, 李舜尧2, 陈明雨1, 高彦征2
, 司友斌11. 安徽农业大学资源与环境学院, 农田生态保育与污染防控安徽省重点实验室, 合肥 230036;
2. 南京农业大学资源与环境科学学院, 南京 210095
收稿日期: 2018-09-26; 修回日期: 2018-10-22; 录用日期: 2018-10-22
基金项目: 安徽省自然科学基金（No.1808085QD104）；安徽农业大学稳定和引进人才科研项目（No.yj2018-31）
作者简介: 孙凯(1987-), 男, E-mail:sunkai@ahau.edu.cn
通讯作者（责任作者）: 高彦征, E-mail:gaoyanzheng@njau.edu.cn
司友斌, E-mail:youbinsi@ahau.edu.cn
摘要: 本研究通过批量降解试验，探讨了功能菌株Massilia sp.Pn2和Mycobacterium flavescens 033降解菲和芘的基本动力学过程和规律；重点采用温室盆栽试验，研究了接种混合菌株对黑麦草体内PAHs含量及多酚氧化酶（PPO）和过氧化物酶（POD）活性的影响.结果表明，菌株Pn2和033可以分别利用菲和芘作为碳源和能源进行生长；在30℃、pH=7.0条件下，菌株Pn2和033对100 mg·L-1菲和50 mg·L-1芘的降解率分别高达99.7%和98.3%，降解半衰期分别为0.34 d和0.95 d（R2>0.98）.与接种灭活混合菌株对比，接种混合菌株Pn2和033显著地降低了黑麦草体内菲和芘的含量和积累量（p < 0.05），并阻控菲和芘由黑麦草根向茎叶转移.同时，接种混合菌株Pn2和033显著地提高了黑麦草根和茎叶中POD（p < 0.05）活性，该酶能够促进黑麦草体内超氧自由基的清除，并保护细胞免受PAHs损伤，进而影响PAHs在黑麦草体内的代谢过程.研究结果为阐明接种混合功能菌降低植物体内PAHs污染的作用机理提供了一定的参考价值.
关键词:多环芳烃混合功能菌黑麦草接种效能降解机制
Inoculation with mixed PAH-degrading bacteria mitigates the contamination of phenanthrene and pyrene in ryegrass (Lolium multiflorum Lam) using greenhouse pot experiments
SUN Kai1
, LI Shunyao2, CHEN Mingyu1, GAO Yanzheng2 , SI Youbin1 1. Anhui Province Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, Hefei 230036;
2. College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095
Received 26 September 2018; received in revised from 22 October 2018; accepted 22 October 2018
Abstract: Polycyclic aromatic hydrocarbons (PAHs) are a category of persistent organic contaminants extensively found in soil environments. PAHs can be taken up, accumulated, and translocated by plants, which pose a significant risk worldwide to food safety and hence human health due to their high bio-accumulation, carcinogenicity, toxicity, and biodegradation-resistant. It is well documented that inoculation of PAH-degrading bacterium is a simple and effective strategy for mitigating the contamination of PAHs in plants. However, microscopic information is available on inoculation with mixed PAH-degrading bacteria to regulate plant PAH contamination and the activities of related to metabolic enzymes in plants. In this study, the biodegradation kinetics and regularity of phenanthrene and pyrene were respectively investigated using Massilia sp. Pn2 and Mycobacterium flavescens 033 in in vitro. In particular, greenhouse pot experiments of inoculated two PAH-degrading bacteria were conducted to reduce PAHs residues in ryegrass (Lolium multiflorum Lam), and the effect of strains Pn2 and 033 on the activities of polyphenol oxidase (PPO) and peroxidase (POD) was also explored in in vivo. Results indicated that both strains Pn2 and 033 could use phenanthrene and pyrene as the sources of carbon and energy for growth of tested strains. They were effective in biodegrading PAHs in in vitro, and the biodegradation rates of phenanthrene and pyrene were respectively 99.7% and 98.3%, the half-life values were respectively 0.34 d and 0.95 d (R2>0.98). Compared with the mixed dead bacteria control (CRD), inoculation with mixed PAH-degrading strains altered the migration and transformation of PAHs in soil-ryegrass systems, and reduced the concentration and accumulation of PAHs into ryegrass roots and shoots (p < 0.05). In addition, inoculation with mixed PAH-degrading bacteria also impacted the activities of PPO (p>0.05) and POD (p < 0.05) in plants. Thus, strains Pn2 and 033 could be a useful bacterial resource for reducing PAHs contents via regulating the activity of POD inside plants. These findings provide a novel perspective in utilizing plant-bacteria partnerships to reduce plan PAH residues and regulate the activities of enzymes in plants, with ultimate goal of protecting agricultural products safety and human health.
Keywords: PAHsfunctional bacteriaryegrasscolonizationdegradation mechanism
1 引言(Introduction)2016年, 国务院印发了《土壤污染防治行动计划》, 旨在加强我国土壤污染防治, 逐步改善土壤环境质量, 确保农产品安全和人群健康.多环芳烃(PAHs)是土壤环境中普遍存在的一类持久性有机污染物(POPs), 具有高生物富集、致毒、致癌和难降解等特点(Menzie et al., 1992; Wang et al., 2012).人类生产活动中产生的PAHs可以通过污水灌溉、大气沉降、石油泄漏、农业废物燃烧和化肥施用等多种途径进入农田生态系统(Sams?e-Petersen et al., 2002; Cai et al., 2017).PAHs进入农田土壤环境后, 可以被农作物根部吸收、积累并转运至茎叶, 进而沿食物网传递危害野生物种和人群健康(Kim et al., 2013;陈保冬等, 2015; Chen et al., 2015).如何降低土壤和农作物PAHs污染、保护农业耕地、生产出绿色安全的农产品已经成为国内外关注的热点问题之一.
