2021, Vol. 48扩展功能
文章信息
- 王彤, 汪涵, 周明达, 冉小川, 王伟刚, 吴敏, 王亚宜
- WANG Tong, WANG Han, ZHOU Mingda, RAN Xiaochuan, WANG Weigang, WU Min, WANG Yayi
- 污水脱氮功能微生物的组学研究进展
- Advances in omics of functional microorganisms for nitrogen removal in wastewater
- 微生物学通报, 2021, 48(12): 4844-4870
- Microbiology China, 2021, 48(12): 4844-4870
- DOI: 10.13344/j.microbiol.china.210386
-
文章历史
- 收稿日期: 2021-05-01
- 接受日期: 2021-06-03
- 网络首发日期: 2021-07-06
氮素污染已成为一个日益严重的环境问题，过量氮素会导致水体富营养化，从而威胁人类健康并增加水处理成本[1-2]。污水脱氮对于保护水资源，尤其对于水资源短缺地区极为重要。因此，污水脱氮已成为污水处理领域热点问题之一。由微生物驱动的污水生物脱氮(Biological Nitrogen Removal，BNR)工艺因其高效率、低成本和无二次污染的特点备受关注，已成为污水处理厂主流的脱氮方式[3-4]。现有污水处理厂生物脱氮是通过传统工艺好氧硝化和缺氧反硝化反应来去除氮素[5]。传统脱氮工艺氮去除效能较好，但存在流程长和成本高等缺点。近年来，完全氨氧化(Complete Ammonia Oxidation，Comammox)[6]、厌氧氨氧化(Anaerobic Ammonia Oxidation，Anammox)[7]、厌氧铁氨氧化(Iron-Dependent Anaerobic Ammonium Oxidation，Feammox)、硫酸盐型厌氧氨氧化(Sulfate-Dependent Anaerobic Ammonium Oxidation，Sulfammox)、异化硝酸盐还原成铵(Dissimilatory Nitrate Reduction to Ammonium，DNRA)及亚硝酸盐/硝酸盐型厌氧甲烷氧化(Nitrite/Nitrate-Dependent Anaerobic Methane Oxidation，n-DAMO)[8]等新型工艺因其节约能源、氮去除效能高等优势引起国内外学者广泛关注。这些氮去除微生物在脱氮过程中发挥重要作用，但目前人们对于这些微生物的作用机理、代谢途径及种间种内相互作用的认识仍不完善[9-10]。
分子生物学(Molecular Biology)是一门现代生物学，是在微生物学、遗传学、生物化学与细胞学等学科相结合基础上发展起来的新兴学科[11]。分子生物学方法因其能对样品中微生物进行原位检测，快速、有效获得遗传水平上微生物的多样性信息，近年来在环境领域得到了广泛应用[12-13]。随着研究人员对分子生物学的关注，该技术以脱氧核糖核酸(Deoxyribonucleic Acid，DNA)、核糖核酸(Ribonucleic Acid，RNA)、蛋白质和代谢产物等作为研究对象，在生物脱氮体系中关于微生物多样性、种群结构及功能基因等研究逐渐成为热点[14-16]。过去十几年研究中，分子生物学方法应用于污水生物脱氮的研究主要涉及微生物种群、功能变化及代谢机制等方面[17-18]，通过16S rRNA基因测序和宏组学方法确定“谁在那里” “他们有多少” “他们在做什么” “已经发生过什么”等一系列问题，从而帮助提高关键微生物存活率及工艺脱氮效能[19]。
本文对当前流行的分子生物学方法，包括16S rRNA基因测序、实时荧光定量PCR (Real-Time Quantitative PCR，RT-qPCR)和宏组学(宏基因组学、宏转录组学、宏蛋白质组学和代谢组学)进行介绍。重点综述近年来分子生物学技术在揭示污水生物脱氮过程的功能微生物，即硝化细菌(Nitrifying Bacteria)、反硝化细菌(Denitrifying Bacteria)、Comammox细菌、Anammox细菌、Feammox细菌、Sulfammox细菌及n-DAMO作用机理的研究进展，并提出相应的应用前景，以期为实现分子生物学方法的高效应用提供依据，为未来污水生物脱氮过程中相关微生物的研究提供新方向。
1 分子微生物学工具 1.1 16S rRNA基因测序高通量测序技术以样品中提取的核酸为研究对象，以特定核酸片段为生物标志物，从而揭示环境中微生物的组成和多样性[20]。其中，16S rRNA基因序列包括高变区域和保守区域，保守区域反映了生物物种间亲缘关系，而可变区域则反映物种间差异，在微生物进化和生态学研究中占重要地位[21]。该方法主要用来检测微生物系统中的群落组成和生物多样性，测定过程主要包括：样品中DNA的提取、引物及探针的设计、PCR扩增、基因文库的构建、序列测定、序列分析及系统进化树构建等过程(图 1)[20, 22]。近年来，特异性标记基因扩增子测序技术已经被广泛应用于活性污泥系统，特别是硝化细菌、反硝化细菌和Anammox细菌等功能微生物[22-23]。有学者认为检测结果可靠性主要取决于PCR引物的特异性，并总结和评估了Anammox细菌功能基因在不同引物下的测定效率[24]。随后，有研究人员将新改进的靶向rRNA寡核苷酸探针成功应用于7个实际市政污水处理厂活性污泥样品中氨氧化细菌的量化研究[25]。可见，引物及探针的设计是测定过程中的重要步骤。表 1总结了脱氮微生物16S rRNA基因和功能基因的常用引物，为后续研究学者进行相关检测提供了依据。其次，16S rRNA基因测序方法所测定数据的分析也是一项重要步骤。数据分析过程中，包含用碱基序列的拼接按照97%相似度将序列划分为可操作分类单元(Operational Taxonomic Unit，OTU)或按100%相似度划分为扩增序列变体(Amplicon Sequence Variants，ASV)[33]。之后在Quantitative Insights Into Microbial Ecology (QIIME)或QIIME2平台对序列进行物种分类注释，分析群落丰富度、多样性和覆盖度[34]并得出差异性物种[21, 35]，为后续宏基因组学和宏转录组学分析提供基础。
| 引物名称 Primers name |
序列 Sequences |
目标微生物/基因 Targeting microorganism/gene |
参考文献 References |
| A438F | GTCRGGAGTTADGAAATG | 厌氧氨氧化菌 Anammox bacteria |
[26] |
| A684R | ACCAGAAGTTCCACTCTC | ||
| AMX818F | ATGGGCACTMRGTAGAGGGGTTT | 厌氧氨氧化菌 Anammox bacteria |
[27] |
| AMX1066R | AACGTCTCACGACACGAGCTG | ||
| 343F | TACGGRAGGCAGCAG | 通用高通量测序Universal high-throughput sequencing | [24] |
| 338R | GCTGCCTCCCGTAGGAGT | ||
| 515F | GTGYCAGCMGCCGCGGTA | 通用高通量测序 Universal high-throughput sequencing |
[24] |
| 806R | GGACTACCVGGGTATCTAAT | ||
| nirKF | GTGGATGTTATTAGCAACGTTGC | 亚硝酸盐还原酶基因 Nitrite reductase genes, nirK |
[28] |
| nirKR | ATTTTACGTGCAGTAAACCTCC | ||
| nirSF | GAACATCATTGCCTATGGC | 亚硝酸盐还原酶基因 Nitrite reductase genes, nirS |
[29] |
| nirSR | TTCCAGTTGTGCTCCTTGTA | ||
| nosZF | GGTAACCTTGACAACACCGA | 氧化亚氮还原酶基因 Nitrous oxidoreductase genes, nosZ |
[29] |
| nosZR | ATGACGAAGCCGTGAGACA | ||
| hzsB_396F | ARGGHTGGGGHAGYTGGAAG | 联氨合成酶基因 Hydrazine synthetase gene, hzsB |
[30] |
| hzsB_742R | GTYCCHACRTCATGVGTCTG | ||
| hzsC205F | AYCARTCDGGDCAYAGYGTDWC | 联氨合成酶基因 Hydrazine synthetase gene, hzsC |
[31] |
| hzsC745R | CAHACMGGHARCCAGTTCTTYGG | ||
| hzocl1F1 | TGYAAGACYTGYCAYTGG | 联氨氧化还原酶基因 Hydrazine oxidoreductase gene, hzo |
[32] |
| hzocl1R2 | ACTCCAGATRTGCTGACC |
实时荧光定量PCR (RT-qPCR)技术是一种常见的检测核酸分子数量的分子生物学技术，攻破了PCR只能定性而无法定量分析的瓶颈。该技术自动化程度高、特异性强并可解决PCR易污染等问题，在微生物数量和群落功能基因定量分析中得到了应用广泛[36]。RT-qPCR测定原理是在PCR反应体系中加入荧光染料或荧光探针，通过荧光信号积累实时监测整个PCR反应过程，利用DNA模板起始浓度与循环阈值数(Cycle Threshold，Ct)之间存在线性关系，通过Ct值的测定，根据标准曲线或内参基因对未知模板进行定量分析[37]。RT-qPCR技术的关键因素主要取决于DNA提取效率和适当引物的选择[38]。RT-qPCR技术在环境微生物领域已有广泛应用，但仍存在不能统一标准曲线、不同PCR检测的Ct值无法直接比较等缺陷。相信随着未来研究技术方法的不断改进与完善，RT-qPCR技术在生物脱氮领域将会有更广阔的应用空间。
1.3 宏组学技术宏组学(Metaomics)指特定生物体系内的基因、蛋白质和代谢水平所发生的反应、相关性及对所涉及生物过程进行整体性认识，是目前新兴分子生物学方法之一。其包含宏基因组学、宏转录组学、宏蛋白质组学和代谢组学[39]，简易测定流程和原理如图 1所示。
