多环芳烃、多氯联苯优良降解菌的分离鉴定及降解特性研究
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摘要
作为持久性有机污染物(Persistent organic pollutants,POPs)的典型代表,多环芳烃(polycyclic aromatic hydrocarbons,PAHs)和多氯联苯(polychlorinatedbiphenyls,PCBs)的分析方法、环境影响及其污染修复技术是目前国内外热点研究领域,生物降解特别是微生物降解被认为是去除环境中PAils、PCBs的主要途径。本文在全面综述国内外相关研究的基础上,通过调查和监测选择并确定了PAHs、PCBs污染的土壤样品,筛选得到的一株可以高效降解PAHs、PCBs的菌株,基于形态学特征、生理生化试验、细胞化学成分鉴定及16S rDNA序列比对分析等手段,鉴定该菌株属于两面神菌属(Janibacter sp.),命名为JY11。以此为基础,系统研究了JY11对PAHs、PCBs的降解效果和影响因素以及与Phanerochaete.chrysosporium JY16联合降解效率,并借助气相色谱—质谱联用技术对降解中间产物的定性分析,初步探讨了该菌株对菲、芘、苯并(α)芘和PCB18、PCB77的代谢途径。论文主要研究内容和结果如下:
1.污染土壤中PAHs和变压器油中PCBs的提取及分析
(1)采用索氏提取、超声提取及超临界CO_2提取三种样品前处理技术,结合GC-FID分析技术,研究获得了济南炼油厂及胜利油田土壤中PAHs的污染特征。济南炼油厂土壤中检出全部16种PAHs,浓度范围为5.0～593.2μg/kg;胜利油田采油厂土壤中检出12种PAHs,浓度范围为227.7～1107.3μg/kg,两处土壤均在一定程度上受到了PAHs的污染。三种提取技术均有很好的提取效率和重现性,相对标准偏差(RSD%)小于5%(n=3),PAHs加标回收率为88.7%～102.6%,符合美国EPA标准中环境样品回收率(70%-140%)及RSD%(＜20%)的要求。
(2)采用液液萃取、超声萃取及固相萃取等三种样品前处理技术,结合本实验室前期建立的PCBs分析方法,研究建立了简便快速的PCBs提取及GC-ECD分析技术。研究测定了废弃国产变压器油中类二噁英类及阻转类PCBs的成分与含量,检出10种类二噁英类PCBs(PCB77,8l,105,114,118,123,126,156,167,189)和4种阻转类PCBs(PCB45,95,132,149)。实验还结合Areolor 1242标准品对变压器油中PCBs组分进行了分析,得到PCBs的分布特征:二氯联苯含量为14.2%;三氯联苯含量为51.2%;四氯联苯含量为29.8%;五氯和六氯联苯含量为4.8%。由此可见,国产变压器油以三氯联苯为主,高氯代PCBs所占比例很少。三种提取方法的RSD%均小于5%(n=3),内标PCB209的回收率为82.3%～91.6%,符合美国EPA标准中回收率及相对标准偏差的要求。
2.PAHs、PCBs降解菌的分离与鉴定
(1) PAHs、PCBs优势降解菌的分离。采集济南市炼油厂、胜利油田采油厂及原济南市变压器厂的土样,以菲、蒽、芘的混合样品为唯一碳源,采用定时定量转接、逐步提高碳源浓度的方法,共获得125个单菌落,经降解效率初步测定,选取降解能力较强的两株细菌JY11和JY3A进行鉴定研究。
(2) JY11和JY3A的鉴定。通过形态特征描述,综合运用生理生化检测、脂肪酸分析、甲基萘醌分型及GC摩尔百分含量测定等方法,结合16S rDNA序列分析、构建系统进化树,JY3A被认为属于Bacillus菌属的B.subtilis类群,由于Bacillus菌属目前定名的复杂性和不确定性,尚需进一步PFGE和DNA-DNA杂交实验数据支持鉴定到种。
(3) JY11被鉴定为Janibacter菌属。该属1997年方予以命名,确定模式种为Janibacter limosus,至今仅有5个种的报道。但JY11与该属模式种有较大差异,与其遗传距离最近的为Janibacter anophelis。细胞脂肪酸组成及含量分析结果表明,JY11在脂肪酸化学分类特征上表现出独特的菌株特异性,不同于Janibacter limosus和Janibacter anophelis的已有报道。初步实验表明JY11在表现出良好的PAHs降解能力的同时,对PCBs亦有较好的降解能力,因此选择JY11菌作为后续研究的出发菌株。
3.Janibacter sp.JY11对PAHs、PCBs的降解效果及影响因素研究
(1) Janibacter sp.JY11对PAHs的降解效果
研究了液体培养条件下,Janibacter sp.JY11对初始浓度为100 mgl PAHs的降解效果。结果表明,Janibacter sp.JY11不仅对低分子量PAHs如菲、芴、苊等有很好的降解效果,对四环的芘、荧蒽、苯并(α)蒽等也有较好的降解效果,五环和六环的高分子量PAHs也有不同程度的降解。由此可见,JY11表现出非常宽的降解底物谱及很强的降解能力。
(2)影响Janibacter sp.JY11对PAHs的降解效率的因素
实验中分别以三环的菲、四环的芘和五环的苯并(α)芘为代表,研究了不同PAHs初始浓度、不同浓度的表面活性剂Tween 80和Triton X-100及水杨酸诱导作用对PAHs降解效率的影响。结果表明:随着初始浓度的增加,芘和苯并(α)芘的降解效率逐渐降低,而菲则可以在很宽的浓度范围内全部降解;表面活性剂Tween 80对PAHs降解有一定的促进作用;通过用水杨酸做共代谢底物,相比用菲作共代谢底物,苯并(α)芘的降解效率提高了28%。
(3) Janibacter sp.JY11对PCBs的降解效果
实验研究了在液相纯培养条件下,JY11对初始浓度分别10 mg/l及100 mg/lPCBs的降解效果。结果表明,随着初始浓度增加,降解效率略有下降,但总体上JY11对PCBs具有较好的降解效果。对于初始浓度为10 mg/l的PCBs,二氯及三氯PCBs的降解效率为83%～100%,多数高于90%;四氯PCBs的降解效率为78%～94%;五氯及六氯PCBs的降解效率也达到50～455%。JY11对邻位、对位取代PCBs均可降解,只是随着氯原子数增加,降解效率降低。由此可见,氯原子取代位置并不是影响JY11对PCBs降解的主要因素。
