磺胺二甲基嘧啶(SM2)高效降解菌J2的分离筛选及降解特性研究
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摘要
从城市污水处理厂活性污泥中分离得到一株能以磺胺二甲基嘧啶(SM2)为唯一碳源的菌株,经生理生化鉴定和16S rRNA基因序列同源性分析,将此菌鉴定为蜡样芽孢杆菌(Bacillus cereus),命名为J2.不同条件下的降解特性研究结果表明,J2菌株具有极高的SM2耐受性(100 mg·L~(-1))、较广的温度(20～30℃)和pH(6～8)适用范围;在温度30℃、pH=8.0、初始OD_(600)=0.1、SM2起始浓度为50 mg·L~(-1)的条件下,J2菌株在36 h内对SM2的降解率可达100%,降解效率远超目前已报道的其他SM2降解菌株,展现出了良好的应用潜力.J2菌株降解SM2过程中产生了5种主要中间代谢产物,分析推断其降解SM2的途径分为两条:①磺胺二甲基嘧啶分子首先在酶促反应作用下脱除SO_2,生成嘧啶环和苯胺环自由基,这两种自由基再经过环间耦合生成N-(4,6-二甲基嘧啶-2基)-1,4-二苯胺,该分子中的C—N键在活性氧物种的作用下断开生成苯胺和2-氨基-4,6-二甲基嘧啶;②在漆酶的作用下N~4键断裂,产生N-(3,5-二甲基嘧啶)-苯磺酰胺,之后N-(3,5-二甲基嘧啶)-苯磺酰胺的N—S键断裂,进一步形成2-氨基-4,6-二甲基嘧啶和苯亚砜.
In the present study, a bacterial strain isolated from the activated sludge of an urban wastewater treatment plant was employed to degrade sulfamethazine. According to the phenotype, physiological, biochemical analyses and 16 S rRNA sequence analysis, the strain was preliminary identified as Bacillus cereus and designated as J2. J2 has high SM2 tolerance(100 mg·L~(-1)), the broad temperature(20～30 ℃) and pH(6～8) adaptability. Results show that 50 mg·L~(-1) SM2 was completely degraded in 36 h at 30 ℃, pH 7.0 with OD_(600) at 0.1. J2 also exhibits higher tolerance and degradation efficiency than other SM2-degrading strains. Five intermediates were identified by LC-MS/MS analysis, two probable degradation pathways of SM2 by J2 was tentatively proposed: ①SO_2 was firstly removed from the SM2 molecules under the action of enzymatic reaction to generate pyrimidine and aniline radicals and then N-(4,6-dimethylpyrimidine-2)-1,4-diphenylamine was generated through ring coupling reaction between the two radicals, and the C—N bond subsequently broke off to the form phenylamine and 2-amino-4,6-dimethylpyrimidine by the attack of reactive oxygen species. ②The N~4 bond was broken through the action of laccase, producing N-(4-hydrosulfonylphenyl)-3,5-dimethylaniline, and then the N—S bond was broken to form 2-amino-4,6-dimethylpyrimidine and hydrosulfonylbenzene.
In the present study, a bacterial strain isolated from the activated sludge of an urban wastewater treatment plant was employed to degrade sulfamethazine. According to the phenotype, physiological, biochemical analyses and 16 S rRNA sequence analysis, the strain was preliminary identified as Bacillus cereus and designated as J2. J2 has high SM2 tolerance(100 mg·L~(-1)), the broad temperature(20～30 ℃) and pH(6～8) adaptability. Results show that 50 mg·L~(-1) SM2 was completely degraded in 36 h at 30 ℃, pH 7.0 with OD_(600) at 0.1. J2 also exhibits higher tolerance and degradation efficiency than other SM2-degrading strains. Five intermediates were identified by LC-MS/MS analysis, two probable degradation pathways of SM2 by J2 was tentatively proposed: ①SO_2 was firstly removed from the SM2 molecules under the action of enzymatic reaction to generate pyrimidine and aniline radicals and then N-(4,6-dimethylpyrimidine-2)-1,4-diphenylamine was generated through ring coupling reaction between the two radicals, and the C—N bond subsequently broke off to the form phenylamine and 2-amino-4,6-dimethylpyrimidine by the attack of reactive oxygen species. ②The N~4 bond was broken through the action of laccase, producing N-(4-hydrosulfonylphenyl)-3,5-dimethylaniline, and then the N—S bond was broken to form 2-amino-4,6-dimethylpyrimidine and hydrosulfonylbenzene.
