CN104560791B - 一株降解农药的苏云金芽孢杆菌及其应用 - Google Patents
一株降解农药的苏云金芽孢杆菌及其应用 Download PDFInfo
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- CN104560791B CN104560791B CN201410774202.1A CN201410774202A CN104560791B CN 104560791 B CN104560791 B CN 104560791B CN 201410774202 A CN201410774202 A CN 201410774202A CN 104560791 B CN104560791 B CN 104560791B
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Abstract
本发明公开了一株降解农药的苏云金芽孢杆菌及其应用。所述菌株为苏云金芽孢杆菌(Bacillus thuringiensis) Bt‑1,菌种保藏号为CCTCC M 2013680。Bt‑1在20~40 °C、pH 6.0~9.0和接种量0.05~0.25 g/L下能有效降解嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯和甲氰菊酯等农药,降解率达到80%以上。Bt‑1安全性高,环境适应性强,降解效率高,使用方便,可用于去除蔬菜、果树、茶叶、棉花、烟草等农作物中的农药残留,解决农业生产中农药残留超标问题,生产无公害绿色农产品;也可用于修复农药污染的土壤、水体等自然环境,解决环境污染问题,保护生态环境和人类健康。
Description
技术领域
本发明涉及应用环境微生物和农业领域,具体涉及一株降解农药的苏云金芽孢杆菌及其应用。具体用于农作物、农产品中农药残留的去除,也可用于修复农药污染的土壤、水体等自然环境。
背景技术
农药残留污染给生态环境和人类健康带来严重危害,特别是甲氧基丙烯酸酯类和拟除虫菊酯类农药的广泛使用导致的农药残留污染。甲氧基丙烯酸酯类农药主要包括嘧菌酯、醚菌酯和吡唑醚菌酯等,拟除虫菊酯类农药主要包括高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯和甲氰菊酯等。这两类农药化学结构上均带有酯键,是目前市场上的主要农药类型。其大量、频繁的使用已经严重危害到生态环境的安全,引发食品安全问题,进而损害人体健康。大量研究表明,这两类农药对一些有益昆虫如蜜蜂等造成了极大危害,对鱼类和水生无脊椎动物具有很高的毒性。此外,这两类农药具有环境激素效应,长期接触或摄入将导致慢性疾病,对人类、鸟类和其他哺乳动物具有慢性毒性,甚至有致癌、致畸、致突变的危险。这表明拟除虫菊酯类农药和甲氧基丙烯酸酯类农药并不像先前人们所认为的那样对非靶标生物安全和环境友好,不会造成污染或中毒。随着高灵敏度生物检测技术的发明和人们对农药毒理的进一步研究,这两类农药的危害还会被人们更深入地认识和了解。因此,消除农产品及环境中的拟除虫菊酯类和甲氧基丙烯酸酯类农药残留污染已成为科研工作者亟待解决的具有重大经济和社会意义的科研命题。
微生物降解以其高效、廉价、安全等特点,成为处理各类有机污染物的有效措施。近年来,研究利用降解微生物治理农药残留污染已引起广泛关注。目前许多发达国家和大公司都投入巨资以从事农药残留高效降解菌的研究和开发,如美国BCI公司、美国工程服务生物修复公司已经将降解微生物及其制剂产品规模化生产,国内的北京佳农新贸易发展有限公司已成功生产“比亚”生物降解剂可用于去除有机磷类农药残留。但是,利用微生物实现对农药进行降解,专一性高,往往一种微生物不能同时降解多类农药。而污染环境通常是多农药残留并存,因此研究开发能同时降解多类农药的降解微生物具有非常重要的理论和应用价值。特别是,目前还没有可同时降解甲氧基丙烯酸酯类和拟除虫菊酯类农药残留的降解微生物的报道。