宏基因组学(Metagenomics)是由美国威斯康星大学Handelaman课题组在1988年首次提出，最初指的是生态环境中全部微小生物遗传物质的总和[11]。如今，宏基因组学以特定环境中全部微生物遗传物质的总和为研究对象，通过功能基因筛选和测序分析，进一步研究微生物群落组成结构和多样性及功能，并预测其进化关系和代谢途径等[43]。该方法无需对微生物进行分离和纯化，可通过提取样品中DNA直接构建宏基因组文库，利用高通量测序对测序结果进行比对来研究样品中的微生物特性[34]。检测过程包括从样品中提取DNA、剪切DNA片段化、对DNA片段进行测序、将所有序列组装成一个微生物种群宏基因组组装基因组(Metagenome Assembly Genome，MAG)，从而获得微生物群落组成图；对组装的MAG进行功能注释，得出微生物群落功能组成图，进一步预测微生物的生理生化特性、代谢途径及其在生物地球化学循环中的相关作用[40]。宏基因组常见的功能注释平台有基因、酶通路Kyoto Encyclopaedia of Genes and Genomes (KEGG)、直系同源蛋白Clusters of Orthologous Groups (COG)、碳水化合物活性酶Carbohydrate Active Enzymes Database (CAZy)、抗生素抗性基因Antibiotic Resistance Database (ARDB)、致病菌毒力因子Virulence Factor Database (VFDB)、基因本体论分类汇总Gene Ontology (GO)、蛋白结构域Protein Family (Pfam)、Ⅲ型分泌系统(Type Ⅲ Secretion System，T3SS)效应蛋白和细胞色素P450等，这些平台为揭示环境中微生物的功能代谢提供了强大数据库。
宏转录组学(Metatranscriptomics)由美国约翰霍普金斯大学医学院Velculescu团队所提出的转录组延展而来，指对特定时刻、特定环境中全部微生物的RNA进行测序，代表微生物系统转录水平上功能和分类的表达及调控，提供特定时间和特定空间微生物群落功能活性信息[44]。该过程包括从微生物群落中提取总RNA，去除核糖体RNA以获得高水平的信使RNA (mRNA)，然后将mRNA反向转录成互补DNA (cDNA)，进行高通量测序之后对序列进行物种、功能注释，获得活性物种组成谱及各功能类群丰度图谱，并进行差异比较、物种组成和代谢通路富集等分析[39]。宏转录组学通常与宏基因组学并用，由宏基因组学提供组装基因组作为转录分析方法的模板[45]。宏转录组学已被广泛应用于各种生态环境，包括土壤、沉积物、肠道微生物群和活性污泥等，是深入揭示群落功能和活动、识别未培养微生物新途径的一个强有力工具[16, 46]。
蛋白质组概念由澳大利亚悉尼大学Wasinger等在1994年首次提出。蛋白质组是指一个基因组所表达的全部蛋白质，可定义为细胞或组织所表达的全部蛋白质；2004年，西班牙米格尔·埃尔南德斯·德埃尔切大学Rodriguez-Valera团队首次提出了宏蛋白质组学(Metaproteomics)，即微生物群落中所有蛋白质组的总和，该方法可探究微生物组成及代谢方式，揭示微生物群落动态发展、种内相互关系和营养竞争关系等[42, 47]。宏蛋白质组学研究策略通常有多重液相色谱串联质谱(Liquid Chromatography-Mass Spectrometry，LC-MS)和双向凝胶电泳(Two-Dimensional Polyacrylamide Gel Electrophoresis，2-D PAGE)联合质谱(Mass Spectrometry，MS)技术。常见的分析过程从提取蛋白质开始，然后通过LC或2-D PAGE技术分离蛋白，再通过质谱技术进行蛋白鉴定，从而确定样品蛋白质属于哪个物种，进一步分析群落之间相互作用[48]。宏蛋白质组学是一种比宏基因组学和宏转录组学更直接地揭示不同环境中微生物代谢过程的强大工具，已被广泛应用于各种不同复杂环境，包括土壤、海洋系统、淡水系统和活性污泥等[49]。然而该测定方法也有一定限制因素，如蛋白质分离和鉴定较难、检测技术要求较高、蛋白丰度差异较大、稳定性差和大规模使用较为困难等[42]，因此，后续关于蛋白质分离纯化、检测方法及数据分析等方面的研究至关重要。
1999年，英国伦敦大学Nicholson课题组首次提出代谢组学(Metabolomics)，其致力于分析特定条件下微生物在特定生理期内所有的代谢产物，涉及微生物信号传递和应激反应等功能[50]。该方法的研究策略主要有2种：非靶向代谢组学(Untargeted Metabolomics)和靶向代谢组学(Targeted Metabolomics)，前者是非特异性的，允许对所有代谢物进行检测，旨在同时分析尽可能多的代谢物，有助于揭示新代谢途径或生物标志物；后者具有高度特异性，可对预先确定代谢物进行定量检测[51]。代谢物分离与检测是代谢组学研究的关键步骤，目前常用的揭示代谢物结构和测定分子浓度的分析技术包含核磁共振(Nuclear Magnetic Resonance，NMR)、LC-MS、气相色谱串联质谱(Gas Chromatography-Mass Spectrometry，GC-MS)和傅里叶红外转换光谱[39-41]。代谢组学变化反过来可以调节基因和蛋白质活性，从而揭示复杂反馈机制和各组学之间的相互关系。代谢物提取和检测的精准性和稳定性还有待提升，此限制仍是该方法被广泛应用的一项重要挑战。
宏组学方法常见数据分析包括物种组成分析、α多样性、β多样性、差异比较、相关分析、网络分析和系统发育树分析等。主要统计方法包括主成分分析(Principal Component Analysis，PCA)、主坐标分析(Principal Coordinate Analysis，PCoA)、非度量多维尺度分析(Non-Metric Multidimensional Scaling，NMDs)、冗余分析(Redundancy Analysis，RDA)和典范对应分析(Canonical Correspondence Analysis，CCA)等。常用计算工具包括R语言Vegan包、PC-ORD软件和MATLAB软件等。近期，有研究团队总结了关于微生物扩增测序和宏基因组方法实用操作指南，介绍了适用于微生物分析的统计学和可视化方法，为研究人员在研究过程中选择合适的分析工具、更有效地分析数据、高效挖掘数据背后的生物学意义提供了一定的科学理论支撑[34]。
分子生物学测定方法各有优势及不足(表 2)，在未来水处理技术研究中还需要根据具体问题具体分析，运用正确的分析测定工具解决合适问题才能实现对这些新兴技术的有效利用。
| 分子生物学方法 Molecular biology methods |
优点 Advantages |
不足 Disadvantages |
| 16S rRNA基因测序 | 所需生物量少，快速测定，成本相对较低 Less biomass, rapid determination, and relatively low cost |
DNA提取、PCR偏差较大，无法定量 The deviation of DNA extraction and PCR was large, and the result could not be quantified |
| 实时荧光定量PCR RT-qPCR | 灵敏度高、定量准确和特异性强，不易被污染 High sensitivity, quantitative accuracy and specificity, and the sample not easily contaminated |
标准曲线制作复杂，荧光探针设计较难，实验费用较高 The standard curve was complicated, the design of fluorescent probe was difficult, and the cost was high |
| 宏基因组学 Metagenomics |
具有高效性和准确性，可进行基因预测，更全面地反映微生物代谢特定底物信息 Metagenomics was efficient and accurate, and predicted gene, and more comprehensive reflection of microbial metabolism of specific substrate information |
成本较高，分析过程复杂 The cost was high, and the analysis process was complex |
| 宏转录组学 Metatranscriptomics |
原位衡量微生物群落宏基因组表达水平，易发现新基因和微生物 In situ measurement of metagenomic expression levels of microbial communities, and new genes and microorganisms was easily discovered |
RNA纯化过程困难，测序成本较高 The RNA purification process was difficult and the sequencing cost was high |
| 宏蛋白质组学 Metaproteomics |
认识微生物群落的发展、种内相互关系和营养竞争关系 Realized the development of microbial communities, intraspecific interrelationships and nutritional competition |
蛋白质分离和鉴定较难，对检测技术要求较高 Protein isolation and identification was difficult, and the requirement of detection technology was high |
| 代谢组学 Metabolomics |
了解生物体基因型和表型，揭示新代谢途径或生物标志物 Understood the genotype and phenotype of organisms, and revealed new metabolic pathways or biomarkers |
依赖代谢物分离与检测，数据处理较为复杂 Relied on the isolation and detection of metabolites, and the data analysis was complex |
生物脱氮过程中所涉及的氮循环途径如图 2所示，主要包含硝化反应、Comammox、反硝化反应、Anammox、Feammox、Sulfammox、n-DAMO和DNRA等，涉及的主要微生物有氨氧化细菌(Ammonia-Oxidizing Bacteria，AOB)、氨氧化古菌(Ammonia-Oxidizing Archaea，AOA)、亚硝酸盐氧化细菌(Nitrite-Oxidizing Bacteria，NOB)、Comammox细菌、反硝化细菌、Anammox细菌、Feammox细菌、Sulfammox细菌和n-DAMO微生物等[9-10]。
|
| 图 2 污水处理过程中各种微生物介导的氮转化途径 Figure 2 Nitrogen transformation pathways mediated by various microorganisms in wastewater treatment processes |