引文
Baran W,Adamek E,Ziemiańska J,et al.2011.Effects of the presence of sulfonamides in the environment and their influence on human health[J].Journal of Hazardous Materials,196(1):1-15
Bing L,Tong Z.2010.Biodegradation and adsorption of antibiotics in the activated sludge process [J].Environmental Science & Technology,44(9):3468-3473
Cecinato A,Romagnoli P,Perilli M,et al.2017.Pharmaceutical substances in ambient particulates:A preliminary assessment [J].Chemosphere,183:62-68
Dmitrienko S G,Kochuk E V,Apyari V V,et al.2014.Recent advances in sample preparation techniques and methods of sulfonamides detection-A review[J].Analytica Chimica Acta,850:6-25
Essam T,Amin M A,Tayeb O E,et al.2010.Kinetics and metabolic versatility of highly tolerant phenol degrading Alcaligenes strain TW1[J].Journal of Hazardous Materials,173(1):783-788
付泊明,陈立伟,蔡天明,等.2017.诺氟沙星降解菌NOR-36的分离筛选及降解特性研究[J].环境科学学报,37(2):576-584
郭晓丹娜,郭夏丽.2017.白腐真菌共培养对磺胺二甲基嘧啶降解的影响[J].生物技术通报,33(5):197-202
Hsu J T,Chen C Y,Young C W,et al.2014.Prevalence of sulfonamide-resistant bacteria,resistance genes and integron-associated horizontal gene transfer in natural water bodies and soils adjacent to a swine feedlot in northern Taiwan[J].Journal of Hazardous Materials,277(4):34-43
Jiang L,Hu X,Yin D,et al.2011.Occurrence,distribution and seasonal variation of antibiotics in the Huangpu River,Shanghai,China[J].Chemosphere,82(6):822-828
Klavarioti M,Mantzavinos D,Kassinos D.2009.Removal of residual pharmaceuticals from aqueous systems by advanced oxidation processes[J].Environment International,35(2):402-417
Ma Jesús G,Carlos E R,Teresa V,et al.2011.Biodegradation of sulfamethazine by Trametes versicolor:Removal from sewage sludge and identification of intermediate products by UPLC-QqTOF-MS[J].Science of the Total Environment,409(24):5505-5512
Huang M,Tian S,Chen D,et al.2012.Removal of sulfamethazine antibiotics by aerobic sludge and an isolated Achromobacter sp.S-3[J].Journal of Environmental Sciences,24(9):1594-1599
Mao F,Liu X,Wu K,et al.2018.Biodegradation of sulfonamides by Shewanella oneidensis MR-1 and Shewanella sp.strain MR-4[J].Biodegradation,29(11):1-12
Michael C D,Ching-Hua H.2004.Transformation of the antibacterial agent sulfamethoxazole in reactions with chlorine:kinetics,mechanisms,and pathways[J].Environmental Science & Technology,38(21):5607-5615
Moore J E,Moore P J A,Millar B C,et al.2010.The presence of antibiotic resistant bacteria along the River Lagan[J].Agricultural Water Management,98(1):217-221
梅子凡.2017.多级A/O工艺处理养猪废水中抗生素的试验研究[D].重庆:重庆大学
Na Y,Junfeng W,Shiju Z,et al.2015.Removal of concentrated sulfamethazine by acclimatized aerobic sludge and possible metabolic products[J].Peerj,DOI:10.7717/peerj.1359
Pan L,Tang X,Li C,et al.2017.Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp.S-07[J].World Journal of Microbiology & Biotechnology,33(5):85
Rysz M,Alvarez P J J.2004.Amplification and attenuation of tetracycline resistance in soil bacteria:aquifer column experiments[J].Water Research,38(17):3705-3712
任南琪,李建政.2005.污染控制微生物生态学[M].哈尔滨:哈尔滨工业大学出版社
Sanderson H,Brain R A,Johnson D J,et al.2004.Toxicity classification and evaluation of four pharmaceuticals classes:antibiotics,antineoplastics,cardiovascular,and sex hormones[J].Toxicology,203(1):27-40
Shi B J,Wang Y,Geng Y K,et al.2018.Application of membrane bioreactor for sulfamethazine-contained wastewater treatment[J].Chemosphere,193:840-846
?inigoj-Ga.Contamination of farm animals and fishes from slovenia with heavy metals and sulfonamides[J].Bulletin of Environmental Contamination & Toxicology,64(2):235-241
