摘要
多环芳烃(PAHs)是一类广泛存在环境中的典型持久性有机有毒污染物。大多数的多环芳烃具有“三致”作用,通过食物链的传递会对生态环境和人体健康造成极大危害,因而这类化合物引起了人们的关注。生物修复特别是利用微生物降解被认为是去除环境中PAHs的主要途径,具有处理形式多样、成本低、对环境影响小等优点。尽管目前已发现环境中存在许多可降解PAHs的微生物,但也存在一些问题：一些以某种PAHs为唯一碳源筛选出来的单一优势菌种往往只能降解特定类型污染物且微生物活性受各种环境因素(如温度、酸度、盐度和湿度)的影响较大；或者将几种优势菌简单的混合构建高效菌群,多种菌的优化组合是一个很复杂的课题,这样的菌群有时因为种间的抑制作用很难实现作用最大化,有些菌株代谢PAHs的途径中产生了比母本毒性更高的中间产物。因此,如何获得高效降解PAHs的、作用底物范围广、环境适应性更强、具有积累少甚至不积累有毒中间代谢产物降解途径的菌株是值得研究的课题,将有助于提供PAHs污染环境的生物修复
为此,本论文的主要目的是期望获得环境适应能力强,能高效降解PAHs,并且具有积累有毒中间代谢产物较少的降解途径的一个新菌种。具体研究内容包括：以原生质体融合技术为基础,通过使用环境中重要的污染物降解者——假单胞菌(Pseudomonas sp)和鞘氨醇单胞菌(Sphingomonas sp)(二者皆为革兰氏阴性菌)作为亲本进行融合,研究了原生质体形成和再生的最佳条件、融合的方法和筛选鉴定融合子的方法；通过和不同菌种包括亲本对比降解PAHs的效果研究,分析了融合菌株的高效降解性能和环境的广泛适应性；通过研究融合菌株降解不同底物及多种PAHs的性能、代谢途径及代谢产物的产生和积累的影响,探讨了其降解PAHs的机理。主要研究结果如下：
(1)确定了亲本GP3A (Pseudomonas sp)和GY2B (Sphingomonas sp)原生质体最适形成和再生条件。选择对数生长期的菌体,经过青霉素G钠和EDTA的预处理,可以更容易地得到原生质体。当酶解温度为37℃,酶解浓度为5mg/L, GP3A的酶解时间为100min, GY2B的酶解时间为80min时,GP3A和GY2B的原生质体形成率最高。采用轻微摇动的酶解方式时菌株的原生质体形成率和再生率明显比水浴静置的方式时菌株的原生质体形成率和再生率高。添加一定浓度的Mg2+, Ca2+和L-丝氨酸有助于原生质体的再生。采用夹层培养方式的再生率明显高于单层培养和混合培养方式的再生率。
(2)结合抗药性实验和菲、芘的初步降解实验筛选了融合子并进行了形态学和分子生物学鉴定。通过对GP3A和GY2B进行抗药性研究,发现利用80μg/ml哌拉西林+80μg/ml头孢他啶或80μg/ml哌拉西林+(100～150μg/ml)红霉素的再生培养基可以筛选出融合子；进一步利用含有菲或芘的平板筛选出对菲和芘都有降解效果的融合子,再对这些融合子进行初步生物降解摇瓶实验,最后筛选出一株对菲和芘降解效果较高的融合子,并将其命名为F14。通过平板菌落形态、电子显微镜与扫描电子显微镜(SEM)观察和分子生物学技术PCR-RFLP分析鉴定出融合菌株F14与亲本菌株GP3A和GY2B为不同的菌种,是二者的融合子。
(3)F14降解菲、芘及混合PAHs的研究。F14可以将初始浓度为100mg/L的菲在24h降解99%以上,通过对比其它菌包括亲本GY2B,发现F14具有较高的降解菲的能力。F14在温度为20-40℃和pH为6.5-9的范围内对菲都有很好的降解效果。通过非竞争性抑制动力学方程对各初始浓度S0及对应比降解速率Rxo进行非线性拟合,得到的动力学参数降解动力学常数k=134.77mg菲g X-1h-1,饱和系数Ks=77.50mg/L,抑制系数KsI=742mg/L。较大的k和KSI值表明融合菌株F14对菲具有快速降解以及能耐受高浓度菲的抑制作用的能力。F14对初始浓度为15,50和100mg/L的芘在第10d的降解率分别为46%,37.1%和18%。芘的降解速率常数(k1)随着浓度的增加下降,芘降解速率常数和初始浓度有很好的线性关系。当菲和芘一起存在时,和单独降解菲和芘比较发现菲的存在对芘的降解有促进作用而芘的存在对菲有抑制的作用。
F14具有和亲本GY2B不同的菲降解途径。F14降解菲的代谢途径有两条：在双加氧酶的作用下,菲开环生成cis-3,4-phenanthrenedihydrodiol和cis-1,2-phenanthrenedihydrodiol然后分别转化为phenanthrene-3,4-diol和phenanthrene-1,2-diol。Phenanthrene-3,4-diol开环生成1-羟基-2-萘酸,再脱羧形成1-萘酚,开环后生成乙酰水杨酸。Phenanthrene-1,2-diol开环生成2-羟基-1-萘酸甲酯,再转化为乙酰水杨酸。乙酰水杨酸再生成水杨酸,然后通过邻苯二酚最后进入三羧酸循环(TCA)彻底降解为CO2和H2O。经过2-羟基-1-萘酸甲酯代谢是主要的代谢途径,2-羟基-1-萘酸的毒性比1-羟基-2-萘酸小,F14在降解菲的过程中积累的有毒的中间代谢产物少。F14降解混合的菲和芘,检测到了一种芘中间代谢产物4,5-二氢芘。
F14能够利用一定浓度的萘,水杨酸,2-羟基-1-萘酸和邻苯二酚为唯一碳源和能源进行生长和繁殖。芘的存在和芴、蒽、菲的存在对苊的降解有促进作用。其它三种三环芳烃的存在能分别促进蒽和芴的降解,芘的存在和其它六种PAHs的同时存在则分别对蒽和芴的降解有抑制作用。其它低环的多环芳烃的存在能够促进对芘的降解。F14对萘,菲和芘的混合,苊,蒽,菲和芴的混合,萘、苊、蒽、菲、芴、荧蒽和芘的混合都有一定的降解效果。
