环境科学研究  2020, Vol. 33 Issue (3): 668-676  DOI: 10.13198/j.issn.1001-6929.2019.09.04

引用本文  

闫苗苗, 张海涵, 钊珍芳, 等. 生物脱氮技术中好氧反硝化细菌的代谢及应用研究进展[J]. 环境科学研究, 2020, 33(3): 668-676.
YAN Miaomiao, ZHANG Haihan, ZHAO Zhenfang, et al. Research Progress of Metabolism and Application of Aerobic Denitrifying Bacteria in Biological Denitrification Technology[J]. Research of Environmental Sciences, 2020, 33(3): 668-676.

基金项目

国家自然科学基金面上项目(No.51978561);陕西省重点研发计划项目(No.2018ZDXM-SF-020);陕西省国际科技合作计划项目(No.2018KW-011)
Supported by National Natural Science Foundation of China (No.51978561); Key Research and Development Program Projects in Shaanxi Province, China (No.2018ZDXM-SF-020); Shaanxi International Science and Technology Cooperation Program, China (No.2018KW-011)

责任作者

张海涵(1981-), 男, 陕西户县人, 教授, 博士, 主要从事环境工程微生物研究, zhanghaihan@xauat.edu.cn.

作者简介

闫苗苗(1994-), 女, 陕西榆林人, Ymmzsj@163.com

文章历史

收稿日期:2019-04-10
修订日期:2019-08-27
生物脱氮技术中好氧反硝化细菌的代谢及应用研究进展
闫苗苗1,2, 张海涵1,2, 钊珍芳1,2, 李苏霖1,2, 黄廷林1,2, 杨尚业1,2    
1. 西安建筑科技大学, 陕西省环境工程重点实验室, 陕西 西安 710055;
2. 西安建筑科技大学环境与市政工程学院, 陕西 西安 710055
摘要:微生物脱氮是水体氮素去除最常用的有效方式,具有高效、经济和二次污染小等特点.好氧反硝化细菌的发现是微生物脱氮技术的重要里程碑,为新型生物脱氮领域的研发提供了新出路.当前,探究好氧反硝化细菌的脱氮特性及各环境因子(如碳源种类、C/N、温度、pH、DO、盐度、重金属含量等)在好氧条件下对反硝化过程的影响已成为污染水体氮素逸出的研究热点.通过总结好氧反硝化细菌的脱氮机制、筛选来源和种类、鉴定、脱氮特性与环境影响因素及其在实际含氮污染水体中的应用,结果表明,好氧反硝化细菌种类丰富、存在环境广泛,脱氮效率一般在50%以上;目前,好氧反硝化细菌脱氮机理的研究主要停留在氮的转化方面,对其中心碳的代谢研究以及碳氮比对反应机理的具体影响尚未明确,需要进一步探究;有关好氧反硝化细菌的脱氮特性研究尚处于实验室小试或中试阶段.建议进一步筛选高效菌群,进行碳氮代谢途径研究,优化好氧反硝化细菌固定化技术,并将其应用于实际工程中,减少水体中的氮素污染,对于实现高效、经济的脱氮具有重要社会价值.
关键词好氧反硝化细菌    机制    筛选    影响因素    应用    
Research Progress of Metabolism and Application of Aerobic Denitrifying Bacteria in Biological Denitrification Technology
YAN Miaomiao1,2, ZHANG Haihan1,2, ZHAO Zhenfang1,2, LI Sulin1,2, HUANG Tinglin1,2, YANG Shangye1,2    
1. Shaanxi Key Laboratory of Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China;
2. School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China
Abstract: Microbial denitrification is the most common and effective way to remove nitrogen in water. It has the advantage of high efficiency, low cost and low secondary pollution. The discovery of aerobic denitrifying bacteria is an important milestone in biological denitrification technology, and opens up a new approach in the biological denitrification field. At present, it has become a research hotspot to explore the denitrification characteristics of aerobic denitrifying bacteria and the influence of environmental factors (such as carbon source, C/N, temperature, pH, dissolved oxygen, salinity, heavy metals and so on) on denitrification process under aerobic conditions in polluted water. This paper mainly introduces the denitrification mechanisms, screening sources and species, identification and denitrification characteristics of aerobic denitrifying bacteria, and its influencing factors and applications in nitrogen-rich polluted water. The results show that there are abundant aerobic denitrifying bacteria species and they widely exist in the environment, and their nitrogen removal efficiency are generally above 50%. Currently, the research on aerobic denitrifying bacteria is mainly focused on the transformation of nitrogen. However, the central metabolism and the influence of C/N are not clear and need to be further explored. The research on the denitrification characteristics of aerobic denitrifying bacteria is in the stage of laboratory test or pilot test. It will be highly valuable to further screen highly-efficient denitrifying bacteria, study the carbon and nitrogen metabolic pathways, optimize the immobilization technology of aerobic denitrifying bacteria, and apply it to practical projects. By these means, the outcome will be a significant reduction in nitrogen pollution in water and a high-efficiency and economical denitrification.
Keywords: aerobic denitrifying bacteria    mechanisms    screening    influencing factors    application    