Tao L,Shilin Y,Yanqiu C,et al.2014.Integrated biomarker responses in zebrafish exposed to sulfonamides[J].Environmental Toxicology & Pharmacology,38(2):444-452
Topp E,Chapman R,Devers-Lamrani M,et al.2013.Accelerated biodegradation of veterinary antibiotics in agricultural soil following long-term exposure,and isolation of a sulfamethazine-degrading sp[J].Journal of Environmental Quality,42(1):173-178
Wang D,Qian S,Zhao W T,et al.2014.Pharmaceutical and personal care products in the surface water of China:A review[J].Chinese Science Bulletin,59(59),DOI:10.1360/972013-370
Wang J,Zhou A,Zhang Y,et al.2016.Research on the adsorption and migration of sulfa antibiotics in underground environment[J].Environmental Earth Sciences,75(18),DOI:10.1007/s12665-016-6056-9
Wang S,Hu Y,Wang J.2018.Biodegradation of typical pharmaceutical compounds by a novel strain Acinetobacter sp[J].Journal of Environmental Management,217:240-246
Weiwei B,Zhimin Q,Xun P,et al.2009.Removal of veterinary antibiotics from sequencing batch reactor (SBR) pretreated swine wastewater by Fenton's reagent[J].Water Research,43(17):4392-4402
王强锋,朱彭玲,夏中梅,等.2018.三种农用抗生素降解真菌的筛选及其降解性能[J].农业资源与环境学报,35(6):533-539
Yang J F,Ying G G,Zhao J L,et al.2011.Spatial and seasonal distribution of selected antibiotics in surface waters of the Pearl Rivers,China[J].Journal of Environmental Science and Health,Part B,46(3):272-280
Yi L,Daqing M,Michal R,et al.2010.Trends in antibiotic resistance genes occurrence in the Haihe River,China.[J].Environmental Science & Technology,44(19):7220-7225
杨基先,马放,任南琪.2002.污染控制微生物学实验[M].哈尔滨:哈尔滨工业大学出版社
Zhang Y,Marrs C F,Simon C,et al.2009.Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp.[J].Science of the Total Environment,407(12):3702-3706
赵方.2012.磺胺二甲基嘧啶的微波与微生物降解研究[D].郑州:郑州大学
赵锐.2012.青岛近海两种生态环境可培养细菌多样性研究及3株海洋新菌的分类鉴定[D].青岛:中国海洋大学
Bing L,Tong Z.2010.Biodegradation and adsorption of antibiotics in the activated sludge process [J].Environmental Science & Technology,44(9):3468-3473
Cecinato A,Romagnoli P,Perilli M,et al.2017.Pharmaceutical substances in ambient particulates:A preliminary assessment [J].Chemosphere,183:62-68
Dmitrienko S G,Kochuk E V,Apyari V V,et al.2014.Recent advances in sample preparation techniques and methods of sulfonamides detection-A review[J].Analytica Chimica Acta,850:6-25
Essam T,Amin M A,Tayeb O E,et al.2010.Kinetics and metabolic versatility of highly tolerant phenol degrading Alcaligenes strain TW1[J].Journal of Hazardous Materials,173(1):783-788
付泊明,陈立伟,蔡天明,等.2017.诺氟沙星降解菌NOR-36的分离筛选及降解特性研究[J].环境科学学报,37(2):576-584
郭晓丹娜,郭夏丽.2017.白腐真菌共培养对磺胺二甲基嘧啶降解的影响[J].生物技术通报,33(5):197-202
Hsu J T,Chen C Y,Young C W,et al.2014.Prevalence of sulfonamide-resistant bacteria,resistance genes and integron-associated horizontal gene transfer in natural water bodies and soils adjacent to a swine feedlot in northern Taiwan[J].Journal of Hazardous Materials,277(4):34-43
Jiang L,Hu X,Yin D,et al.2011.Occurrence,distribution and seasonal variation of antibiotics in the Huangpu River,Shanghai,China[J].Chemosphere,82(6):822-828
Klavarioti M,Mantzavinos D,Kassinos D.2009.Removal of residual pharmaceuticals from aqueous systems by advanced oxidation processes[J].Environment International,35(2):402-417
Ma Jesús G,Carlos E R,Teresa V,et al.2011.Biodegradation of sulfamethazine by Trametes versicolor:Removal from sewage sludge and identification of intermediate products by UPLC-QqTOF-MS[J].Science of the Total Environment,409(24):5505-5512
Huang M,Tian S,Chen D,et al.2012.Removal of sulfamethazine antibiotics by aerobic sludge and an isolated Achromobacter sp.S-3[J].Journal of Environmental Sciences,24(9):1594-1599
