环境科学  2023, Vol. 44 Issue (10): 5490-5497   PDF    
引用本文
薛慧, 林辉, 王智, 杨玉义. 人工湿地去除抗生素抗性基因的研究进展[J]. 环境科学, 2023, 44(10): 5490-5497.
XUE Hui, LIN Hui, WANG Zhi, YANG Yu-yi. Research Progress on Removing Antibiotic Resistance Genes in Constructed Wetlands[J]. Environmental Science, 2023, 44(10): 5490-5497.
人工湿地去除抗生素抗性基因的研究进展
薛慧1,2,3, 林辉4, 王智5, 杨玉义1,2,3     
1. 中国科学院武汉植物园水生植物与流域生态重点实验室, 武汉 430074;
2. 中国科学院大学, 北京 100049;
3. 中国科学院丹江口湿地生态系统野外科学观测研究站, 武汉 430074;
4. 浙江省农业科学院环境资源与土壤肥料研究所省部共建农产品质量安全危害因子与风险防控国家重点实验室, 杭州 310021;
5. 中国科学院精密测量科学与技术创新研究院环境与灾害监测评估湖北省重点实验室, 武汉 430077
收稿日期: 2022-10-12; 修订日期: 2022-12-14
基金项目: 国家自然科学基金项目（32071614）
作者简介: 薛慧(1998~), 女, 硕士研究生, 主要研究方向为环境微生物, E-mail: xuehui99127@163.com
通信作者: 杨玉义, E-mail: yangyy@wbgcas.cn
摘要: 抗生素抗性基因（ARGs）在环境中的污染现状和危害逐渐受到重视.常规的水处理工艺在减少抗生素耐药菌（ARB）方面表现出非常好的效果.然而，即使在消毒过程中ARB完全失活，产生游离的ARGs也可能通过转化或转导等手段整合到其他微生物体内，使ARGs在环境中传播和扩散.因此，需要有特定的工艺处理废水中的ARGs.人工湿地是目前有效且经济环保的废水处理工艺，并且已有大量研究表明在去除抗生素和ARGs方面有显著效果.通过综述目前国内外人工湿地水处理系统对ARGs的去除效率的研究进展，结果表明，潜流人工湿地相较于表面流人工湿地对ARGs的去除效率更高.人工湿地对ARGs的去除效率因人工湿地强化类型、植物、温度和pH等因素而异，在去除环境中ARGs的应用有广阔的前景，同时也面临挑战.
关键词: 人工湿地      抗生素抗性基因(ARGs)      抗生素耐药菌(ARB)      去除效率      影响因素     
Research Progress on Removing Antibiotic Resistance Genes in Constructed Wetlands
XUE Hui1,2,3 , LIN Hui4 , WANG Zhi5 , YANG Yu-yi1,2,3     
1. Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China;
3. Wetland Ecosystem Field Research Station of Danjiangkou, Chinese Academy of Sciences, Wuhan 430074, China;
4. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Environment, Resource, Soil and Fertilizers, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
5. Key Laboratory for Environment and Disaster Monitoring and Evaluation of Hubei, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430077, China
Abstract: Recently, the issue of environmental pollution and emerging antibiotic resistance genes (ARGs) has gradually gained attention. Antibiotic resistant bacteria (ARB) can be effectively reduced via the conventional water treatment processes. Although the ARB are completely inactivated during the disinfection process, the free ARGs can be incorporated into other microorganisms through transformation or transduction, allowing the ARGs to spread and propagate. Therefore, ARGs in wastewater must be handled by a specific process. It has been demonstrated in several studies that treatment using constructed wetland is an effective, economical, and environmentally friendly method of removing antibiotics and resistance genes from wastewater. Here, the research progress on the removal effect of constructed wetland water treatment systems on ARGs at home and abroad was reviewed. The results revealed that the removal efficiency of ARGs in subsurface flow constructed wetland was higher than that in surface flow-constructed wetlands. The composite-constructed wetland had significantly improved removal efficiency of ARGs compared to that in the ordinarily constructed wetland; however, the parameter setting of the composite process still requires further research. Several studies have reported that the removal efficiency of ARGs using constructed wetlands varies depending on the type of constructed wetland enhancement, plant, temperature, pH, and other factors. The results of the current study revealed that cross-mixing was the best way to combine plants, whereas the selection of plant species has not yet shown a clear dominant species. Temperature and pH affected the removal of ARGs by altering the microbial community in constructed wetlands. Although longer hydraulic residence time could increase the removal efficiency of ARGs, it also increased the enrichment risk of ARGs. The selection of constructed wetland substrate type should focus on fillers with a high specific surface area; the flow direction of the up-flow type was generally more efficient than the down-flow type in removing ARGs. In conclusion, the various factors (such as, the constructed wetland type, substrate type, hydraulic retention time, ambient temperature, and plant species) need to be integrated into the design of the constructed wetland system parameters to achieve the most effective treatment effect. The application of constructed wetlands in removing ARGs from the environment has broad prospects but also faces challenges.
Key words: constructed wetlands      antibiotics resistance genes(ARGs)      antibiotic resistant bacteria(ARB)      removal efficiency      influence factor     