研究指出, 接种功能细菌不仅可以促进植物生长(?akmak?i et al., 2006; Glick, 2014), 也能够降低植物PAHs污染(Arslan et al., 2017; Waigi et al., 2017).例如, Liu等(2014; 2017)从看麦娘(Alopecurus aequalis Sobol)体内获得一株具有菲降解特性的菌株Massilia sp. Pn2, 该菌能够重新定殖在黑麦草(Lolium multiflorum Lam)和小麦(Triticum aestivum L. cv. Yangmai-16)体内, 促进植物生长并降低植物体内菲含量.然而, 关于接种混合功能菌降低植物PAHs污染的作用机理仍不清楚(刘爽等, 2013;孙凯等, 2014; Liu et al., 2017).已有资料显示, 植物吸收PAHs后, 其体内过氧化物酶(POD)可以清除植物体内超氧自由基, 控制膜脂的过氧化作用并保护细胞膜的正常代谢(Kvesitadze et al., 2009;夏红霞等, 2013)；而植物体内多酚氧化酶(PPO)能够催化氧化PAHs开环, 生成易降解的中间产物(Iwabuchi et al., 1998).由此可见, 植物体内POD和PPO活性大小可能影响其对PAHs的应答反应和代谢水平.能否通过接种功能菌改变植物体内POD和PPO活性, 调节植物体内PAHs的降解代谢？该想法值得深入探究.
菲和芘作为3环和4环PAHs的典型代表, 常被用于研究PAHs的微生物降解和植物代谢过程(Barnier et al., 2014; Zielińska et al., 2015).本研究选择实验室前期获得的两株分别具有高效菲和芘降解特性的Massilia sp. Pn2和Mycobacterium flavescens 033作为供试菌株, 通过批量降解试验方法, 探讨菌株Pn2和033降解菲和芘的基本动力学过程和规律；重点采用温室盆栽试验方法, 研究接种混合菌株Pn2和033对黑麦草体内PAHs含量及PPO和POD活性的影响.以期从植物体内PAHs含量及其PPO和POD活性方面阐明接种混合菌降低植物PAHs污染的作用机制, 为利用植物-功能菌的微生态调控技术规避作物PAHs污染风险和保障人群健康提供理论依据.
2 材料与方法(Materials and methods)2.1 化学试剂和培养基菲和芘购自德国Fluka公司, 纯度>98%, 其理化性质如图 1所示.甲醇为色谱醇, 其它试剂为分析纯.利用丙酮制备浓度分别为4 mg·mL-1和2 mg·mL-1的菲和芘母液, 置于4 ℃冰箱保存备用.
图 1(Fig. 1)
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| 图 1 菲和芘理化性质 Fig. 1Selected physicochemical properties of phenanthrene and pyrene in this study |
LB培养基：蛋白胨10.0 g·L-1, 酵母粉5.0 g·L-1, NaCl 10.0 g·L-1, pH=7.0.
无机盐培养基(MSM)：(NH4)2SO4 1.50 g·L-1, K2HPO4·3H2O 1.91 g·L-1, KH2PO4 0.50 g·L-1, MgSO4·7H2O 0.20 g·L-1, 2 mL微量元素溶液(CoCl2·6H2O 0.1 mg·L-1, MnCl2·4H2O 0.425 mg·L-1, ZnCl2 0.05 mg·L-1, NiCl2·6H2O 0.01 mg·L-1, CuSO4·5H2O 0.015 mg·L-1, Na2MoO4·2H2O 0.01 mg·L-1, Na2SeO4·2H2O 0.01 mg·L-1), pH=7.0.
固体培养基：添加1.8%琼脂, 制备固体LB培养基和固体MSM.
霍格兰营养液：Ca(NO3)2·4H2O 945 mg·L-1, KNO3 506 mg·L-1, NH4NO3 80 mg·L-1, KH2PO4 136 mg·L-1, MgSO4 493 mg·L-1, 2.5 mL铁盐溶液(FeSO4·7H2O 2.78 g, EDTA·Na2 3.73 g), 500 mL去离子水(18.2 MΩ·cm), 5 mL微量元素溶液(KI 0.83 mg·L-1, H3BO3 6.2 mg·L-1, MnSO4 22.3 mg·L-1, ZnSO4 8.6 mg·L-1, Na2MoO4 0.25 mg·L-1, CuSO4 0.025 mg·L-1, CoCl2 0.025 mg·L-1, pH 6.0), pH=5.5.