Mao F,Liu X,Wu K,et al.2018.Biodegradation of sulfonamides by Shewanella oneidensis MR-1 and Shewanella sp.strain MR-4[J].Biodegradation,29(11):1-12
Michael C D,Ching-Hua H.2004.Transformation of the antibacterial agent sulfamethoxazole in reactions with chlorine:kinetics,mechanisms,and pathways[J].Environmental Science & Technology,38(21):5607-5615
Moore J E,Moore P J A,Millar B C,et al.2010.The presence of antibiotic resistant bacteria along the River Lagan[J].Agricultural Water Management,98(1):217-221
梅子凡.2017.多级A/O工艺处理养猪废水中抗生素的试验研究[D].重庆:重庆大学
Na Y,Junfeng W,Shiju Z,et al.2015.Removal of concentrated sulfamethazine by acclimatized aerobic sludge and possible metabolic products[J].Peerj,DOI:10.7717/peerj.1359
Pan L,Tang X,Li C,et al.2017.Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp.S-07[J].World Journal of Microbiology & Biotechnology,33(5):85
Rysz M,Alvarez P J J.2004.Amplification and attenuation of tetracycline resistance in soil bacteria:aquifer column experiments[J].Water Research,38(17):3705-3712
任南琪,李建政.2005.污染控制微生物生态学[M].哈尔滨:哈尔滨工业大学出版社
Sanderson H,Brain R A,Johnson D J,et al.2004.Toxicity classification and evaluation of four pharmaceuticals classes:antibiotics,antineoplastics,cardiovascular,and sex hormones[J].Toxicology,203(1):27-40
Shi B J,Wang Y,Geng Y K,et al.2018.Application of membrane bioreactor for sulfamethazine-contained wastewater treatment[J].Chemosphere,193:840-846
?inigoj-Ga.Contamination of farm animals and fishes from slovenia with heavy metals and sulfonamides[J].Bulletin of Environmental Contamination & Toxicology,64(2):235-241
Tao L,Shilin Y,Yanqiu C,et al.2014.Integrated biomarker responses in zebrafish exposed to sulfonamides[J].Environmental Toxicology & Pharmacology,38(2):444-452
Topp E,Chapman R,Devers-Lamrani M,et al.2013.Accelerated biodegradation of veterinary antibiotics in agricultural soil following long-term exposure,and isolation of a sulfamethazine-degrading sp[J].Journal of Environmental Quality,42(1):173-178
Wang D,Qian S,Zhao W T,et al.2014.Pharmaceutical and personal care products in the surface water of China:A review[J].Chinese Science Bulletin,59(59),DOI:10.1360/972013-370
Wang J,Zhou A,Zhang Y,et al.2016.Research on the adsorption and migration of sulfa antibiotics in underground environment[J].Environmental Earth Sciences,75(18),DOI:10.1007/s12665-016-6056-9
Wang S,Hu Y,Wang J.2018.Biodegradation of typical pharmaceutical compounds by a novel strain Acinetobacter sp[J].Journal of Environmental Management,217:240-246
Weiwei B,Zhimin Q,Xun P,et al.2009.Removal of veterinary antibiotics from sequencing batch reactor (SBR) pretreated swine wastewater by Fenton's reagent[J].Water Research,43(17):4392-4402
王强锋,朱彭玲,夏中梅,等.2018.三种农用抗生素降解真菌的筛选及其降解性能[J].农业资源与环境学报,35(6):533-539
Yang J F,Ying G G,Zhao J L,et al.2011.Spatial and seasonal distribution of selected antibiotics in surface waters of the Pearl Rivers,China[J].Journal of Environmental Science and Health,Part B,46(3):272-280
Yi L,Daqing M,Michal R,et al.2010.Trends in antibiotic resistance genes occurrence in the Haihe River,China.[J].Environmental Science & Technology,44(19):7220-7225
杨基先,马放,任南琪.2002.污染控制微生物学实验[M].哈尔滨:哈尔滨工业大学出版社
Zhang Y,Marrs C F,Simon C,et al.2009.Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp.[J].Science of the Total Environment,407(12):3702-3706
赵方.2012.磺胺二甲基嘧啶的微波与微生物降解研究[D].郑州:郑州大学
赵锐.2012.青岛近海两种生态环境可培养细菌多样性研究及3株海洋新菌的分类鉴定[D].青岛:中国海洋大学
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