几种污染环境下五氯酚的微生物修复研究
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摘要
五氯酚(pentachlorophenol,PCP)作为有机氯农药广泛地用作杀菌剂、杀螺剂、杀藻剂、杀虫剂和除草剂,此外也用作木材防腐剂等,从而进入水体、河流与湖泊的沉积物、土壤等环境。由于其化学性质稳定、残效期长、毒性高,对生物和人体具有广谱毒性和致突变性,五氯酚被认为是环境中主要的持久性有机污染物(POPs)及优先监测污染物之一,而其生物危害性及其在自然环境中的迁移、转化与降解等规律与降解和生物修复研究也一直是全世界研究和关注的热点。
白腐菌在对五氯酚这类难降解有机物的生物修复中显示出良好的应用前景。因为这种担子菌类降解污染物具有非专一性、彻底性、广谱性及适于固液两种基质等特点,它还有着独特的胞外酶系,对许多异生物质有独特的降解能力和降解机理,其中对白腐菌研究得最多的是黄孢原毛平革菌(Phanerochaetechrysosporium)。
本课题针对五氯酚污染的几种典型环境:农业废物、污水、河流底泥和土壤,模拟其污染清情况,分别用黄孢原毛平革菌的游离菌、所产漆酶和固定化菌对其进行处理和生物修复。而后探讨了湘江河流底泥吸附五氯酚的动力学和热力学特点,为评价和预测五氯酚在水环境中的迁移、归宿及人工控制这类污染物提供了可靠的理论依据。
本课题首先选取典型农业废物——稻草,利用稻草基质在固态发酵条件下研究白腐菌降解五氯酚的效果。通过研究发酵过程中黄孢原毛平革菌的生物量、酶活变化及降解五氯酚和木质素的降解情况发现黄孢原毛平革菌的生长趋势、酶活变化以及五氯酚和木质素的降解速度都呈基本一致的趋势,前10d生物量、酶活增长非常迅速,到第10d,达到最高,之后逐渐下降,五氯酚和木质素的降解率也随之变化,证明了微生物的生长情况、酶系的活性是和降解率紧密相关的。同时紫外光谱和红外光谱分析结果也表明在白腐菌的作用下,稻草中的木质素结构遭到严重破坏,难降解的大分子长键烃被切断成易降解的小分子短键烃。因而,白腐菌在降解五氯酚的同时,也达到了处理难降解稻草木质素的独特效果。
其次研究了黄孢原毛平革菌产漆酶的优化条件,以及所产出的漆酶对水中五氯酚的降解。黄孢原毛平革菌虽然长时间被认为是不能产漆酶的白腐真菌,但本研究证明它在特定培养基上——利用农、林、食品工业的残渣(香蕉皮和玉米芯)作为固态发酵的底物,具有较好的产漆酶能力。这样既解决了这些含碳水化合物高的废物引起的环境的问题,又能生产更有利用价值的真菌漆酶。当香蕉皮与玉米芯混合比例为1∶2、诱导剂为0.4mMCuSO4时,黄孢原毛平革菌能获得最高漆酶活,使用硫酸铜和藜芦醇做诱导剂都可提高漆酶酶活。同时利用该条件下所产的漆酶酶液对五氯酚污染的污水进行了处理,比较了粗酶液与纯化酶液以及添加介质与不添加介质体的处理效果,发现纯化漆酶优于粗酶液,添加介质ABTS能提高漆酶活性,提高处理率。利用低成本的农林业残渣作为发酵底物生产真菌漆酶并应用到污染物修复中去,这将是一条既经济又高效的途径,对环境污染治理产生一定意义。
然后探讨了固定化和游离黄孢原毛平革菌对五氯酚降解效果及堆制过程中添加黄孢原毛平革菌和未加菌时各种参数的变化,包括有机质、碳氮比、木质纤维素、腐殖质、种子发芽、微生物量碳等。在经过近两个月的堆制后,堆料达到腐熟和稳定并基本消除五氯酚的毒害。实验结果说明了堆肥是对五氯酚污染的土壤进行生物修复的一种有效方法,在堆肥中添加白腐菌有助于木质纤维素的降解和腐殖质的形成,而且固定化技术也是生物修复中可行的方法之一。
最后对湘江长沙河段5个断面沉积物样品进行了初步分析,研究了样品的物理化学性质,吸附动力学性质,吸附等温线类型以及吸附热力学性质。研究分别用抛物线模型和线性模型对五氯酚吸附动力学进行拟合,结果是线性方程对湘江沉积物的拟合情况更好,实验还分别测定了五氯酚在湘江不同沉积物上的吸附等温线,并分别用线性吸附模型、非线性的Freundlich吸附模型、双模式吸附模型行了拟合,可以推断出在沉积物吸附五氯酚过程中发挥主要作用的并不是线性分配作用,而是非线性吸附,其符合Freundlich吸附等温式。由吸附热力学实验得到五氯酚在湘江沉积物上的吸附热值,由此推断五氯酚在湘江沉积物的吸附主要为物理吸附,主要吸附机理为疏水键力,同时一定程度上存在范德华力、偶极间力和氢键力。这一研究对评价和预测五氯酚在水环境中的迁移、归宿及处理这类污染物可提供可靠的理论依据和参考。
Pentachlorophenol(PCP),as one of the organochlorine pesticides,has been widely used as fungicide,molluscicide,algaecide,pesticide,herbicide and wood preservatives,etc.,thereafter it enters the environment such as water,soil and the sediment of the rivers and lakes.But due to its steady chemical feature,long persistence and high toxicity,it is of wide toxicity and mutagenicity to human being and animals.PCP is considered as the one of the main POPs in the environment and one of the priority pollutants.Therefore the biohazards,transportation,transformation and degradation of PCP have been the hot topic all over the world.