2.2 供试植物和菌株选取生长旺盛、根系发达且脂肪含量高的黑麦草(Lolium multiflorum Lam)作为供试植物.本实验室前期从南京某PAHs污染区的健康植物看麦娘(Alopecurus aequalis)体内获得一株具有高效菲降解特性的革兰氏阴性菌株Massilia sp. Pn2, 该菌在含有50 mg·L-1芘的固体MSM上生长良好, 且能够定殖在黑麦草体内(Liu et al., 2014).芘降解菌株Mycobacterium flavescens 033购自美国菌种保藏中心, 属于革兰氏阳性菌，该菌对100 mg·L-1菲具有较强的耐受性, 并能够在植物体内定殖(Dean-Ross et al., 1996; Wang et al., 2017).分别采用含有高浓度菲和芘的MSM扩大培养菌株Pn2和033, 当菌株生长至对数期时, 将培养液置于高速离心机, 8000 r·min-1下离心5 min, 反复用无菌水清洗菌体表面吸附的PAHs后, 用灭菌的MSM制备菌悬液.
2.3 批量降解试验采用批量降解试验方法, 分别测定功能菌株Pn2和033对菲和芘的降解效率和基本动力学参数(Zhu et al., 2016).将0.5 mL菲和芘的丙酮母液加入到50 mL灭菌三角瓶中, 置于无菌操作台, 待丙酮挥发后, 向三角瓶中添加20 mL灭菌MSM, 分别获得以100 mg·L-1菲和50 mg·L-1芘作为碳源和能源的PAHs降解培养基.按照5%接种量分别在含有菲和芘的MSM中加入OD600=1.0、处于对数生长期的菌株Pn2(菌密度为1.26×107 CFU·mL-1)和033(菌密度为1.17×107 CFU·mL-1)菌悬液(菌悬液为灭菌的MSM), 置于30 ℃、150 r·min-1避光摇床培养, 定时取样, 测定培养液中细菌数量及PAHs残留浓度.设置接种灭活菌株处理组作为空白对照, 每组实验重复3次.
采用整瓶提取方法测定培养基中PAHs残留浓度.向培养基中加入等体积色谱甲醇, 超声萃取30 min, 8000 r·min-1高速离心后, 过0.22 μm滤膜, 高效液相色谱(HPLC, Shimadzu LC-20AT, Japan)测定溶液中PAHs残留浓度.HPLC设定参数：Inertsil ODS-SP-C18反相色谱柱(150 mm×4.6 mm, 5 μm), UV/Vis检测器(SPD-20A), 流动相为甲醇/水=90:10(V/V), 流速为1.0 mL·min-1, 柱温为40 ℃, 检测波长为245 nm, 进样量为20 μL.
2.4 温室盆栽试验采用温室盆栽试验方法, 研究接种混合菌株对植物体内PAHs含量及其PPO和POD活性的影响.以南京江宁区距离土壤表面0~20 cm农田黄棕壤作为供试土壤，土壤理化性质为：pH=6.02, 有机碳含量14.3 g·kg-1, 黏土含量24.7%, 沙含量67.9%.土壤样品风干后, 过20目筛, 用含有菲和芘的丙酮母液污染.将污染土壤置于黑暗室温老化30 d后, 测得土壤中菲和芘的含量分别为84.65和43.58 mg·kg-1.试验设置6种不同处理, 具体如表 1所示.黑麦草种子经75%乙醇表面消毒5 min后, 用去离子水清洗干净, 置于30 ℃恒温培养箱催芽48 h.每盆(350 g土壤)种植定量经过催芽的黑麦草种子, 待黑麦草生长至幼苗期进行间苗, 每盆留取10株黑麦草幼苗继续生长.人工气候箱温度设置为25/20 ℃(白昼), 湿度50%.培养过程中适量补充半强度的霍格兰营养液, 以保持土壤中养分含量.待黑麦草植株生长至10 cm高时, 采用灌根法, 在其根部土壤接种10 mL OD600=2.0的混合菌悬液(菌悬液为灭菌的半强度霍格兰营养液, 菌株Pn2菌密度为5.63×108 CFU·mL-1, 菌株033菌密度为4.29×108 CFU·mL-1), 培养30 d后, 收集黑麦草根和茎叶样品, 测定其鲜重和干重并分析PAHs含量.每组实验重复3次.
表 1(Table 1)
| 表 1 温室盆栽试验中6种不同处理的黄棕壤 Table 1 Yellow-brown soil of six different treatments in the greenhouse pot experiments | ||||||||||||||||||||||||||
表 1 温室盆栽试验中6种不同处理的黄棕壤 Table 1 Yellow-brown soil of six different treatments in the greenhouse pot experiments
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2.5 土壤/植物样品中PAHs含量分析土壤和黑麦草样品中菲和芘的提取和测定方法参照文献(Gao et al., 2004).具体操作方法如下：①黑麦草样品冷冻干燥后, 用研钵研磨粉碎, 称取一定量的黑麦草干重样品置于30 mL玻璃离心管中, 用30 mL的二氯甲烷和正己烷(1:1, V/V)溶液分3次, 每次10 mL超声萃取30 min(土壤样品每次超声萃取60 min)；②将萃取液收集后过无水硫酸钠-硅胶柱净化, 再次用5 mL的二氯甲烷和正己烷(1:1, V/V)洗脱净化柱中残留PAHs；③洗脱液收集至旋转蒸发瓶, 40 ℃恒温浓缩至干, 甲醇定容到1 mL, 过0.22 μm孔径滤膜后, HPLC定量分析土壤/黑麦草样品中菲和芘残留浓度.
