两株优势菌对多环芳烃的降解机理研究
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摘要
多环芳烃(polycycli aromatic hydrocarbon,PAHs)是环境中普遍存在的一类具有“三致”作用的污染物,主要来源于石化产品的不完全燃烧和利用。随着现代工业的迅速发展,多环芳烃对环境的污染日益严重。目前各国科学工作者普遍认为,微生物降解法是去除环境中多环芳烃的最有效途径。
实验室在前期工作中,以长期被焦化废水污染的污泥为优良菌源、利用平板划线分离法筛选出一批可有效降解葸、菲、芘的多环芳烃优势降解菌。本论文以实验室保存的两株多环芳烃优势降解菌——黄杆菌CN4和芽胞杆菌CN2为研究对象,研究了两株菌的生物降解特性、生物降解动力学特征、生物降解过程中产生的中间产物、微生物的产酶特征、酶促降解特性及酶促降解动力学特征;对两株菌降解PAHs的生物降解和酶促降解影响因素进行了分析比较；讨论了菌体降解动力学和酶促降解动力学的区别与联系。研究结果对深入理解多环芳烃的生物降解机理及加速环境中多环芳烃的去除提供了一定的理论基础。
论文采用培养基扩大培养法获取两株菌的菌悬液,利用紫外光度法研究了影响多环芳烃在水相中生物降解的环境因素、不同碳源培养条件下收集的菌体对多环芳烃的降解影响及微生物降解动力学特征。研究结果表明,在MSM培养基中单独以葡萄糖作为碳源,以及以葡萄糖和芘作为共同碳源进行CN4菌、CN2菌培养,发现共同碳源培养条件下得到的菌体对多环芳烃的降解效果更好。以葸的降解为例,利用共同碳源培养基培养48h后的CN4菌对多环芳烃进行降解,反应120h后,葸的去除率为57.4%,是相同条件下以葡萄糖为单一碳源时降解效果的1.5倍；利用共同碳源培养96h的CN2菌进行降解反应72h后,蒽的去除率达到70%,是以葡萄糖为单一碳源时降解效率的1.8倍。
CN4菌降解多环芳烃的最适pH为5,在pH=5时,CN4对葸、菲、芘的去除率分别为27.6%、33.5%、26.4%;CN2菌降解多环芳烃的最适pH为6,在pH=6时,CN2对葸、菲、芘的去除率分别为33.5%、46.5%、36.5%;pH为5-7时,两株茵对多环芳烃的降解效果较好：在碱性条件下两株菌对多环芳烃的降解受到抑制；在菌量为1.0×107CFUs·mL-1、当体系中葸、菲、芘浓度超过200mg/L时,降解反应受到抑制；在相同菌量及最佳pH条件下,CN2菌对同种PAH的降解效果优于CN4菌。
分别利用吹扫捕集进样-GC/MS法、液液萃取-GC/MS法及红外光谱法分析了CN4菌、CN2降解菲的中间产物。并根据中间产物对多环芳烃的降解途径进行了推测。结果表明,黄杆菌CN4菌降解菲时,产生香豆素、邻苯二甲酸甲酯、苯乙烯酸、羟基萘酸、原儿茶酸、二氢醇菲、菲二醇等中间代谢物质,芽孢杆菌CN2降解菲的中性部分中,主要检测到的中间产物包括邻苯二甲酸甲酯和菲醌类物质。根据降解时有菲二醇或菲醌产生可推测两种菌在降解菲时,都是首先通过双加氧酶的作用在芳环上加氧,然后发生环裂解反应生成有机羧酸类物质,最终生成C02和水；黄杆菌CN4代谢菲时,通过双加氧酶的作用产生菲二醇类物质,最终发生环裂解反应生成原儿茶酸进行进一步的代谢；而芽孢杆菌CN2代谢菲时,通过双加氧酶的作用产生菲醌,然后形成邻苯二甲酸甲酯物质进行代谢。中间产物的不同说明两株菌中存在着不同的酶系。
对两株菌所产活性酶进行了定位,确定了两株菌所产活性酶为胞内酶。并确定了用超声波破碎法获得胞内酶的工艺。超声波破碎法提取酶的条件为：破碎功率为260W,最大不超过280W。破碎总时间为15min,工作／间隙时间为30s/30s。
利用紫外光度法研究了水相中多环芳烃酶促降解的动力学特性。研究结果表明,两株菌所产酶降解PAH的最佳pH条件为弱酸性至中性(5.0-7.0),最适温度为32℃,在30℃-35℃之间胞内酶保持较高的降解活性,反应温度超过50℃时,蛋白酶迅速失活。在最适pH和温度条件下,同种酶对不同浓度的相同底物酶促反应的表观反应级数基本相同。CN2酶促降解体系中,蒽、菲、芘的平均表观反应级数分别为0.933,0.995,0.93；CN4酶促降解体系中,葸、菲、芘的平均表观反应级数分别为0.93,0.76,0.958。一定条件下,CN4胞内酶对葸的降解速率大于对菲的降解速率,对芘的降解速率最小；CN2胞内酶对蒽和菲的降解速率基本相等,对芘的降解速率最小。
研究了金属离子对多环芳烃生物降解和酶促降解的影响。实验结果表明Mn2+离子的投加能够提高两株菌对蒽、菲、芘的去除率。Ca2+的加入对两株菌所产酶降解蒽、菲、芘均可起到促进作用。在外加Ca2+的浓度为0.5 mmol/L时,CN4所产胞内酶对葸、菲、芘的去除率分别为不加金属离子时的1.3倍、1.9倍、1.8倍；CN2所产胞内酶对蒽、菲、芘的去除率分别为不加金属离子时的1.4倍、1.6倍、1.4倍。激活作用的机理主要是由于金属离子在底物与酶之间起到了某种搭桥作用,它先与酶结合,再与底物结合,形成酶-金属-底物的复合物,从而对酶促降解起到激活作用。
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, some of which are highly carcinogenic, genotoxic, and a threat to public health. The major sources of PAHs in the environment are the combustion of organic matter and the processing and use of fossil fuels. Chemical-physical as well as biological methods are used to remove PAHs, Above all biological methods are favored because of good results and low costs. In our previous research work, we have obtained a set of dominant bacteria from sludge contaminated by coke plant wastewater which could degrade PAHs effectively. On that basis, we further researched biodegradation and enzymatic degradation characteristics for Flavobacterium sp CN4 and Bacillus CN2, which were isolated from coke plant sludge. The results provide theoretical basis for understanding PAHs biodegradation mechanism.
