头孢菌素类降解菌Achromobacter sp.YF-1的筛选与功能鉴定
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摘要
为合理、无污染地利用头孢菌素类废弃药渣,以长期堆放的头孢菌素类药渣土壤为样品源,从中获得浸出液,通过第三代头孢药品头孢克肟驯化、筛选出具有头孢菌素类抗生素降解能力的菌株;利用革兰氏染色法、电镜超薄切片技术双染色法对其进行形态学观察,16S rDNA进行分子生物学序列分析;采用单因素控制变量法对菌株的生长特性及最佳降解条件进行了初步探讨。结果表明:该菌为杆状细菌,无芽孢;经16S rDNA序列比对,进一步确定该菌株属于Achromobacter属,并命名为Achromobacter sp. YF-1;该降解菌在温度37℃、pH 6～7、转速120 r/min培养条件下培养7 d,头孢菌素类抗生素降解率达92.71%;该菌株对头孢菌素类抗生素的降解能力稳定,可用于处理药渣中残留的头孢菌素类抗生素。
In order to conduct rational and pollution-free utilization of waste cephalosporin drug residues,this paper took the soil loaded for a long time by cephalosporin drug residues as specimen source; obtained leaching liquid from the soil; and screened out the strains with ability of cephalosporin antibiotic degradation by the 3rd generation of cephalosporin drug cefixime. Morphological observation was carried on this strain by gram staining and double staining electron microscope ultrathin section technique,and gene sequence analysis was conducted on 16S rDNA. The growth characteristics and optimum degradation conditions of the strain were studied by single factor control variable method.The results showed that: This strain was a bacillus without spore; it was confirmed as genus Achromobacter and named as Achromobacter sp. YF-1 through 16S rDNA sequence alignment; under the conditions of temperature 37℃,p H 6 ～ 7,revolving speed 120 r/min for 7 d cultivation,the degradation rate of cephalosprins could reach 92.71%; the degradation ability to cephalosprins of this strain was stable,and could be used to treat cephalosporin antibiotics residual.
In order to conduct rational and pollution-free utilization of waste cephalosporin drug residues,this paper took the soil loaded for a long time by cephalosporin drug residues as specimen source; obtained leaching liquid from the soil; and screened out the strains with ability of cephalosporin antibiotic degradation by the 3rd generation of cephalosporin drug cefixime. Morphological observation was carried on this strain by gram staining and double staining electron microscope ultrathin section technique,and gene sequence analysis was conducted on 16S rDNA. The growth characteristics and optimum degradation conditions of the strain were studied by single factor control variable method.The results showed that: This strain was a bacillus without spore; it was confirmed as genus Achromobacter and named as Achromobacter sp. YF-1 through 16S rDNA sequence alignment; under the conditions of temperature 37℃,p H 6 ～ 7,revolving speed 120 r/min for 7 d cultivation,the degradation rate of cephalosprins could reach 92.71%; the degradation ability to cephalosprins of this strain was stable,and could be used to treat cephalosporin antibiotics residual.
引文
[1]刘元望,李兆君,冯瑶,等.微生物降解抗生素的研究进展[J].农业环境科学学报,2016,35(2):212-214.Liu Y W,Li Z J,Feng Y,et al..Research progress in microbial degradation of antibiotics[J].J.Agro-environ.Sci.,2016,35(2):212-214.
[2]任妙贤,陈良柱,潘志坤,等.头孢维星研究概况[J].动物医学进展,2015,36(2):106-110.Ren M X,Chen L Z,Pan Z K,et al..In troduction to cefovecin[J].Prog.Vet.Med.,2015,36(2):106-110.
[3]孙样,王宇驰,张春然,等.头孢菌素类抗生素的发现与发展[J].国外医药(抗生素分册),2014,35(4):154-158,181.Sun Y,Wang Y C,Zhang C R,et al..Discovery and development of cephalosporin antibiotics[J].World Notes Antibiotics,2014,35(4):154-158,181.