2.6 黑麦草体内PPO和POD活性测定采用Shah等(2001)研究方法, 测定黑麦草体内PPO和POD活性, 并略做改进.分别称取0.1 g黑麦草根和茎叶样品, 置于冰浴研钵中, 加入0.05 g聚乙烯吡咯烷酮(PVP)和3 mL pH=7.8的磷酸盐缓冲溶液(PBS), 充分研磨均匀.采用高速离心机在4 ℃、10000 r·min-1下离心10 min, 上清液为粗酶提取液.利用邻苯二酚显色法测定PPO活性, 向玻璃试管中快速添加2 mL 0.01 mol·L-1 PBS(pH=6.0)、1.0 mL 11 g·L-1邻苯二酚和40 μL粗酶液, 充分摇匀后, 置于30 ℃水浴锅中水浴10 min, 于420 nm波长下测定其吸光度值.利用愈创木酚显色法测定POD活性, 向玻璃试管中迅速加入2 mL 0.1 mol·L-1醋酸缓冲液(pH=5.0)、1.0 mL 2.5 g·L-1愈创木酚、0.1 mL 3% H2O2和20 μL粗酶液.将混合液摇匀后, 置于37 ℃水浴锅水浴15 min, 在470 nm波长下测定其吸光度值.每组实验设置5个重复.酶活性(R)单位定义为每分钟D值增加0.01, 单位为U·g-1·min-1.
 | (1) |
3 结果和讨论(Results and discussion)3.1 菲和芘的降解动力学菌株Pn2和033在30 ℃、pH=7.0条件下能够分别以菲和芘作为碳源和能源进行生长繁殖, 且对菲和芘具有高效的降解效能(图 2).接种0~1.5 d, 菌株Pn2的细胞数量(N)快速增加, 菲降解率也逐渐增加, 表明该菌能够以菲作为碳源和能源进行生长繁殖(图 2a)；接种1.5 d后, 培养液中菌株Pn2的细菌数量呈现下降趋势, 菲降解率增加缓慢, 主要是因为降解液中菲含量较低, 菌株没有足够的碳源和能源进行生长繁殖；接种3 d后, 菌株Pn2对菲的降解率高达99.7%.菌株033对芘的降解动力学过程和规律与菌株Pn2类似, 接种6 d后, 其对芘的降解率高达98.3%(图 2b).依据培养液中PAHs残留浓度, 拟合降解动力学方程如下：C菲=134.49e-2.028t, 半衰期为0.34 d(R2=0.9889)；C芘=57.226e-0.728t, 半衰期为0.95 d(R2=0.9861)；其中, C表示培养基中PAHs残留浓度, t表示接种时间.
图 2(Fig. 2)
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| 图 2 PAHs降解菌的生长曲线及其对PAHs的降解动力学 (a.菲降解菌Pn2, b.芘降解菌033) Fig. 2The biodegradation dynamics of PAHs and growth curve of single strain Pn2 or 033 with PAHs as their carbon source |
目前, 研究者已经从环境介质中筛选出多种具有PAHs降解特性的功能菌(徐成斌等, 2015; Bacosa et al., 2015; Liu et al., 2016;刁硕等, 2017).例如, 倪雪等(2013)从PAHs污染植物体内获得两株菲降解菌Stenotrophomonas sp. P1和Pseudomonas sp. P2, 其对菲的降解率均高达90%以上.李全霞等(2008)从土壤中分离一株高效芘降解菌Mycobacterium sp. M11, 该菌对菲、蒽和荧蒽等PAHs具有良好的降解效能.本研究中, 菌株Pn2和033分别对菲和芘具有高效的降解效率.已有资料显示, 功能菌株降解PAHs的过程主要包含2种关键酶, 一是起始反应阶段的双加氧酶, 它能够完成PAHs羟基化；二是邻苯二酚双加氧酶, 它可以催化单环芳烃氧化, 使苯环裂解并通过三羧酸循环矿化为CO2和H2O(Dean-Ross et al., 1996;王涛等, 2016;Waigi et al., 2017).菌株Pn2能够将菲的2个相邻碳原子羟基化形成顺-二醇, 并在多种酶系共同调节下导致苯环逐一裂解产生水杨酸和邻苯二酚(Liu et al., 2014)；菌株033代谢芘的产物主要包括芘-4, 5-二醇、4, 5-二羧基-菲、4-菲甲酸和邻苯二甲酸(Dean-Ross et al., 1996).
3.2 接种混合菌降低黑麦草体内PAHs含量植物主要通过根部吸收POPs, 多数POPs的logKow值在3.0~8.3之间, 其值越低越容易被植物根吸收(Takaki et al., 2014).菲和芘的logKow值分别为4.57和5.18(图 1), 它们能够被黑麦草根高效地吸收积累, 并与根部脂质膜结合(Chaudhry et al., 2002).由于黑麦草体内缺少相应的PAHs转运蛋白, 其主要通过扩散作用将细胞壁中的菲和芘运输到木质部, 并由植物蒸腾拉力作用实现黑麦草根部PAHs向茎叶迁移(Campos et al., 2008).当黑麦草吸收转运PAHs时, 为避免PAHs对自身的毒害作用, 其能够通过蒸发或降解作用降低PAHs毒性(Arslan et al., 2017).本研究中, 接种混合菌株Pn2和033(CRB)可以作为一种新型有效途径用于增强黑麦草对PAHs的耐性, 甚至降低黑麦草体内PAHs污染.