Using beef extract peptone medium to culture two strains which were maintained on beef-peptone agar slants, and the bacteria-suspended buffer of two strains were respectively prepared. Two bacteria also could grow with pyrene or glucose as alternative carbon sources. And pyrene-grown strains produced higher levels of PAHs degradation activity than glucose-grown bacteria. After 5-d degradation, the removal efficiency of anthracene by CN4 which were grown with pyrene as the sole carbon source for 48-h was 1.50-fold more than the bacteria were cultured with glucose as the sole carbon source. And 96-h incubation CN2 by using pyrene and glucose as carbon source, the removal efficiency of anthracene within 72-h by CN2 was 1.80-fold more than the bacteria grown on glucose. The main influence factors of PAHs biodegradation and kinetic characteristics in aqueous were investigated by using UV spectrophotometer. The results showed CN4 and CN2 had higher activity for degrading anthracene, phenanthrene, pyrene at pH 5-7. At pH 5, the removal efficiency of anthracene, phenanthrene, pyrene by CN4 was 27.6%、33.5%、26.4% within 24h, respectively,. At pH 6, the removal efficiency of anthracene, phenanthrene, pyrene by CN2 was 33.5%、46.5%、36.5% within 24h, respectively. Biodegradation was inhibited When PAHs concentrations was over 200mg/L and cell concentration was 1.0×107CFUs·mL-1.
Phenanthrene metabolites by Flavobacterium sp CN4 were assayed by using Purge and Trap combing with gas chromatography-mass spectrometry (GC-MS). Metabolism of phenanthrene by Bacillus CN2 were analyzed using liquid-liquid extraction combing with GC-MS and Fourier transform infrared spectroscopy (FTIR). In phenathrene degradation by CN4, the major metabolites include Coumarin, Phthalates, cinnamate, hydroxy-naphthoic acid, protocatechuic acid, phenanthrenedihydrodiol, phenanthrenediol. And the main metabolites were Phthalates and phenanthrenequinone when phenathrene was degraded by CN2. The change of functional groups between biodegradation metabolites and phenanthrene was given by comparison of FTIR spectra of phenanthrene with metabolites. Degradation pathway was proposed by identifying metabolites of phenanthrene. Degradation of phenanthrene by CN4 and CN2 were both initiated by dioxygenation, and then ring cleavages were happened to produce carboxylic acid. However, the metabolites of phenathrne by two strains were not quite the same. This indicated two strains had different enzyme system.