[4]李月海,刘冬玲,谢幼梅.抗生素菌渣的综合利用[J].山东畜牧兽医,2000(6):28-31,39.
[5]潘永全,何明忠,谭毅,等.四环素药渣对幼兔的增重试验[J].粮食与饲料工业,1996(7):28-30.Pan Y Q,He M Z,Tan Y,et al..Rabbit gain traits with wastes of tetracycline[J].Cereal Feed Ind.,1996(7):28-30.
[6]Sassman S A,Lee L S.Sorption of three tetracyclines by several soils:Assessing the role of p H and cation exchange[J].Environ.Sci.Technol.,2005,39(19):7452-7459.
[7]Crane M,Watts C,Boudard T.Chronic aquatic environmental risks from exposure to human pharmaceuticals[J].Sci.Total Environ.,2006,367(1):23-41.
[8]赵楠.减少抗生素使用,德国EW营养公司是认真的[J].中国畜牧杂志,2018,54(5):157-160.
[9]Kumar K,Gupta S C,Baidoo S K,et al..Antibiotic uptake by plants from soil fertilized with animal manure[J].J.Environ.Qual.,2005,34(6):2082-2085.
[10]王敏,唐景春.土壤中的抗生素污染及其生态毒性研究进展[J].农业环境科学学报,2010,29:261-266.Wang M,Tang J C.Research of antibiotics pollution in soil environments and its ecological toxicity[J].J.Agro-Environ.Sci.,2010,29:261-266.
[11]Prado N,Ochoa J,Amrane A.Biodegradation and biosorption of tetracycline and tylosin antibiotics in activated sludge system[J].Proc.Biochem.,2009,44(11):1302-1306.
[12]Kay P,Blackwell P A,Boxall A B.A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with field data[J].Environ.Pollut.,2005,134(2):333-341.
[13]苏家宜,姬玉娇,张婷,等.发酵中药渣对断奶仔猪血浆生化参数和抗氧化指标的影响[J].天然产物研究与开发,2017,29(2):284-289.Su J Y,Ji Y J,Zhang T,et al..Effects of fermented Chinese herb residues on plasma biochemical parameters and antioxidant indexes of weaned piglets[J].Nat.Product Res.Dev.,2017,29(2):284-289.
[14]孙绍锋,郝永利,李玉爽.解析《国家危险废物名录》[J].中国环境管理,2013,5(2):46-48.Sun S F,Hao Y L,Li Y S.The national hazardous waste list analysis[J].Chin.J.Environ.Manage.,2013,5(2):46-48.
[15]Costanzo S D,Murby J,Bates J.Ecosystem response to antibiotics entering the aquatic environment[J].Marine Pollut.Bull.,2005,51(1-4):218-223.
[16]冯宝佳,曾强,赵亮,等.水环境中抗生素的来源分布及对健康的影响[J].环境监测管理与技术,2013,25(1):14-17,21.Feng B J,Zeng Q,Zhao L,et al..Source distribution of antibiotics in water environment and its impact on human health[J].Admin.Tech.Environ.Monitor.,2013,25(1):14-17,21.
[17]Bu Q,Wang B,Huang J,et al..Pharmaceuticals and personal care products in the aquatic environment in China:A review[J].J.Hazard.Mater.,2013,15:189-211.
[18]Brandt K K,Amézquita A,Backhaus T,et al..Ecotoxicological assessment of antibiotics:A call for improved consideration of microorganisms[J].Environ.Int.,2015,85(11):189-205.
[19]Junker T,Alexy R,Knacker T,et al..Biodegradability of14C-labelled antibiotics in a modified laboratory sewage treatment plant at environmentally relevant concentrations[J].Environ.Sci.Technol.,2006,40(1):318-324.
[20]Dolliver H,Gupta S,Noll S.Antibiotic degradation during manure composting[J].J.Environ.Qual.,2008,37(3):1245-1253.