抗生素耐药性是指微生物对抗生素对其抑制和致死作用有耐受性, 进而存活和繁殖.抗生素抗性基因(antibiotics resistance genes, ARGs), 是导致微生物产生耐药性的重要原因.Pruden等[1]首次在科罗拉多州北部的环境中将ARGs作为新兴污染物进行研究, 环境中的ARGs逐渐引起重视.污水处理厂、动物养殖和制药厂废水及含抗生素残留的固体废弃物(污泥、畜禽粪便等)是土壤和水体环境中ARGs和抗生素污染的主要来源[2~4].ARGs造成环境污染且风险较高的原因在于其传播性[5], 转移的复杂性[6]以及从环境中转移到生物群落中的风险[7], 使得ARGs的去除成为当前环境领域的研究热点.

传统水处理技术(氯化消毒、臭氧消毒、紫外线照射)、电离辐射技术、好氧堆肥和厌氧消化和膜处理工艺等对ARGs处理的效率已经得到研究[8].城市污水处理厂中经传统水处理技术处理后的出水中抗生素耐药菌(antibiotic resistant bacteria, ARB)的表型和基因型均受到一定影响[9, 10], 但有研究表明大多数污水处理厂中ARGs去除效率较低, 甚至会增加ARGs的丰度[11, 12].膜处理工艺对抗生素有显著的去除效果[6, 13, 14], 但同时诱导产生了ARGs, 处理后的污水排入环境中反而增加了ARGs传播风险[15].进一步的研究证实处理过的废水排放会导致受纳河流和水体中的ARGs富集和可移动遗传因子发生显著变化[16, 17].

20世纪80年代以来, 具有环保和经济等优点的人工湿地逐步进入工程应用.人工湿地在去除废水中的抗生素和ARGs等新型复杂污染物的作用逐渐引起关注(图 1).已有研究表明人工湿地对ARGs有较高的去除效率[18, 19].García等[20]综述了人工湿地(CWs)处理新型有机污染物的研究进展, 包括不同的抗生素、ARB和ARGs, 并比较了不同类型人工湿地对抗生素的去除效果, 但未比较不同类型人工湿地对ARGs的去除效率以及影响因素.因此, 本文综述了人工湿地对ARGs去除应用的研究进展, 比较了不同类型人工湿地对ARGs的去除效果, 总结了影响去除效率的因素, 并阐述了人工湿地在去除ARGs中的发展方向及挑战, 以期为去除环境中的ARGs提供参考.

图 1 人工湿地去除环境中的ARGs Fig. 1 Removal of ARGs from the environment using constructed wetlands

1 人工湿地对ARGs的去除

人工湿地是通过模拟自然湿地、人为设计和建造的具有可控性和工程化特点, 以基质、植物和微生物协同, 通过物理、化学和生物作用进行污水处理的人工生态系统.人工湿地对环境中ARGs的去除工艺研究逐渐引起国内外学者的注意.本文依据人工湿地基础类型(表面流人工湿地、潜流人工湿地和复合型人工湿地)总结ARGs的去除效果(表 1).

表 1 不同人工湿地处理系统对ARGs去除效果 Table 1 Removal of ARGs via constructed wetland treatment systems

1.1 表面流人工湿地

表面流人工湿地, 指水面在基质表面以上, 水从进水端流向出水端的人工湿地, 在流动过程中, 与土壤、植物及植物根部的生物膜接触, 通过物理、化学以及生物反应, 污水得到净化, 并在终端流出.表面流人工湿地通常与其他类型的人工湿地综合应用.Fang等[21]研究了稳定运行10余年的综合表面流人工湿地中14种ARGs的浓度变化, 发现可以有效去除ARGs, 但在冬季观察到5个ARGs (sul1、tetA、tetC、tetE和qnrS)和夏季观察到6个ARGs (sul1、sul3、tetA、tetC、tetE和qnrS)在特定湿地子系统中的浓度显著增加.该研究还观察到移动遗传元件intI1和几个ARGs的浓度之间很强的正相关, 因此长期表面流人工湿地作业可能增加下游水体中ARGs污染物的水平.目前单独针对表面流人工湿地的研究较少[22], 且仅有研究未能表现出较好的去除率, 可能该人工湿地类型不是去除ARGs的有效工艺, 因此需要结合其他类型的工艺深入研究对ARGs的去除效率和机制.