To detect active enzymes which were produced by two strains, salting out method and ultrasonication were respectively used to obtained extracellular enzyme and endoenzyme, and PAH enzyme degradation experiments were executed. The results showed Coarse endoenzyme excreted from two strains could degrade pyrene effectively. Within 30 minutes, the removal rates of the pyrene were 26.5% and 20.8% by endoenzyme produced from CN4 and CN2, respectively. However, the extracellular enzyme didn't show degradation activity toward pyrene within 6 hours. These results showed endoenzymes excreted from two strains were active enzyme. To obtained endoezyme, the optimum ultrasonication conditions were as follows:CN4 or CN2 bacterial suspensions were subjected to sonication for 15 min on ice with a cell ultrasonifier, the amplitude 260W(280W at most) and 30-s pulse intervals. The lysate was centrifuged for 20min at 11000 r·min-1 to remove cell debris.
Enzymatic-degradation kinetic characteristics of PAHs in aqueous were investigated. At 32℃and pH 6, the endoenzyme was the most active. The coarse endoenzyme of two strains had higher activity for degrading anthracene, phenanthrene, pyrene in weak acid medium at a temperature between 30℃and 35℃. The endoenzyme enzyme lost degrading activity when temperatures exceed 50℃. Enzymatic degradation reaction rate keep linear relation with PAH concentrations when substrate concentrations were not more than 200mg/L. The average initial rates were increasing with PAH initial concentration, but the average initial rates were decreasing with time. The apparent reaction orders were roughly the same when enzymatic degradation time was different.
Various metal ions were investigated as additives which could potentially enhance enzyme activity. It was found that Ca2+ could markedly activate the coarse enzymes. As 0.5mmol/L Ca2+ was added, the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN4 were improved by 1.3,1.9 and 1.8 times, respectively. And the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN2 were improved by 1.4,1.6 and 1.4 times, respectively. The mechanism of this result is that metal ion provide a bridge for enzyme to substrate, Which bind enzyme to form ion-enzyme complex. Then bind substrate to form enzyme-ion-substrate complex.
实验室在前期工作中,以长期被焦化废水污染的污泥为优良菌源、利用平板划线分离法筛选出一批可有效降解葸、菲、芘的多环芳烃优势降解菌。本论文以实验室保存的两株多环芳烃优势降解菌——黄杆菌CN4和芽胞杆菌CN2为研究对象,研究了两株菌的生物降解特性、生物降解动力学特征、生物降解过程中产生的中间产物、微生物的产酶特征、酶促降解特性及酶促降解动力学特征;对两株菌降解PAHs的生物降解和酶促降解影响因素进行了分析比较；讨论了菌体降解动力学和酶促降解动力学的区别与联系。研究结果对深入理解多环芳烃的生物降解机理及加速环境中多环芳烃的去除提供了一定的理论基础。