(4)天然植物组分诱导Janibacter sp.JY11降解PCBs
通过在液体培养基中添加九州虫草子座挥发油、唐古特青兰挥发油、草果挥发油、香芹酮、柠檬烯等天然植物组分,考察了其对Jannibactor sp.JY11降解PCBs效率的影响。结果表明九州虫草子座挥发油、香芹酮能显著提高绝大多数PCBs的降解效率,柠檬稀也有一定的促进作用。
4.Janibacter sp.JY11-P.chrysosporium JY16联合降解PAHs、PCBs
利用白腐真菌胞外氧化还原酶系丰富,底物专一性低,对外源物质的降解不受其浓度大小的影响等优点,在Janibacter sp.JY11中加入一株白腐真菌—黄孢原毛平革菌(P.chrysosporium JY16),研究其联合降解作用。实验结果表明:液体培养条件下,混合菌对PAHs的降解效果明显好于单一菌降解效果,尤其是四环的芘和五环的苯并(α)蒽、苯并(κ)荧蒽的降解效率相比JY11单独降解提高了20%以上。对于模拟污染土壤环境,混合菌对芘和苯并(α)芘的降解效率分别为73%和60%,相比用单一菌降解也得到很大程度的提高。同样液体培养条件下,混合菌对PCBs的降解效果明显优于单一菌降解效果。尤其是对于类二噁英类的六氯联苯PCB156,167和七氯联苯PCB189,单一菌并不利用;而当两株菌联合降解时,三种高氯PCBs降解了10%左右。在此实验基础上,用混合菌直接降解变压器油及模拟污染土壤中的PCBs。结果表明:变压器油中PCBs的降解效率为8.7%～83.3%;对于模拟污染土壤样品,除PCB74,类二噁英类PCB105,118和阻转类PCB132,149没有降解外,其余PCBs的降解效率为18.4%～72.6%。
5.Janibacter sp.JY11降解PAHs、PCBs中间产物及代谢途径初步分析
以三环的菲、四环的芘和五环的苯并(α)芘作为JY11降解PAHs代谢途径分析的研究对象;以三氯PCB18及类二噁英类PCB77作为JY11降解PCBs代谢途径分析的研究对象,利用GC-MS技术,通过对中间产物的检测,初步推导了JY11降解PAHs、PCBs的代谢途径。
(1)菲:菲在双加氧酶的作用下生成顺式-2,3-二氢-二羟基菲,然后转化成1-羟基-二萘酸,脱羧后生成1-萘酚,最后开环生成水杨酸。
(2)芘:芘在双加氧酶的作用下生成顺式-4,5-二氢-二羟基芘,然后在脱氢酶作用下重新芳香化生成顺式-4,5-二羟基芘,该产物进一步通过双加氧酶开环生成4,5-二羧酸菲,最终在脱羧酶的作用下生成4-羟基菲,最后开环生成邻苯二甲酸。
(3)苯并(α)芘:苯并(α)芘在双加氧酶的作用下生成顺式-7,8-二氢-二羟基苯并(α)芘,然后在脱氢酶作用下重新芳香化生成顺式7,8-二羟基苯并(α)芘,该产物进一步通过双加氧酶开环生成7-羟基-8-羧酸芘。
(4)三氯联苯PCB18和四氯联苯PCB77代谢途径基本相同,即JY11进攻PCBs的2,3位,生成2,3.二氢二羟基-PCBs,该产物在脱氢酶的作用下生成2,3-二羟基-PCBs;然后在第二个双加氧酶作用下开环生成2,5-二氯-2-羟基-6-氧-6-2,5-二氯苯基-2,4-己二烯酸,该产物在水解酶的作用下生成氯代苯甲酸。
As the representative substances of persistent organic pollutants(POPs),study on the analysis method,environmental impact and pollution repairing of polycyclic aromatic hydrocarbons(PAHs) and polychlorinated biphenyls(PCBs) is a research emphasis in all over the world.Especially microbiodegradation is considered to be a major way to wipe off PAHs and PCBs in the environment.Based on the review of related study in the worldwide,a highly effective PAHs and PCBs-degrading bacterium was isolated.The isolate was identified as Janibacter sp.with respect to its 16S rDNA sequence and fatty acid profiles,as well as various physiological characteristics,and named JY11.Further study has been done systematically on degradation ability and relevant factors that affect the degradation performance. Co-degradation of PAHs and PCBs with JY11 and P chrysosporium JY16 was also researched.The probable pathway of PAHs and PCBs degraded by JY11 were proposed with detecting the metabolites by GC-MS.The main content and experimental results are as follow:
1.Extraction and analysis of PAHs from polluted soil and PCBs from transformer oil