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分子生物学方法已成为研究污水生物脱氮过程中微生物作用的主要技术之一，近年来关于污水生物脱氮处理领域文章发表数量逐年递增，研究热点如图 3所示。16S rRNA基因测序已经被广泛应用于研究污水脱氮过程中微生物群落、分类、功能基因变化、微生物代谢机理和调控机制等方面。同时，宏组学方法的应用也在逐年递增。近年来，借助于分子生物学方法，研究人员对污水处理厂中微生物群落组成和功能基因的表达进行了一系列探索。2012年，有学者利用高通量测序研究了14个污水处理厂活性污泥中微生物多样性，初步得出活性污泥系统中微生物群落组成[52]；随后，宏基因组学和宏转录组学被用来分析活性污泥中微生物群落组成和基因表达，首次从DNA和cDNA层面揭示了污水处理厂中微生物相关基因的表达[45]；2015年有研究使用Illumina测序对市政污水处理厂A2O处理工艺活性污泥进行了分析，揭开了活性污泥组成的“黑箱”，使人们深入认识了污水处理厂A2O反应器内活性污泥生物群落的组成和功能[53]。以上研究表明，变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和疣微菌门(Verrucomicrobia)是活性污泥体系中丰度较高的细菌门，绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)和浮霉菌门(Planctomycetes)等丰度有所下降；主要的细菌菌属包括硝化螺菌属(Nitrospira)、索氏菌属(Thauera)、亚硝化螺菌属(Nitrosospira)、拟衣藻属(Dechloromonas)、烟杆菌属(Ignavibacterium)、亚硝化单胞菌属(Nitrosomonas)、不动杆菌属(Acinetobacter)和丛毛单胞菌属(Comamonas)等；进一步关注与氮代谢相关的微生物发现，反硝化相关基因序列数最多(76.74%)，其次是氨化(15.77%)、固氮(3.88%)和硝化(3.61%)；也有研究表明，活性污泥中与反硝化相关的基因(膜结合/周质硝酸盐还原酶基因narG/napA、亚硝酸盐还原酶基因nirB和一氧化氮还原酶基因norB)序列在DNA和cDNA数据库中均占主导地位，而硝化相关基因(氨单加氧酶基因amoA和羟胺氧化还原酶基因hao)也以相对较高的水平表达[53-55]。氮循环相关微生物在不同污水处理厂发挥着不同作用，可见亟须通过分子生物学方法探究脱氮微生物在污水处理厂具体的基因表达、代谢途径和差异分析。
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| 图 3 分子生物学方法在污水生物脱氮领域近10年发表文章统计及研究热点 Figure 3 Publication statistics and research hotspot of molecular biology methods for wastewater biological nitrogen removal in recent 10 years |
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生物处理过程中，硝化反应由2个生物氧化过程组成(图 2)：(1) 氨氮氧化为亚硝态氮，该反应由AOB或AOA催化氨单加氧酶(Ammonia Monooxygenase，Amo)形成中间体羟胺(NH2OH)，再经羟胺氧化还原酶(Hydroxylamine Oxidoreductase，Hao)形成一氧化氮，从而进一步氧化成亚硝态氮；(2) 由NOB催化亚硝酸盐氧化还原酶(Nitrite Oxidoreductase，Nxr)在好氧条件下将亚硝态氮氧化为硝态氮[10]。有研究学者基于cDNA和DNA序列丰度发现，硝化作用关键功能基因amoA和hao的表达远高于其他代谢过程功能基因的表达，表明硝化作用在活性污泥体系中具有较高活性[45]，是污水生物脱氮过程中的重要步骤。
如今，已报道的AOB属包括亚硝化单胞菌属(Nitrosomonas)、亚硝化螺菌属(Nitrosospira)、亚硝化弧菌属(Nitrosovibrio)、亚硝化叶状菌属(Nitrosolobus)和亚硝化球菌属(Nitrosococcus)[56]，其中污水处理厂最常见的AOB是Nitrosomonas和Nitrosospira[25]。AOB几乎存在于所有污水处理厂中，有研究应用16S rRNA基因测序技术和荧光原位杂交(Fluorescence In Situ Hybridization，FISH)技术检测生物污水处理系统中AOB的丰度，发现在活性污泥中AOB含量可达1.0×1010 cells/mL，在污水脱氮过程发挥着重要作用[36]。目前，研究人员对AOB代谢机制及其与其他种类微生物之间相互作用的理解并不深入。近年来，污水处理过程中一种新型生物脱氮工艺——部分亚硝化-厌氧氨氧化(Partial Nitritation-Anammox，PN/A)工艺由于其不可替代的优越性得到研究学者的广泛关注。在PN/A工艺运行过程中，AOB为Anammox菌提供反应所需的亚硝氮，为实现高效经济的生物脱氮提供了保障[57]。然而，使AOB在PN/A工艺运行过程中保持稳定作用仍具有挑战性，AOB同时受到溶解氧(Dissolved Oxygen，DO)、曝气速率、污泥停留时间(Sludge Retention Time，SRT)和温度等环境因子的影响[57]。采用PN/A工艺序批式反应器(Sequencing Batch Reactor，SBR)处理高盐高氨氮废水时发现，微生物群落组成结构、功能基因及相互作用会显著影响氮去除效率和N2O、NO排放性能[58]。虽然AOB可在低DO、高pH值(7.0−8.5)条件下生存，但在实际高氨氮废水处理过程中仍会有亚硝酸盐累积不充分、异养菌大量繁殖等问题，并且不同种类微生物之间相互作用(如竞争、互惠共生和协同作用等)还需借助分子生物学技术来进一步探究。
污水处理厂活性污泥系统内AOB对污水中氨氮氧化成亚硝氮过程的贡献远高于AOA，但AOA应对极端条件的适应性要强于AOB[53]。近年来，有研究证实了在土壤海洋环境生态系统中AOA含量和古菌amoA基因丰度高于AOB，并在温泉和淡水系统中普遍存在[59]。AOA在氨限制条件下能适应环境，维持细胞合成和生长，这主要因为其可通过高亲和力氨转运蛋白Amt2和S层蛋白优先利用同化和分解代谢途径进行反应[46]。随后，有学者通过宏基因组方法确定了AOA中编码羟基丙酸/羟基丁酸(Hydroxypropionate/Hydroxybutyrate，HP/HB)循环途径的关键酶是3-Hydroxypropionyl-CoA脱水酶，该酶使CO2固定途径具有多样化，可催化HP/HB循环多个反应，使AOA具备更高能源效率并降低了蛋白质生物合成成本[60]。越来越多的研究表明，AOA在污水生物脱氮过程中可能发挥着重要作用。因此，对于AOA在污水处理厂的高度富集、代谢机理及其在不利条件下的生存机制应予以重视。
目前研究认为NOB分为4个系统发育不同的类群：硝化球菌属(Nitrococcus)、硝化杆菌属(Nitrobacter)、硝化螺菌属(Nitrospira)和硝化刺菌属(Nitrospina)[56]。在污水处理厂中发现NOB优势属是Nitrospira和Nitrobacter，而且Nitrospira相比Nitrobacter更具代谢多样性，是污水处理厂存在的主要NOB[48]。NOB的丰度受DO、温度、pH和SRT影响较大。例如，有课题组利用16S rRNA基因测序技术测定发现低DO (0.2−1.0 mg-O2/L)条件下运行的SBR反应器中Nitrospira丰度从3.1%提高至53.0%[61]；也有学者利用宏基因组学检测到移动床生物反应器(Moving Bed Biofilm Reactor，MBBR)中温度从26 ℃降至20 ℃后，Nitrospira相对丰度从6.2%下降到5.2%[62]。研究表明不同污水处理厂硝化微生物群落存在显著差异[63]。市政污水处理厂A2O工艺中Nitrospira相对丰度为3.3%左右[64]；有学者检测了14个污水处理厂活性污泥中微生物的构成，发现仅1个样品主要NOB是Nitrospira[52]。可见，NOB在不同污水处理厂生物脱氮循环过程中发挥着不同作用，造成其丰度的差异性原因还需进一步探究。近年分子生物学分析结果表明，NOB具有较多样的代谢途径，除了将亚硝酸盐转化为硝酸盐，还具有编码氰化酶(可将氰酸盐转化为NH3和CO2)、尿素转运蛋白和胞质尿素酶的基因及降解尿素的能力，从而进一步与AOB相互作用产生新的“互惠取食效应”。亚硝氮氧化并不是NOB的主要代谢方式，还具有其他生存机制，这也取决于其生存条件[65]。随着PN/A等新型自养脱氮技术研究的推进，近年关于NOB的研究则主要致力于自养反应体系中NOB抑制策略[57]。NOB和AOB的环境耐受性有所差异，通过调节pH和DO等环境因子可实现有效的NOB抑制。
在污水生物氮去除过程中，与常规硝化工艺相比，通过提高AOB和AOA活性、抑制NOB增殖，从而将硝化反应控制在部分亚硝化阶段，其具有显著优势：(1) 减少40%化学需氧量(Chemical Oxygen Demand，COD)、亚硝氮生成量增加1.5−2.0倍；(2) 节省25%耗氧量、减少300%污泥生成、CO2排放量降低20%[3]。近年来，关于部分亚硝化研究也逐渐增多，其中PN/A工艺和短程硝化反硝化工艺适用性的探索已逐步成为热点。因此，如何利用分子生物学方法揭示AOB和AOA微生物的活性维持技术、NOB抑制机理、微生物之间的交互作用、反应过程中代谢途径和调控机制将成为后续研究重点。
Comammox菌能够独立执行整个硝化过程，将氨直接氧化成硝酸盐(图 2)[66]。近年来，MAG分析技术为Comammox菌的发现和环境分布提供了有利的基因组学证据。有研究人员利用宏基因组学方法首次定量检测了16个主流及侧流污水BNR反应器内Comammox菌丰度并阐明了其潜在功能，结果表明Comammox菌基因组在所有BNR系统中占总编码DNA序列的0.28%−0.64%，其对污水处理厂生物脱氮的贡献还需进一步量化[67]。澳大利亚Daims课题组发现Comammox菌来自Nitrospira，分离出了第一株Comammox菌Candidatus Nitrospira inopinata[68]。随后，Ca. Nitrospira nitrosa、Ca. Nitrospira nitrificans和Nitrospira sp. strain Ga0074138被相继报道[66, 69]，同时Lawson等证实了所有已知Comammox菌都属于NitrospiraⅡ亚分支，其基因组均含有参与氨和亚硝氮氧化过程的功能基因[70]。有学者专门针对Comammox菌中amoA基因设计引物，研究了废水处理厂中Comammox菌amoA基因的多样性和丰富度，结果表明Comammox菌中amoA基因丰度很高，较AOB中amoA基因丰度高182.7倍[6]。Comammox菌包含高亲和力尿素转运蛋白(Urea Transporters)、铜稳态蛋白、外膜孔蛋白(FmdC)和尿素羧化酶相关转运蛋白(UctT)，有助于Comammox菌在低浓度基质条件下利用尿素进行代谢；同时也检测到胍丁胺酶(Agmatinase，Agm)，该酶可以水解胍丁胺产生尿素，可见Comammox菌可利用不同底物进行反应，从而使Comammox菌代谢方式更加多样[71]。有学者指出Comammox菌产生N2O可能显著低于AOB。类似于AOB，Ca. Nitrospira inopinata含有完整的编码amo和β亚基基因，但缺乏编码完整的亚硝酸盐还原酶基因，这表明Ca. Nitrospira inopinata产生的N2O可能来自NH2OH降解[72]。宏组学结果表明，Ca. Nitrospira编码双向氢化酶基因、甲酸盐吸收和氧化基因(可将氢或甲酸盐氧化产生替代能源)及高亲和力SulP/SLC26型转运蛋白(可以转运各种底物，包括硫酸盐、碳酸氢盐和氯化物)，具有较强的基因组和代谢通用性[73]。随后有研究报道在低氨氮和微氧条件下Ca. Nitrospira成为反应器中主要微生物群落(94%)，氨氮平均去除速率为58.6 mg-N/(L·d)[61]；并且发现Ca. Nitrospira可在有限铜元素存在环境下保持活性，具有降解尿素的代谢能力。Comammox菌的发现改变了学界对硝化过程的认识，在污水生物除氮方面有一定应用基础，但是其在废水处理领域的作用还尚待研究，与其他微生物之间的相互作用及调控机理还需进一步探究[74]。
