacteria and the methanogens. The acid-producing bacteria are capableof degrading organics into volatile fatty acids, from which the methanogens utilize anddegrade into methane, carbon dioxide and other gases in order to achieve the reductionof sludge. Besides these two major bacterial populations, the system contained variousmicroorganisms. The stability and efficiency of the reactor depended largely on theactivity of microbial diversity and dominant species. The stability and efficiency of thereactor under different operation conditions has certain correlation with the change ofthe microbial structure in the system. In this study, the traditional isolation and culturemethods and16S rDNA sequence analysis were used to study the distribution ofmicroorganisms and the predominant species under the optimal operating conditions inthe TISTD reactor. The sludge samples were taken during the start-up period and thesteady state with sludge feeding rate of20%,30%,40%and50%. Then the wholegenome was analyzed by PCR-TGGE fingerprinting technology to acquire themicrobial structure and ecological changes in the reactor at various stages. Additionally, dehydrogenase activity of anaerobic sludge was measured to reveal the dynamicvariation of the metabolic processes in the reactor at different stages. All the resultscomprehensively unraveled the mechanisms of the microbiological reactions in theTISTD under moderate temperature and provided some insights for the furtherapplication of the reactor and process improvement. The main results are as follows:
①The efficacy under different sludge feeding rate (20%,30%,40%and50%)showed that the optimal sludge feeding rate was30%, and the hydraulic retention timewas3.33d. When the water content and the ratio of VS/TS were in the range of99.4~99.6%and0.62~0.77and the water content and the ratio of VS/TS of effluentsludge was in the range89.5%~93.5%and0.21~0.28respectively and the suspendedsolids in effluent water were in the range of0.15~0.6g/L, the thickening and digestionefficacy of the TISTD were way better than conventional gravitational thickening tankand anaerobic digester.
②Under an optical microscope, many protozoan amoeba, bean-shaped insect,flagellates and ciliates can be detected in the outer reaction chamber; Scanning electronmicroscope provided us with the complicated microbial community structure and cellmorphology. The predominant strains, enriched by anaerobic morphology isolation andculture, were shown to be short rod-shaped, filamentous and spherical. Threepredominant bacteria were identified as Bacillus and methanogenic genus by16S rDNAsequence analysis, indicating that TISTD realized biophase separation effectively duringthe anaerobic digestion in the reactor making the outer reaction chamber favorable forBacillus to thicken and acidify the sludge while the inner chamber suitable for archaeaand methanogens to digest the sludge and produce methane.
③TGGE profiles with many a significantly strips at different positions duringdifferent stages illustrated that the microbial populations inside the reactor were quitediverse under the moderate temperature condition and also the predominant populationsvaried. As the load of the reactor changed, the microbial community structure andpopulation showed a significant change dynamically, which indicated the significanceof the cooperation of all the microbial populations in the achievement of the efficiencyof TISTD. The analysis of the typical bands tapping recovery showed that, the strains inclose relationship with the predominant ones in anaerobic sludge TISTD reactor systemwere uncultured strains.
④The anaerobic sludge dehydrogenase activity was measured for acomprehensive understanding of the structural and functional diversity of the microbial community in the reactor system. Dehydrogenase activity showed the similar trend inthe outer and inner chamber of the reactor, which increased first and then decreased. Butthe dehydrogenase activity of the sludge from the inner reaction chamber was alwayslower than that from the outer reaction chamber. According to the dehydrogenaseactivity curve, the optimal sludge-feeding rate of TISTD reactor was30%, at whichpoint the dehydrogenase activity was80.1μg L~(-1) h~(-1)in the inner reaction chamber and97.7μg L~(-1) h~(-1)in the outer reaction chamber.
引文
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