(1) Soxhlet extraction,supereritical CO_2 extraction and ultrasonic extraction were selected to extract PAHs from polluted soil gathered from Jinan Oil Refinery Factory and Shengli Oil Field.And the extracts were analyzed by GC-FID.The results showed that:16 kinds of PAHs in Jinan Oil Refinery Factory were detected with a concentration of 5.0～593.2μg/kg;12 kinds of PAHs in Shengli Oil Field were detected with a concentration of 227.7～1107.3μg/kg.The spiked recovery of each method was in the range of 88.7～102.6%and the relative standard deviation(RSD%) was in the range of 1.5～4.4%,meeting the US EPA standard of recoveries(70～140%) and RSD of repeated samples(＜20%).
(2) Liquid-liquid extraction,ultrasonic extraction and solid phase extraction were selected to extract PCBs from Chinese transformer oil and the extracts were analyzed by GC-ECD.Based on the former study results on PCBs analysis in our laboratory,10 kinds of dioxin-like PCBs(PCB77,81,105,114,118,123,126,156, 167 and 189) and four common exist atropisomeric-PCBs were detected in the Chinese transformer oil.Other PCBs components were also analyzed with Arcolor 1242 standard.The recovery of internal standard of PCB209 with different extraction method was in the range of 82.3%～91.6%and the RSD%less than 5%,meeting the US EPA standard of recoveries and RSD of repeated samples.
2.Isolation and identification of PAHs and PCBs-degrading bacteria
16 PAHs-degrading strains were newly isolated from the polluted soil in Jinan Oil Refinery Factory,Shengli Oil Field and former Jinan Power Transformer Factory, Shandong Province of China.Two bacteria,JY11 and JY3A,were investigated. Strain JY11 was identified as Janibacter sp.with respect to its morphology,16S rDNA sequence,phylogenetic analysis,and fatty acid profiles,as well as various physiological and biochemical characteristics.The strain was Gram-positive, non-motile,non-spore-forming,short rods in young culture,0.8-1.0μm in diameter and 1.3-1.6μm long,and coccoid cells in the stationary phase of growth that are 1.0-1.2μm in diameter and 1.3-1.5μm long,occurred in pairs and sometimes in chains or in group,aerobic,oxidase-week positive,catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY11 was shown to be most closely related to Janibacter anophelis(99.93%),J.terrae(98.48%),J.marinus(98.38%),J. limosus(98.34%),J.melonis(98.20%) and J.corallicola(97.79%). Chemotaxonomic data(menaquinone,GC content and major fatty acids) supported the allocation of the strain to the genus Janibacter.
Strain JY3A was characterized as motile,rod-shaped,Gram-positive,endospore forming,single or in pairs and sometimes in chains,aerobic,oxidase-positive, catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY3A was shown to be most closely related to B.vallismortis(99.69%),B.subtile (99.65%),B.amyloliquefaciens(99.60%),B.mojavensis(99.57%) and B.atrophaeus (99.42%).Chemotaxonomic data supported the proposal to assign the strain to the B. subtilis group of the genus Bacillus.