大量生活污水与工业废水的排放、农药和化肥的过量使用均造成水体的严重污染[1-2].各类型水体的污染源不同,污染状况也存在一定差异,但氮素含量过高是引起水体富营养化的关键因素之一[3].如何去除水体中过量氮素已成为控制水体污染的重要问题之一[4].传统生物脱氮技术因构筑物多、运行费用高、系统抗负荷冲击能力低、硝化/反硝化条件控制复杂等缺点需要进一步改进[5].而好氧反硝化脱氮技术的硝化和反硝化过程均在好氧环境中发生,减少了构筑物;另外,在同一构筑物中,反硝化反应产生的碱度可将硝化反应产生的酸中和,不需另外投加碱度[6].近年来国内外大量文献报道[7-12]发现了可在好氧环境下进行反硝化脱氮的微生物,其将有机物及氮素作为自身碳氮源,在除氮的同时还可去除部分有机物.好氧反硝化细菌的发现和研究是对生物脱氮理论的进一步探索,为开发新型生物脱氮技术奠定了重要的基础和平台.

自1984年Robertson等[8]发现首株异养硝化-好氧反硝化细菌Thiosphaera pantotrophus (后被命名为Paracoccus denitrificans)以来,众多研究人员就致力于从各种环境(如土壤[9]、水库[7]、湖泊[1]、河流[10]、海洋[11]及污水处理厂[12]等)中筛分好氧反硝化细菌,并探索其在不同环境因子中的脱氮方式与脱氮特性.好氧反硝化细菌的探索与研究为处理氮污染水体开拓了新途径.该文主要从好氧反硝化细菌的脱氮机理、筛选与鉴定、脱氮特性及影响因素等方面进行综述,并对其在实际氮污染水体中的应用和今后的研究方向进行展望.

1 好氧反硝化细菌的作用机理

好氧反硝化细菌在好氧环境中依次使用硝酸还原酶(Nap)、亚硝酸盐还原酶(Nir)、一氧化氮还原酶(Nor)和一氧化二氮还原酶(Nos),将硝酸盐氮转化为气态氮(NO3-→NO2-→NO→N2O→N2)[13],其在好氧环境中的主要作用机理如图 1所示.

图 1 好氧反硝化细菌的脱氮过程[13-17] Fig.1 The denitrification progress of aerobic denitrifiers[13-17]

首先,NO3--N进入细胞发生硝酸还原反应.硝酸盐还原酶有两种位于不同区域的基因表达形式,即nar基因表达的膜结合硝酸盐还原酶和nap基因表达的周质硝酸盐还原酶[13-14].在好氧环境下,Nar的活性因氧存在而受到抑制;Nap的活性受氧分子的抑制性较小,能够优先表达,是好氧反硝化过程所必需的[14].作为Nap蛋白的末端还原酶,napA基因的扩增可以决定好氧反硝化过程是否由Nap催化[13]. nap基因表达非常重要,为好氧环境下反硝化细菌的反硝化脱氮提供了必要条件.