发明内容
本发明的目的在于针对现有技术的上述不足,提供一株可同时降解甲氧基丙烯酸酯类和拟除虫菊酯类农药的苏云金芽孢杆菌菌株Bt-1。
本发明另一目的是提供上述菌株在制备降解甲氧基丙烯酸酯类和拟除虫菊酯类农药的制剂中的应用。
本发明另一目的是提供一种降解甲氧基丙烯酸酯类和拟除虫菊酯类农药的制剂。
本发明的另一目的是提供该苏云金芽孢杆菌Bt-1的应用,Bt-1可用于农作物、农产品中农药残留的去除,生产无公害绿色农产品;同时可用于修复农药污染的土壤、水体等自然环境,保护生态环境和人类健康。
本发明的目的通过以下技术方案予以实现:
一株降解农药的苏云金芽孢杆菌(Bacillus thuringiensis)Bt-1,菌种保存在中国典型培养物保藏中心(CCTCC),地址为湖北省武汉市武汉大学,保藏号为CCTCC M2013680,保藏日期为2013年12月20日。
所述菌株经鉴定为苏云金芽孢杆菌(Bacillus thuringiensis),在LB固体平板中呈现淡黄色,菌落较大、边缘不整齐,具芽孢。通过生理生化特性测定,显示该菌株属于革兰氏阳性菌;可利用葡萄糖、甘油、核糖、果糖、麦芽糖、淀粉;不利用木糖、半乳糖、甘露醇、鼠李糖、乳糖、海藻糖、阿拉伯糖。该菌株16S rDNA的GenBank登录号为KF290569。
所述苏云金芽孢杆菌Bt-1在降解农药方面的应用。
优选地,所述苏云金芽孢杆菌Bt-1在降解农作物、土壤或水体中农药的应用。
优选地,所述农药选自嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯或甲氰菊酯中的一种或几种。
更优选地,所述的苏云金芽孢杆菌Bt-1菌体数量为1.0×107CFU/mL。
与现有技术相比,本发明具有以下有益效果:
1.本发明筛选获得一株苏云金芽孢杆菌Bt-1(CCTCC M 2013680),该菌株安全性高,环境适应性强,在20~40℃、pH 6.0~9.0和接种量0.05~0.25g/L下能有效降解嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯和甲氰菊酯等农药残留,降解率达到80%以上,降解效率高,具有非常重要的应用价值。
2.苏云金芽孢杆菌Bt-1使用方便,直接施用可在短时间内有效降解农作物、土壤和水体环境中的甲氧基丙烯酸酯类和拟除虫菊酯类农药残留,解决农业生产中农药残留超标问题,同时保护生态环境和人类健康。
附图说明
图1.苏云金芽孢杆菌Bt-1的菌落形态。
图2.苏云金芽孢杆菌Bt-1的16S rDNA系统进化分析。
图3.苏云金芽孢杆菌Bt-1降解50mg/L嘧菌酯的HPLC色谱图。
图4.苏云金芽孢杆菌Bt-1降解50mg/L溴氰菊酯的HPLC色谱图。
具体实施方式
下面结合说明书附图和具体实施例来进一步详细阐述本发明。以下实施例为本发明较佳的实施方式,但并不对本发明的保护范围做任何形式的限定。本发明主要阐述所述菌株以及基于所述菌株的应用思想,实施方式中简单参数的替换不能一一在实施例中赘述,但并不因此限制本发明,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,应被视为等效的置换方式,都应包含在本发明范围内。
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,本发明所用试剂和材料均为市购。
实施例1苏云金芽孢杆菌Bt-1的获得