3.Study of degradation ability and related factors that affect the degradation performance of PAHs and PCBs by Janibacter sp.JY11
(1) Degradation of PAHs by Janibacter sp.JY11
PAHs with initial concentration of 100 mg/l were degraded with JY11 in liquid culture.The result showed that not only the low molecular PAHs,such as phenanthrene,fluorene and acenaphthene,but also pyrene,fluoranthene and benzo(a)anthracene have a good degradation result.Also the high molecular PAHs degraded in different degree.JY11 presented a wide degradation spectrum and high effective degradation ability.
(2) Effects of different initial concentrations,surfactant and salicylic acid on the degradation efficiency of PAHs were studied taking phenanthrene,pyrene and benzo(a)pyrene as representative.The results showed that the degradation efficiency of pyrene and benzo(a)pyrene decreased with the increase of initial concentration, while phenanthrene can be degraded completely with different initial concentrations. Tween 80 is helpful for the degradation of PAHs.Through adding salicylic acid in the medium,the degradation efficiency of benzo(a)pyrene increased 28%compared with phenanthrene as co-metabolized substance.
(3) Degradation of PCBs by Janibacter sp.JY11
The degradation efficiency of PCBs with a concentration of 10 mg/l and 100 mg/l was studied separately.The result showed that the degradation efficiency decreased in some degree with the increase of initial concentration of PCBs.But JY11 presented a good performance as a whole.For PCBs with an initial concentration of 10 mg/l,the degradation efficiencies of the di and triclorine-CBs were 83%～100%;the degradation efficiencies of the tetraclorine-CB were 78%～94%; and the degradation efficiencies of penta and hexaclorine-CB were 50～65%.The degradation efficiencies of PCBs decreased along with the increase of the number of chlorine atoms.
(4) In the inducing experiment by natural plant constituent,essential oil of Cordyceps kyushuensis and carvone can enhance the degradation efficiency notably; also,cymene is helpful in some degree.While,essential oil from Dracocephalum tanguticum and biphenyl nearly have no effect on the degradation efficiency.But essential oil from Amomum tsao-ko crevost et lemaire is harmful for degradation of PCBs by JY11.
4.Co-degradation of PAHs and PCBs by Janibacter sp.JY11 and P. chrysosporium JY16
Through adding white rot fungi,P.chrysosporium JY16,to the medium of JY11, the degradation threshold of PAHs and PCBs decreased and the degradation efficiency increased according to their co-degradation effect.
In liquid culture,the degradation efficiency of the mixed bacterium and fungi is much better than JY11 and JY16.Especially the degradation efficiency of four ring-pyrene and five ring-benzo(a)anthracene.Benzo(k)fluoranthene increased at least 20%compared with JY11.For the modified polluted soil sample,73%of pyrene and 60%of benzo(a)pyrene was degraded with mixed bacterium and fungi after thirty days cultivation.The degradation efficiency increase a lot compared with the sole bacterium or fungi.Similar result was got with PCBs.The degradation result with mixed culture is better than any sole bacterium or fungi.Especially for dioxin like PCB156,167 and 189,which could not be utilized by sole JY11 or JY16, decreased by 10 percent with co-culture.PCBs in the transformer oil and modified polluted soil were degraded by the mixed bacterium and fungi.The result showed that the degradation efficiencies of PCBs in transformer oil were 8.7%～83.3%.And the degradation efficiencies of PCBs in the modified polluted soil were 18.4%～72.6%.While PCB74,dl-PCB105,118 and atropisomeric-PCB132,149 showed no degradation.
5.Analysis of metabolites and proposed pathway of PAHs and PCBs degraded by Janibacter sp.JY11
Phenanthrene,pyrene and benzo(a)pyrene were selected as the research targets of PAHs degradation pathway.PCB 18 and PCB77 were selected as the targets of PCBs degradation pathway study.The proposed pathway of PAHs and PCBs was concluded by detecting the metabolites by GC-MS.
(1) Phenanthrene was transformed to 2,3-dihydroxy-2,3-dihydrophenanthrene firstly with dioxygenase of JY11.2,3-dihydroxy-2,3-dihydrophenanthrene was metabolized to 1-hydroxy-2-naphthoic acid,which transformed to 1-naphthol. 1-naphthol was cleaved to salicylic acid at last.
(2) Pyrene was transformed to 4,5-dihydroxy-4,5-dihydropyrene firstly with dioxygenase of JY11.Then 4,5-dihydroxy-4,5-dihydropyrene was metabolized to phenanthrene 4,5-dicarboxylic acid,which transformed to 4-hydroxyphenanthrene. 4-hydroxyphenanthrene was cleaved to phthalic acid at last.
