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阿特拉津(2-Chloro-4-ethylamino-6-isopropylamino-s-triazine,ATZ)是一种通过阻断叶绿体中质体醌结合蛋白和抑制光合作用来防治阔叶和禾草类杂草的选择性除草剂,土壤对其吸附性较低,因此极易向地表水、深层土壤和地下中迁移[1]. 低浓度的阿特拉津显著降低作物的株高、根长、根干重,高浓度的阿特拉津甚至会导致植物死亡[2];阿特拉津影响雄性斑马鱼的神经发育和神经功能[3],已成为一种污染源并对动植物和人类健康造成不可忽视的风险[4-5]. 由于阿特拉津严重的危害及影响,2004年在欧盟被禁止使用,但由于它价格低廉而在世界其他地区被广泛使用,仍是国际上销售的主要除草剂之一.
目前,环境中阿特拉津的去除方法主要有吸附法[6]、光催化法[7]、高级氧化法[8]、植物修复法[9-10]、生物法[11]等,其中基于微生物降解的生物修复法不仅效率高,而且几乎不损害生态环境,并且可将ATZ转化为无毒或低毒的物质.很多研究者已筛选出能将阿特拉津作为唯一氮源或碳源的微生物,如细菌Pseudomonas sp. strain AKN5[12]、Klebsiella sp. FH-1[13]、Citricoccus sp. strain TT3[14]、Arthrobacter sp. 30、Pseudomonas sp. AD39[15]和真菌Pleuroyus ostreatus INCQS 40310[16]等. 这些菌株对阿特拉津具有耐受性并能降解高浓度的阿特拉津,Enterobacter sp.LY-2[17]对浓度为100 mg·kg−1的污染土壤具有修复效果,14 d后阿特拉津浓度降低为9.9 mg·kg−1;产脲节杆菌(Arthrobacter ureafaciens)CS3,培养2 d 可将50 mg·L−1的阿特拉津完全降解[18]. 虽然目前报道的去除阿特拉津的微生物种类较多,但仍然存在停滞期长,降解耗时较长,耐受性差等不足。而且大部分菌株来源于中国北部寒冷地区,东北多为黑土,呈中性或偏碱性、有机质及氮磷含量丰富,而长三角地区雨量充沛、气温较高,土壤通透性差、呈弱酸性、有机质及氮磷含量较低,这些菌株可能不适应长三角的土壤环境,因此有必要寻找适合长三角地区的高效阿特拉津降解微生物资源.
本研究从江苏省5个不同地区的农田土壤中筛选分离能适应长三角地区阿特拉津污染土壤的菌株,研究ATZ初始浓度、温度和pH对菌株繁殖和降解效果的影响,判断菌株的适用环境范围,并采用HPLC-MS测定ATZ的降解产物,推断可能存在的降解转化途径.
一株高效阿特拉津降解菌株的筛选及其降解能力和机理
Screening and identification of an atrazine-degrading strain and its degradation capacity and mechanism on atrazine
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摘要: 在江苏省某玉米农田土壤中筛选出以阿特拉津(ATZ)为唯一碳源和氮源的菌株D2,经16S rDNA基因序列分析,将其鉴定为土壤芽孢杆菌(Solibacillus),菌株D2的生长曲线符合SGompertz模型和Slogistic模型. 不同ATZ初始浓度、pH和培养温度条件下对ATZ的降解实验表明,菌株D2对ATZ具有极高的耐受性(>200 mg·L−1),在温度为20—30 ℃、pH值为5—9的条件下降解率均能达100%,可将100 mg·L−1的ATZ在18 h内完全去除,ATZ去除量与D2菌株的数量呈显著的负相关(r=−0.983,P<0.01). 对ATZ的降解中间产物测定表明,菌株D2可通过脱氯羟基化、加氢脱烷基化、甲基化、脱烷基化和水解等过程将阿特拉津转化为羟基阿特拉津(HA)、阿特拉通(atraton)、脱乙基阿特拉津(DEA)、西玛津(DMA)、羟基西玛津(HDMA)和脱乙基脱异丙基阿特拉津(DACT). 因此,D2是一株高效降解菌株,环境适应能力高于大部分已报道菌株,能够广泛应用于ATZ污染废水和污染土壤修复等领域.Abstract: Strain D2, using atrazine(ATZ) as the only carbon source and nitrogen source, was isolated from a maize field in Jiangsu Province. It was preliminarily identified as Solibacillus by 16S rDNA gene sequence analysis, and its growth curve both fit SGompertz model and Slogistic model. Experiments demonstrated that under different initial ATZ concentration, pH and temperature, strain D2 could tolerate high concentration of ATZ (>200 mg·L−1), wider pH and temperature, compared with reported strains. Strain D2 degraded ATZ from 100 mg·L−1 to 0 mg·L−1 within 18 h at 20—30 ℃, pH 5—9, and there was a significant negative correlation between the amount of ATZ removal and the population of D2 strain (r=−0.983, P<0.01). Analysis of the degradation intermediates of ATZ revealed that strain D2 transformed atrazine into hydroxy atrazine (HA), atraton, simazine (DMA), deethyl atrazine (DEA), hydroxy simazine (HDMA) and desethyl-desisopropyl atrazine(DACT)through dechlorination hydroxylation, hydrodealkylation, methylation, dealkylation and hydrolysis. Therefore, strain D2 is a strong degrading strain with better environmental adaptability than most reported strains, and could be used widely in the remediation of ATZ wastewater and contaminated soil.
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Key words:
- atrazine /
- strain /
- degradation characteristics /
- degradation pathway /
- Yangtze River Delta
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表 1 菌株D2的生理生化特性
Table 1. Physiological characteristic of strain D2
特征
Characteristic结果
Results革兰氏染色 + 淀粉水解 − 吲哚 − 甲基红 − V-P产生 − 柠檬酸盐利用 − 硝酸盐还原 + 过氧化氢酶 + 注:“+”为阳性;“−”为阴性. Note:“+” means positive and “−” means negative. 表 2 菌株生长曲线的SGompertz模型和Slogistic模型拟合参数对比
Table 2. Comparative list of bacteria growth due to SGompertz and Slogistic model
名称
NameSGompertz model Slogistic model R2 a1 K1 XC/h μ1/h−1 R2 a2 K2 X0/h μ2/h−1 D2 0.991 1.995 0.279 9.647 0.214 0.992 1.958 0.454 10.950 0.222 表 3 浓度与OD600的相关性分析
Table 3. Correlation analysis between concentration and OD600
平均值
Average value标准差
Standard deviation浓度
ConcentrationOD600 浓度 41.093 44.110 1 OD600 0.080 0.026 −0.983** 1 注:* P<0.05,** P<0.01. 表 4 已报道菌株对阿特拉津的降解效果的比较
Table 4. Comparison of atrazine degradation by strains has been reported
菌种
Strains浓度/(mg·L−1)Concentation 温度/℃Temperature pH 地区Region 降解率/%
Degradation时间/h Time 文献来源
Literature sourcesKlebsiella sp. FH1 50 25 9 吉林 81.5% 264 [13] LY-2 100 25 — 35 6 — 9 哈尔滨 98.7% 48 [17] CS3 50 30 7 河北 100% 48 [18] Arthrobacter sp. ZXY-2 50 30 — 35 8 — 9 哈尔滨 100% 6 [35] Arthrobacter sp. DNS10 100 30 7.5 哈尔滨 99.41% 24 [36] Paenarthrobacter sp. W11 100 30 7 吉林 97.1% 60 [37] Paenarthrobacter sp. W24 100 30 7 吉林 94.2% 72 [38] Arthrobacter sp. C2 100 30 7 — 9 吉林 100% 72 [33] Pseudomonas sp. 20 30 / 巴西 99% 24 [39] Achromobacter sp. 20 30 / 巴西 39% 48 [39] Pencillium sp. yz11-22N2 8 28 7 / 91.2% 120 [40] D2 100 20 — 30 5 — 9 江苏 100% 18 本研究 -
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