Polycyclic aromatic hydrocarbons (PAHs) are a class of organic pollutants which attractincreasing attention in recent years for their widespread occurrence in the environment. PAHsare of great environmental and human health concerns since many PAHs have been shown tobe toxic, mutagenic and carcinogenic, and can be accumulated and transferred throughout thefood chain. Bioremediation is considered as a cost-effective and environment-friendly processfor removing PAHs and is a major way in the environment. Though a large number ofbacteria capable of biodegrading PAHs have been isolated from the natural environment,there are still some problems. The bacteria utilizing one kind of PAHs as the sole carbonsource isolated from environment usually have high selectivity for compounds of PAHs andare not very flexible to the changes of their environmental variables. In addition, thehigh-efficiency bacteria can be constructed by mixing the dominant bacteria simply. However,the optimum combination of a variety of bacteria is a very complex and difficult subject. Forexample, it is difficult to achieve the maximum efficiency because of the interspecificinhibition, and some metabolites produced during the degradation process are more toxic thanPAHs themselves. Therefore, it is urgent to obtain a high-efficiency PAHs-degradingbacterium with high environmental adaptability and by which less or even non toxicmetabolites are produced during the degradation process.
The purpose of this study was to construct a high-efficiency PAHs-degrading bacteriumby protoplast fusion between Sphingomonas sp. GY2B and Pseudomonas sp. GP3A, which isflexible to the changes of their environmental variables, and capable of degrading multiplePAHs simultaneously. To improve the formation and regeneration frequency of protoplasts forGramnegative bacteria, the effects of some factors on protoplast isolation and regenerationwere investigated in present study. The degradation capability and environmental adaptabilityof the fusant strain were also tested by comparing with its parents and other strains. Finally,the PAHs degradation mechanism by the fusant strain was analyzed and discussed. The mainresearch results are as the followings:
(1) Pretreatment with penicillin G sodium salt and EDTA enhanced the protoplastformation of Bacterial strains of GY2B and GP3A. The optimal condition for GY2B andGP3A protoplast formation were in5mg/ml lysozyme at37°C for80min and100min in ashaking culture, respectively. The protoplast formation frequency was95.2%and99.6%forGP3A and GY2B in shaking cultures, while only28.1%and34.2%in the static culture. Areasonable concentration of calcium ions, magnesium ions and L-serine increased the regeneration frequency of protoplasts. Double layers culture (soft-agar overlayers) methodcould also significantly increase the regeneration frequency as compared with single or mixedculture method.