[21]张红娟.抗生素菌渣堆肥化处理研究[D].郑州:郑州大学,硕士学位论文,2010.Zhang H J.Research of the composting treatment of antibiotic mushroom dregs[D].Zhengzhou:Zhengzhou University,Master Disertation,2010.
[22]Wu X F,Wei Y S,Zheng J X,et al..The behavior of tetracyclines and their degradation products during swine manure composting[J].Bioresour.Technol.,2011,102(10):5924-5931.
[23]马玉龙,张作义,刘宁普,等.废弃药渣中残留泰乐菌素降解菌的筛选[J].环境科学研究,2010,23(8):1076-1080.Ma Y L,Zhang Z Y,Liu N P,et al..Screening of degrading bacteria for tylosin residue in pharmaceutical waste[J].Res.Environ.Sci.,2010,23(8):1076-1080.
[24]刘力嘉,谢丽,张作义,等.泰勒菌素高效降解菌的筛选及降解特性研究[J].农业环境科学学报,2011,30(5):1027-1030.Liu L J,Xie L,Zhang Z Y,et al..Isolation and degradation characteristics of a tylosin-degrading strain[J].J.AgroEnviron.Sci.,2011,30(5):1027-1030.
[25]曾丽璇,曾宝强,张秋云.头孢拉定降解菌的驯化、筛选及降解性能[J].华南师范大学学报(自然科学版),2008(4):99-102.Zeng L X,Zeng B Q,Zhang Q Y.Screening,isolation and biodegradation ability of cefradine degradable bacteria[J].J.South China Norm.Univ.(Nat.Sci.),2008(4):99-102.
[26]刘元望,李兆君,冯瑶,等.微生物降解抗生素的研究进展[J].农业环境科学学报,2016,35(2):212-224.Liu Y W,Li Z J,Feng Y,et al..Research progress in micobial degradation of antibiotics[J].J.Agro-Environ.Sci.,2016,35(2):212-224.
[27]Erickson B D,Elkins C A,Mulli L B,et al..A metallo-betalactamase is responsible for the degradation of ceftiofur by the bovine intestinal baterium Bacillus cereus P41[J].Vet.Microbiol.,2014,172(3/4):499-504.
[28]Selvi A,Salam J A,Das N.Biodegradation of cefdinir by a novl yeast strain,Ustilago sp.SMN03 isolated from pharmaceutical wastewater[J].World J.Microbiol.Biotechnol.,2014,30(11):2839-2850.
[29]Lin B K,Lyu J I,Lyu X J,et al..Characterization of cefalexin degradation capabilities of two Pseudomonas strains isolated from activated sludge[J].J.Hazard.Mater.,2015,282:158-164.
[30]胡秀红,李景壮,叶胜蓝,等.一株阿维菌素降解菌AW1-18的筛选与分类鉴定[J].生物技术通报,2012(10):223-228.Hu X H,Li J Z,Ye S L,et al..Screening and identification of avermectin-degrading bacterium strain AW1-18[J].Biotechnol.Bull.,2012(10):223-228.
[31]蔡刚.木糖氧化无色杆菌(Achromobacter xylosoxydans)中几种β-内酰胺酰化酶的性质与结构研究[D].上海:中国科学院上海生命科学研究院植物生理生态研究所,博士学位论文,2006.Cai G.Characterazation and structural studies of severalβ-laetam acylases from Achromobacter xylosoxydans[D].Shanghai:Institute of Plant Physiology and Ecology,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Doctor Dissertation,2006.
[32]梁亮.木糖氧化无色杆菌研究进展[J].右江医学,2017,45(1):112-115.
[2]任妙贤,陈良柱,潘志坤,等.头孢维星研究概况[J].动物医学进展,2015,36(2):106-110.Ren M X,Chen L Z,Pan Z K,et al..In troduction to cefovecin[J].Prog.Vet.Med.,2015,36(2):106-110.
