CN104877934B - 一株耐盐多环芳烃降解菌Tzymx及其在海洋污染环境中的应用 - Google Patents
一株耐盐多环芳烃降解菌Tzymx及其在海洋污染环境中的应用 Download PDFInfo
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Abstract
一株耐盐的多环芳烃降解菌，该菌株为寡养单胞菌(Stenotrophomonas sp.)Tzymx，在中国典型培养物保藏中心的保藏号为CCTCC NO:M 2014626，具备在高盐环境下降解多环芳烃的能力。本发明具有以下优点：本发明提供的Tzymx对高盐环境中的多环芳烃有快速降解能力，且降解底物范围广，在短时间内降解率高。由于该菌具有耐高盐作用，使其在海洋等高盐环境中多环芳烃的修复中具有广阔的应用潜力。
Description
技术领域
本发明属于环境工程和微生物工程技术领域，尤其涉及一株耐盐多环芳烃的降解菌寡养单胞菌Stenotrophomonas sp.Tzymx及在多环芳烃污染环境修复中的应用
背景技术
多环芳烃(PAHs)是一类持久性有机污染物(POPs)。海洋环境中PAHs的来源主要是海底原油渗漏、海上运输、油轮事故、油井渗漏、陆源入海污染等。随着人类海洋活动的日益频繁，PAHs污染水平呈现逐年加重趋势。苯并芘、芘、苯并荧蒽等强致癌的高分子量PAHs都有检出。PAHs通过黏附于海洋沉积物经年累月地在海洋中积累，会在海洋生物体内会富集放大，对高营养级生物造成危害。在受PAH污染的海域进行渔业养殖，其产品的食用品质会受到严重影响，一旦被人体摄入则会严重危害健康。
多环芳烃(PAHs)污染修复是当前环境修复中研究的热点。微生物降解是对PAHs污染土壤进行修复的重要途径之一。对于多环芳烃污染，国内外进行了大量多环芳烃降解微生物的筛选工作。到目前为止，已分离出多个具有多环芳烃降解能力的菌株，如假单胞菌属(Pseudomonas putida G7)、分枝杆菌属(Mycobacterium vanbaalenii PYR-1)、芽孢杆菌属(Bacillus sp.P1)等。
传统分离出的多环芳烃降解菌在遇到高盐环境时，会抑制微生物的代谢作用，从而使微生物失去降解能力，并且由于条件限制，一般海洋等高盐环境在进行多环芳烃降解时不会进行 脱盐处理，因此在实际应用中，筛选出耐盐降解菌为高盐环境多环芳烃生化降解提供了可行性。
发明内容
本发明针对现有技术中多环芳烃降解菌不耐高盐的不足之处，提供一种降解多环芳烃菌株，该菌株可以应用于海洋等高盐环境中多环芳烃的污染治理。
为达到上述目的，本发明是通过以下技术方案得以实现的：
本发明提供的寡养单胞菌Stenotrophomonas sp.，命名为Stenotrophomonassp.Tzymx，于2014年12月4日保藏于“中国典型培养物保藏中心”，其保藏号CCTCC NO:M2014626。地址：中国武汉武汉大学。
所述菌株具有以多环芳烃为唯一碳源和能源进行生长的能力。
所述降解多环芳烃的菌株在被多环芳烃污染的海洋等高盐环境的生物修复中的应用。
所述菌株的16SrDNA序列如SEQ NO.1所示。
利用所述耐高盐的多环芳烃降解菌进行海洋等高盐环境生物修复的方法，其特征在于该方法由以下步骤组成：选取浙江台州湾的滩涂围塘养殖沉积泥100g，寡养单胞菌Stenotrophomonas sp.Tzymx按质量百分比2.0％与沉积泥均匀，加水使泥土含水率保持在15％，放入恒温振荡器中，控制培养温度在25℃的恒温条件下，振荡培养修复5天至20天。
Tzymx是从浙江省台州市的滩涂围塘养殖沉积泥中分离并筛选得到。本发明Tzymx可在25～35℃，pH值7.0～8.5培养条件下较好生长；菌株形态特征为菌株形态特征为杆状,革兰氏染色阴性；无芽孢；无荚膜,有鞭毛；在LB固体平板上生长菌落形态为圆形,黄色,微隆起,边缘整齐,湿润。
