方文雯(1998—),女,安徽池州人,硕士研究生,主要从事环境化学研究。E-mail:
为研究腐殖酸(Humic acid,HA)对微生物降解环境中多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)的影响,以四环芘为典型PAHs及HA为代表性溶解性有机质,选取芘高效降解菌
Pollution by Polycyclic Aromatic Hydrocarbons(PAHs)in the soil environment has become a serious problem. In recent years, microbial degradation of PAHs to achieve soil remediation has become an important method. However, microbial degradation is not a very efficient and ideal process for the remediation of PAHs-polluted environments. In this study, humic acid(HA)was added during the degradation process to explore its effect on the microbial degradation of PAHs.
Using pyrene as a typical PAH and HA as representative dissolved organic matter,
Results show: 1)The presence of HA significantly accelerated pyrene biodegradation. Approximately 88.33%±3.40% of pyrene was biodegraded within 7 days of incubation with the addition of HA, but only 35.33%±3.27% was biodegraded without HA; 2)The FT-ICR-MS results indicated that addition of HA induced two biodegradation pathways - attacking of 1, 2-positions and 4, 5-positions of pyrene by dioxygenase, whereas in the absence of HA only the 4, 5-position was attacked; 3)The CLSM results showed that HA enhanced the growth of
HA can improve the degradation efficiency by increasing the degradation pathway of pyrene and inducing the interaction of functional groups on the cell surface to form a microbial membrane. Meanwhile, the addition of exogenous HA could also promote the interactions among colonies and accelerate the degradation.
随着我国经济的快速发展,人为活动对环境造成了很多的影响,其中煤、石油、木材等不完全燃烧以及石油加工过程中产生的多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)的激增严重威胁了土壤的生态环境、农产品安全和人体健康[
微生物转化是自然环境中有机污染物消除的主要途径之一,也是有机污染物控制与环境修复的核心技术方法[
溶解性有机质(Dissolved organic matter,DOM)是土壤和自然水体中的一种重要组分,由碳含量约50%的有机分子和氧、氮、磷和硫等各种杂原子按照一定比例组成[
基于以上所述,本研究以污染土壤中最常见的四环芘作为代表性PAHs,以腐殖酸(Humic acid,HA)为代表性DOM设置添加和未添加实验,利用高效菌株
芘降解菌
腐殖酸的成分及元素组成
The molecular class and element composition of humic acid
样品 |
碳水化合物 |
氨基糖 |
蛋白质+脂质 |
单宁 |
木质素 |
不饱和烃 |
腐殖酸 |
0.20±0.04 | 0.55±0.06 | 67.71±3.22 | 0.37±0.01 | 30.15±1.00 | 0.95±0.03 |
样品 |
稠环芳烃 |
C/% | H/% | O/% | N+P+S/% | |
腐殖酸 |
0.07±0.008 | 62.85 | 9.02 | 18.01 | 10.12 |