[3]孙样,王宇驰,张春然,等.头孢菌素类抗生素的发现与发展[J].国外医药(抗生素分册),2014,35(4):154-158,181.Sun Y,Wang Y C,Zhang C R,et al..Discovery and development of cephalosporin antibiotics[J].World Notes Antibiotics,2014,35(4):154-158,181.
[4]李月海,刘冬玲,谢幼梅.抗生素菌渣的综合利用[J].山东畜牧兽医,2000(6):28-31,39.
[5]潘永全,何明忠,谭毅,等.四环素药渣对幼兔的增重试验[J].粮食与饲料工业,1996(7):28-30.Pan Y Q,He M Z,Tan Y,et al..Rabbit gain traits with wastes of tetracycline[J].Cereal Feed Ind.,1996(7):28-30.
[6]Sassman S A,Lee L S.Sorption of three tetracyclines by several soils:Assessing the role of p H and cation exchange[J].Environ.Sci.Technol.,2005,39(19):7452-7459.
[7]Crane M,Watts C,Boudard T.Chronic aquatic environmental risks from exposure to human pharmaceuticals[J].Sci.Total Environ.,2006,367(1):23-41.
[8]赵楠.减少抗生素使用,德国EW营养公司是认真的[J].中国畜牧杂志,2018,54(5):157-160.
[9]Kumar K,Gupta S C,Baidoo S K,et al..Antibiotic uptake by plants from soil fertilized with animal manure[J].J.Environ.Qual.,2005,34(6):2082-2085.
[10]王敏,唐景春.土壤中的抗生素污染及其生态毒性研究进展[J].农业环境科学学报,2010,29:261-266.Wang M,Tang J C.Research of antibiotics pollution in soil environments and its ecological toxicity[J].J.Agro-Environ.Sci.,2010,29:261-266.
[11]Prado N,Ochoa J,Amrane A.Biodegradation and biosorption of tetracycline and tylosin antibiotics in activated sludge system[J].Proc.Biochem.,2009,44(11):1302-1306.
[12]Kay P,Blackwell P A,Boxall A B.A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with field data[J].Environ.Pollut.,2005,134(2):333-341.
[13]苏家宜,姬玉娇,张婷,等.发酵中药渣对断奶仔猪血浆生化参数和抗氧化指标的影响[J].天然产物研究与开发,2017,29(2):284-289.Su J Y,Ji Y J,Zhang T,et al..Effects of fermented Chinese herb residues on plasma biochemical parameters and antioxidant indexes of weaned piglets[J].Nat.Product Res.Dev.,2017,29(2):284-289.
[14]孙绍锋,郝永利,李玉爽.解析《国家危险废物名录》[J].中国环境管理,2013,5(2):46-48.Sun S F,Hao Y L,Li Y S.The national hazardous waste list analysis[J].Chin.J.Environ.Manage.,2013,5(2):46-48.
[15]Costanzo S D,Murby J,Bates J.Ecosystem response to antibiotics entering the aquatic environment[J].Marine Pollut.Bull.,2005,51(1-4):218-223.
[16]冯宝佳,曾强,赵亮,等.水环境中抗生素的来源分布及对健康的影响[J].环境监测管理与技术,2013,25(1):14-17,21.Feng B J,Zeng Q,Zhao L,et al..Source distribution of antibiotics in water environment and its impact on human health[J].Admin.Tech.Environ.Monitor.,2013,25(1):14-17,21.
[17]Bu Q,Wang B,Huang J,et al..Pharmaceuticals and personal care products in the aquatic environment in China:A review[J].J.Hazard.Mater.,2013,15:189-211.
[18]Brandt K K,Amézquita A,Backhaus T,et al..Ecotoxicological assessment of antibiotics:A call for improved consideration of microorganisms[J].Environ.Int.,2015,85(11):189-205.
