石化源污水中混合酚细菌降解过程及机理研究
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摘要
酚类化合物作为重要的工业原料和很多工业企业生产的副产物,具有使用范围广、对环境危害性大的特点,成为常见的工业污染物。在所有酚类污染物的排放源中,石化污水是最严重的排放源之一。含酚废水的处理方法较多,生物法以其经济有效且无二次污染的特点,越来越受到重视。由于含酚废水的难降解特性,寻找高效的降解菌种成为一个重要的研究问题。目前的研究主要集中在利用微生物降解单一目标的酚类化合物,这过度的简化了实际状态,因为废水中的酚类常常成分复杂,且各成分之间存在相互影响。
基于以上问题,本文研究了微生物降解混合酚类的过程及机理。本研究利用传统的富集、筛选、纯化的方法,从受含酚废水污染的土壤中筛选出了8种酚类化合物的10株降解菌。研究10株降解菌对混合酚类的降解发现,假单胞菌XQ23具有最好的降解能力。能在24小时内降解除2,6-二甲基酚和2,4,6-三甲基酚外的其余10种混合酚,总降解量为400 mg/L。而在单一酚的降解实验中,菌株XQ23能在10小时内分别降解350 mg/L的苯酚、2-甲基苯酚、3-甲基苯酚、4-甲基苯酚、3-乙基苯酚、2,3-二甲基苯酚和3,4-二甲基苯酚,部分降解2,5-二甲基苯酚,但不能降解3,5-二甲基苯酚、2,4-二甲基苯酚、2,6-二甲基苯酚和2,4,6-三甲基苯酚。实验说明各酚类化合物混合后,促进了菌株XQ23对3,5-二甲基苯酚、2,4-二甲基苯酚和2,5-二甲基苯酚的降解。同时实验也检测了菌株XQ23对实际粗酚产品和实验室模拟粗酚产品的降解,表明模拟粗酚可以很好的模拟实际粗酚样品的降解情况。
通过16s rDNA序列分析和系统发育分析,鉴定10株降解菌中7株属于假单胞菌属,2株属于鞘氨醇杆菌属,1株属于芽孢杆菌属,降解能力与菌种的分类无必然联系。实验对菌株XQ23的温度、pH值、溶解氧、盐度、传代系数等特性进行了研究,结果表明菌株XQ23为好氧菌,盐度的耐受度较好,在42.40 g/L的高盐浓度下依然能较好生长。最适温度为30℃,最适pH值为6。在丰富培养基中连续传代12代后,对酚类化合物的降解性能没有改变。
As an important industrial raw materials and by-products of many industrial enterprises, phenolic compounds are now common industrial pollutants with features of a wide range and detrimental. Petrochemical wastewater is one of the most serious sources in all pollution sources of phenolic compounds. The method of microorganism biodegradation is an economic and effective way to treat phenolic wastewater. As the biodegradable characteristics of phenolic wastewater, to find efficient phenol-degradation bacteria becomes a major research question. Current bioremediation researches mostly concentrate on microbial degradation of single target phenolic compound, which over-simplifies the natural status, because the pollutants, commonly existing in complex mixtures, often affect the biodegradation kinetics each other.
Based on the above issues, this paper studied the process and mechanism of biodegradation of mixed phenols. We use traditional enrichment, screening, purification methods to select 10 degrading bacteria of 8 kinds of phenolic compounds from a soil sample polluted by phenol wastewater. In the 10 strains on the degradation of mixed phenols, we found that Pseudomonas sp. XQ23 has the best performances. XQ23 can degrade all the 10 mixed phenols except for 2,6-dimethylphenol and 2,4,6-trimethylphenol in 24 hours with the total phenol concentration 400 mg/L. In the degrading experiments for single phenols, Pseudomonas sp. XQ23 can degrade phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 3-ethylphenol and 2,3-dimethylphenol, partially degrade 2,5-dimethylphenol, but not 3,5-dimethylphenol、2,4-dimethylphenol、2,6-dimethylphenol and 2,4,6-trimethylphenol. Experiments illustrate that the mixture of phenolic compounds promote strain XQ23’s degradation on 3,5-dimethylphenol, 2,4-dimethylphenol and 2,5-dimethylphenol. Furthermore, we found that there were no differences for the degradation of phenols by XQ23 on a crude phenol product and a simulation of the product by pure phenols, indicating that the degradation of mixed phenolic compounds can well simulate real crude phenol products.