1.2 潜流人工湿地

潜流人工湿地, 指水面在基质表面以下, 水从进水端水平或垂直流向出水端的人工湿地, 包括水平潜流人工湿地、向上垂直流人工湿地、向下垂直流人工湿地.垂直流潜流式湿地具有占地面积较其它形式湿地小, 处理效率高等优点.Nõlvak等[23]发现水平潜流人工湿地对ARGs去除的效果欠佳, 仅能更有效的去除磺胺类抗性基因sul1. Liu等[24]发现垂直流人工湿地对废水中四环素类抗性基因tetM、tetO和tetW具有较高的去除效率, 去除率最高达90%左右.而Huang等[25]的研究进一步证实垂直上流式人工湿地对含四环素抗性基因tet的养猪废水具有良好的处理性能, tet丰度减少0.26~3.3个数量级.Chen等[26]发现6个中尺度人工湿地(表面流、水平潜流和垂直潜流等)不仅对于磺胺类抗性基因(sul1、sul2和sul3), 四环素抗性基因(tetG、tetM、tetO和tetX)和大环内酯类抗性基因(ermB和ermC)等的总去除效率在63.9%~84.0%之间, 而且对抗生素的总去除率在75.8%~98.6%之间.该研究还发现植物的存在有利于污染物的去除, 而且潜流人工湿地的污染物去除率明显高于表面流人工湿地.Liu等[27]研究表明水平潜流人工湿地对ARGs的去除效率优于垂直潜流人工湿地(50%以上), 尤其是对磺胺类ARGs的去除效果更好.Huang等[28]发现垂直潜流人工湿地中四环素抗性基因(tet)和Ⅰ类整合子整合酶基因(intI1)的去除效率在33.2%~99.1%之间.但研究中上流式处理出水的tet基因相对丰度大多高于进水处理, 可能由于抗生素浓度压力, 因此人工湿地处理含高浓度抗生素废水有潜在风险, 出水和基质中ARGs相对丰度的变化则取决于水力流向.综上所述, 潜流人工湿地相较于表面流人工湿地对ARGs的去除效率更高.

1.3 复合型人工湿地

复合型人工湿地, 包括表面流潜流复合人工湿地、塔式复合人工湿地、微生物燃料电池耦合人工湿地、廊道循环人工湿地等.复合型人工湿地综合各种类型和方法强化了基础类型的功能, 其去除效率也较普通人工湿地高[29].Chen等[30]研究了由1个调节池、4个表层和潜流人工湿地和1个稳定单元组成的复合人工湿地, 发现对农村生活污水中ARGs的去除率较高, 降低了1~3个数量级.Chen等[31]研究表明人工曝气混合型人工湿地对ARGs总去除率达到87.8%~99.1%.Huang等[32]发现下行垂直潜流-上行垂直潜流湿地(DVF-UVF)对ARGs(intI1、ermB、ermC、ermF、tetW和tetG)的去除效率显著高于卧式潜流-上行垂直潜流湿地(HF-UVF), 原因在于DVF-UVF可以去除和抑制细菌生长.Zhang等[33]设计的堆叠式微生物燃料电池-人工湿地耦合生物膜电极反应器能够有效去除持续高浓度的磺胺甲唑(SMX)99.29%, 但持续高浓度SMX提高了生物膜介质和出水中sul基因的相对丰度.Wen等[34]发现在微生物燃料电池耦合人工湿地的阳极区添加海绵铁能减少ARGs积累, 原因是电极层的强化对微生物群落组成和功能有显著影响.Li等[35]的研究表明, 锌(Zn)的持续积累会降低ARGs的丰度, 可能是Zn的持续大量积累抑制ARGs的增殖.综上表明, 复合型人工湿地相较于普通型人工湿地对ARGs的去除效率有显著提升, 但复合工艺的参数设置仍需进一步地研究.