[19]Junker T,Alexy R,Knacker T,et al..Biodegradability of14C-labelled antibiotics in a modified laboratory sewage treatment plant at environmentally relevant concentrations[J].Environ.Sci.Technol.,2006,40(1):318-324.
[20]Dolliver H,Gupta S,Noll S.Antibiotic degradation during manure composting[J].J.Environ.Qual.,2008,37(3):1245-1253.
[21]张红娟.抗生素菌渣堆肥化处理研究[D].郑州:郑州大学,硕士学位论文,2010.Zhang H J.Research of the composting treatment of antibiotic mushroom dregs[D].Zhengzhou:Zhengzhou University,Master Disertation,2010.
[22]Wu X F,Wei Y S,Zheng J X,et al..The behavior of tetracyclines and their degradation products during swine manure composting[J].Bioresour.Technol.,2011,102(10):5924-5931.
[23]马玉龙,张作义,刘宁普,等.废弃药渣中残留泰乐菌素降解菌的筛选[J].环境科学研究,2010,23(8):1076-1080.Ma Y L,Zhang Z Y,Liu N P,et al..Screening of degrading bacteria for tylosin residue in pharmaceutical waste[J].Res.Environ.Sci.,2010,23(8):1076-1080.
[24]刘力嘉,谢丽,张作义,等.泰勒菌素高效降解菌的筛选及降解特性研究[J].农业环境科学学报,2011,30(5):1027-1030.Liu L J,Xie L,Zhang Z Y,et al..Isolation and degradation characteristics of a tylosin-degrading strain[J].J.AgroEnviron.Sci.,2011,30(5):1027-1030.
[25]曾丽璇,曾宝强,张秋云.头孢拉定降解菌的驯化、筛选及降解性能[J].华南师范大学学报(自然科学版),2008(4):99-102.Zeng L X,Zeng B Q,Zhang Q Y.Screening,isolation and biodegradation ability of cefradine degradable bacteria[J].J.South China Norm.Univ.(Nat.Sci.),2008(4):99-102.
[26]刘元望,李兆君,冯瑶,等.微生物降解抗生素的研究进展[J].农业环境科学学报,2016,35(2):212-224.Liu Y W,Li Z J,Feng Y,et al..Research progress in micobial degradation of antibiotics[J].J.Agro-Environ.Sci.,2016,35(2):212-224.
[27]Erickson B D,Elkins C A,Mulli L B,et al..A metallo-betalactamase is responsible for the degradation of ceftiofur by the bovine intestinal baterium Bacillus cereus P41[J].Vet.Microbiol.,2014,172(3/4):499-504.
[28]Selvi A,Salam J A,Das N.Biodegradation of cefdinir by a novl yeast strain,Ustilago sp.SMN03 isolated from pharmaceutical wastewater[J].World J.Microbiol.Biotechnol.,2014,30(11):2839-2850.
[29]Lin B K,Lyu J I,Lyu X J,et al..Characterization of cefalexin degradation capabilities of two Pseudomonas strains isolated from activated sludge[J].J.Hazard.Mater.,2015,282:158-164.
[30]胡秀红,李景壮,叶胜蓝,等.一株阿维菌素降解菌AW1-18的筛选与分类鉴定[J].生物技术通报,2012(10):223-228.Hu X H,Li J Z,Ye S L,et al..Screening and identification of avermectin-degrading bacterium strain AW1-18[J].Biotechnol.Bull.,2012(10):223-228.
[31]蔡刚.木糖氧化无色杆菌(Achromobacter xylosoxydans)中几种β-内酰胺酰化酶的性质与结构研究[D].上海:中国科学院上海生命科学研究院植物生理生态研究所,博士学位论文,2006.Cai G.Characterazation and structural studies of severalβ-laetam acylases from Achromobacter xylosoxydans[D].Shanghai:Institute of Plant Physiology and Ecology,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Doctor Dissertation,2006.
[32]梁亮.木糖氧化无色杆菌研究进展[J].右江医学,2017,45(1):112-115.