如表 2所示, 接种混合PAHs降解菌株能够有效地减少黑麦草体内菲和芘的含量.与CRD(污染土壤+黑麦草+灭活的混合菌)处理组对比, 接种混合菌株Pn2和033促使黑麦草根和茎叶中菲含量分别由59.72和10.50 mg·kg-1降低至48.92和6.65 mg·kg-1, 芘含量分别由114.43和12.96 mg·kg-1降低至88.41和9.73 mg·kg-1(p < 0.05).黑麦草是畜牧业中常见牧草, 降低黑麦草体内PAHs污染对促进畜牧业发展、阻隔PAHs在植物-动物的食物链中传递及保护人群健康等具有重要意义(Sun et al., 2014a; Zhu et al., 2014).本研究中, 与CRD处理组对比, 接种混合菌株Pn2和033显著地降低了黑麦草体内21.63%的总PAHs含量.
表 2(Table 2)
| 表 2 在黄棕壤中种植黑麦草并接菌30 d后黑麦草体内菲和芘的含量 Table 2 The concentration of phenanthrene and pyrene in ryegrass planted in yellow-brown soil for 30 d | |||||||||||||||||||||||||||
表 2 在黄棕壤中种植黑麦草并接菌30 d后黑麦草体内菲和芘的含量 Table 2 The concentration of phenanthrene and pyrene in ryegrass planted in yellow-brown soil for 30 d
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黑麦草生物量的增加可能导致其体内PAHs含量减少, 计算黑麦草体内PAHs积累量可以排除该因素干扰(Sun et al., 2014b).黑麦草体内PAHs积累量(B)计算公式见式(2).
 | (2) |
由图 3可知, 与CRD处理组对比, 接种混合菌株Pn2和033(CRB)促使黑麦草根中菲和芘的积累量分别由1.69和4.18 μg·盆-1减少至1.34和2.37 μg·盆-1, 黑麦草茎叶中菲和芘的积累量分别由1.95和3.79 μg·盆-1减少至1.58和2.13 μg·盆-1.该研究结果进一步表明, 接种混合菌株Pn2和033能够降低黑麦草体内PAHs的含量.
图 3(Fig. 3)
 |
| 图 3 黑麦草根(a)和茎叶(b)中菲和芘的积累量 Fig. 3The accumulation amounts of phenanthrene and pyrene in ryegrass roots (a) and shoots (b) |
转移系数(TF)的大小能够说明PAHs从黑麦草根向茎叶的传输能力, 进而反映植物地上部位PAHs污染风险(Sun et al., 2014b).TF计算公式如下所示：
 | (3) |
由图 4可知, 与CRD处理组对比, 接种混合菌株Pn2和033(CRB)促使黑麦草体内菲和芘的TF分别由0.1692和0.1216降低至0.1388和0.1191.这些结果表明, 接种混合菌株Pn2和033不仅能够降低黑麦草体内PAHs含量和积累量, 也可以降低PAHs由黑麦草根向茎叶的转移作用, 从而降低黑麦草地上部位PAHs污染风险.
图 4(Fig. 4)
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| 图 4 菲和芘在黑麦草体内的转移系数(TF) Fig. 4The translocation factor (TF) of phenanthrene and pyrene in ryegrass |
3.3 接种混合菌促进土壤中PAHs降解接种混合功能菌株Pn2和033不仅能够降低植物体内PAHs污染, 也可以促进土壤中PAHs降解.与污染土壤接种灭活的混合菌相比(CD), CB(污染土壤+混合菌)、CRD和CRB(污染土壤+黑麦草+混合菌)均能够减少土壤中PAHs含量, 其中, CRB处理效果最佳, 致使土壤中菲和芘的去除率分别增加了37.3%和59.8%(图 5).研究人员采用PCR-DGGE技术证实, 不同浓度PAHs污染土壤中Massilia spp.的数量在微生物群落中始终占据主导地位, 其能够有效地降解土壤中PAHs(Zhang et al., 2010).Sun等(2014b)研究指出, 采用灌根法接种植物内生菌株Staphylococcus sp. BJ06能够促进黑麦草生长, 并有效地降低土壤-植物体系中芘含量.这些究结果表明, 植物-混合功能菌的联合作用能够高效地去除土壤中混合PAHs污染.土壤中PAHs含量的降低直接减少了植物对土壤中PAHs的吸收和积累, 导致植物体内PAHs含量下降(Sun et al., 2014b; Wang et al., 2017).
图 5(Fig. 5)
 |
| 图 5 不同处理土壤中菲和芘的含量 Fig. 5The concentrations of phenanthrene and pyrene in soil by different treatments |
另有研究表明, 接种的外来功能菌株可能与土著微生物竞争或协同降解土壤中PAHs(Afzal et al., 2014).PAHs在植物体内的含量与土壤中PAHs含量呈正相关, 接种混合功能菌株Pn2和033可能在黑麦草根际土壤释放分泌液, 促进土壤中PAHs降解；同时, 两株菌的PAHs降解基因在土著微生物群落间的水平迁移也可以加速土壤中PAHs降解, 从而减少黑麦草对土壤中PAHs的吸收和积累(Taghavi et al., 2005).此外, 黑麦草根系产生的有机酸、根际氧化、共代谢诱导和生物表面活性剂等物质及功能, 不仅可以为混合功能菌株提供良好的生长环境, 也能够促进根际土壤中PAHs降解和矿化(Afzal et al., 2014; Sprocati et al., 2014).