2.2 反硝化细菌反硝化反应是由反硝化细菌催化反硝化酶系[硝酸盐还原酶(Nitrate Reductase，Nar)、亚硝酸盐还原酶(Nitrite Reductase，Nir)、一氧化氮还原酶(Nitric-Oxide Reductase，Nor)和氧化亚氮还原酶(Nitrous-Oxide Reductase，Nos)]将硝酸盐还原至N2的过程[10] (图 2)。值得注意的是，Nir同时可将亚硝酸盐还原为NH3，从而进一步同化为有机氮。反硝化细菌在污水生物处理厂中广泛存在，该细菌在生物化学和分类学上都具有多样性，按营养物种类可分为异养反硝化细菌和自养反硝化细菌，按溶解氧条件可分为缺氧反硝化细菌和好氧反硝化细菌[28]。近年来，分子生物学在反硝化细菌方面的应用主要包括纯菌株代谢途径探究、微生物群落组成及变化、与其他工艺耦合时反硝化细菌的变化和作用机制及极端条件(低温、高盐度、高负荷及贫营养等)对反硝化细菌代谢通路的影响等方面[18, 75-76]，具体案例见表 3。
| Research object | Research method | Research contents | Research results | References |
| Biofilm Reactors | 16S rRNA Metagenomics Metaproteomics | Multiple molecular omics approaches were used to determine the microbial community composition, co-occurrence network and metabolic pathways | The microorganisms in the biofilm reactors had more nodes but less interactions than those in floc reactors. And a lower proportion of denitrifiers and higher resistance to oxygen and salinity perturbation were observed in the biofilm reactors when compared with the floc reactors | [18] |
| Enhanced biological phosphorus removal (EBPR) Bioreactors (SBR) | 16S rRNA Metagenomics | The nitrogen conversion pathways in the EBPR bioreactors were studied | The result suggested that N2O production within this denitrifying polyphosphate accumulating organisms (DPAO) enriched microbial consortium likely derives at least in part from flanking non-PAO denitrifying bacteria, and the N2O reducing genes were lacking in the community genome | [77] |
| Denitrification reactor (expanded granular sludge bed, EGSB) | 16S rRNA Metagenomics | Effects of salinity on denitrifying bacteria, gene diversity and abundance and nitrogen metabolism pathway treated high nitrate wastewater | Halomonas and Marinobacter were the main denitrifying bacteria, and Marinobacter played an important role in nitrate reduction under high NaCl stress | [43] |
| Sulfur autotrophic denitrification reactor (Sulfur-based packed bed reactor, SPBR) | 16S rRNA | Microbial interactions and metabolic pathways of coupled anammox and sulfur autotrophic/mixotrophic denitrification systems under different conditions were clarified | In SPBR, the richness and diversity of microbial community decreased after the addition of glucose, and the addition of organic carbon significantly changed the overall structure of molecular ecological network and the complexity of microbial interaction | [78] |
| Denitrification reactor (EGSB) | 16S rRNA Metagenomics | The effects of 1-hydroxyethane-(1, 1-bis- phosphonic acid) (HEDP) concentration on nitrogen removal performance and narG, nirK and nosZ in a heterotrophic denitrification reactor were investigated | When sludge was exposed to different concentrations of HEDP, narG and nirK were up-regulated, while napA and nirS were down-regulated. Thaurea, Azoarcus, Pseudomonas and Halomonas were the main microorganisms in the reactor | [79] |
| 25 strains of heterotrophic nitrification aerobic denitrifying (HNAD) bacteria | 16S rRNA Metagenomics | The cell culture and metagenomics were used to identify cultivable HNAD bacteria from seawater and analyzed their interspecific interactions and denitrification pathways | HNAD bacteria converted inorganic nitrogen into N2 and organic nitrogen through nitrogen metabolism pathways such as assimilation, partial nitrification, nitroalkylation oxidation, nitrate/nitrite dissimilation reduction and denitrification, and had interspecies coexistence and cooperation relationship | [80] |
| Heterotrophic metal reducing bacteria Shewanella oneidensis MR-1 | 16S rRNA | The mechanism of Shewanella oneidensis MR-1 eliminated nitrite accumulation and N2O emission to improve denitrification performance was studied | The strain Shewanella oneidensis MR-1 promoted electron transfer activity by forming nanotubes between cells, which leaded to an increase in denitrification enzyme activity, carbon source metabolism, adenosine triphosphate (ATP) levels and cell activity | [81] |
| Aerobic denitrifying bacteria Zobellella sp. B307 | Metabolomics | Through bacterial growth, denitrification capacity, related enzyme activity and metabolic pathways to study the toxic effects of nano-zinc oxide on Zobellella sp. B307 | The result suggested that the nano-zinc oxide can change the membrane permeability of bacteria, affect related protein synthesis and gene expression and other metabolic pathways, inhibit its reductase activity and denitrification ability, and the higher the concentration, the more obvious the toxic effect | [82] |