(3) Benzo(a)pyrene was transformed to benzo(a)pyrene-7,8-dihydrodiol firstly with dioxygenase of JY11.And then benzo(a)pyrene-7,8-dihydrodiol was metabolized to benzo(a)pyrene-7,8-dihydroxy,which transformed to pyrene-7-hydroxy-8-carboxylic acid at last.
(4) PCB18 and PCB77 have a similar probable pathway,that is JY11 attacked the 2,3 position of PCBs and which metabolized to 2,3-dihydroxy-2,3-dihydro-PCB, then transformed to 2,3-dihydroxy-PCB,and then yielded to a yellow color product, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid,which was transformed to ehlorbenzoic acid under a hydrolase.
1.污染土壤中PAHs和变压器油中PCBs的提取及分析
(1)采用索氏提取、超声提取及超临界CO_2提取三种样品前处理技术,结合GC-FID分析技术,研究获得了济南炼油厂及胜利油田土壤中PAHs的污染特征。济南炼油厂土壤中检出全部16种PAHs,浓度范围为5.0～593.2μg/kg;胜利油田采油厂土壤中检出12种PAHs,浓度范围为227.7～1107.3μg/kg,两处土壤均在一定程度上受到了PAHs的污染。三种提取技术均有很好的提取效率和重现性,相对标准偏差(RSD%)小于5%(n=3),PAHs加标回收率为88.7%～102.6%,符合美国EPA标准中环境样品回收率(70%-140%)及RSD%(＜20%)的要求。
(2)采用液液萃取、超声萃取及固相萃取等三种样品前处理技术,结合本实验室前期建立的PCBs分析方法,研究建立了简便快速的PCBs提取及GC-ECD分析技术。研究测定了废弃国产变压器油中类二噁英类及阻转类PCBs的成分与含量,检出10种类二噁英类PCBs(PCB77,8l,105,114,118,123,126,156,167,189)和4种阻转类PCBs(PCB45,95,132,149)。实验还结合Areolor 1242标准品对变压器油中PCBs组分进行了分析,得到PCBs的分布特征:二氯联苯含量为14.2%;三氯联苯含量为51.2%;四氯联苯含量为29.8%;五氯和六氯联苯含量为4.8%。由此可见,国产变压器油以三氯联苯为主,高氯代PCBs所占比例很少。三种提取方法的RSD%均小于5%(n=3),内标PCB209的回收率为82.3%～91.6%,符合美国EPA标准中回收率及相对标准偏差的要求。
2.PAHs、PCBs降解菌的分离与鉴定
(1) PAHs、PCBs优势降解菌的分离。采集济南市炼油厂、胜利油田采油厂及原济南市变压器厂的土样,以菲、蒽、芘的混合样品为唯一碳源,采用定时定量转接、逐步提高碳源浓度的方法,共获得125个单菌落,经降解效率初步测定,选取降解能力较强的两株细菌JY11和JY3A进行鉴定研究。
(2) JY11和JY3A的鉴定。通过形态特征描述,综合运用生理生化检测、脂肪酸分析、甲基萘醌分型及GC摩尔百分含量测定等方法,结合16S rDNA序列分析、构建系统进化树,JY3A被认为属于Bacillus菌属的B.subtilis类群,由于Bacillus菌属目前定名的复杂性和不确定性,尚需进一步PFGE和DNA-DNA杂交实验数据支持鉴定到种。
(3) JY11被鉴定为Janibacter菌属。该属1997年方予以命名,确定模式种为Janibacter limosus,至今仅有5个种的报道。但JY11与该属模式种有较大差异,与其遗传距离最近的为Janibacter anophelis。细胞脂肪酸组成及含量分析结果表明,JY11在脂肪酸化学分类特征上表现出独特的菌株特异性,不同于Janibacter limosus和Janibacter anophelis的已有报道。初步实验表明JY11在表现出良好的PAHs降解能力的同时,对PCBs亦有较好的降解能力,因此选择JY11菌作为后续研究的出发菌株。
3.Janibacter sp.JY11对PAHs、PCBs的降解效果及影响因素研究
(1) Janibacter sp.JY11对PAHs的降解效果
研究了液体培养条件下,Janibacter sp.JY11对初始浓度为100 mgl PAHs的降解效果。结果表明,Janibacter sp.JY11不仅对低分子量PAHs如菲、芴、苊等有很好的降解效果,对四环的芘、荧蒽、苯并(α)蒽等也有较好的降解效果,五环和六环的高分子量PAHs也有不同程度的降解。由此可见,JY11表现出非常宽的降解底物谱及很强的降解能力。
(2)影响Janibacter sp.JY11对PAHs的降解效率的因素
实验中分别以三环的菲、四环的芘和五环的苯并(α)芘为代表,研究了不同PAHs初始浓度、不同浓度的表面活性剂Tween 80和Triton X-100及水杨酸诱导作用对PAHs降解效率的影响。结果表明:随着初始浓度的增加,芘和苯并(α)芘的降解效率逐渐降低,而菲则可以在很宽的浓度范围内全部降解;表面活性剂Tween 80对PAHs降解有一定的促进作用;通过用水杨酸做共代谢底物,相比用菲作共代谢底物,苯并(α)芘的降解效率提高了28%。
(3) Janibacter sp.JY11对PCBs的降解效果
实验研究了在液相纯培养条件下,JY11对初始浓度分别10 mg/l及100 mg/lPCBs的降解效果。结果表明,随着初始浓度增加,降解效率略有下降,但总体上JY11对PCBs具有较好的降解效果。对于初始浓度为10 mg/l的PCBs,二氯及三氯PCBs的降解效率为83%～100%,多数高于90%;四氯PCBs的降解效率为78%～94%;五氯及六氯PCBs的降解效率也达到50～455%。JY11对邻位、对位取代PCBs均可降解,只是随着氯原子数增加,降解效率降低。由此可见,氯原子取代位置并不是影响JY11对PCBs降解的主要因素。
(4)天然植物组分诱导Janibacter sp.JY11降解PCBs
通过在液体培养基中添加九州虫草子座挥发油、唐古特青兰挥发油、草果挥发油、香芹酮、柠檬烯等天然植物组分,考察了其对Jannibactor sp.JY11降解PCBs效率的影响。结果表明九州虫草子座挥发油、香芹酮能显著提高绝大多数PCBs的降解效率,柠檬稀也有一定的促进作用。