3.4 接种混合菌影响植物体内PPO和POD活性植物体内POPs的降解主要通过氧化反应、水解作用和环氧化物的形成等途径实现(Chaudhry et al., 2002), 而这些过程需要POPs降解酶参与.研究指出, 植物体内的氧化酶、还原酶和酯酶等可以直接参与植物体内POPs的降解代谢过程(Kvesitadze et al., 2009)；而接种功能菌可以通过诱导这些酶活性, 影响植物体内POPs代谢转移(Sinsabaugh, 2010;Afzal et al., 2014).如图 6所示, 所有种植黑麦草的处理组, 植物根中PPO和POD活性均高于茎叶.与CRD处理组对比, 接种混合菌株Pn2和033能够影响黑麦草体内PPO(p > 0.05)和POD(p < 0.05)活性.PAHs污染降低了黑麦草根中PPO活性, 可能是由于黑麦草根中PAHs含量过高, 对PPO活性起到抑制作用；而接种混合菌株显著地促进了黑麦草根和茎叶中POD活性, 其中, 根中POD活性最大, 为1611.62 U·g-1·min-1.这些结果表明, 当黑麦草受到PAHs污染胁迫时, 其体内PPO和POD活性发生改变, 从而介导植物对PAHs的应答反应和代谢作用；而接种混合功能菌株能够改变植物体内PAHs降解酶PPO和POD活性, 影响植物体内PAHs的降解代谢.
图 6(Fig. 6)
 |
| 图 6 菲和芘污染条件下黑麦草体内PPO(a)和POD酶活性(b) Fig. 6The activities of PPO (a) and POD (b) in ryegrass under phenanthrene- and pyrene-contaminated conditions |
Weyens等(2009)研究指出, 接种菌株Burkholderia cepacia VM1468能够降低三氯乙烯污染的杨树体内接触酶和超氧化物歧化酶(SOD)活性.另有研究表明, 某些功能菌也能产生加氧酶作用于PAHs等有机污染物, 促使苯环裂解代谢(雷东锋等, 2004).因此, 通过接种混合功能菌群改变植物体内PAHs降解酶活性, 有望促进植物体内PAHs降解代谢.本研究中, 接种混合菌株Pn2和033可能通过诱导植物体内PAHs降解酶PPO和POD活性, 促进植物体内PAHs降解代谢.分析原因可能是由于POD活性的升高促进了植物体内超氧自由基的清除, 并保护植物细胞膜免受PAHs损伤、能够正常代谢, 进而直接增强植物对PAHs的利用效率(Kvesitadze et al., 2009;夏红霞等, 2013;Afzal et al., 2014).尽管如此, 今后的研究应当深入挖掘接种混合功能菌株对植物体内PAHs降解和相关酶活性变化的内在关系, 系统地揭示接种功能菌促进植物体内PAHs降解代谢的作用机理.
3.5 接种混合功能菌降低植物PAHs污染的作用机制结合本研究结果和已有的文献报道, 总结混合功能菌降低植物PAHs污染的作用机制主要包括以下5点：①接种混合功能菌直接降低了土壤中PAHs含量, 导致植物对土壤中PAHs的吸收和积累量降低(图 5)；②接种混合功能菌显著地影响植物体内PAHs降解酶POD活性, 强化植物-混合功能菌对PAHs的降解代谢潜能(图 6)；③混合功能菌定殖在植物细胞间隙和维管束导管中, 直接利用植物体内PAHs作为碳源和能源进行生长繁殖, 或以共代谢方式降解植物体内PAHs(Afzal et al., 2014; Sun et al., 2014a)；④在植物根际或体内, 混合功能菌可以将PAHs降解基因水平迁移到其他土著菌群中, 促进整个根际或内生菌群对PAHs的降解代谢作用(Wilson et al., 2003; Wang et al., 2007)；⑤混合功能菌PAHs降解基因在植物根际或体内的大量表达, 增加了PAHs降解基因的丰度和表达量, 促进了植物体内PAHs降解代谢潜能(Siciliano et al., 2001).
4 结论(Conclusions)1) 在30 ℃、pH=7.0条件下, 接种菌株Pn2和033对菲和芘的降解效率分别高达99.7%和98.3%, 降解半衰期分别为0.34 d和0.95 d.
2) 与CRD处理组对比, 接种混合菌株Pn2和033显著地降低了黑麦草体内PAHs含量, 并阻控黑麦草地上部位PAHs污染风险.
3) 接种混合菌株Pn2和033可增强黑麦草体内POD活性, 可能影响黑麦草体内PAHs的降解代谢.