2 影响人工湿地去除ARGs的因素

环境中ARGs的发生和积累可导致微生物间耐药基因的水平转移[46].在环境中存在的大量未被利用的抗生素造成的选择压力下, ARGs通过质粒、转座子和整合子系统进行水平转移, 使环境中微生物获得抗生素耐药性[47], 因此环境中ARGs的去除具有一定难度.根据人工湿地特性, 可以将影响人工湿地对ARGs去除效率的因素概括为植物、温度、pH、水力停留时间和人工湿地强化类型等.

2.1 植物

植物是人工湿地中生物群落的主要成分, 与系统中ARGs的维持与传播有密切联系.人工湿地中大量的附着位点、快速的氧损失和密集根系的分泌物为废水和基质中微生物群落提供了适宜的环境, 通过水平基因转移和垂直基因转移促进了人工湿地内部ARGs的传播[48].Yi等[49]发现大多数ARGs的相对丰度在湿地芦苇床上显著增加, 尤其是磺胺类抗性基因sul1. Huang等[50]设计了4种不同植物种植模式[S1:单种黄菖蒲(Iris pseudacorus), S2:单种芦苇(Phragmites australis), M1:黄菖蒲和芦苇连续混种和M2:黄菖蒲和芦苇交叉混种]的水平潜流人工湿地, 发现交叉混种(M2)比连续混种(M1)对总ARGs去除率高50%左右.且研究中4种人工湿地对总ARGs的去除效率顺序为: S1>S2>M2>M1, 说明单一种植模式比混合种植模式对ARGs的去除效率高.但Abou-Kandil等[51]研究小型湿地不同种植模式对ARGs的去除效率的研究表明, 混合连续种植[芦苇(Phragmites australis)+窄叶香蒲(Typha angustifolia)]相较单一栽培模式[无植物区段+芦苇(Phragmites australis)]对ARGs的去除效率更高, 总ARGs去除效率在74.3%~94.7%之间, 该研究指出芦苇和窄叶香蒲的根沉积产物对ARGs的转移和扩散有一定的抑制作用[52, 53].然而, 也有研究表明添加大型植物[香蒲(Typha spp.)、西伯利亚狐尾藻(Myriophyllum sibiricum)和狸藻(Utricularia vulgaris)]与否对ARGs的去除效率没有显著影响[54], Vivant等[55]对芦苇(Phragmites australis)的研究也有类似的结果.总体而言, 植物通过根际效应改变微生物群落的结构, 从而改变抗生素耐药性[56], 而人工湿地中植物的组合方式、生理特性和植物根际环境等均能影响ARGs的去除效率.目前研究结果表明, 交叉混种是最佳的植物组合方式, 而在植物种类的选择上尚未表现出明显的优势物种, 可因地制宜.

2.2 温度和pH

Zhang等[57]对抗性基因和微生物群落之间的共生模式进行了网络分析, 发现属的组成与土壤中的ARGs和MRGs丰度具有相关性.且温度和pH会改变环境中ARGs的持久性[58].因此人工湿地中温度和pH会通过改变微生物群落而影响ARGs的去除.基于温度的研究有季节性研究, 例如Fang[21]和范增增等[37]的研究结果均表明冬季比夏季去除效率高约30%.Sabri等[59]研究发现ARGs丰度与温度呈负相关.另外, Li等[60]对污水处理工艺(缺氧-好氧活性污泥法结合氯化工艺)的研究表明, 细菌群落受pH的影响, 其相对丰度变化呈正相关.Wu等[61]对养猪场周边土壤四环素ARGs丰度及多样性的研究表明, 四环素ARGs与pH值呈负相关.而且Liu等[62]通过4种不同的基质类型模拟的人工湿地中发现, ARGs总相对丰度与基质pH呈显著负相关.研究表明, pH是微生物群落中ARGs的潜在宿主改变的重要驱动因素[63, 64].而pH对抗生素的影响进一步会对系统中ARGs的命运产生影响, 较宽pH范围可能促进了某些抗生素的微生物降解[65].由于温度和pH对微生物群落的影响较大且因群落组成和性质而异, 因此温度和pH的影响也因人工湿地种类而异, 在具体人工湿地中应具体考虑.