其次,NO3--N发生还原反应.亚硝酸盐还原酶(Nir)位于细胞外膜与内膜之间的周质中,可以在有氧和无氧条件下表达[13].该酶制约着反硝化脱氮过程的关键步骤,有两种非同源性的基因表达,即含铜的nirS和含细胞色素的nirK[15-16].其中,nirS型反硝化细菌在环境中种类丰富,分布广泛,被广泛用于评价水库、湖泊、污水处理厂等各类型污染水体,对污染水体的净化处理有着巨大的应用潜力. NO在细胞内膜上被转化为N2O.一氧化氮还原酶(Nor)在细胞内膜上发挥作用,对NO的亲和力较高,可将NO几乎全部转化为N2O[13].该酶主要包括含b型和c型的膜结合酶NorB和NorC.在此反应过程中,NorB从单血红素细胞色素c亚单位NorC接收电子,从而进行反应[17].

最后,从细胞内膜释放或者环境中进入周质中的N2O被还原成N2.一氧化二氮还原酶(Nos)位于细胞周质中,对氧气的敏感性并不强,在有氧和缺氧条件下均能表达,作用产物为气态氮(N2)[13].反硝化细菌典型的Nos是一种名为NosZ的同二聚铜蛋白,是一种位于细胞膜外的周质酶.对于一些物种(如脱氮副球菌),N2O的还原与质子跨胞质膜的转移相互耦合(即电子转移到Nos是通过细胞色素C进行的),该过程也有细胞色素BC1复合体的参与[18].

目前对好氧反硝化细菌机理的研究主要停留在氮的转化方面,如研究好氧反硝化细菌脱氮基因丰度、好/缺氧条件下脱氮效果对比及氮的最终转化形态等;但是对其中心碳代谢过程的相关研究及碳氮比、氮磷比对其反应机理的具体影响尚未明确,需要进一步探究.

2 好氧反硝化细菌的筛选

好氧反硝化细菌具有对环境的适应性较强、分布广泛、反硝化速率较快等特征.目前,众多研究者从各环境中筛选出了不同种类的好氧反硝化细菌,以期运用到实际环境中,有效治理含氮污染水体[8, 19]. HUANG等[19]从海产养殖污染水体中筛分得到一株芽孢杆菌Bacillus strain N31,添加不同的氮源后发现,该菌株对NH4+-N、NO2--N、NO3--N的去除率分别达86.3%、86.4%、89.4%,硝化/反硝化效果均较好. HUANG等[7]从山东省枣庄市周村水源水库中筛选得到的好氧反硝化细菌Pseudomonas stutzeri strain ZF31,在氮平衡中有75%的初始氮经好氧反硝化作用,生成气态氮释放;低C/N条件下总氮去除率为73.30%,低温(10 ℃)条件下总氮去除率为60.08%,菌株Pseudomonas stutzeri strain ZF31在水源水库的生物原位修复中有着潜在的应用价值.

2.1 好氧反硝化细菌的分离与脱氮特性研究

有关好氧反硝化细菌的探索与研究日益增多,筛分得到的好氧反硝化菌种类与数量也逐渐增多.好氧反硝化细菌的筛选与鉴定方法日益成熟,发现的属种范围逐步扩大,脱氮性能普遍较高,在氮污染水体处理方面应用前景广泛.近年来从不同环境中筛选的一些好氧反硝化细菌如表 1所示,主要有副球菌属(Paracoccus sp.)、芽孢杆菌属(Bacillus sp.)、假单胞菌属(Pseudomonas sp.)、产碱杆菌属(Alcaligenes sp.)、不动杆菌属(Acinetobacter sp.)、克雷伯氏菌属(Klebsiella sp.)、红球菌属(Rhodococcus sp.)以及卤单胞菌属(Halomonas sp.)等.