参考文献
| Arslan M, Imran A, Khan Q M, et al. 2017. Plant-bacteria partnerships for the remediation of persistent organic pollutants[J]. Environmental Science and Pollution Research, 24(5): 4322–4336.DOI:10.1007/s11356-015-4935-3 |
| Afzal M, Khan Q M, Sessitsch A. 2014. Endophytic bacteria:Prospects and applications for the phytoremediation of organic pollutants[J]. Chemosphere, 117: 232–242.DOI:10.1016/j.chemosphere.2014.06.078 |
| Bacosa H P, Inoue C. 2015. Polycyclic aromatic hydrocarbons (PAHs) biodegradation potential and diversity of microbial consortia enriched from tsunami sediments in Miyagi, Japan[J]. Journal of Hazardous Materials, 283: 689–697.DOI:10.1016/j.jhazmat.2014.09.068 |
| Barnier C, Ouvrard S, Robin C, et al. 2014. Desorption kinetics of PAHs from aged industrial soils for availability assessment[J]. Science of the Total Environment, 470: 639–645. |
| ?akmak?i R, D?nmez F, Ayd?n A, et al. 2006. Growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions[J]. Soil Biology & Biochemistry, 38(6): 1482–1487. |
| Cai C Y, Li J Y, Wu D, et al. 2017. Spatial distribution, emission source and health risk of parent PAHs and derivatives in surface soils from the Yangtze River Delta, eastern China[J]. Chemosphere, 178: 301–308.DOI:10.1016/j.chemosphere.2017.03.057 |
| Campos V, Merino I, Casado R, et al. 2008. Phytoremediation of organic pollutants[J]. Spanish Journal of Agricultural Research, 6: 38–47.DOI:10.5424/sjar/200806S1-372 |
| Chen Z X, Ni H G, Jing X, et al. 2015. Plant uptake, translocation, and return of polycyclic aromatic hydrocarbons via fine root branch orders in a subtropical forest ecosystem[J]. Chemosphere, 131: 192–200.DOI:10.1016/j.chemosphere.2015.03.045 |
| Chaudhry Q, Schr?der P, Werck-Reichhart D, et al. 2002. Prospects and limitations of phytoremediation for the removal of persistent pesticides in the environment[J]. Environmental Science and Pollution Research, 9: 4–17.DOI:10.1007/BF02987313 |
| 陈保冬, 赵方杰, 张莘, 等. 2015. 土壤生物与土壤污染研究前沿与展望[J]. 生态学报, 2015, 35(20): 6604–6613. |
| 刁硕, 王红旗, 许洁, 等. 2017. 低温耐盐芘降解菌的筛选鉴定及降解特性研究[J]. 中国环境科学, 2017, 37(2): 677–685. |
| Dean-Ross D, Cerniglia C E. 1996. Degradation of pyrene by Mycobacterium flavescens[J]. Applied Microbiology and Biotechnology, 46: 307–312.DOI:10.1007/s002530050822 |
| Glick B R. 2014. Bacteria with ACC deaminase can promote plant growth and help to feed the world[J]. Microbiological Research, 169(1): 30–39.DOI:10.1016/j.micres.2013.09.009 |
| Gao Y, Zhu L. 2004. Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils[J]. Chemosphere, 55(9): 1169–1178.DOI:10.1016/j.chemosphere.2004.01.037 |
| Iwabuchi T, Harayama S. 1998. Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp.KP7[J]. Journal of Biological Chemistry, 273(14): 8332–8336.DOI:10.1074/jbc.273.14.8332 |
| Kim K H, Jahan S A, Kabir E, et al. 2013. A review of airborne polycyclic aromatic hydrocarbons (PAHs) and their human health effects[J]. Environment International, 60: 71–80.DOI:10.1016/j.envint.2013.07.019 |
| Kvesitadze E, Sadunishvili T, Kvesitadze G. 2009. Mechanisms of organic contaminants uptake and degradation in plants[J]. World Academy of Science, Engineering and Technology, 55(6): 458–468. |
| Liu J, Liu S, Sun K, et al. 2014. Colonization on root surface by a phenanthrene-degrading endophytic bacterium and its application for reducing plant phenanthrene contamination[J]. PloS One, 9(9): e108249.DOI:10.1371/journal.pone.0108249 |
| Liu J, Xiang Y, Zhang Z, et al. 2017. Inoculation of a phenanthrene-degrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat[J]. Applied Microbiology and Biotechnology, 101(12): 5199–5212.DOI:10.1007/s00253-017-8247-z |
| Liu S H, Zeng G M, Niu Q Y, et al. 2016. Bioremediation mechanisms of combined pollution of PAHs and heavy metals by bacteria and fungi:A mini review[J]. Bioresource Technology, 224: 35–33. |
| 雷东锋, 冯怡, 蒋大宗. 2004. 植物中多酚氧化酶的特征[J]. 自然科学进展, 2004, 14(6): 606–614.DOI:10.3321/j.issn:1002-008X.2004.06.002 |
| 李全霞, 范丙全, 龚明波, 等. 2008. 降解芘的分枝杆菌M11的分离鉴定和降解特性[J]. 环境科学, 2008, 29(3): 763–768.DOI:10.3321/j.issn:0250-3301.2008.03.037 |
| 刘爽, 刘娟, 凌婉婷, 等. 2013. 一株高效降解菲的植物内生细菌筛选及其生长特性[J]. 中国环境科学, 2013, 33(1): 95–102.DOI:10.3969/j.issn.1000-6923.2013.01.014 |
| Menzie C A, Potocki B B, Santodonato J. 1992. Exposure to carcinogenic PAHs in the environment[J]. Environmental Science & Technology, 26: 1278–1284. |
| 倪雪, 刘娟, 高彦征, 等. 2013. 2株降解菲的植物内生细菌筛选及其降解特性[J]. 环境科学, 2013, 34(2): 746–752. |