The 10 strains were identified by 16s rDNA sequence analysis and phylogenetic analysis. Among them seven strains belong to Pseudomonas strains, two are Sphingobacterium strains, and one is Bacillus strain. The phenol degradation abilities have corelation with taxonomy. Strain XQ23’s characteristics of temperature, pH, dissolved oxygen, salinity, and generations were tested. The results showed that XQ23 is an aerobic bacterium. It has good tolerance on salinity (growth in 42.40 g/L salt). The optimum growth temperature is 30oC, pH is 6. After 12 generations of serial cultivation in rich medium, the degradation ability of phenolic compounds has not changed.
基于以上问题,本文研究了微生物降解混合酚类的过程及机理。本研究利用传统的富集、筛选、纯化的方法,从受含酚废水污染的土壤中筛选出了8种酚类化合物的10株降解菌。研究10株降解菌对混合酚类的降解发现,假单胞菌XQ23具有最好的降解能力。能在24小时内降解除2,6-二甲基酚和2,4,6-三甲基酚外的其余10种混合酚,总降解量为400 mg/L。而在单一酚的降解实验中,菌株XQ23能在10小时内分别降解350 mg/L的苯酚、2-甲基苯酚、3-甲基苯酚、4-甲基苯酚、3-乙基苯酚、2,3-二甲基苯酚和3,4-二甲基苯酚,部分降解2,5-二甲基苯酚,但不能降解3,5-二甲基苯酚、2,4-二甲基苯酚、2,6-二甲基苯酚和2,4,6-三甲基苯酚。实验说明各酚类化合物混合后,促进了菌株XQ23对3,5-二甲基苯酚、2,4-二甲基苯酚和2,5-二甲基苯酚的降解。同时实验也检测了菌株XQ23对实际粗酚产品和实验室模拟粗酚产品的降解,表明模拟粗酚可以很好的模拟实际粗酚样品的降解情况。
通过16s rDNA序列分析和系统发育分析,鉴定10株降解菌中7株属于假单胞菌属,2株属于鞘氨醇杆菌属,1株属于芽孢杆菌属,降解能力与菌种的分类无必然联系。实验对菌株XQ23的温度、pH值、溶解氧、盐度、传代系数等特性进行了研究,结果表明菌株XQ23为好氧菌,盐度的耐受度较好,在42.40 g/L的高盐浓度下依然能较好生长。最适温度为30℃,最适pH值为6。在丰富培养基中连续传代12代后,对酚类化合物的降解性能没有改变。
As an important industrial raw materials and by-products of many industrial enterprises, phenolic compounds are now common industrial pollutants with features of a wide range and detrimental. Petrochemical wastewater is one of the most serious sources in all pollution sources of phenolic compounds. The method of microorganism biodegradation is an economic and effective way to treat phenolic wastewater. As the biodegradable characteristics of phenolic wastewater, to find efficient phenol-degradation bacteria becomes a major research question. Current bioremediation researches mostly concentrate on microbial degradation of single target phenolic compound, which over-simplifies the natural status, because the pollutants, commonly existing in complex mixtures, often affect the biodegradation kinetics each other.
Based on the above issues, this paper studied the process and mechanism of biodegradation of mixed phenols. We use traditional enrichment, screening, purification methods to select 10 degrading bacteria of 8 kinds of phenolic compounds from a soil sample polluted by phenol wastewater. In the 10 strains on the degradation of mixed phenols, we found that Pseudomonas sp. XQ23 has the best performances. XQ23 can degrade all the 10 mixed phenols except for 2,6-dimethylphenol and 2,4,6-trimethylphenol in 24 hours with the total phenol concentration 400 mg/L. In the degrading experiments for single phenols, Pseudomonas sp. XQ23 can degrade phenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 3-ethylphenol and 2,3-dimethylphenol, partially degrade 2,5-dimethylphenol, but not 3,5-dimethylphenol、2,4-dimethylphenol、2,6-dimethylphenol and 2,4,6-trimethylphenol. Experiments illustrate that the mixture of phenolic compounds promote strain XQ23’s degradation on 3,5-dimethylphenol, 2,4-dimethylphenol and 2,5-dimethylphenol. Furthermore, we found that there were no differences for the degradation of phenols by XQ23 on a crude phenol product and a simulation of the product by pure phenols, indicating that the degradation of mixed phenolic compounds can well simulate real crude phenol products.