论文采用培养基扩大培养法获取两株菌的菌悬液,利用紫外光度法研究了影响多环芳烃在水相中生物降解的环境因素、不同碳源培养条件下收集的菌体对多环芳烃的降解影响及微生物降解动力学特征。研究结果表明,在MSM培养基中单独以葡萄糖作为碳源,以及以葡萄糖和芘作为共同碳源进行CN4菌、CN2菌培养,发现共同碳源培养条件下得到的菌体对多环芳烃的降解效果更好。以葸的降解为例,利用共同碳源培养基培养48h后的CN4菌对多环芳烃进行降解,反应120h后,葸的去除率为57.4%,是相同条件下以葡萄糖为单一碳源时降解效果的1.5倍；利用共同碳源培养96h的CN2菌进行降解反应72h后,蒽的去除率达到70%,是以葡萄糖为单一碳源时降解效率的1.8倍。
CN4菌降解多环芳烃的最适pH为5,在pH=5时,CN4对葸、菲、芘的去除率分别为27.6%、33.5%、26.4%;CN2菌降解多环芳烃的最适pH为6,在pH=6时,CN2对葸、菲、芘的去除率分别为33.5%、46.5%、36.5%;pH为5-7时,两株茵对多环芳烃的降解效果较好：在碱性条件下两株菌对多环芳烃的降解受到抑制；在菌量为1.0×107CFUs·mL-1、当体系中葸、菲、芘浓度超过200mg/L时,降解反应受到抑制；在相同菌量及最佳pH条件下,CN2菌对同种PAH的降解效果优于CN4菌。
分别利用吹扫捕集进样-GC/MS法、液液萃取-GC/MS法及红外光谱法分析了CN4菌、CN2降解菲的中间产物。并根据中间产物对多环芳烃的降解途径进行了推测。结果表明,黄杆菌CN4菌降解菲时,产生香豆素、邻苯二甲酸甲酯、苯乙烯酸、羟基萘酸、原儿茶酸、二氢醇菲、菲二醇等中间代谢物质,芽孢杆菌CN2降解菲的中性部分中,主要检测到的中间产物包括邻苯二甲酸甲酯和菲醌类物质。根据降解时有菲二醇或菲醌产生可推测两种菌在降解菲时,都是首先通过双加氧酶的作用在芳环上加氧,然后发生环裂解反应生成有机羧酸类物质,最终生成C02和水；黄杆菌CN4代谢菲时,通过双加氧酶的作用产生菲二醇类物质,最终发生环裂解反应生成原儿茶酸进行进一步的代谢；而芽孢杆菌CN2代谢菲时,通过双加氧酶的作用产生菲醌,然后形成邻苯二甲酸甲酯物质进行代谢。中间产物的不同说明两株菌中存在着不同的酶系。
对两株菌所产活性酶进行了定位,确定了两株菌所产活性酶为胞内酶。并确定了用超声波破碎法获得胞内酶的工艺。超声波破碎法提取酶的条件为：破碎功率为260W,最大不超过280W。破碎总时间为15min,工作／间隙时间为30s/30s。
利用紫外光度法研究了水相中多环芳烃酶促降解的动力学特性。研究结果表明,两株菌所产酶降解PAH的最佳pH条件为弱酸性至中性(5.0-7.0),最适温度为32℃,在30℃-35℃之间胞内酶保持较高的降解活性,反应温度超过50℃时,蛋白酶迅速失活。在最适pH和温度条件下,同种酶对不同浓度的相同底物酶促反应的表观反应级数基本相同。CN2酶促降解体系中,蒽、菲、芘的平均表观反应级数分别为0.933,0.995,0.93；CN4酶促降解体系中,葸、菲、芘的平均表观反应级数分别为0.93,0.76,0.958。一定条件下,CN4胞内酶对葸的降解速率大于对菲的降解速率,对芘的降解速率最小；CN2胞内酶对蒽和菲的降解速率基本相等,对芘的降解速率最小。
研究了金属离子对多环芳烃生物降解和酶促降解的影响。实验结果表明Mn2+离子的投加能够提高两株菌对蒽、菲、芘的去除率。Ca2+的加入对两株菌所产酶降解蒽、菲、芘均可起到促进作用。在外加Ca2+的浓度为0.5 mmol/L时,CN4所产胞内酶对葸、菲、芘的去除率分别为不加金属离子时的1.3倍、1.9倍、1.8倍；CN2所产胞内酶对蒽、菲、芘的去除率分别为不加金属离子时的1.4倍、1.6倍、1.4倍。激活作用的机理主要是由于金属离子在底物与酶之间起到了某种搭桥作用,它先与酶结合,再与底物结合,形成酶-金属-底物的复合物,从而对酶促降解起到激活作用。
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, some of which are highly carcinogenic, genotoxic, and a threat to public health. The major sources of PAHs in the environment are the combustion of organic matter and the processing and use of fossil fuels. Chemical-physical as well as biological methods are used to remove PAHs, Above all biological methods are favored because of good results and low costs. In our previous research work, we have obtained a set of dominant bacteria from sludge contaminated by coke plant wastewater which could degrade PAHs effectively. On that basis, we further researched biodegradation and enzymatic degradation characteristics for Flavobacterium sp CN4 and Bacillus CN2, which were isolated from coke plant sludge. The results provide theoretical basis for understanding PAHs biodegradation mechanism.