4.Janibacter sp.JY11-P.chrysosporium JY16联合降解PAHs、PCBs
利用白腐真菌胞外氧化还原酶系丰富,底物专一性低,对外源物质的降解不受其浓度大小的影响等优点,在Janibacter sp.JY11中加入一株白腐真菌—黄孢原毛平革菌(P.chrysosporium JY16),研究其联合降解作用。实验结果表明:液体培养条件下,混合菌对PAHs的降解效果明显好于单一菌降解效果,尤其是四环的芘和五环的苯并(α)蒽、苯并(κ)荧蒽的降解效率相比JY11单独降解提高了20%以上。对于模拟污染土壤环境,混合菌对芘和苯并(α)芘的降解效率分别为73%和60%,相比用单一菌降解也得到很大程度的提高。同样液体培养条件下,混合菌对PCBs的降解效果明显优于单一菌降解效果。尤其是对于类二噁英类的六氯联苯PCB156,167和七氯联苯PCB189,单一菌并不利用;而当两株菌联合降解时,三种高氯PCBs降解了10%左右。在此实验基础上,用混合菌直接降解变压器油及模拟污染土壤中的PCBs。结果表明:变压器油中PCBs的降解效率为8.7%～83.3%;对于模拟污染土壤样品,除PCB74,类二噁英类PCB105,118和阻转类PCB132,149没有降解外,其余PCBs的降解效率为18.4%～72.6%。
5.Janibacter sp.JY11降解PAHs、PCBs中间产物及代谢途径初步分析
以三环的菲、四环的芘和五环的苯并(α)芘作为JY11降解PAHs代谢途径分析的研究对象;以三氯PCB18及类二噁英类PCB77作为JY11降解PCBs代谢途径分析的研究对象,利用GC-MS技术,通过对中间产物的检测,初步推导了JY11降解PAHs、PCBs的代谢途径。
(1)菲:菲在双加氧酶的作用下生成顺式-2,3-二氢-二羟基菲,然后转化成1-羟基-二萘酸,脱羧后生成1-萘酚,最后开环生成水杨酸。
(2)芘:芘在双加氧酶的作用下生成顺式-4,5-二氢-二羟基芘,然后在脱氢酶作用下重新芳香化生成顺式-4,5-二羟基芘,该产物进一步通过双加氧酶开环生成4,5-二羧酸菲,最终在脱羧酶的作用下生成4-羟基菲,最后开环生成邻苯二甲酸。
(3)苯并(α)芘:苯并(α)芘在双加氧酶的作用下生成顺式-7,8-二氢-二羟基苯并(α)芘,然后在脱氢酶作用下重新芳香化生成顺式7,8-二羟基苯并(α)芘,该产物进一步通过双加氧酶开环生成7-羟基-8-羧酸芘。
(4)三氯联苯PCB18和四氯联苯PCB77代谢途径基本相同,即JY11进攻PCBs的2,3位,生成2,3.二氢二羟基-PCBs,该产物在脱氢酶的作用下生成2,3-二羟基-PCBs;然后在第二个双加氧酶作用下开环生成2,5-二氯-2-羟基-6-氧-6-2,5-二氯苯基-2,4-己二烯酸,该产物在水解酶的作用下生成氯代苯甲酸。
As the representative substances of persistent organic pollutants(POPs),study on the analysis method,environmental impact and pollution repairing of polycyclic aromatic hydrocarbons(PAHs) and polychlorinated biphenyls(PCBs) is a research emphasis in all over the world.Especially microbiodegradation is considered to be a major way to wipe off PAHs and PCBs in the environment.Based on the review of related study in the worldwide,a highly effective PAHs and PCBs-degrading bacterium was isolated.The isolate was identified as Janibacter sp.with respect to its 16S rDNA sequence and fatty acid profiles,as well as various physiological characteristics,and named JY11.Further study has been done systematically on degradation ability and relevant factors that affect the degradation performance. Co-degradation of PAHs and PCBs with JY11 and P chrysosporium JY16 was also researched.The probable pathway of PAHs and PCBs degraded by JY11 were proposed with detecting the metabolites by GC-MS.The main content and experimental results are as follow:
1.Extraction and analysis of PAHs from polluted soil and PCBs from transformer oil
(1) Soxhlet extraction,supereritical CO_2 extraction and ultrasonic extraction were selected to extract PAHs from polluted soil gathered from Jinan Oil Refinery Factory and Shengli Oil Field.And the extracts were analyzed by GC-FID.The results showed that:16 kinds of PAHs in Jinan Oil Refinery Factory were detected with a concentration of 5.0～593.2μg/kg;12 kinds of PAHs in Shengli Oil Field were detected with a concentration of 227.7～1107.3μg/kg.The spiked recovery of each method was in the range of 88.7～102.6%and the relative standard deviation(RSD%) was in the range of 1.5～4.4%,meeting the US EPA standard of recoveries(70～140%) and RSD of repeated samples(＜20%).