The 10 strains were identified by 16s rDNA sequence analysis and phylogenetic analysis. Among them seven strains belong to Pseudomonas strains, two are Sphingobacterium strains, and one is Bacillus strain. The phenol degradation abilities have corelation with taxonomy. Strain XQ23’s characteristics of temperature, pH, dissolved oxygen, salinity, and generations were tested. The results showed that XQ23 is an aerobic bacterium. It has good tolerance on salinity (growth in 42.40 g/L salt). The optimum growth temperature is 30oC, pH is 6. After 12 generations of serial cultivation in rich medium, the degradation ability of phenolic compounds has not changed.
引文
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[3] Annadurai G., Ling L. Y., Lee J. F.. Statistical optimization of medium components and growth conditions by response surface methodology to enhance phenol degradation by Pseudomonas putida[J]. Hazard Mater, 2008,V151(1):171-178
[4]张锦,李圭白,马军.含酚废水的危害及处理方法的应用特点[J].化学工程师.2001(2):36-37
[5] Enroth C., Neujahr H., Schneider G., et al. The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis[J]. Structure, 1998,V6(5):605-617
[6] Kirchner U., Westphal A. H., Müller R., et al. Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD[J]. J Biol Chem, 2003,V278(48):45-53
[7] Beetymoters, WU J Y S. Removal of organic Precursors by permangnanate oxidation and alum coagulation[J]. Water Research, 1985,V19(3):309-314
[8]张自杰.排水工程下册[M].第四版.北京:中国建筑工业出版社,2002
[9]于萍,姚琳,罗运柏.高浓度含酚废水处理的新工艺[J].工业水处理,2002,22(9A):5-8
[10]Tang Shouyin, Jin Yizhong, Dai Youzhi, et al. GC/MS analysis of organic constituents in refinery waste water[J]. Journal of Chinese Mass Spectrometry, 1994,V15(3):36-41
[11]Broholm M. M., Arvin E.. Biodegradation of phenols in a sandstone aquifer under aerobic conditions and mixed nitrate and iron reducing conditions[J]. Journal of Contaminant Hydrology, 2000,V44:239-273
[12]Peters M., Heinaru E., Talpsep E., et al. Acquisition of a deliberately introduced phenol degradation operon pheBA by different indigenous Pseudomonas species.[J]. Appl Environ Microbiol, 1997,V63(12):4899-4906
[13]刘琼玉,李太友.含酚废水的无害化处理技术进展[J].环境污染治理技术与设备,2002,3(2A):62-66
[14]贾太轩,冯世宏,杜惠玲.含酚废水的氧化法处理[J].天津化工,2005,19(2A):12-14
[15]蒋展鹏.环境工程学[M].北京:高等教育出版社,1992
[16]夏北成.环境污染物生物降解[M].北京:化学工业出版社,2002
[17]王莉莉,杨孙楷.我国高浓度含酚废水的治理技术近况[J].环境污染与防治,17(5):29-30
[18]姜金华,王勇.从煤焦油酚水中回收粗酚的研究[J].煤炭技术,2002,21(1A):36-37
[19]Christoskova S. T., Stoyanova M.. Degradation of phenolic waste water over Ni- Oxide[ J ]. Wat. Res, 2001,V15(8):2073-2077
[20]雷乐成.水处理高级氧化技术[M].北京:化学工业出版社,2001
[21]李建政.环境工程微生物学[M].北京:化学工业出版社,2004
[22]Leslie.废水生物处理[M].第二版.张锡辉,刘勇弟.北京:化学工业出版社,2003:246
[23]Quan Xiangchun, Shi Hanchang, Liu Hong, et al. Removal of 2,4-dichlorophenol in a conventional activated sludge system through bioaugmentation[J]. Process Biochem, 2004,V39:1701–1707
[24]Jenkins D., Garrison M. G.. The Causes and cures of activated sludge bulking and foaming[M]. 2nd ed. Michigan,Ann Arbor: Lewis Publishers, 1993
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