Using beef extract peptone medium to culture two strains which were maintained on beef-peptone agar slants, and the bacteria-suspended buffer of two strains were respectively prepared. Two bacteria also could grow with pyrene or glucose as alternative carbon sources. And pyrene-grown strains produced higher levels of PAHs degradation activity than glucose-grown bacteria. After 5-d degradation, the removal efficiency of anthracene by CN4 which were grown with pyrene as the sole carbon source for 48-h was 1.50-fold more than the bacteria were cultured with glucose as the sole carbon source. And 96-h incubation CN2 by using pyrene and glucose as carbon source, the removal efficiency of anthracene within 72-h by CN2 was 1.80-fold more than the bacteria grown on glucose. The main influence factors of PAHs biodegradation and kinetic characteristics in aqueous were investigated by using UV spectrophotometer. The results showed CN4 and CN2 had higher activity for degrading anthracene, phenanthrene, pyrene at pH 5-7. At pH 5, the removal efficiency of anthracene, phenanthrene, pyrene by CN4 was 27.6%、33.5%、26.4% within 24h, respectively,. At pH 6, the removal efficiency of anthracene, phenanthrene, pyrene by CN2 was 33.5%、46.5%、36.5% within 24h, respectively. Biodegradation was inhibited When PAHs concentrations was over 200mg/L and cell concentration was 1.0×107CFUs·mL-1.
Phenanthrene metabolites by Flavobacterium sp CN4 were assayed by using Purge and Trap combing with gas chromatography-mass spectrometry (GC-MS). Metabolism of phenanthrene by Bacillus CN2 were analyzed using liquid-liquid extraction combing with GC-MS and Fourier transform infrared spectroscopy (FTIR). In phenathrene degradation by CN4, the major metabolites include Coumarin, Phthalates, cinnamate, hydroxy-naphthoic acid, protocatechuic acid, phenanthrenedihydrodiol, phenanthrenediol. And the main metabolites were Phthalates and phenanthrenequinone when phenathrene was degraded by CN2. The change of functional groups between biodegradation metabolites and phenanthrene was given by comparison of FTIR spectra of phenanthrene with metabolites. Degradation pathway was proposed by identifying metabolites of phenanthrene. Degradation of phenanthrene by CN4 and CN2 were both initiated by dioxygenation, and then ring cleavages were happened to produce carboxylic acid. However, the metabolites of phenathrne by two strains were not quite the same. This indicated two strains had different enzyme system.
To detect active enzymes which were produced by two strains, salting out method and ultrasonication were respectively used to obtained extracellular enzyme and endoenzyme, and PAH enzyme degradation experiments were executed. The results showed Coarse endoenzyme excreted from two strains could degrade pyrene effectively. Within 30 minutes, the removal rates of the pyrene were 26.5% and 20.8% by endoenzyme produced from CN4 and CN2, respectively. However, the extracellular enzyme didn't show degradation activity toward pyrene within 6 hours. These results showed endoenzymes excreted from two strains were active enzyme. To obtained endoezyme, the optimum ultrasonication conditions were as follows:CN4 or CN2 bacterial suspensions were subjected to sonication for 15 min on ice with a cell ultrasonifier, the amplitude 260W(280W at most) and 30-s pulse intervals. The lysate was centrifuged for 20min at 11000 r·min-1 to remove cell debris.
Enzymatic-degradation kinetic characteristics of PAHs in aqueous were investigated. At 32℃and pH 6, the endoenzyme was the most active. The coarse endoenzyme of two strains had higher activity for degrading anthracene, phenanthrene, pyrene in weak acid medium at a temperature between 30℃and 35℃. The endoenzyme enzyme lost degrading activity when temperatures exceed 50℃. Enzymatic degradation reaction rate keep linear relation with PAH concentrations when substrate concentrations were not more than 200mg/L. The average initial rates were increasing with PAH initial concentration, but the average initial rates were decreasing with time. The apparent reaction orders were roughly the same when enzymatic degradation time was different.
Various metal ions were investigated as additives which could potentially enhance enzyme activity. It was found that Ca2+ could markedly activate the coarse enzymes. As 0.5mmol/L Ca2+ was added, the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN4 were improved by 1.3,1.9 and 1.8 times, respectively. And the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN2 were improved by 1.4,1.6 and 1.4 times, respectively. The mechanism of this result is that metal ion provide a bridge for enzyme to substrate, Which bind enzyme to form ion-enzyme complex. Then bind substrate to form enzyme-ion-substrate complex.
引文
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