| Aerobic denitrifying bacteria Acinetobacter WGX-9 | 16S rRNA | The effect of natural organic matter and algae organic matter with different molecular weights on the denitrification efficiency of aerobic denitrifying bacteria was explored | The strain WGX-9 expressed nitrate reductase, and preferentially used small molecules of tryptophan and tyrosine to promote the denitrification performance of microorganisms in actual water | [83] |
| Aerobic denitrifying bacteria Pseudomonas stutzeri T13 | Metagenomics Metatranscriptomics | To prove the distribution of strains in the assimilation/alienation metabolic pathways and reveal the coordinated effect of strains on the assimilation/alienation metabolism of nitrogen | The ammonia nitrogen metabolism pathway of strain Pseudomonas stutzeri T13 was an assimilation pathway, and the nitrate metabolism pathway was composed of denitrification and DNRA. And there was a significant interaction between different nitrogen metabolism pathways | [84] |
有研究学者在2012年首次利用宏基因组学和宏转录组学对污水处理厂的活性污泥中微生物群落进行表征分析，发现反硝化相关基因序列占主导地位[45]。在污水处理系统中，检测到主要反硝化细菌包括假单胞菌属(Pseudomonas)、土生单胞菌属(Terrimonas)、副球菌属(Paracoccus)、陶厄氏菌属(Thauera)、脱氯单胞菌属(Dechloromonas)、生丝微菌属(Hyphomicrobium)、丛毛单胞菌属(Comamonas)和不动杆菌属(Acinetobacter)。也有学者利用宏基因组学和宏转录组学研究了反硝化反应器中微生物群落功能物种和代谢特征，指出反硝化菌Thauera aminoaromatica是消耗乙酸盐、积累聚羟基丁酸酯和硝酸盐还原的主要菌株，通过三羧酸(Tricarboxylic Acid，TCA)循环产生的能量主要用于内源聚β-羟基丁酸酯(Poly-β-Hydroxybutyrate，PHB)合成和硝酸盐还原[85]。
好氧反硝化工艺由荷兰学者Robertson和Kuenen共同提出，该工艺凭借其不受氧浓度限制、成本低、不产生二次污染及能同步去除水体中碳和氮污染物等优势，受到国内外研究学者的关注[86]。研究表明，Nar可分为膜结合硝酸盐还原酶(NarG)、周质硝酸盐还原酶(NapA)和同化硝酸盐还原酶(Nas)，NapA在好氧、缺氧和厌氧环境下均能表达，是好氧反硝化工艺关键酶；近年来，利用分子生物学技术研究好氧反硝化菌的报道逐渐增多[87]。有学者利用qPCR技术在mRNA水平探究了好氧反硝化菌Klebsiella sp. KSND的代谢途径，结果表明该菌具有3种硝酸盐还原酶，NapA主要负责调控反硝化和同化过程；也有学者利用16S rRNA基因测序和宏组学的方法分析好氧反硝化菌Pseudomonas indoloxydans YY-1在低温条件下的代谢途径，发现低温条件下TCA循环、细胞色素还原酶、转氢酶和三磷酸腺苷合成酶上调，而包含烟酰胺腺嘌呤二核苷酸脱氢酶和硝酸盐同化功能蛋白下调，进一步扩展了对好氧反硝化细菌Pseudomonas indoloxydans YY-1在低温条件下代谢策略的理解[87]。分子生物学技术在好氧反硝化中的应用目前仍主要集中在菌株筛选等方面，对于微生物代谢机理、电子传递能力、不同微生物之间相互作用和实际工程应用下的调控策略还需进一步研究。相信未来通过分子生物学技术手段可进一步提高好氧反硝化工艺脱氮效能，从而扩展好氧反硝化技术的实际应用。
自养反硝化脱氮技术是一种无需外加有机碳源即可实现水中硝态氮深度去除的新型氮去除工艺。该工艺相比于常规反硝化工艺具有脱氮效率高和运行成本低等优势。其中硫自养反硝化(Sulfur- Driven Autotrophic Denitrification，SDAD)工艺和铁自养反硝化(Nitrate-Dependent Fe(Ⅱ) Oxidation，NDFO)工艺是污水处理领域研究较多的自养反硝化工艺[88]。SDAD工艺指SDAD菌利用还原硫化合物(如硫单质、硫化物和硫代硫酸盐等)作为电子供体将硝态氮还原为氮气的过程。目前已知的SDAD菌大部分属于Proteobacteria，在属水平上，脱氮硫杆菌(Thiobacillus denitrificans)和脱氮硫单胞菌(Sulfurimonas denitrificans)是2种最常见的SDAD菌[89]。大多数SDAD菌具有较宽pH适应范围和温度适应性，环境适应能力较强。在SDAD反应过程中，硫化物醌氧化还原酶(Sulfide-Quinone Oxidoreductase，SQR)和硫氧化还原酶(Sulfur Oxygenase-Reductase，SOR)功能酶发挥着关键作用。有学者利用16S rRNA基因测序和宏蛋白质组学方法研究Pseudomonas sp. C27代谢途径，发现磷酸腺苷酸硫酸还原酶使Pseudomonas sp. C27在不同底物下表达出不同硫代谢途径[90]。近年来，有研究表明SDAD和Anammox工艺耦合可显著提高一体化反应系统的脱氮效能，利用16S rRNA基因测序和RT-qPCR分析可知微生物群落之间相互作用支持了耦合系统的稳定性[91]。SDAD工艺作为一种自养脱氮工艺，可显著去除水体中硝酸盐且经济成本较低，有较大潜力被广泛应用，但仍存在出水中硫酸盐含量较高、反应速率较慢等不足。因此，未来研究应侧重于提高微生物代谢活性、减少反应副产物并探究耦合工艺稳定性，从而促进SDAD脱氮工艺在处理高硝酸盐废水方面的应用。1996年由德国学者Straub首次观察到Fe(Ⅱ)氧化和硝酸盐还原同时发生，并提出微生物驱动的NDFO工艺，即以Fe(Ⅱ)为电子供体，通过微生物的氧化还原反应将硝态氮还原为氮气的一种新型代谢途径[92]。NDFO菌在环境中分布广泛、种类众多，近年来受到研究者的广泛关注。NDFO菌大多属于Proteobacteria，同时利用16S rRNA基因测序方法可知铁氧化副球菌(Paracoccus ferrooxidans)、食酸菌属(Acidovorax)和施氏假单胞菌(Pseudomonas stutzeri)是常见NDFO菌种属。现有研究表明：NDFO菌在反应过程中可通过生物(如铁氧化酶作用)或化学(如硝酸盐还原产物化学作用) 2种不同方式氧化Fe(Ⅱ)形成Fe(Ⅲ)矿物沉淀，并去除水体中氮素，2种机制在NDFO过程中同时发生、互相影响，使反应机理更加复杂；有学者对NDFO过程中的生物反应和非生物反应进行了量化，结果表明细菌分泌的胞外聚合物可增强Fe(Ⅱ)非生物氧化，60%−75%的Fe(Ⅱ)氧化是通过菌株Acidovorax BoFeN1、Acidovorax ebreus TPSY、Paracoccus denitrificans Pd 1222和Pseudogulbenkiania sp. 2002实现的[92]。然而对于反应过程中相关酶的探究还鲜有报道，因此，在定量解析2种不同反应机制和探索代谢机理方面，分子生物学技术对研究NDFO过程有重要的应用前景，可能是区分其代谢机理的有效手段。NDFO过程所生成的Fe(Ⅲ)矿物具有较高的生物利用性和较大的比表面积，可有效促进污染物降解，进一步可作为吸附剂和絮凝剂去除磷酸盐和重金属。同时Fe(Ⅲ)还可作为Feammox反应电子受体，促进氨氮进一步去除。自养反硝化脱氮工艺电子供体的选择取决于微生物对底物生物利用度，微生物亲和力、水质参数(pH、温度和化学组成)、经济成本及环境条件等因素。如何有效利用这些过程和机制进行高效污水脱氮需要进一步探索。
结合表 3可知，分子生物学方法实现了对反硝化细菌既全面又系统的认识，从分子学层面解释了反硝化细菌代谢机理及在不同条件下微生物群落的变化机制，为提高污水中脱氮性能提供了科学指导。
2.3 厌氧氨氧化细菌厌氧氨氧化Anammox反应指Anammox菌在缺氧条件下，以亚硝酸盐作为电子受体将氨氧化为N2的过程(图 2)[93]，反应方程式见公式(1)。在过去20年中，与传统好氧硝化-缺氧反硝化过程相比，Anammox反应由于无需添加有机碳源、无需曝气，显著降低了能耗和剩余污泥产生等优势，已发展成为一种经济高效的污水脱氮技术，深受研究学者青睐。
| $ \begin{array}{l} \mathrm{NH}_{4}{ }^{+}+1.32 \mathrm{NO}_{2}{ }^{-}+0.066 \mathrm{HCO}_{3}{ }^{-}+0.12 \mathrm{H}^{+} \rightarrow 1.02 \mathrm{~N}_{2}+ \\ 0.26 \mathrm{NO}_{3}{ }^{-}+0.066 \mathrm{CH}_{2} \mathrm{O}_{0.5} \mathrm{~N}_{0.15}+2.03 \mathrm{H}_{2} \mathrm{O} \end{array} $ | (1) |