While white rot fungi have shown great potential in the degradation of the hard-to-be-degraded organic matters like PCP.Because this kind of Basidiomycetes has very special features,such as non-specific,degrading pollutants completely,wide range of target pollutants,suitable for both liquid and solid substrate and excreting special extracellular enzymes,it can degrade many kinds of xenobiotics.Among the researched fungi,Phanerochaete chrysosporium is the one receiving the most concern.
According to different typical environments for PCP,that is agricultural waste, wastewater,river sediment and soil,free Phanerochaete chrysosporium,laccase generated from Phanerochaete chrysosporium and immobilized Phanerochaete chrysosporium were respectively added for the treatment and bioremediation.The adsorption kinetics and thermodynamic of Xiang river sediment on PCP was also investigated,which can provide the theoretical reference for the assessment and prediction of the transportation,treatment of PCP in water environment.
Firstly,the degradation effect of Phanerochaete chrysosporium on PCP and lignin in agricultural waste with straws through solid-state fermentation was investigated.It is found that the change of biomass,enzyme activities and degradation ratio of PCP and lignin are in a similar tendency.In the first 10 days the biomass and enzyme activity increased quite fast and reached the maximum on the 10~(th)day and afterwards they decreased gradually.The degradation ration of PCP and lignin changed correspondingly.It means there's close relationship among the strain growth and enzyme activity and degradation ratio.By ultraviolet spectrum and infrared spectrum analysis it can be seen that the lignin structure in the straw has been destroyed by P.chrysosporium,the difficult-to-be-degraded big-molecule long-bond hydrocarbons were cutted into small-molecule short-bond hydrocarbons.That means P. chrysosporium can degrade both PCP and straw lignin.
Secondly,the optimum condition for the laccase generation by P.chrysosporium was investigated,and the laccase generated under the optimum condition was applied in the treatment of the PCP-polluted water.Although it has been considered in the past that P.chrysosporiumon can not generate laccase,the result obtained in the experiment proved that under specific condition laccase can be generated by P. chrysosporium.The optimum condition for the laccase generation is as follows:the mixture of banana skin and corncob is 1∶2 and with the inducer of 0.4mM CuSO_4. Using either CuSO_4 or veratryl alcohol as inducer the laccase activity will be enhanced.The draft and purified laccase obtained under the above condition were applied for the PCP-contaminated water treatment.The purified laccase functioned better than draft laccase,and the medium-ABTS could also help the function of laccase.Making use of low-cost agriculture waste as the fermentation substrate to produce enzyme is a both economical and high-efficient method,that is it can not only solve the environmental problems caused by the agriculture waste,but also produce valuable enzyme.
Besides,the treatment effect of immobilized and free P.chrysosporium on PCP and the change of parameters,such as organic matters,C/N,lignin,cellulose,humus, seed germination and microbe biomass in the composting were investigated.After 2-month composting,the compost is mature and the toxicity of PCP is also almost eliminated.It can be concluded from the experiment that composting is an effective bioremediation method for the PCP-polluted soil and adding P.chrysosporium will also facilitate the degradation of lignin,cellulose and the generation of humus,and the immobilization method is also feasible in the bioremediation.
Finally five sediment samples from Xiang river in Changsha was analyzed and the physical and chemical features and adsorption kinetics and thermodynamic of Xiang river sediment on PCP was also investigated.The linear model can fit the adsorption kinetics better than Parabola model.The adsorption isotherm was also obtained and fitting with linear adsorption model,non-linear Freundlich adsorption model,Dual Reactive Domain Model,from which it can be concluded that the adsorption of PCP on sediment complies with Freundlich adsorption model,which is non-linear.The adsorption calorific value was obtained by the thermodynamic experiment and it can be concluded that the main adsorption mechanism is the physical adsorption.The adsorption is mainly the action of hydrophilic bond, meanwhile van der waals forces,hydrogen bond forces and dipole bond forces may also exist.The result can be provided as a reference for the estimation,assessment and prediction of the transportation,artificial control of PCP in water environment.