本发明人通过富集培养法筛选获得苏云金芽孢杆菌Bt-1。用来富集的土壤样品采集自新加坡。取土壤样品5g加入50mL基础培养基(配方为:(NH4)28O4,2g;MgSO4,0.2g;CaCl2,0.01g;FeSO4,0.005g;MnCl2,0.002g;K2HPO4,10.5g;KH2PO4,4.5g,蒸馏水1000mL,pH7.5)中。同时加入嘧菌酯原药母液(DMSO为溶剂),使嘧菌酯最终质量浓度为50mg/L,于30℃、150r/min摇床下进行降解菌富集培养。培养7d后,按10%的接种量转接到第二批含100mg/L嘧菌酯的基础培养基中。相同条件培养7d后,再按10%的接种量转接到含嘧菌酯为200mg/L的基础培养基中,继续培养7d。以此类推,不断增加嘧菌酯的质量浓度。最后取0.2mL基础培养基发酵液均匀涂抹到含嘧菌酯的固体平板中反复进行划线分离,直至得到单菌落。采用上述方法从土壤中成功筛选获得一株农药高效降解菌,命名为Bt-1。
实施例2苏云金芽孢杆菌Bt-1的鉴定
所述菌株在LB固体平板中呈现淡黄色,菌落较大、边缘不整齐,具芽孢。通过生理生化特性测定,显示该菌株属于革兰氏阳性菌;可利用葡萄糖、甘油、核糖、果糖、麦芽糖、淀粉;不利用木糖、半乳糖、甘露醇、鼠李糖、乳糖、海藻糖、阿拉伯糖。该菌株16SrDNA的GenBank登录号为KF290569。因此,经形态学特征、生理生化特性及16S rDNA系统发育分析,菌株Bt-1鉴定为苏云金芽孢杆菌(Bacillus thuringiensis)。
将所述菌株保藏于中国典型培养物保藏中心(CCTCC),保藏日期是2013年12月20,保藏号是CCTCC M 2013680,所述菌株命名为苏云金芽孢杆菌(Bacillus thuringiensis)菌株Bt-1。
所述中国典型培养物保藏中心的地址为湖北省武汉市武汉大学。
实施例3苏云金芽孢杆菌Bt-1对不同农药的生物降解效果
在基础培养基(配方同实施例1)中分别加入嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯和甲氰菊酯,使其终浓度为50mg/L;接种浓度为1.0×107CFU/mL的菌剂,并设不接菌的培养基为对照,31℃培养0~5d,定期取样,应用HPLC检测农药的残留量并计算降解率,结果见表1所示。结果表明,苏云金芽孢杆菌Bt-1在短时间内可有效降解嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯和甲氰菊酯,5d内对上述农药的降解率分别达到82.5%、86.2%、85.4%、97.1%、90.9%、82.4%和86.5%,降解效率高。
表1苏云金芽孢杆菌Bt-1对不同农药的降解效果
注:
Claims (7)
1.一株降解农药的苏云金芽孢杆菌菌株(Bacillus thuringiensis) Bt-1,其特征在于,所述菌株于2013年12月27日保藏于中国典型培养物保藏中心(CCTCC),菌种保藏号为CCTCC M 2013680。
2.根据权利要求1所述的菌株,其特征在于,所述菌株在LB固体平板中呈现淡黄色,菌落较大、边缘不整齐,具芽孢。
3.权利要求1所述菌株在降解农药中的应用,所述的农药为嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯或甲氰菊酯中的一种或几种。
4.根据权利要求3所述的应用,其特征在于,所述菌株在降解农作物、土壤或水体中农药的应用。
5.权利要求1所述菌株在制备降解农药的制剂中的应用,所述的农药为嘧菌酯、醚菌酯、吡唑醚菌酯、高效氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯或甲氰菊酯中的一种或几种。
6.根据权利要求3所述的应用,其特征在于,所述的苏云金芽孢杆菌Bt-1菌体的数量为1.0×107 CFU/mL。
7.一种降解农药的制剂,其特征在于,含有苏云金芽孢杆菌Bt-1和其他辅剂,所述苏云金芽孢杆菌Bt-1于2013年12月27日保藏于中国典型培养物保藏中心(CCTCC),菌种保藏号为CCTCC M 2013680。
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