表 1 不同来源中筛选的好氧反硝化细菌 Table 1 Aerobic denitrifying bacteria screened from different sources
2.2 好氧反硝化细菌的鉴定

为了更系统地研究不同种类的好氧反硝化细菌,有必要对高效菌进行鉴定和分类.鉴定方法主要有生理生化特性研究、形态鉴定和基因测序[47-51].其中,形态鉴定和生理化特性研究主要是观察菌落生长状态和单个菌体形状,得到高效菌株的表征,其种类确定需用基因测序技术进一步鉴定.目前鉴定高效菌株的手段通常是用16S rRNA(16S rDNA)基因引物判定其种类,而nosZnirS / nirK是检测好氧反硝化细菌的常用基因.基因测序首先是从克隆文库发展而来,随后逐步发展了限制性片段长度多态性分析(RFLP)、末端限制性片段长度多态性分析(T-RFLP)、变性梯度凝胶电泳/温度梯度凝胶电泳(DGGE/TGGE)等技术.随着分析方法的逐渐完善,目前主要利用高通量测序技术(454焦磷酸测序、qPCR、Illumina MiSeq DNA测序)对菌株进行鉴定并构建系统发育树.通过扫描电镜并结合高通量测序技术得到不同种属的好养好硝化细菌鉴定状况(见表 2).

表 2 不同菌属分类下的鉴定状况 Table 2 Identification of different genus of denitrifying bacteria
3 好氧反硝化脱氮过程中的影响因素

好氧反硝化细菌因在好氧环境中进行反硝化这一独特优势而被重点关注,目前的研究仍处于实验室阶段.研究者虽然分离出了大量好氧反硝化细菌,但不同菌种对环境的要求与耐受度差异较大,研究各因素对好氧反硝化过程的影响,对氮污染水体的治理极其重要.影响好氧反硝化细菌反硝化过程的主要因素通常有碳源、C/N、温度、酸碱度(pH)、DO(由摇瓶来控制)以及盐度、重金属含量等.

3.1 碳源种类对好氧反硝化脱氮的影响

碳是生物有机结构组成的最基本元素.在脱氮过程中,碳源不仅可作为好氧反硝化细菌生长代谢的营养物质,还为其反硝化过程(氧化还原过程)提供能量和电子,故碳源的种类与相对含量均会对好氧反硝化速率产生一定影响.白洁等[51]从胶州湾海底沉积物中分离出好氧反硝化细菌Zobellella sp. B307,探究柠檬酸三钠、琥珀酸钠、乙酸钠、丁二酸钠对该菌株反硝化效率的影响,发现丁二酸钠作为碳源时可去除91.39%的NO3--N,反硝化效率最高. GUO等[46]从活性污泥中筛选出一株阴沟肠杆菌——Enterobacter cloacae strain HNR,分别以乙二胺四乙酸、柠檬酸盐、葡萄糖、醋酸盐、琥珀酸盐为碳源探究其反硝化效果,发现葡萄糖是该菌株的最有利碳源,可基本将全部NO3--N进行反硝化反应.李健[41]在不加碳源或分别单独添加蔗糖、醋酸钠、葡萄糖、丁二酸钠时,卤单胞菌Halomonas sp. B02在以丁二酸钠为碳源时的反硝化效率最高,可达82.25%.各碳源对不同菌株的影响效果有所差异,但具体影响机理尚不明确,需进一步探究.