(2) Liquid-liquid extraction,ultrasonic extraction and solid phase extraction were selected to extract PCBs from Chinese transformer oil and the extracts were analyzed by GC-ECD.Based on the former study results on PCBs analysis in our laboratory,10 kinds of dioxin-like PCBs(PCB77,81,105,114,118,123,126,156, 167 and 189) and four common exist atropisomeric-PCBs were detected in the Chinese transformer oil.Other PCBs components were also analyzed with Arcolor 1242 standard.The recovery of internal standard of PCB209 with different extraction method was in the range of 82.3%～91.6%and the RSD%less than 5%,meeting the US EPA standard of recoveries and RSD of repeated samples.
2.Isolation and identification of PAHs and PCBs-degrading bacteria
16 PAHs-degrading strains were newly isolated from the polluted soil in Jinan Oil Refinery Factory,Shengli Oil Field and former Jinan Power Transformer Factory, Shandong Province of China.Two bacteria,JY11 and JY3A,were investigated. Strain JY11 was identified as Janibacter sp.with respect to its morphology,16S rDNA sequence,phylogenetic analysis,and fatty acid profiles,as well as various physiological and biochemical characteristics.The strain was Gram-positive, non-motile,non-spore-forming,short rods in young culture,0.8-1.0μm in diameter and 1.3-1.6μm long,and coccoid cells in the stationary phase of growth that are 1.0-1.2μm in diameter and 1.3-1.5μm long,occurred in pairs and sometimes in chains or in group,aerobic,oxidase-week positive,catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY11 was shown to be most closely related to Janibacter anophelis(99.93%),J.terrae(98.48%),J.marinus(98.38%),J. limosus(98.34%),J.melonis(98.20%) and J.corallicola(97.79%). Chemotaxonomic data(menaquinone,GC content and major fatty acids) supported the allocation of the strain to the genus Janibacter.
Strain JY3A was characterized as motile,rod-shaped,Gram-positive,endospore forming,single or in pairs and sometimes in chains,aerobic,oxidase-positive, catalase-positive.On the basis of 16S rDNA sequence similarity studies,strain JY3A was shown to be most closely related to B.vallismortis(99.69%),B.subtile (99.65%),B.amyloliquefaciens(99.60%),B.mojavensis(99.57%) and B.atrophaeus (99.42%).Chemotaxonomic data supported the proposal to assign the strain to the B. subtilis group of the genus Bacillus.
3.Study of degradation ability and related factors that affect the degradation performance of PAHs and PCBs by Janibacter sp.JY11
(1) Degradation of PAHs by Janibacter sp.JY11
PAHs with initial concentration of 100 mg/l were degraded with JY11 in liquid culture.The result showed that not only the low molecular PAHs,such as phenanthrene,fluorene and acenaphthene,but also pyrene,fluoranthene and benzo(a)anthracene have a good degradation result.Also the high molecular PAHs degraded in different degree.JY11 presented a wide degradation spectrum and high effective degradation ability.
