CN115094009B - 一种降解3,5,6-三氯-2-吡啶酚的枯草芽孢杆菌制剂及其制备方法与应用 - Google Patents
一种降解3,5,6-三氯-2-吡啶酚的枯草芽孢杆菌制剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种用于生物降解3,5,6‑三氯‑2吡啶酚(TCP)的枯草芽孢杆菌制剂及其制备方法与应用,所述菌株为枯草芽孢杆菌ZW,保存编号为CGMCC25210。本发明枯草芽孢杆菌Bacillus subtilis.ZW,具有生长迅速、易分离培养、抗逆行强和对人、植物、动物没有致病性,不污染环境,生物安全性高等优点。同时,所述枯草芽孢杆菌ZW菌体细胞在21小时内能将初始浓度为50mg/L的TCP降解率达72%,具有较高的TCP降解效率,本发明对于微生物法降解毒死蜱或TCP尤其是生物修复毒死蜱污染的土壤及水体具有较高的应用价值。
Description
技术领域
本发明属于微生物降解农药残留领域,涉及一种用于生物降解3,5,6-三氯-2吡啶酚(TCP)的枯草芽孢杆菌制剂及其制备方法与应用。
背景技术
毒死蜱(Chlorpyrios)是一种中等毒性的有机磷农药,主要用于水稻、玉米、蔬菜、果树及茶树的病虫害防治。由于毒死蜱的大量生产和超量使用,在农业生产环境和农产品中,毒死蜱的高毒、难降解残留物TCP的污染问题日趋严重。
TCP具有环境毒性,其对土壤微生物的毒性是毒死蜱的2.5倍。研究发现,如果TCP含量过高(>100 mg/L)会阻滞毒死蜱以及其它有机污染物的降解,进而影响生态环境系统的自我调节及修复功能。TCP对非靶标生物存在潜在危险,低浓度TCP便对水生生物和陆生生物产生中等毒性。TCP对人体也会产生危害,它不仅能够降低男性精子的质量和体内睾丸素合成的速率从而影响人体的生殖性能,还能引发青春期女性的睡眠呼吸障碍,最终影响其后代在婴儿期及以后的睡眠。目前,TCP已被美国环境保护局认定为持久性和移动性有毒农药代谢产物,并且在英国TCP也被列入危险指数最高的有毒代谢产物。
微生物降解是消除农药残留的有效手段,它具有安全、成本低、无二次污染等优点。目前国内外已有TCP降解菌株的报导。如,徐明刚等从山东某农药污水处理厂活性污泥中筛选出一株能够利用毒死蜱和TCP作为碳、磷和氮源用于自身生长的菌株副球菌属Paracoccus sp. strain TRP,李晓楼在对微生物降解甲基毒死蜱的研究中分离得到一株能够降解TCP的菌株荧光假单胞菌Pseudomonas fluorescens ZHLXL-2,目前国内外分离的TCP降解菌株少,降解能力偏低等问题。本发明以长期施用毒死蜱农药的韭菜田土壤作为菌种来源,从中筛选高效降解TCP的菌株。枯草芽孢杆菌(Bacillus subtilis)是一类产生芽孢的革兰氏阳性菌株,具有生长迅速、易分离培养、抗逆行强和对人、植物、动物没有致病性,不污染环境,生物安全性高等优点。因此,本发明对于微生物法降解毒死蜱或TCP尤其是生物修复毒死蜱污染的土壤及水体具有较高的应用价值。
发明内容
本发明的第一个目的是针对现有的TCP高效降解菌株少,降解能力低等问题,提供一种用于高效降解TCP的枯草芽孢杆菌,该菌株的菌体细胞或芽孢对环境安全,且均能高效降解TCP,在修复毒死蜱或TCP污染的土壤及水体方面具有重要的应用前景。
本发明的第二个目的是提供一种降解TCP的枯草芽孢杆菌制剂及其应用,所述的枯草芽孢杆菌制剂由上述用于降解TCP的芽孢杆菌制成,能够高效生物降解TCP。
本发明第一方面提供了一种用于降解TCP的枯草芽孢杆菌,该菌株为革兰氏阳性,好氧,没有荚膜,有周生鞭毛,可以运动;生长较迅速、代谢旺盛、抗逆性强;在固体培养基上生长时,菌落的表面粗糙,呈污白或微黄色,有时会形成褶皱;在液体培养基中生长时,有时会形成絮状物。其所述的芽孢杆菌为枯草芽孢杆菌ZW,保藏编号为CGMCC 25210。在本发明的一些实施例中,所述枯草芽孢杆菌ZW菌体细胞在21小时内能将初始浓度为50mg/L的TCP降解率达72%。