截至目前，利用16S rRNA基因测序技术已报道的Anammox菌有6属19种(Candidatus Brocadia、Candidatus Kuenenia、Candidatus Jettenia、Candidatus Scalindua、Candidatus Anammoxglobus、Candidatus Anammoximicrobium)。Anammox菌在自然界的分布十分广泛，不仅存在于海洋、淡水、陆地、污水处理厂和土壤等各种生态系统中，甚至在极端温度或者极端pH环境中也有Anammox菌分布[9, 94]。宏组学研究表明，不同Anammox菌可能存在不同的代谢途径。2006年，荷兰Strous课题组从环境DNA中获得了Candidatus Kuenenia stuttgartiensis (Ca. K. stuttgartiensis)相对完整的基因序列；根据nirS的出现，Strous等提出在厌氧氨氧化反应中，可能存在以NO为中间产物的新型代谢途径[95]。Hu等研究表明Ca. K. stuttgartiensis含有kuste3160，编码nor基因，进一步揭示了Ca. K. stuttgartiensis的代谢途径[96]。相反，Candidatus Brocadia (Ca. Brocadia)缺乏典型nir基因，其可能含有未知基因而不是nir将亚硝酸盐还原成羟胺，然后由羟胺和铵进一步反应生成N2。研究证明Anammox菌可还原氧化石墨烯，具有胞外电子传递能力。在微生物电解池中，Anammox菌可使用工作电极作为电子受体氧化氨氮，对使用生物电化学系统高效处理高氮废水具有重要意义[97]。由此可见，Anammox菌是一种多功能性微生物，具有不同的代谢途径，在实际市政污水处理厂工程应用过程中可能发挥不同氮转化途径。
有学者研究了市政污水处理厂接种外源厌氧氨氧化颗粒的可行性及微生物氮循环途径，结果证实了Anammox菌在富氨体系中生长的可行性，并从组学方面提升了研究人员对常规污水处理厂中厌氧氨氧化氮循环的认识[98]。也有研究人员利用宏组学方法研究了市政污水处理厂中缺氧-载体生物膜上厌氧氨氧化作用机理，以及厌氧氨氧化与反硝化作用之间相互作用关系。narG高丰度结果表明，在缺氧载体生物膜中硝酸盐向亚硝酸盐转化潜力更高，缺氧MBBR与A2O系统的结合有助于污水处理过程中Anammox菌原位富集，从而增加污水中氮素去除效率[99]。本课题组利用宏蛋白质组学方法首次探究了低温条件下Anammox菌潜在的蛋白质组学调控机制，结果表明当温度从35 ℃降至20 ℃时，Anammox菌蛋白质的完整性通过降解和再循环反应来维持，而不是通过修复有故障的蛋白质，并且在20 ℃和15 ℃的较低温度下，Anammox菌中参与厌氧氨氧化反应的大多数关键酶蛋白保持恒定水平，从而使得Anammox菌适应了低温环境，为厌氧氨氧化工艺在低温条件下的应用调控提供了一定理论基础。该团队还进一步发现不同Anammox菌在低温下的反应调控机制不同，反应过程中Ca. Jettenia相较于Ca. Kuenenia和Ca. Brocadia的蛋白质丰度变化更显著[100]。
分子生物学方法在Anammox菌近年的研究主要致力于Anammox菌代谢机理[96-97]、厌氧氨氧化反应器中微生物种间代谢关系[101-102]、反应器不同阶段微生物功能变迁[103]、厌氧氨氧化颗粒形成机理[104-106]、絮体污泥形态变化和微生物群落组成变化[107]、Anammox菌快速定量测定[108]和不利条件下(低温、饥饿、重金属和高负荷等) AnAOB的胁迫效应[109]等，具体案例见表 4。这些研究结果为厌氧氨氧化过程中脱氮性能和群落结构及功能提供了更深层次的见解，有助于厌氧氨氧化技术的工程应用。
| Research object | Research method | Research contents | Research results | References |
| Anammox reactor (MBRs) | 16S rRNA Metatranscriptomics Metabolomics | Analyzed the differences in the metabolic pathways of anammox bacteria in the membrane bioreactor | The mixed anammox bacteria showed higher nitrogen removal rate, biomass and extracellular polymer secretion under the condition of C/N of 0.3, mainly due to changes in reduced coenzyme I, coenzyme I and ATP consumption | [110] |
| Anammox reactor (SBR) | Metagenomics Metatranscriptomics | The response of Ca. Jettenia caeni and Ca. Brocadia sinica to acetate was investigated | COG analysis showed that B. sinica was more competitive than J. caeni after the addition of acetate, showing advantages in metabolic activity and growth, mainly attribute to the up-regulation of genes involved in the metabolic process | [111] |
| Anammox reactor (SBR) | Metagenomics Metatranscriptomics | Combining metabolic networks and metatranscriptomics to detect gene expression and potential interactions between anammox bacteria and heterotrophic bacteria | Chlorella bacteria may be highly active protein degradation products that decomposed extracellular peptides during the denitrification process. Other heterotrophic microorganisms may also help to remove debris and peptides produced by anammox bacteria during the reaction metabolism process | [112] |
| Xi’an wastewater treatment plant (AM-AAO) | 16S rRNA Metagenomics | Quantifying the contribution percentage of anammox to the improvement of nitrogen removal efficiency of wastewater treatment plant | The abundance of narG indicated that the potential of nitrate to nitrite conversion in anoxic carrier biofilms was higher, which promoted the growth of anammox bacteria in mainstream processes | [99] |
| Anammox reactor (EGSB) | Metagenomics | Differences in microbial communities and nitrate production in the anammox reactors under different nitrogen loads were studied | In the lower load anammox reactor, the total nitrogen removal efficiency was low, and the nitrite oxidoreductase gene content was relatively high | [113] |
| Anammox reactor (SBR) | 16S rRNA Metagenomics | The mechanism of anammox and denitrification coupled system in low-temperature conditions for low-concentration wastewater treatment was explored | The interaction between flocculent sludge microbial communities promoted the interaction between heterotrophic denitrifying bacteria and anammox bacteria, as well as the activity of anammox bacteria at low temperatures | [114] |
| Anammox reactor (EGSB) | 16S rRNA Metagenomics Metatranscriptomics | The complex nitrogen metabolism pathway in the integrated reactor of partial denitrification and anammox was investigated | Most of the nitrogen was removed by the coupling system (92.1%), but part of the nitrogen was also removed by dissimilation denitrification. Ca. Jettenia caeni was the most important anaerobic bacteria, and Thaurea was the main denitrifying bacteria in the coupled reaction system | [115] |
| Anammox reactor (SBR) | 16S rRNA Metabolomics | The response mechanism of anammox bacteria to temperature was inquired | Low temperature leaded to the down-regulation of CO2 fixation, tricarboxylic acid cycle and pyruvate metabolism of the anammox bacteria, resulting in a significant decrease in nitrogen removal activity, but the up-regulation of the RNA synthesis level, putrescine and signal molecule synthesis of the flora, thereby regulating the metabolism of the microorganism to adapt to the low temperature environment | [116] |