(2) Effects of different initial concentrations,surfactant and salicylic acid on the degradation efficiency of PAHs were studied taking phenanthrene,pyrene and benzo(a)pyrene as representative.The results showed that the degradation efficiency of pyrene and benzo(a)pyrene decreased with the increase of initial concentration, while phenanthrene can be degraded completely with different initial concentrations. Tween 80 is helpful for the degradation of PAHs.Through adding salicylic acid in the medium,the degradation efficiency of benzo(a)pyrene increased 28%compared with phenanthrene as co-metabolized substance.
(3) Degradation of PCBs by Janibacter sp.JY11
The degradation efficiency of PCBs with a concentration of 10 mg/l and 100 mg/l was studied separately.The result showed that the degradation efficiency decreased in some degree with the increase of initial concentration of PCBs.But JY11 presented a good performance as a whole.For PCBs with an initial concentration of 10 mg/l,the degradation efficiencies of the di and triclorine-CBs were 83%～100%;the degradation efficiencies of the tetraclorine-CB were 78%～94%; and the degradation efficiencies of penta and hexaclorine-CB were 50～65%.The degradation efficiencies of PCBs decreased along with the increase of the number of chlorine atoms.
(4) In the inducing experiment by natural plant constituent,essential oil of Cordyceps kyushuensis and carvone can enhance the degradation efficiency notably; also,cymene is helpful in some degree.While,essential oil from Dracocephalum tanguticum and biphenyl nearly have no effect on the degradation efficiency.But essential oil from Amomum tsao-ko crevost et lemaire is harmful for degradation of PCBs by JY11.
4.Co-degradation of PAHs and PCBs by Janibacter sp.JY11 and P. chrysosporium JY16
Through adding white rot fungi,P.chrysosporium JY16,to the medium of JY11, the degradation threshold of PAHs and PCBs decreased and the degradation efficiency increased according to their co-degradation effect.
In liquid culture,the degradation efficiency of the mixed bacterium and fungi is much better than JY11 and JY16.Especially the degradation efficiency of four ring-pyrene and five ring-benzo(a)anthracene.Benzo(k)fluoranthene increased at least 20%compared with JY11.For the modified polluted soil sample,73%of pyrene and 60%of benzo(a)pyrene was degraded with mixed bacterium and fungi after thirty days cultivation.The degradation efficiency increase a lot compared with the sole bacterium or fungi.Similar result was got with PCBs.The degradation result with mixed culture is better than any sole bacterium or fungi.Especially for dioxin like PCB156,167 and 189,which could not be utilized by sole JY11 or JY16, decreased by 10 percent with co-culture.PCBs in the transformer oil and modified polluted soil were degraded by the mixed bacterium and fungi.The result showed that the degradation efficiencies of PCBs in transformer oil were 8.7%～83.3%.And the degradation efficiencies of PCBs in the modified polluted soil were 18.4%～72.6%.While PCB74,dl-PCB105,118 and atropisomeric-PCB132,149 showed no degradation.
5.Analysis of metabolites and proposed pathway of PAHs and PCBs degraded by Janibacter sp.JY11
Phenanthrene,pyrene and benzo(a)pyrene were selected as the research targets of PAHs degradation pathway.PCB 18 and PCB77 were selected as the targets of PCBs degradation pathway study.The proposed pathway of PAHs and PCBs was concluded by detecting the metabolites by GC-MS.
(1) Phenanthrene was transformed to 2,3-dihydroxy-2,3-dihydrophenanthrene firstly with dioxygenase of JY11.2,3-dihydroxy-2,3-dihydrophenanthrene was metabolized to 1-hydroxy-2-naphthoic acid,which transformed to 1-naphthol. 1-naphthol was cleaved to salicylic acid at last.
(2) Pyrene was transformed to 4,5-dihydroxy-4,5-dihydropyrene firstly with dioxygenase of JY11.Then 4,5-dihydroxy-4,5-dihydropyrene was metabolized to phenanthrene 4,5-dicarboxylic acid,which transformed to 4-hydroxyphenanthrene. 4-hydroxyphenanthrene was cleaved to phthalic acid at last.
(3) Benzo(a)pyrene was transformed to benzo(a)pyrene-7,8-dihydrodiol firstly with dioxygenase of JY11.And then benzo(a)pyrene-7,8-dihydrodiol was metabolized to benzo(a)pyrene-7,8-dihydroxy,which transformed to pyrene-7-hydroxy-8-carboxylic acid at last.
(4) PCB18 and PCB77 have a similar probable pathway,that is JY11 attacked the 2,3 position of PCBs and which metabolized to 2,3-dihydroxy-2,3-dihydro-PCB, then transformed to 2,3-dihydroxy-PCB,and then yielded to a yellow color product, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid,which was transformed to ehlorbenzoic acid under a hydrolase.
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