菌株保藏:枯草芽孢杆菌Bacillus subtilis. ZW ,由聊城大学分离、鉴定,已在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)保藏,保藏日期:2022年06月29日,保藏编号:CGMCC.No25210. 本发明中该菌株被命名为枯草芽孢杆菌ZW(Bacillus subtilis. ZW)。
本发明第二方面提供了一种降解TCP的枯草芽孢杆菌制剂,其含有如本发明第一方面所述的芽孢杆菌的菌体细胞或芽孢。
在本发明的一些实施例中,所述降解TCP的枯草芽孢杆菌制剂为液态制剂,优选的,在所述降解TCP的液态制剂中,芽孢杆菌菌体细胞或芽孢浓度为1.0 x 1010 -3.0 x1010 /mL。
在本发明的另一些实施例中,所述降解TCP的枯草芽孢杆菌制剂为固体粉末制剂,优选的,在所述降解TCP的固体粉末制剂中,芽孢杆菌菌体细胞或芽孢的含量为1.0 x 1010-3.0 x 1010 /g。
本发明第三方面提供了一种如本发明第二方面所述的降解TCP的枯草芽孢杆菌制剂的制备方法,其包括:
1)菌种的活化培养;2)对活化菌种进行发酵培养获得发酵培养物;3)对发酵培养物离心处理,收获枯草芽孢杆菌的菌体细胞或芽孢;
所述发酵菌株与本发明第一方面所述的枯草芽孢杆菌菌株的16s rDNA的同源性至少为95%的菌株。
根据本发明,所述发酵培养基组分为:蛋白胨:10 g/L;酵母膏:5 g/L;NaCl:10 g/L;pH:7.0;所述的优选培养温度为35℃。
本发明的第四个方面提供了本发明第二方面所述的降解TCP的枯草芽孢杆菌制剂或如本发明第三方面所述的制备方法制备的降解TCP的枯草芽杆菌制剂在毒死蜱或TCP污染土壤及水体修复中的应用,其包括:
1)使用PBS缓冲液对冷冻干燥后的枯草芽孢杆菌制剂进行稀释,制成用于降解TCP的液态菌剂;
2)使用海藻糖、甘油和可溶性淀粉对冷冻干燥后的枯草芽孢杆菌制剂进行复配稀释,制成用于降解TCP的固态菌剂。
与现有技术相比,本发明具有如下有益效果:本发明枯草芽孢杆菌Bacillussubtilis. ZW ,具有生长迅速、易分离培养、抗逆行强和对人、植物、动物没有致病性,不污染环境,生物安全性高等优点。同时,所述枯草芽孢杆菌ZW菌体细胞在21小时内能将初始浓度为50mg/L的TCP降解率达72%,具有较高的TCP降解效率,本发明对于微生物法降解毒死蜱或TCP尤其是生物修复毒死蜱污染的土壤及水体具有较高的应用价值。
附图说明
图1为本发明枯草芽孢杆菌ZW的菌落形态。
图2为枯草芽孢杆菌ZW在TCP为唯一碳源的培养液中的生长曲线。
图3为枯草芽孢杆菌ZW降解TCP的高效液相色谱图,TCP出峰时间为2.5min。
图4为枯草芽孢杆菌ZW对TCP的降解曲线,其中对照组为未接种菌体的处理。
具体实施方式
以下结合实施例进一步说明本发明,所举之例不应视为对本发明保护范围的限制。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用的设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例用于说明本发明,但不要来限制本发明的范围。
1.术语
本发明中所述用语“菌体”是指细菌的菌体细胞。
本发明所述用语:“芽孢”是指芽孢杆菌在一定条件下形成的抗逆行强的休眠体。
2. 本发明中的相关检测方法
1)本发明中菌体细胞或芽孢浓度测定:取枯草芽孢杆菌ZW培养物,经磷酸缓冲液稀释一定倍数,采用平板涂布法测定菌体细胞或芽孢的浓度。
2)本发明中TCP浓度测定方法:采用高效液相色谱检测,检测条件:色谱柱C18反相柱,流动相(甲醇:水)7:3,流速0.4ml/min,检测波长265nm,进样体积10uL。首先制作一系列不同浓度的TCP溶液,采用液相色谱检测TCP的峰面积,绘制峰面积与TCP浓度的标准曲线。
3.实施例