| Anammox reactor (SBR) | Metatranscriptomics | Metatranscriptomics revealed the reaction mechanism and recovery mechanism of anammox bacteria to the inhibition of dissolved oxygen | Appropriate addition of nano-zero-valent iron can effectively restore the activity of anammox bacteria. And the overall transcriptional central metabolism, transcription cofactor binding, protein folding and oxidoreductase activity of anammox bacteria were down-regulate under low DO conditions | [117] |
| Anammox reactor (SBR) | Metatranscriptomics | The survival mechanism of Ca. K. stuttgartiensis under Zn(Ⅱ) stress was studied | Ca. K. stuttgartiensis up-regulated many functional genes to form a regulatory network, including functions related to substrate degradation, Zn(Ⅱ) efflux chelation, DNA repair, protein degradation, synthesis and signal transduction processes | [118] |
| Anammox continuous flow reactor | 16S rRNA | From the aspects of extracellular polymer composition and microbial community structure, the difference between partial nitrification and anammox (PN/A) granular sludge cultured under medium high temperature and low temperature was analyzed | Low-temperature acclimation did not change the types of AOB and anammox bacteria in the PN/A granular sludge, but the diversity of the microorganism was improved; and the effective enrichment of Ca. Kuenenia in granular sludge was an important basis for the adaptation of the bacterial microorganism to the low-temperature environment | [119] |
| Anammox reactor (MBR) | Metabolomics | The carbon and nitrogen metabolism pathways of Ca. K. stuttgartiensis was explored | The result showed that the Ca. K. stuttgartiensis can use formic acid but can’t use acetic acid for metabolism. And during the reaction, energy was obtained through the oxidative tricarboxylic acid cycle | [120] |
| Anammox reactor (MBR) | Metatranscriptomics | To understand the glycogen metabolism process of Ca. Brocadia sinica in a continuous membrane bioreactor using bicarbonate as a carbon source | During the growth phase, Ca. Brocadia sinica synthesized glycogen through three known glycogen biosynthetic pathways and used it as energy storage; when the cells were hungry, the stored glycogen was converted to trehalose as a stress protector | [121] |
2005年，罗格斯大学Clément等首次在厌氧湿地中观察到氨氮氧化和Fe(Ⅲ)还原现象[122]。随后，2006年日本学者Sawayama首次证实在厌氧条件下Feammox菌以Fe(Ⅲ)为电子受体将氨氮氧化成硝态氮、亚硝态氮和N2，并将该过程称为Feammox (图 2)[123]。近年来，Feammox已在各种不同生态环境如水稻土、湿地和沉积物中被检测到。Feammox反应过程复杂，方程式如公式(2)–(4)所示，但该反应由于无需曝气和添加有机碳源、污泥产量低等优势，为污水处理厂自养生物脱氮带来了新途径[124]。
| $ 8 \mathrm{Fe}(\mathrm{OH})_{3}+14 \mathrm{H}^{+}+\mathrm{NH}_{4}^{+} \rightarrow \mathrm{Fe}^{2+}+21 \mathrm{H}_{2} \mathrm{O}+\mathrm{NO}_{3}^{-} $ | (2) |
| $ \begin{array}{l} 6 \mathrm{Fe}(\mathrm{OH})_{3}+10 \mathrm{H}^{+}+\mathrm{NH}_{4}^{+} \rightarrow \mathrm{Fe}^{2+}+9 \mathrm{H}_{2} \mathrm{O}+\mathrm{NO}_{2}^{-} \end{array} $ | (3) |
| $ \begin{array}{l} 3 \mathrm{Fe}(\mathrm{OH})_{3}+5 \mathrm{H}^{+}+\mathrm{NH}_{4}^{+} \rightarrow \mathrm{Fe}^{2+}+9 \mathrm{H}_{2} \mathrm{O}+0.5 \mathrm{~N}_{2} \end{array} $ | (4) |
Feammox反应所涉及相关性功能微生物目前尚未统一。截至目前，Feammox菌主要包含有铁还原菌、Acidimicrobiaceae sp. A6和Anammox菌[125]。有学者利用分子生物学技术发现铁还原菌和Feammox反应有密切联系，其中地杆菌属(Geobacter)和厌氧粘细菌(Anaeromyxobacter)是驱动Feammox过程的主要微生物[126]。2016年，有研究人员在实验室分离出一株具有Feammox作用的菌株，该细菌为杆状，长1.5 μm，通过16S rRNA基因测序该菌株属于酸化菌科(Acidimicrobiaceae)，因此命名为Acidimicrobiaceae sp. A6[127]。随后，大量研究报道在Feammox过程中Acidimicrobiaceae sp. A6发挥着重要作用，但对其具体作用机理还需结合分子生物学技术进一步探索。也有学者在厌氧氨氧化污泥中培养Feammox，采用高通量测序技术发现Anammox菌(Ca. K. stuttgartiensis、Ca. Brocadia sinica和Ca. Brocadia fulgida)在Feammox过程中发挥着关键作用，但对于具体反应机理尚未可知[128]。在污水处理过程中，Feammox也可能与其他过程耦合反应进行脱氮。有研究人员将Feammox、Anammox和NDFO结合进行同步脱氮，总氮去除率达63.2%，但耦合反应中铁元素和氮元素归趋及各微生物之间的相互作用还不明确[129]。
Feammox反应目前的研究主要集中在湿地、湖泊沉积物生态系统中，在污水处理厂研究较少。但Feammox作为一种新型脱氮途径，对污水处理厂氮去除具有潜在作用。有关Feammox反应作用机理研究尚需从宏基因组学及宏蛋白质组学角度进行探讨。与此同时，Feammox和其他工艺如Anammox和反硝化等耦合还需进一步探索，从而拓宽Feammox工艺在污水脱氮过程中的应用。
2.5 硫酸盐型厌氧氨氧化2001年，西班牙Fdz-Polanco教授在处理甜菜酒糟废水的活性炭厌氧流化床中首次发现了硫酸盐与氨氮的同步脱除，并生成元素硫和氮气，将此过程称为Sulfammox[130]。此后，Sulfammox反应指以氨氮为电子供体、硫酸盐为电子受体生成硫单质和氮气的过程(图 2)。然而，最新研究表明Sulfammox可能是好氧氨氧化、Anammox和异养硫酸盐还原过程的耦合反应[131]。由此可见，Sulfammox反应机理目前还不明确，其中间产物还存在争议。
迄今为止，关于Sulfammox菌研究还鲜有报道。2008年，有学者利用高通量测序技术发现Brocadia Anammoxoglobus Sulfate在Sulfammox反应过程中得到富集[132]；随后有研究人员分离出可进行Sulfammox反应的纯菌食苯芽孢杆菌ASR (Bacillus benzoevorans ASR)，该菌株为杆状，最适pH值和温度分别为8.5和30 ℃[133]；有研究利用16S rRNA基因测序技术在Sulfammox反应器中鉴定到疣微菌门(Verrucomicrobia)相对丰度较高，推测该类微生物是Sulfammox菌[134]。可见，Sulfammox微生物群落的结构研究目前还大多数处于推测阶段。有学者推测Sulfammox和SDAD工艺耦合系统可实现微生物之间的动态平衡，减少反应副产物，保证出水水质达标。耦合工艺中相互作用机制还处于未知，而分子生物学技术具有巨大潜力来确定功能细菌、识别关键生物并探究微生物之间的相互作用。可见，对于Sulfammox新型工艺未来在污水处理厂的广泛应用，分子生物学技术将发挥不可替代的作用。