另外,一些好氧反硝化细菌不仅可以利用常规的碳源,还能将芳香类化合物及其衍生物等(如苯[52]、苯酚[53]、硝基苯酚[54])作为碳源,既进行好氧反硝化脱氮,又可除去难降解的有毒害物质. LI等[52]以苯为碳源,将苯降解的好氧反硝化菌株Pseudomonas sp. BN5在初始苯浓度为52.37 mg/L、NH4+-N浓度为16.13 mg/L条件下培养72 h,苯去除率为100%,NH4+-N去除率为70.86%.王国英等[53]从活性污泥中筛选的一株有降解苯酚作用的好氧反硝化细菌Diaphorobacter sp. PDB3,在C/N为7、摇床转速为160 r/min下,以苯酚为唯一碳源,有机物去除率可达90.4%;在TN去除过程中,有52.3%转化为生物量、37.2%转化为氮气释放. Kulkarni[54]在SBR反应器中加入含有Thiosphaera pantotropha 的单一污泥生物质,以硝基苯酚为唯一氮源,添加适量酵母提取物和蛋白胨,结果表明,反应器中好氧反硝化细菌对硝基苯酚具有良好的降解效果.通过专门驯化筛分出具有降解有毒害作用或难降解物质能力的好氧反硝化细菌,在脱氮的同时还可以除去大量难降解的有机物,对氮污染水体的治理有着重大潜在价值.

3.2 C/N、温度、pH和DO对好氧反硝化脱氮的影响

在探讨环境因素时,通常采用单因素控制变量的试验探究C/N、温度、酸碱度和DO(通过摇瓶转速来控制)对好氧反硝化细菌反硝化效率的影响.另外,一些研究中也会利用正交试验或者设计响应曲面(RSM)方法构建脱氮特性模型,得出最佳优化条件下的脱氮效果.好氧反硝化细菌在优化条件下的脱氮效率如表 3所示.

表 3 好氧反硝化细菌在不同条件下的脱氮效率 Table 3 Denitrification efficiency of aerobic denitrifying bacteria under optimized conditions

C/N、温度、pH和DO是影响反硝化脱氮的重要指标,筛选出不同条件下的好氧反硝化细菌并将其应用到不同污染区域中去(如筛选耐低温的细菌处理高冷地区废水、筛选贫营养细菌处理微污染水源水体、筛选富营养细菌处理高浓度废水、确定最佳DO以减少污水处理厂工艺流程等),对以后脱氮处理工艺的发展具有重要科学意义.

3.3 其他因素对好氧反硝化的影响

氮污染水体成分较复杂,在实际处理过程中不仅要考虑营养、温度等对好氧反硝化细菌的影响,还要考虑菌株对盐度和重金属等有毒有害物质的耐受性[58-59].潘玉瑾等[58]研究了好氧反硝化菌株Pseudomonas chengduensis ZPQ2对NaCl的耐受性,发现在最佳好氧反硝化条件下,该菌株在盐度为2%时,仍能分别去除98.1%的COD和93.4%的NO3--N. CHEN等[59]研究了氧化锌纳米颗粒对好氧反硝化细菌Pseudomonas stutzeri PCN-1的抑制作用,结果表明,加入1 mg/L氧化锌纳米颗粒时脱氮效率为100%,逐渐增至128 mg/L时脱氮效率降至1.70%.可见,氧化锌纳米颗粒的存在对硝酸还原酶和亚硝酸盐还原酶的基因表达和催化活性有明显的抑制作用,最终导致NO3--N还原延迟,NO2--N累积量增加,该结果对评估纳米材料潜在的生态毒性和风险十分重要[59].研究好氧反硝化细菌对于不同氮污染水体的原水适应性,制备特别菌剂处理不同类型污染水体,在氮污染水体的微生物治理应用方面有重大意义.