白腐菌在对五氯酚这类难降解有机物的生物修复中显示出良好的应用前景。因为这种担子菌类降解污染物具有非专一性、彻底性、广谱性及适于固液两种基质等特点,它还有着独特的胞外酶系,对许多异生物质有独特的降解能力和降解机理,其中对白腐菌研究得最多的是黄孢原毛平革菌(Phanerochaetechrysosporium)。
本课题针对五氯酚污染的几种典型环境:农业废物、污水、河流底泥和土壤,模拟其污染清情况,分别用黄孢原毛平革菌的游离菌、所产漆酶和固定化菌对其进行处理和生物修复。而后探讨了湘江河流底泥吸附五氯酚的动力学和热力学特点,为评价和预测五氯酚在水环境中的迁移、归宿及人工控制这类污染物提供了可靠的理论依据。
本课题首先选取典型农业废物——稻草,利用稻草基质在固态发酵条件下研究白腐菌降解五氯酚的效果。通过研究发酵过程中黄孢原毛平革菌的生物量、酶活变化及降解五氯酚和木质素的降解情况发现黄孢原毛平革菌的生长趋势、酶活变化以及五氯酚和木质素的降解速度都呈基本一致的趋势,前10d生物量、酶活增长非常迅速,到第10d,达到最高,之后逐渐下降,五氯酚和木质素的降解率也随之变化,证明了微生物的生长情况、酶系的活性是和降解率紧密相关的。同时紫外光谱和红外光谱分析结果也表明在白腐菌的作用下,稻草中的木质素结构遭到严重破坏,难降解的大分子长键烃被切断成易降解的小分子短键烃。因而,白腐菌在降解五氯酚的同时,也达到了处理难降解稻草木质素的独特效果。
其次研究了黄孢原毛平革菌产漆酶的优化条件,以及所产出的漆酶对水中五氯酚的降解。黄孢原毛平革菌虽然长时间被认为是不能产漆酶的白腐真菌,但本研究证明它在特定培养基上——利用农、林、食品工业的残渣(香蕉皮和玉米芯)作为固态发酵的底物,具有较好的产漆酶能力。这样既解决了这些含碳水化合物高的废物引起的环境的问题,又能生产更有利用价值的真菌漆酶。当香蕉皮与玉米芯混合比例为1∶2、诱导剂为0.4mMCuSO4时,黄孢原毛平革菌能获得最高漆酶活,使用硫酸铜和藜芦醇做诱导剂都可提高漆酶酶活。同时利用该条件下所产的漆酶酶液对五氯酚污染的污水进行了处理,比较了粗酶液与纯化酶液以及添加介质与不添加介质体的处理效果,发现纯化漆酶优于粗酶液,添加介质ABTS能提高漆酶活性,提高处理率。利用低成本的农林业残渣作为发酵底物生产真菌漆酶并应用到污染物修复中去,这将是一条既经济又高效的途径,对环境污染治理产生一定意义。
然后探讨了固定化和游离黄孢原毛平革菌对五氯酚降解效果及堆制过程中添加黄孢原毛平革菌和未加菌时各种参数的变化,包括有机质、碳氮比、木质纤维素、腐殖质、种子发芽、微生物量碳等。在经过近两个月的堆制后,堆料达到腐熟和稳定并基本消除五氯酚的毒害。实验结果说明了堆肥是对五氯酚污染的土壤进行生物修复的一种有效方法,在堆肥中添加白腐菌有助于木质纤维素的降解和腐殖质的形成,而且固定化技术也是生物修复中可行的方法之一。
最后对湘江长沙河段5个断面沉积物样品进行了初步分析,研究了样品的物理化学性质,吸附动力学性质,吸附等温线类型以及吸附热力学性质。研究分别用抛物线模型和线性模型对五氯酚吸附动力学进行拟合,结果是线性方程对湘江沉积物的拟合情况更好,实验还分别测定了五氯酚在湘江不同沉积物上的吸附等温线,并分别用线性吸附模型、非线性的Freundlich吸附模型、双模式吸附模型行了拟合,可以推断出在沉积物吸附五氯酚过程中发挥主要作用的并不是线性分配作用,而是非线性吸附,其符合Freundlich吸附等温式。由吸附热力学实验得到五氯酚在湘江沉积物上的吸附热值,由此推断五氯酚在湘江沉积物的吸附主要为物理吸附,主要吸附机理为疏水键力,同时一定程度上存在范德华力、偶极间力和氢键力。这一研究对评价和预测五氯酚在水环境中的迁移、归宿及处理这类污染物可提供可靠的理论依据和参考。
Pentachlorophenol(PCP),as one of the organochlorine pesticides,has been widely used as fungicide,molluscicide,algaecide,pesticide,herbicide and wood preservatives,etc.,thereafter it enters the environment such as water,soil and the sediment of the rivers and lakes.But due to its steady chemical feature,long persistence and high toxicity,it is of wide toxicity and mutagenicity to human being and animals.PCP is considered as the one of the main POPs in the environment and one of the priority pollutants.Therefore the biohazards,transportation,transformation and degradation of PCP have been the hot topic all over the world.