Sulfammox作为一种新型脱氮工艺，可利用污水中硫酸盐将氨氮转化为氮气，为污水生物脱氮节约了大量能源，具有重要的工程意义。目前关于Sulfammox研究仍存在诸多问题，如反应机理不明确、功能微生物和功能基因不清晰、反应过程中氮和硫元素循环不了解、反应过程控制策略和应用潜力不清楚、Sulfammox工艺和其他工艺耦合协同工作机制不明晰和Sulfammox实际应用较少等。然而分子生物学方法的发展为解决这些问题提供了科学依据，为污水处理厂自养生物脱氮提供了基础。
2.6 n-DAMO微生物亚硝酸盐/硝酸盐型厌氧甲烷氧化(n-DAMO)反应[135]：利用n-DAMO微生物，以硝酸盐/亚硝酸盐作为电子受体、以甲烷作为电子供体反应生成N2的过程(图 2)，其反应方程式如公式(5)和公式(6)。n-DAMO微生物生长缓慢(倍增时间为数周至数月，较Anammox菌更长)，当以悬浮形式生长在反应体系中时，其代谢活性受到限制。但n-DAMO微生物与电子受体亲和力较高，从而利于提高总氮去除效能，并可直接利用温室气体甲烷来进行反应，将甲烷氧化为二氧化碳，降低了温室效应[9]。因此，近年来得到研究学者们的进一步关注。
| $ \mathrm{CH}_{4}+4 \mathrm{NO}_{3}{ }^{-} \rightarrow \mathrm{CO}_{2}+4 \mathrm{NO}_{2}{ }^{-}+2 \mathrm{H}_{2} \mathrm{O} \mathrm{n}-\mathrm{DAMO} \text { 古菌 } $ | (5) |
| $ \begin{array}{l} 3 \mathrm{CH}_{4}+8 \mathrm{NO}_{2}^{-}+8 \mathrm{H}^{+} \rightarrow 3 \mathrm{CO}_{2}+4 \mathrm{~N}_{2}+10 \mathrm{H}_{2} \mathrm{O} \text { n-DAMO 细菌 } \end{array} $ | (6) |
n-DAMO微生物由n-DAMO古菌(Ca. Methanoperedens nitroreducens)和n-DAMO细菌(Cadidatus Methylomirabilis oxyfera、Ca. Methylomirabilis lanthanidiphila)组成。根据16S rRNA基因序列发现，目前所报道的n-DAMO细菌Ca. M. oxyfera和Ca. M. lanthanidiphilan均属于NC10门[9]。宏基因组学研究结果表明，narG/napA、nir、norB基因均存在于n-DAMO细菌中；而n-DAMO古菌宏基因组研究表明，Ca. M. nitroreducens含有编码膜/周质结合硝酸还原酶narGHJ和napH基因，可利用硝酸盐作为末端电子受体进行反应[135-136]。
目前关于n-DAMO工艺的研究主要集中在该工艺可行性和去除效能方面，而对于该微生物具体代谢机制和其他微生物之间相互作用研究甚少。有学者发现在反应过程中n-DAMO微生物成为反应器中优势微生物群落(20%−30%)，其对亚硝酸盐还原为N2具有显著影响[137]。随后有研究人员发现MBR反应器在不同温度(25−10 ℃)下均具有较好的氮去除性能(总氮去除率为90%−94%)，n-DAMO微生物丰度均在7.9%−10.6%之间，而低温使n-DAMO古菌失去竞争力，从而使其从悬浮系统中淘洗出；他们进一步指出，微生物对低温的耐受性是由于生物膜产能过剩造成[138]。膜生物反应器中还包括一定数量其他微生物，这些微生物对总氮去除的贡献率及与n-DAMO微生物的相互作用还需进一步探究。随后也有课题组进一步分析了硫酸盐浓度对n-DAMO工艺的影响机理，发现当硫酸盐浓度为80 mg/L时促进了脱氮效能。微生物群落变化结果表明，菌株Unclassfied_c_ABY1和Norank_f_LD-RB-34比例的变化是影响氮去除性能的主要因素[139]。近期有报道基于16S rRNA基因扩增子测序方法检测了5个污水处理厂中微生物群落组成，发现Anammox菌含量为2.64×106−1.73×1013 copies/g干污泥，而n-DAMO微生物的含量为2.52×107−2.19×108 copies/g干污泥，证明了n-DAMO微生物在污水处理厂氮和碳去除过程中起重要作用[140]。同时，n-DAMO古菌宏基因组结果表明，他们具有将甲烷厌氧氧化与DNRA耦合的潜力[8]。n-DAMO古菌在生物膜中产生的亚硝酸盐会通过DNRA过程生成铵，进一步促使Anammox菌消耗亚硝酸盐，从而帮助n-DAMO古菌抵抗亚硝酸盐胁迫。
在微生物生态研究领域，利用分子生物学方法分析不可纯培养的n-DAMO微生物在不同生境中群落构成、丰度和分布及探讨环境因子(如pH、碳源、氮源、温度和DO等)对其群落结构和微生物代谢机制的影响已逐渐成为研究热点。对污水生物处理系统中n-DAMO微生物进行深入探究十分必要，研究n-DAMO微生物在系统中的相互作用，深入了解微生物作用机制，可进一步为污水处理厂高效脱氮提供新途径。
3 展望近年来，分子生物学方法的快速发展使生物脱氮微生物群落的组成与功能基因信息更趋于全面，但分子生物学方法在污水生物处理系统应用过程中仍面临诸多挑战，而且不同方法有其固有的局限性。因此，在今后发展中，分子生物学方法可致力于以下几个方面的发展，从而最大限度为污水处理厂生物脱氮提供科学基础。
3.1 标准测定方法的构建虽然分子生物学方法在污水处理领域已被广泛应用，但不同方法之间的研究结果差异性也不可忽视。近年来，有课题组研究了标准引物对于厌氧氨氧化细菌测定的影响，提出了针对厌氧氨氧化细菌和功能基因(hzo、nir和hzs)的特异性引物[24]。随后有研究人员利用ARB软件包和SILVA数据库的“探针设计”和“探针匹配”功能并通过程序迭代设计新型靶向16S rRNA基因的寡核苷酸探针来检测AOB，表明新型靶向rRNA寡核苷酸探针覆盖率和特异性更加精细化，有助于AOB原位分析和研究群落组成与动态变化[25]。也有学者研究了不同反应器类型(SBR、MBR和生物转盘反应器)、不同DNA提取方法(Fast DNA Spin试剂盒和Qiagen QIAamp DNA试剂盒)和不同qPCR扩增方法在6个不同实验室检测情况下对PN/A反应器内微生物群落组成和功能的影响。结果表明，不同实验室提取的DNA浓度存在显著差异(介于2.7−328 ng/mg之间)，也检测到不同qPCR扩增方法使测序结果显示出较高差异性(1−7 Log10倍)；同时也发现不同DNA提取试剂盒对某一种特定类型样品和微生物表现更好，但是不适用于其他类型微生物，提出需进一步标准化DNA提取和qPCR扩增方法使得qPCR测序结果更具可重复性及有效性[38]。可见，标准化的分子生物学方法对实现重复实验、跨样品和跨研究之间的差异性对比至关重要。因此，今后研究中需开发标准的分子生物学测定分析技术，使其满足不同测定条件下的需求，保证测定结果可重复，减少由于方法差异所造成的结果误差，从而使得不同研究人员之间的数据具有可对比性，进一步挖掘实验结果背后的科学意义。
3.2 不同方法的联用及跨学科结合分子生物学方法近年来在污水处理领域已得到应用，但其大规模解析微生物群落结构与功能关联能力仍受到部分限制，如微生物之间相互作用机制、复杂环境下微生物代谢途径和微生物种群结构变化及功能基因预测等。鞠峰等总结了宏组学方法在揭示活性污泥微生物群落结构和功能的研究进展，为研究学者探索活性污泥微生物群落蕴藏不可培养新物种和基因多样性提供了基础，并提出多组学和交叉学科的方法可以更好地阐明微生物之间相互作用、基因表达活性及与环境效能之间的关系[54]。单一分子生物学方法具有一定的局限性，在分析具体问题的过程中可结合不同方法使得研究结果更加系统。同时，微生物的研究不仅仅依赖分子生物学工具，还需结合不同学科(如微生物学、环境科学、基因组学、系统生物学、数据统计、计算机分析和高级成像等)和不同表征方法(如扫描透射X射线显微镜、互补金属氧化物半导体、微型计算机断层扫描、纳米级二次离子质谱、纳米级稳定同位素探测等)进行协同研究。将这些学科和分子生物学方法学有效地结合起来研究污水处理厂体系中氮循环的微生物资源，从不同方面解析微生物组结构组成、研究复杂体系下微生物之间相互作用并预测微生物代谢功能等，进一步加强分子生物学技术在污水生物脱氮领域的应用。
3.3 检测方法的简易化分子生物学方法对样品要求较高，不同测定方法对样品的处理有所差异。对于水样采集，样品制备方法的简易化还需在今后研究中进一步重视。分子生物学方法测定过程涉及的操作步骤复杂且对操作者要求也较高，这也使得很多研究学者望而止步。因此，从本质上简化操作流程亟须研究。近年来，有课题组使用便携式宏基因组设备对水质进行宏基因组技术可行性的分析，该设备包含小型真空泵和过滤装置、小型离心机、小型PCR机和存储卡大小的MinION (Oxford Nanopore Technologies)，用于16S rRNA基因文库制备和测序；在一个工作日内完成了16S rRNA基因测序工作流程，包括DNA提取、PCR扩增、测序文库制备和测序工作；根据互联网速度，测序数据可在24−72 h内获得；同时验证了便携式设备测定结果和qPCR结果之间有很好的一致性[141]。简化分子生物学测定流程对其测序结果影响较小，但可促进该方法在实际工程中的应用。
本课题组也在总结和整理分子生物学方法实验步骤，通过实验结果得出较为广泛应用的分子生物学测定过程，并不断优化分子生物学方法测定分析流程，为该技术的应用提供依据。
4 小结微生物在污水处理厂脱氮过程中发挥着重要作用，决定着污水处理厂的出水水质。在污水处理厂生物脱氮效能研究过程中，进一步理解脱氮体系中微生物种类和功能及微生物和环境之间相互作用至关重要。分子生物学方法通过DNA、RNA、蛋白质和代谢产物4个层面揭示了污水生物脱氮系统中相关微生物群落结构、系统发生、功能基因、相互作用、代谢途径和调控规律等，有助于加深研究学者对生物脱氮体系中微生物的全面理解。近年来，研究人员利用分子生物学技术不仅进一步探究了AOB、AOA和反硝化细菌等功能微生物在污水处理厂生物脱氮过程中所发挥的重要作用及代谢机理，同时使得Anammox、Comammox、Feammox、Sulfammox及n-DAMO微生物等新型微生物发挥的作用和研究潜力也逐渐被研究报道，丰富了生物脱氮途径，为实现新型经济有效的生物脱氮工艺提供了基础。本文基于现代分子生物学方法认识了污水处理厂生物脱氮过程中的微生物组成结构，以深入理解生物脱氮过程原理，助力生物脱氮工艺的优化和应用。
随着分子生物学技术的发展，16S RNA基因测序、RT-qPCR技术和宏组学技术在污水生物脱氮领域的应用也会逐渐成熟，但也面临一定挑战。从过去几十年研究来看，微生物会在环境变化过程中迅速做出调控来适应环境。因此，随着分子生物学技术的发展，相信这些新技术将会在生物脱氮领域得到快速发展和应用，使研究者全面了解脱氮微生物群落构成及其功能，利用其研究成果探讨新型微生物菌种及其酶资源，为未来污水处理生物脱氮微生物研究提供新趋势与新研究方向。
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