一株植物内生枯草芽孢杆菌对6种邻苯二甲酸酯的共代谢降解  

Co-metabolic degradation of six phthalic acid esters by an endophytic Bacillus subtilis

作　　者：杨云 肖霞霞[1,2] 陈小龙 程金金[2,3] 余向阳 王亚[2,3] 马桂珍 YANG Yun;XIAO Xia-xia;CHEN Xiao-long;CHENG Jin-jin;YU Xiang-yang;WANG Ya;MA Gui-zhen(School of Food Science and Engineering,Jiangsu Ocean University,Lianyungang 222000,China;Institute of Agricultural Resources and Environment,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of the Environment and Safety Engineering,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏海洋大学食品科学与工程学院,江苏连云港222000 [2]江苏省农业科学院农业资源与环境研究所,江苏南京210014 [3]江苏大学环境与安全工程学院,江苏镇江212013

出　　处：《江苏农业学报》2023年第2期393-404,共12页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(20)1009];江苏中晚熟大蒜产业集群建设项目(22912105)。

摘　　要：从邻苯二甲酸酯(PAEs)污染的青菜(Brassica rapa var.chinensis)中筛选获得1株编号为W34的内生菌。通过生理生化特征和16S rRNA基因测序对该菌进行鉴定,并研究W34对6种PAEs的共代谢降解特性,优化共代谢降解条件,初步探索共代谢基质对W34降解代谢PAEs的影响。结果表明,内生菌W34为枯草芽孢杆菌(Bacillus subtilis),该菌能以6种PAEs为碳源生长,可同时降解邻苯二甲酸二正丁酯(DBP)、邻苯二甲酸丁基苄基酯(BBP)、邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二(2-乙基)己酯(DEHP)和邻苯二甲酸二正辛酯(DnOP)6种PAEs。其中,该菌对DBP和BBP的降解能力较强,20 mg/L质量浓度下DBP和BBP的降解半衰期均小于0.33 d。添加D-纤维二糖为共代谢基质,W34对DMP、DEP、DEHP和DnOP的降解率均显著提升。吐温-80添加量、碳源种类、碳源质量浓度和接菌量对这4种PAEs的降解率均有显著影响。通过单因素试验,得到该菌的吐温-80最佳添加量为0.025%,最佳碳源为蔗糖(浓度为20 mmol/L),最佳接种菌液OD_(600)为0.3。此外,发现菌株W34含有质粒,但其质粒上不含PAEs降解基因,该菌的PAEs降解基因位于细菌的染色体上。菌株W34的粗酶液对6种PAEs均有催化降解活性,蔗糖可显著提高菌株W34胞内酶的催化活性。An endophytic bacterium was isolated from phthalic acid esters(PAEs)contaminated Brassica rapa var.chinensis,and was designated W34.The strain was identified by physiological-biochemical and 16S rRNA gene sequencing analyses,and the co-metabolic degradation characteristics of W34 on six PAEs were investigated to optimize the co-metabolic degradation conditions,so as to make preliminary exploration of the effect of co-substrate on the degradation and metabolism of PAEs by strain W34.The results indicated that,the endophytic bacterium W34 was identified as Bacillus subtilis.The strain could utilize six PAEs as carbon sources for growth and could degrade six PAEs in MSM medium at the same time,such as dibutyl phthalate(DBP),butyl benzyl phthalate(BBP),dimethyl phthalate(DMP),diethyl phthalate(DEP),di-(2-ethylhexyl)phthalate(DEHP)and di-n-octyl phthalate(DnOP).Among them,strain W34 exhibited relative higher degradation ability on DBP and BBP compared with the other four PAEs,and the degradation half-life period of DBP and BBP were all less than 0.33 d at mass concentration of 20 mg/L.The degradation rates of DMP,DEP,DEHP and DnOP by strain W34 were all significantly enhanced in MSM medium by adding D-cellobiose as co-metabolizing matrix.Besides,adding amount of Tween-80,type and mass concentration of the carbon source and inoculation dose all had significant effects on the degradation rates of the four PAEs by strain W34.According to the results of single factor experiment,the optimal adding amount of Tween-80 was 0.025%,the best carbon source was sucrose with a concentration of 20 mmol/L,and the most suitable OD_(600) for bacterial solution used in inoculating was 0.3.Furthermore,it was found that strain W34 contained plasmids,but its plasmids did not contain PAEs degradation gene,and the degradation gene of W34 was located on the chromosome.The crude enzyme solution of strain W34 showed catalytic and degrading activities on the six PAEs,and the addition of sucrose could significantly improve the catalytic activity

关 键 词：内生菌 邻苯二甲酸酯 共代谢 降解途径 

分 类 号：Q939.96[生物学—微生物学]