(2) The study on antibiotic resistance showed that fusants of GP3A and GY2B could bescreened using regeneration culture containing80g/ml piperacillin and80g/ml ceftazidine,or80g/ml piperacillin and erythrocin (100~150g/ml). Fusants were screened successfullythrough this method. Then the selected strains were inoculated in solid mineral mediumconsisting of single phenanthrene and pyrene. Subsequently, the strains with obvious clearzone were inoculated in liquid mineral medium containing phenanthrene and pyrene forscreening. A fusant strain with the highest degradation ability of phenanthrene and pyrene wasselected and named F14. The results of colony morphology, electron microscope, scanningelectron microscope (SEM) and molecular technology (PCR-RFLP) indicated that fusantstrain F14was different from its parents and was the fusant strain of GP3A and GY2B.
(3) Phenanthrene (100mg/L) could be almost completely degraded within24hours by thefusant strain F14, which was much quicker than GY2B and other strains. The fusant strainF14had a wider range of temperature (25-40°C) and pH value (6.5-9.0). Non-competitivesubstrate inhibition kinetics was found to be fit for phenanthrene biodegradation and thebiodegradation rate constant k was134.77mg phenanthrene g/X/h the saturation constant KSwas77.50mg/L and inhibition constant KSIwas742mg/L. A larger k and KSIvalue indicatesthat the culture could endure high concentration of phenanthrene. F14could degrade about46%,37.1%and18%pyrene after10d inoculation at the initial concentration of15,50and100mg/L, respectively. The degradation of phenanthrene and pyrene in mixture compoundsystems showed that phenanthrene degradation was clearly delayed in the presence of pyreneand pyrene degradation was appears enhanced in the presence of phenanthrene.
F14has different degradation pathway with GY2B. There are two pathway of degradingphenanthrene by F14: under the effect of dioxygenase, phenanthrene was cleaved andsubsequently metabolized to cis-3,4-phenanthrenedihydrodiol andcis-1,2-phenanthrenedihydrodiol, then transformed to phenanthrene-3,4-diol andphenanthrene-1,2-diol. Phenanthrene-3,4-diol was then cleaved to1-hydroxy-2-naphthoicacid, which was further decarboxylated to1-naphthol.1-Naphthol was cleaved toacetylsalicylic acid. Phenanthrene-1,2-diol was cleaved to methyl2-hydroxy-1-naphthoic acid,which was transformed to acetylsalicylic acid. Acetylsalicylic acid was transformed tosalicylic acid, which was further transformed to catechol and then accessed TCA-cycle andfinally exhaustive degraded to CO2and H2O. The toxicity of2-hydroxy-1-naphthoic acid was less than1-hydroxy-2-naphthoic acid indicating that phenanthrene was metabolized through apathway having less accumulation of potentially toxic metabolites by F14. An intermediateproduct4,5-dihydropyrene was detected during the degradation of mixture of phenanthreneand pyrene by F14.
The degradation tests of substrates diversity were evidenced that strain F14could usenaphthalene, salicylic acid,2-hydroxy-1-naphthoic acid and catechol as the sole carbon sourceexcept o-phthalate and quinol. The experiment of degrading mixed PAHs showed that thepresence of pyrene or mixture of fluorene, anthracene, and phenanthrene could enhance thedegradation of acenaphthene. The presence of other three PAHs could promote thedegradation of anthracene and fluorene, respectively. However, the presence of pyrene andother six PAHs could inhibit the degradation of anthracene and fluorene, respectively. Pyrenedegradation was enhanced in the presence of low-ring PAHs. F14could degrade the mixtureof naphthalene, phenanthrene and pyrene, the mixture of acenaphthene, anthracene,phenanthrene and fluorene, the mixture of naphthalene, acenaphthene, anthracene, fluorene,phenanthrene, fluoranthene and pyrene.
引文
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