2.3 水力停留时间

水力停留时间对ARGs的去除效率因不同人工湿地类型而异.Huang等[32]研究显示, 混合人工湿地(水平、上行和下行这3种潜流人工湿地)比单一人工湿地的水力停留时间长, 提升了混合人工湿地对intI1和tet的去除效率.但是Zhang等[33]使用堆叠式微生物燃料电池-人工湿地耦合生物膜电极反应器发现, 随着水力停留时间的延长, 生物膜培养基和废水中磺胺类sul基因的相对丰度增加.这与Song等[66]在两种水力停留时间(1.5 d和3 d)和两种循环运行方式(闭路和开路)下对上流式微生物燃料电池耦合人工湿地中磺胺嘧啶的去除及其ARGs的积累的研究结果一致.而且, 水力停留时间增加, 基质中ARGs的表达量逐渐增加[67].Chen等[26]对不同设计参数(水力负荷参数: 10、20和30 cm·d-1)的中尺度水平潜流人工湿地对生活污水中抗生素和ARGs的去除效率进行了优化, 结果表明抗生素和ARGs的去除效率随水力停留时间的增加而增加.综合以上结果, 虽然较长的水力停留时间可增加ARGs的去除效率, 但同时也会增加ARGs的富集风险.因此, 应在设计人工湿地参数时对水力停留时间进行合理优化.

2.4 人工湿地强化类型

人工湿地强化类型主要包括湿地基质类型、水流方向、湿地曝气和湿地复合类型等.已有研究表明人工湿地基质的特性影响系统对ARGs的去除效率[24, 26], 且人工湿地总ARGs水平因基质不同而存在差异.Liu等[62]研究表明, 以牡蛎壳为基质的人工湿地总ARGs相对丰度最低, 与基质体系细菌多样性呈显著正相关.Abou-Kandil等[51]研究表明凝灰岩填充的连续种植人工湿地明显优于砾石填充的连续种植人工湿地, 认为植物连续种植模式结合高比表面积的基质是一种潜在的提高人工湿地去除ARGs效率的策略.在袁涛等[45]设计的亚铁强化人工湿地中, sul1的去除率随着氯化亚铁添加量的增加而提高.而在垂直流人工湿地中, 水流方向比基质类型更能引起ARGs相对丰度的变化[28].此外, 湿地曝气与否也是重要的影响因素, Feng等[68]比较了曝气的人工湿地与不曝气的人工湿地对ARGs的去除率差异, 结果表明, 曝气的人工湿地对ARGs的去除效率高1.6倍左右, 而不曝气的人工湿地增加了ARGs (tetA、tetO和tetW)的增殖.

复合型人工湿地强化类型是影响ARGs去除效果的重要因素, 已有大量针对复合型人工湿地去除环境污染物的研究.Zhang等[69]研究生物电化学系统去除效果与污水处理厂相比, 发现前者通过产生较少的污泥来避免ARGs的增殖, 但是电刺激会影响跨膜通透性和膜电位导致生物电化学系统中ARB和ARGs的增加.Dai等[29]发现微生物燃料电池强化型人工湿地对于ARGs的去除率较普通人工湿地高2.25倍左右.综合以上研究, 人工湿地基质种类选择应侧重选择比表面积高的填充物; 水流方向上流式普遍比下流式去除效率高; 而人工湿地复合类型本质是结合化学手段达到提高效率的目的, 目前电化学方法研究较多, 且能显著提高去除效率.总之, 在设计人工湿地系统参数时需要综合各方面因素(人工湿地类型、基质种类、水力停留时间、环境温度、植物种类等), 以达到最佳的处理效果.

3 人工湿地对ARGs去除的发展方向与挑战

人工湿地被应用于ARGs的去除方面有广阔的前景, 不同设置参数构成要素(植物和基质)、运行参数(水力停留时间等)、运行方式(连续和间歇)和流态(表面流和潜流)等对ARGs的去除效果影响较大.目前的研究主要集中在小型试验规模上, 应增强人工湿地去除ARGs的实际应用, 建立不同湿地的预测模型, 结合实际需求筛选优化人工湿地参数.此外, 明晰ARGs在人工湿地中的行为和去除机制, 也对去除研究至关重要.研究裸露ARGs和胞内ARGs去除效率的区别, 了解ARGs和ARB在不同人工湿地中的行为, 注重ARB的去除是ARGs的去除的重点.通过基质的物理阻隔等方法减少ARGs的水平转移(接合、转导和转化等), 利用微生物有效性和生物降解功能结合物理净化去除环境中ARGs也是人工湿地的研究前景之一.

解决人工湿地基质中ARGs的富集和扩散是一个巨大挑战.由于环境中抗生素以及其他污染物如重金属等的存在, 相应的ARGs可能会发生进化, 因此需要明晰机制.可以考虑将抗生素和ARGs作先后处理, 即通过人工湿地的参数设计和组合先后处理抗生素和ARGs[70], 以避免高浓度抗生素的选择压力影响ARGs的去除效率.同时需注意截留在人工湿地中的ARGs引起二次传播的风险.

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