4 好氧反硝化细菌的实际应用 4.1 单菌在氮污染水体中的应用

好氧反硝化细菌对氮污染水体的生物治理过程发挥着重要重用.但由于实际的氮污染水体处理系统比较复杂,目前关于好氧反硝化细菌的研究主要集中在优势菌的筛选及实验室反硝化性能研究阶段,对治理实际氮污染水体的研究鲜见报道. YANG等[47]筛选出了一株可耐高温的螯台球菌Chelatococcus daeguensis TAD1,当NH4+-N与NO3--N同时存在时,该菌株会优先利用NO3--N;另外,该菌株在50 ℃的高温生物滤池中具有良好的好氧反硝化脱氮性能. WANG等[60]将从沉积物中获得的具有高效反硝化能力(可去除96%的NO3--N)的Pseudomonas sp. HS-N6,利用聚乙烯醇-海藻酸钠和活性炭制成固定化小球,发现固定化颗粒的NO3--N去除能力有所提高,具有稳定良好的反硝化能力.将反硝化细菌进行固定化可防止因流速过快而造成菌种流失,提高氮污染水体中的菌种密度,但关于好氧反硝化细菌固定化技术在实际工程中的应用还不成熟,有待进一步发展.

4.2 混合菌在氮污染水体中的应用

混合培养菌群与单一纯菌种相比具有若干优点,特别是在复杂污染物去除方面(见图 2).从环境样品中分离混合细菌的时间比得到单一菌株要少得多(为了捕获纯菌株,往往需要进行4~5轮纯化).从微生物生态学的角度来看,在混合培养菌群系统中,混合菌群的共存和相互作用具有新颖的生物学功能,如群体感应、欺骗效应和互利共生等.更重要的是,混合好氧反硝化菌群的代谢途径具有多样化,且混合细菌联合体的能力比单一纯菌株强,所以混合菌群对污染物的去除更有效[61-62]. DENG等[63]将筛选到的两株好氧反硝化细菌(Pseudomonas stutzeri SC221-M、Bacillus cereus BSC24)进行混合培养制成菌剂,发现混合菌剂的脱氮效率是单菌的2倍,且添加菌剂后微生物群落结构丰度有所提高. CHEN等[64]将筛选得到的3株种类不同的好氧反硝化细菌——Agrobacterium sp. LAD9、Achromobacter sp. GAD3、Comamonas sp. GAD4,按照质量比为1 :1 :1制成混合菌群PCN,投加到SBR反应器中处理实际氮污染水体,结果表明,在C/N为8时,碳和氮素的去除效率较高且稳定,出水中COD、NH4+-N、TN和TP浓度均满足GB 8978—1996《污水综合排放标准》的一级要求.探索好氧反硝化菌与地域、水体类型之间的关系,将高效菌真正应用于不同类型的氮污染水体处理中还需要进一步努力(如解决反硝化过程中电子供体不足的问题).

图 2 单菌与混合菌的筛选及脱氮过程[58-60] Fig.2 Screening and denitrification of single strain and mixed strains[58-60]
5 结论与展望

a) 各类反硝化细菌的存在环境具有广泛性,在各种环境中(如沉积物、污水处理厂、土壤、垃圾渗滤液、各种生物反应器等)均筛选出了高效好氧反硝化细菌.但在实际应用中,菌种流失、处理实际污水效果较差等问题尚待解决,因此,可进一步优化好氧反硝化细菌固定化技术,并将其应用于实际工程中,实现高效、经济的脱氮.

b) 影响好氧反硝化细菌脱氮性能的因素除了碳源种类、C/N、温度、pH和DO外,还有盐度、重金属含量等.但好氧反硝化细菌在反硝化过程中的代谢途径不同,其原因也尚不明确.利用稳定同位素(13C、15N)和遗传合成生物学对C和N代谢途径的互作分析有待进一步研究.

c) 混合反硝化菌群具有多种代谢途径,其共存和相互作用共同驱动氮和碳的去除,且筛选过程比单菌简便,脱氮效果较单菌好,可作为潜在的反硝化菌剂用于治理实际氮污染水体,有较好的应用前景.

d) 将好氧反硝化细菌运用到微污染水源水体的治理中,探究菌剂活性保持技术和原水适应能力具有重要意义.

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