| Sprocati A, Alisi C, PintoV, et al. 2014. Assessment of the applicability of a "toolbox" designed for microbially assisted phytoremediation:The case study at Ingurtosu mining site (Italy)[J]. Environmental Science and Pollution Research, 21: 6939–6951.DOI:10.1007/s11356-013-2154-3 |
| Sinsabaugh R L. 2010. Phenol oxidase, peroxidase and organic matter dynamics of soil[J]. Soil Biology and Biochemistry, 42(3): 391–404.DOI:10.1016/j.soilbio.2009.10.014 |
| Siciliano S D, Fortin N, Mihoc A, et al. 2001. Selection of specific endophytic bacterial genotypes by plants in response to soil contamination[J]. Applied and Environmental Microbiology, 67(6): 2469–2475.DOI:10.1128/AEM.67.6.2469-2475.2001 |
| Shah K, Kumar R G, Verma S, et al. 2001. Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings[J]. Plant Science, 161(6): 1135–1144.DOI:10.1016/S0168-9452(01)00517-9 |
| Sun K, Liu J, Gao Y, et al. 2014a. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp.Ph6-gfp[J]. Scientific Reports, 4: 5462. |
| Sun K, Liu J, Jin L, et al. 2014b. Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination[J]. Plant and Soil, 374(1/2): 251–262. |
| Sams?e-Petersen L, Larsen E H, Larsen P B, et al. 2002. Uptake of trace elements and PAHs by fruit and vegetables from contaminated soils[J]. Environmental Science & Technology, 36(14): 3057–3063. |
| 盛月慧, 刘娟, 高彦征, 等. 2013. 黑麦草体内POD和PPO活性及可培养内生细菌种群对不同浓度菲污染的响应[J]. 南京农业大学学报, 2013, 36(6): 51–59. |
| 孙凯, 刘娟, 李欣, 等. 2014. 两株具有芘降解功能的植物内生细菌的分离筛选及其特性[J]. 生态学报, 2014, 34(4): 853–861. |
| Taghavi S, Barac T, Greenberg B, et al. 2005. Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene[J]. Applied and Environmental Microbiology, 71(12): 8500–8505.DOI:10.1128/AEM.71.12.8500-8505.2005 |
| Takaki K, Wade A J, Collins C D. 2014. Assessment of plant uptake models used in exposure assessment tools for soils contaminated with organic pollutants[J]. Environmental Science & Technology, 48: 12073–12082. |
| Wilson M S, Herrick J B, Jeon C O, et al. 2003. Horizontal transfer of phnAc dioxygenase genes within one of two phenotypically and genotypically distinctive naphthalene-degrading guilds from adjacent soil environments[J]. Applied and Environmental Microbiology, 69(4): 2172–2181.DOI:10.1128/AEM.69.4.2172-2181.2003 |
| Wang J, Liu J, Ling W, et al. 2017. Composite of PAH-degrading endophytic bacteria reduces contamination and health risks caused by PAHs in vegetables[J]. Science of the Total Environment, 598: 471–478.DOI:10.1016/j.scitotenv.2017.04.126 |
| Waigi M G, Sun K, Gao Y. 2017. Sphingomonads in microbe-assisted phytoremediation:Tackling soil pollution[J]. Trends in Biotechnology, 35(9): 883–899.DOI:10.1016/j.tibtech.2017.06.014 |
| Wang Y, Xiao M, Geng X, et al. 2007. Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere[J]. Applied Microbiology and Biotechnology, 77(3): 733–739.DOI:10.1007/s00253-007-1187-2 |
| Wang Y, Tian Z, Zhu H, et al. 2012. Polycyclic aromatic hydrocarbons (PAHs) in soils and vegetation near an e-waste recycling site in South China:Concentration, distribution, source, and risk assessment[J]. Science of the Total Environment, 439: 187–193.DOI:10.1016/j.scitotenv.2012.08.018 |
| Weyens N, Van Der Lelie D, Artois T, et al. 2009. Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation[J]. Environmental Science & Technology, 43(24): 9413–9418. |
| 王涛, 蓝慧, 田云, 等. 2016. 多环芳烃的微生物降解机制研究进展[J]. 化学与生物工程, 2016, 33(2): 8–14.DOI:10.3969/j.issn.1672-5425.2016.02.002 |
| 夏红霞, 朱启红. 2013. Pb、Zn及其交互作用对蜈蚣草抗氧化酶及叶绿素含量的影响[J]. 环境化学, 2013, 32(6): 959–963. |
| 徐成斌, 王闻烨, 李鲜珠, 等. 2015. 一株菲降解菌的鉴定及降解特性[J]. 环境科学学报, 2015, 35(3): 684–691. |
| Zhu X, Ni X, Liu J, et al. 2014. Application of endophytic bacteria to reduce persistent organic pollutants contamination in plants[J]. CLEAN-Soil Air Water, 42(3): 306–310.DOI:10.1002/clen.v42.3 |
| Zhu X, Ni X, Waigi M G, et al. 2016. Biodegradation of mixed PAHs by PAH-degrading endophytic bacteria[J]. International Journal of Environmental Research and Public Health, 13(8): 805.DOI:10.3390/ijerph13080805 |
| Zielińska A, Oleszczuk P. 2015. Evaluation of sewage sludge and slow pyrolyzed sewage sludge-derived biochar for adsorption of phenanthrene and pyrene[J]. Bioresource Technology, 192: 618–626.DOI:10.1016/j.biortech.2015.06.032 |
| Zhang W, Wang H, Zhang R, et al. 2010. Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China[J]. Ecotoxicology, 19: 96–104.DOI:10.1007/s10646-009-0393-3 |
