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低耗、高效的反硝化除磷技术一直是废水生物脱氮除磷领域的研究热点之一[1-2]。然而, 已有研究[3]过多地关注反硝化除磷工艺效能而忽略了在微生物学方面的深入研究。尽管以往有研究者对反硝化除磷菌(denitrifying polyphosphate-accumulating organisms, DPAOs)进行了研究[4-5], 但受限于当时的微生物检测技术, 对反硝化除磷菌的研究仍不够深入。高通量测序技术作为新型微生物种群鉴定技术, 具有分析结果准确、高速、高灵敏度和高自动化等特点, 可以准确的分析生物多样性, 故广泛应用于环境微生物鉴定领域[6-7]。因此采用高通量测序技术作为考察反硝化除磷微生物群落演替规律的手段不失为一个良好的技术选择, 但同时微生物在系统中的种群特征的研究也有待进一步的深入开展。已有的研究表明, 在反硝化除磷系统中的一些优势菌属, 如气单胞菌属(Aeromonas)、假单胞菌属(Pseudomonas)、肠杆菌科等都具有反硝化除磷功能[8], 它们的存在为解释系统效能的变化提供了理论支撑。
厌氧折流板反应器(anaerobic baffled reactor, ABR)具有构造简单、能耗低、抗冲击负荷能力强、处理效率高以及可实现生物相分离等优点[9-10], 膜生物反应器(membrane bioreactor, MBR)则具有利于世代时间长的硝化细菌的增殖, 从而提高硝化效率的优点[11]。因此, 采用ABR-MBR研究反硝化除磷工艺在培养DPAOs以及研究不同功能区微生物的细菌群落特征方面具有显著优势[12]。
本研究采用Miseq高通量测序手段对ABR-MBR组合工艺中除磷功能区的细菌群落特征进行分析, 以期从微生物多样性、种群丰度及优势菌群分布方面为系统效能的变化提供理论支撑, 为反硝化除磷工艺应用于工程实践提供理论依据, 为开发新型的脱氮除磷工艺提供技术支持。
ABR-MBR工艺反硝化除磷微生物群落特征分析
Analysis of microbial community characteristics of denitrifying phosphorus removal in the ABR-MBR process
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摘要: 为了揭示ABR-MBR组合工艺中反硝化除磷微生物种群演替规律, 采用Miseq高通量测序技术考察了该工艺在不同运行阶段除磷功能区的微生物群落结构。结果表明, 硝化液回流比逐步从150%提升至300%可促进反硝化除磷菌大量富集, 促进系统的启动和稳定运行;系统在运行过程中始终保持较高的微生物多样性;优势微生物种群均以变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)为主, 最大丰度分别为55.13%和7.76%, 且变形菌门功能性微生物主要集中在γ-变形菌纲(Gamaproteobacteria);功能性除磷菌属主要为气单胞菌属(Aeromonas), 假单胞菌属(Pseudomonas)和黄杆菌属(Flavobacterium);其中在逐步提升硝化液回流比过程中气单胞菌属(Aeromonas)被大量富集, 其在γ-变形菌纲(Gamaproteobacteria)的相对丰度由5.30%上升至41.49%并在系统后续运行中维持主导地位。系统除磷效果与功能性除磷微生物相对丰度的变化密切相关。系统中微生物种群的多样性和功能微生物的结构稳定性为ABR-MBR工艺的稳定运行和高效处理提供了保证。Abstract: In order to reveal the microbial population evolution of denitrifying phosphorus removal in a lab-scale ABR-MBR combined process, the Miseq high-throughput sequencing technology was used to identify the microbial community structure in phosphorus removal functional zone at different operation stages of the process. Results indicated that the gradual increase of the nitrifying solution reflux ratio from 150% to 300% could promote a large enrichment of denitrifying phosphorus bacteria (DPBs), as well as the startup and stable operation of the system. The system maintained high microbial diversity throughout the operation. The dominant phyla were Proteobacteria and Bacteroidetes, and their maximum abundances were 55.13% and 7.76%, respectively. The main subgroups of the Proteobacteria were related to Gamaproteobacteria. The functional phosphorus removal bacteria were Aeromonas, Pseudomonas and Flavobacterium. Of which Aeromonas was largely enriched during the gradual increase of the nitrifying solution reflux ratio. Its relative abundance in the γ-Proteobacteria phylum (Gamaproteobacteria) increased from 5.30% to 41.49%, and remained dominant in the subsequent operation of the system. The phosphorus removal efficiency of the system was closely related to the change in the relative abundance of functional phosphorus removal microorganisms. The diversity of microbial population and the structural stability of functional microorganisms in the system provided a guarantee for the stable operation and efficient performance of ABR-MBR process.
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表 1 ABR-MBR组合工艺进水水质
Table 1. Influent wastewater quality in ABR-MBR combination process
水质指标 范围 均值 COD/(mg·L-1) 322~440 379 NH4+-N/(mg·L-1) 47.3~65.3 59.5 NO2--N/(mg·L-1) 0~0.2 0.1 NO3--N/(mg·L-1) 0~0.5 0.2 TN/(mg·L-1) 50.1~67.8 60.6 TP/(mg·L-1) 6.25~8.6 7.6 pH 7.1~7.4 7.2 表 2 ABR反应器除磷功能区微生物种群丰度和多样性分析
Table 2. Analysis of microbial population abundance and diversity in phosphorus removal functional zone of ABR
样品 序列数量/个 OTU数量/个 ACE指数 Chao指数 Shannon指数 Simpson指数 覆盖度/% M1 39 808 651 678 681 4.483 0.052 99.79 M2 33 309 690 744 739 3.619 0.188 99.62 M3 37 197 706 758 770 3.659 0.193 99.64 表 3 除磷功能区变形菌门微生物的群落组成相对百分比
Table 3. Percentage of microbial community of Proteobacteria at class level in phosphorus removal functional zone of ABR
% 菌纲 M1 M2 M3 Bataproteobacteria 74.29 23.23 68.87 Gamaproteobacteria 7.28 65.03 13.79 Deltaproteobacteria 16.38 6.50 14.40 Alphaproteobacteria 1.10 4.87 1.96 -
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