While white rot fungi have shown great potential in the degradation of the hard-to-be-degraded organic matters like PCP.Because this kind of Basidiomycetes has very special features,such as non-specific,degrading pollutants completely,wide range of target pollutants,suitable for both liquid and solid substrate and excreting special extracellular enzymes,it can degrade many kinds of xenobiotics.Among the researched fungi,Phanerochaete chrysosporium is the one receiving the most concern.
According to different typical environments for PCP,that is agricultural waste, wastewater,river sediment and soil,free Phanerochaete chrysosporium,laccase generated from Phanerochaete chrysosporium and immobilized Phanerochaete chrysosporium were respectively added for the treatment and bioremediation.The adsorption kinetics and thermodynamic of Xiang river sediment on PCP was also investigated,which can provide the theoretical reference for the assessment and prediction of the transportation,treatment of PCP in water environment.
Firstly,the degradation effect of Phanerochaete chrysosporium on PCP and lignin in agricultural waste with straws through solid-state fermentation was investigated.It is found that the change of biomass,enzyme activities and degradation ratio of PCP and lignin are in a similar tendency.In the first 10 days the biomass and enzyme activity increased quite fast and reached the maximum on the 10~(th)day and afterwards they decreased gradually.The degradation ration of PCP and lignin changed correspondingly.It means there's close relationship among the strain growth and enzyme activity and degradation ratio.By ultraviolet spectrum and infrared spectrum analysis it can be seen that the lignin structure in the straw has been destroyed by P.chrysosporium,the difficult-to-be-degraded big-molecule long-bond hydrocarbons were cutted into small-molecule short-bond hydrocarbons.That means P. chrysosporium can degrade both PCP and straw lignin.
Secondly,the optimum condition for the laccase generation by P.chrysosporium was investigated,and the laccase generated under the optimum condition was applied in the treatment of the PCP-polluted water.Although it has been considered in the past that P.chrysosporiumon can not generate laccase,the result obtained in the experiment proved that under specific condition laccase can be generated by P. chrysosporium.The optimum condition for the laccase generation is as follows:the mixture of banana skin and corncob is 1∶2 and with the inducer of 0.4mM CuSO_4. Using either CuSO_4 or veratryl alcohol as inducer the laccase activity will be enhanced.The draft and purified laccase obtained under the above condition were applied for the PCP-contaminated water treatment.The purified laccase functioned better than draft laccase,and the medium-ABTS could also help the function of laccase.Making use of low-cost agriculture waste as the fermentation substrate to produce enzyme is a both economical and high-efficient method,that is it can not only solve the environmental problems caused by the agriculture waste,but also produce valuable enzyme.
Besides,the treatment effect of immobilized and free P.chrysosporium on PCP and the change of parameters,such as organic matters,C/N,lignin,cellulose,humus, seed germination and microbe biomass in the composting were investigated.After 2-month composting,the compost is mature and the toxicity of PCP is also almost eliminated.It can be concluded from the experiment that composting is an effective bioremediation method for the PCP-polluted soil and adding P.chrysosporium will also facilitate the degradation of lignin,cellulose and the generation of humus,and the immobilization method is also feasible in the bioremediation.
Finally five sediment samples from Xiang river in Changsha was analyzed and the physical and chemical features and adsorption kinetics and thermodynamic of Xiang river sediment on PCP was also investigated.The linear model can fit the adsorption kinetics better than Parabola model.The adsorption isotherm was also obtained and fitting with linear adsorption model,non-linear Freundlich adsorption model,Dual Reactive Domain Model,from which it can be concluded that the adsorption of PCP on sediment complies with Freundlich adsorption model,which is non-linear.The adsorption calorific value was obtained by the thermodynamic experiment and it can be concluded that the main adsorption mechanism is the physical adsorption.The adsorption is mainly the action of hydrophilic bond, meanwhile van der waals forces,hydrogen bond forces and dipole bond forces may also exist.The result can be provided as a reference for the estimation,assessment and prediction of the transportation,artificial control of PCP in water environment.
引文
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