本发明人首先从长期施用毒死蜱农药的韭菜农田中成功分离筛选除了一株芽孢杆菌,经提取基因组DNA,通过PCR扩增和基于细菌16SrDNA测序的分子鉴定,确定为枯草芽孢杆菌属(Bacillus subtilis)。该菌株为革兰氏阳性,好氧,没有荚膜,有周生鞭毛,可以运动;生长较迅速、代谢旺盛、抗逆性强;在固体培养基上生长时,菌落的表面粗糙,呈污白或微黄色,有时会形成褶皱(图1)。基于上述特征,该菌株被鉴定并命名为枯草芽孢杆菌ZW(Bacillus subtilis. ZW),该菌株已在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为:CGMCC. No25210
以下通过具体实施例对本发明进行具体说明,所述实验方法,如无特殊说明,均为实验室常规方法,下文所述实验材料,无特别说明,均可由商业渠道获得。
实施例1:菌株活化培养
1)配置枯草芽孢杆菌活化培养基,组分(每升):蛋白胨,10.0 g;氯化钠,1.0 g;酵母,5.0 g;蒸馏水,1000 mL;pH:7.0;121℃ 20分钟灭菌。
2)将枯草芽孢杆菌菌株保藏液100uL,无菌条件下接种于上述活化培养基中,35℃,150rpm,摇床培养过夜,菌液浑浊后,再转接新鲜活化培养液,相同条件下培养,获得菌株活化培养液。
实施例2:菌株对TCP的降解
1)配置枯草芽孢杆菌无机盐培养基,组分(每升):硝酸铵,1.0 g;氯化钠,0.5 g;磷酸二氢钾,0.5 g;磷酸氢二钾,1.5 g;硫酸铵,0.5 g;硫酸镁,0.5 g;酵母,0.05 g;蒸馏水,1000 mL;pH:7.0;121℃ 20分钟灭菌。
2)取将上述活化后枯草芽孢杆菌ZW的菌悬液5mL,10000rpm,离心5min,收集菌体细胞
3)将收集的枯草芽孢杆菌ZW菌体细胞接入以TCP(50mg/L)为唯一碳源的无机盐培养液中,无机盐培养液中,35℃,150rpm,摇床培养
4)不同的时间点, 0、3、6、9、12、15、18、21、24 h取样。
5)每次取1mL液体,采用可见分光光度计检测OD600nm的菌体吸光度值,记录菌体在以TCP为唯一碳源的培养液中的生长情况(图2);同时采用液相色谱检测菌体培养液中TCP的浓度(图3),记录菌株对TCP的降解情况(图4)。
6)用于液相色谱检测的样品,需将菌液8000rpm离心5min,后取上清夜,移至干净离心管,冰箱-20℃冷冻,以备真空冷却干燥用于液相。
实施例3:枯草芽孢杆菌ZW液体及固体菌剂的制备
1)使用PBS缓冲液按一定比例对冷冻干燥后的枯草芽孢杆菌制剂进行稀释复配,菌体细胞浓度控制在1.0 x 1010 -3.0 x 1010 /mL,制成用于降解TCP的液态菌剂。
2)将枯草芽孢杆菌发酵液离心,获得菌体。然后按一定比例添加海藻糖、甘油和可溶性淀粉作为保护剂,与菌泥复溶,得到菌悬液。然后经过固定干燥得到制成用于降解TCP的固态菌剂。
Claims (6)
1.一种用于生物降解TCP的芽孢杆菌,其特征在于,所述菌株为枯草芽孢杆菌ZW,保存编号为CGMCC25210。
2. 一种降解TCP的枯草芽孢杆菌菌剂,其特征在于,所述菌剂包括权利要求1所述的芽孢杆菌,所述菌剂为液态制剂,芽孢杆菌菌体细胞或芽孢浓度为1.0 x 1010 -3.0 x 1010 /mL。
3.一种降解TCP的枯草芽孢杆菌菌剂,其特征在于,所述菌剂包括权利要求1所述的芽孢杆菌,所述菌剂为固体粉末制剂,芽孢杆菌菌体细胞或芽孢的含量为1.0 x 1010 -3.0 x1010/g。
4.按照权利要求2或3所述的枯草芽孢杆菌菌剂的制备方法,其特征在于:包括如下步骤:
1)将保藏的如权利要求1所述的枯草芽孢杆菌ZW菌株接种于发酵培养基中活化两代;
2)将活化好的菌株接种于含有50mg/L TCP的发酵液体培养基中,振荡培养至对数生长期,获得枯草芽孢杆菌的发酵培养物;
3)对枯草芽孢杆菌的发酵培养物进行离心分离处理,收获枯草芽孢杆菌的菌体细胞或芽孢。
5.按照权利要求4所述的制备方法,其特征在于:所述的发酵培养基组分为:
蛋白胨:10 g/L;酵母膏:5 g/L;NaCl:10 g/L;pH:7.0;所述培养温度为35℃。
6.如权利要求2或3所述的枯草芽孢杆菌菌剂在降解TCP中的应用。
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