实验分为两组,一组不添加HA,另一组添加HA,评价HA的添加对菌降解芘的效率以及对菌落生长情况的影响;同时设置两个不接种菌的对照组,同样一组不添加HA,另一组添加HA,评价芘在有无HA添加的情况下的挥发损失。分别移取芘浓度为1 g·L–1溶液10 μL、100 μL和1 mL置于每个组的三角瓶中,待丙酮挥发完全后,向添加HA的组分中加入5 mg灭菌后的HA以及90 mL的灭菌无机盐溶液,然后按照10%的菌液接种比例在实验组中加入10 mL
取出培养7 d的菌悬液,加入20 mL正己烷和10 mL 0.1 mol·L–1 HCL溶液,超声10 min(50 Hz)。待液面分层稳定后吸取上层液体,得到有机相正己烷溶液,随后采用氮吹仪换相到乙腈溶液中,最后使用0.45 µm滤头过滤并采用ESI-FT-ICR-MS分析其碎片离子。具体条件如下:毛细管电压-3.8 kV和120 μL·h–1的流量,正离子模式下,为–4.0 kV喷雾电压和-4.5 kV的毛细管柱引入电压;负离子模式下,为4.0 kV喷雾电压和4.5 kV的毛细管柱引入电压和240 V毛细管柱端电压。
向间隔1 d取出的样品中分别添加正己烷20 mL,并超声20 min(50 Hz),移取正己烷相10 mL,采用旋转蒸发浓缩、氮气换相于乙腈的方法进行纯化[
取培养7 d后的样品,离心并收集沉淀,冷冻干燥后,使用共聚焦拉曼光谱仪(HR Evolution,France)测试样品,激发波长为633 nm,扫描范围为100~2 000 cm–1。冷冻后样品采用KBr压片,使用红外光谱测定其官能团变化特征,样品扫描范围为4 000~400 cm–1。瞬态三维荧光光谱(EEM)在室温下测定,将菌液溶液置于四通常量比色皿中。设置测定参数:激发波长(Ex)和发射波长(Em)都为220~550 nm,狭缝宽度为5 nm。
菌落的成活及生长特性采用激光共聚焦显微镜(CLSM,N-STORM,Nikon)检测,取培养7 d后的菌悬液100~150 μL,置于专用培养小皿中心位置,恒温培养24 h。待菌沉降附着至皿底后将多余培养液用移液枪吸掉,然后用100 μL配置好的Calcein/PI染色剂缓慢加进培养皿中,使菌落浸泡且置于暗处染色15 min。随后采用磷酸缓冲液清洗染色液,并置于激光共聚焦下观察。CLSM所设参数:发射激光波长为488 nm和561 nm,物镜倍数为20倍。同时,取10 μL菌悬液梯度稀释后并采用平板计数法统计隔天菌落的生长特征。
ESI-FT-ICR-MS的结果利用Bruker Data Analysis进行分析,高效液相质谱及其他光谱测试结果均由统计软件Microsoft Excel 2016和Origin 2018进行数据处理和作图。
如
培养期间分枝杆菌
Biodegradation of pyrene by
为确定高效菌
ESI-FT-ICR-MS检测芘降解的中间产物信息
The intermediate products of pyrene degradation detected by ESI-FT-ICR-MS
样品类型 |
分子式 |
检测质量 |
理论质量 |
芘 |
C14H10O2 | 421.145([2M+H]+) | 210.068 |
C16H10O2 | 279.040([M+Na2-H]+) | 234.068 | |
腐殖酸-芘 |
C18H14O2 | 566.242([2M+C2H3N+H]+) | 262.099 |
C16H8O2 | 274.085([M+CH3CN+H]+) | 232.052 |
菌落
Biodegradation pathways for pyrene by
为确定菌落在芘降解过程中的作用机制及HA的影响,需要明确菌落的活性及赋存形态。
有无HA添加时菌落
The CLSM images of degradation of pyrene by
然而,添加HA后,生物膜形成速度加快,尤其芘浓度较低时,快速形成了微生物膜(
红外光谱技术能反应细胞表面官能团的变化信息[
菌落
Infrared spectrum of
同时,HA与芘共存时,其红外特征峰变化趋势明显。N-H伸缩振动峰由3 464 cm–1红移至3 227 cm–1;O-H伸缩振动峰由2 335 cm–1蓝移至2 424 cm–1;N-H弯曲振动峰由1 587 cm–1红移至1 537 cm–1;C-O伸缩振动峰由1 098 cm–1红移至1 068 cm–1。由峰位移推断:HA添加诱导了细胞表面、HA、芘之间的相互作用力。而且,在芘降解过程中于1 656 cm–1、1 448 cm–1、1 294 cm–1、1 240 cm–1、1 151 cm–1处出现一系列新峰,它们分别归属于羧酸酯类酰胺谱带的C-O伸缩振动、酰胺Ⅲ带C-N伸缩振动、N-H弯曲振动、芳香类C-N伸缩振动和酸酐C-O伸缩振动。表明HA添加不仅促进芘降解而且有利于菌落
拉曼光谱技术分析生物样品,能给出生物细胞表面蛋白的变化特征[
菌落
Raman spectrum of
当加入HA后,如
为获取微生物降解芘过程中的细胞表面的腐殖化程度,进一步采用三维荧光光谱(EEM)分析了溶液体系及菌落团聚体的谱学变化特征。如
分支杆菌
The EEM images of
添加不同浓度芘后,检测到的EEM图像如
分支杆菌
The EEM images of
溶解性有机质深刻影响着芘的降解。HA不仅能通过增加降解途径的方式促进高效降解菌
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