作为优选，本发明所述菌株能以多环芳烃为唯一碳源和能源生长。
上述耐盐多环芳烃降解菌的筛选方法，可按如下步骤依次实施：
(1)从滩涂围塘养殖沉积泥距地表下5～20cm处取表层泥土，作为菌源进行富集培养；所述富集培养基含有：葡萄糖、牛肉膏、蛋白胨、MgSO4、多环芳烃(包括但不限于萘、菲、荧蒽、芘、苯并蒽、苯并芘)；
(2)取(1)中所述富集培养液接种于液体无机盐选择培养基中进行选择培养，所无机盐选择培养基含有：NaCl、MgCl2﹒6H2O、Na2SO4、CaCl2﹒2H2O、TAPSO、KCl、NH4Cl、Na2HPO4﹒7H2O、NaBr、NaHCO3、H3BO3、SrCl2﹒6H2O、NaF、FeCl2﹒4H2O，将pH调至7.6(此为模拟天然海水组成的培养基)；添加多环芳烃(多环芳烃萘、菲、荧蒽、芘、苯并蒽、苯并芘的浓度均为150mg/L～250mg/L；)；
(3)取(2)中所述选择培养物稀释涂布于固体无机盐选择培养基上，进行分离进行分离即得到多环芳烃降解菌Tzymx。
上述多环芳烃降解菌Tzymx可用于海洋等高盐环境中多环芳烃的生物修复。
本发明Tzymx可在25～35℃，pH值7.0～8.5培养条件下较好生长；菌株形态特征为菌株形态特征为杆状,革兰氏染色阴性；无芽孢；无荚膜,有鞭毛；在LB固体平板上生长菌落形态为圆形,黄色,微隆起,边缘整齐,湿润。
Tzymx在LB(胰蛋白胨1.0％，酵母提取物0.5g％，氯化钠1.0％)固体培养基培养24小时菌落特征为圆形，菌落直径1～2mm，黄色，表面光滑，边缘整齐。
Tzymx的生理生化特征，见表1。
表1 Tzymx的生理生化特征
| 鉴定项目 | 鉴定结果 |
| 吲哚产生 | - |
| M.R实验 | - |
| V.P实验 | - |
| 明胶水解 | + |
| 尿酶实验 | - |
| 酪素水解 | + |
| 苯丙氨酸脱羧酶 | - |
| 精氨酸双水解酶 | - |
| 卵磷脂酶 | - |
| (NH4)2SO4 | - |
| KNO3 | - |
| NH4Cl | - |
| 丙二酸盐利用 | + |
| 柠檬酸盐利用 | + |
| 淀粉水解 | - |
| 纤维素水解 | - |
| 葡萄糖产酸产气 | + |
| 乳糖产酸产气 | + |
| 过氧化氢酶 | - |
| 葡萄糖利用 | + |
| 麦芽糖利用 | ++ |
| 阿拉伯糖利用 | + |
| 木糖利用 | + |
| 甘露醇利用 | + |
| 甘油利用 | + |
| 蔗糖利用 | + |
Tzymx的16SrDNA基因的PCR扩增、测序由上海美吉生物公司完成，得到了长度为1389bp的序列。选择同源性大于97％的基因序列进行系统发育分析，表明菌株与Stenotrophomonas sp.A18同源性最高，结合菌株形态、菌落特征及生理生化特性，鉴定菌株属于寡养单胞菌属(Stenotrophomonas sp.)。
本发明的Tzymx既可以在营养培养基，如：普通牛肉膏蛋白 胨、普通LB、营养琼脂中生长，又可在以多环芳烃为唯一碳源、能源的无机盐基础培养基中生长。
综上所述，本发明具有以下优点：本发明提供的Tzymx对高盐环境中的多环芳烃有快速降解能力，且降解底物范围广，在短时间内降解率高。由于该菌具有耐高盐作用，使其在海洋等高盐环境中多环芳烃的修复中具有广阔的应用潜力。
分类命名：寡养单胞菌
拉丁文名：Stenotrophomonas sp.Tzymx
保藏单位：中国典型培养物保藏中心
地址：中国武汉武汉大学
保藏日期：2014年12月4日
保藏编号：CCTCC NO:M 2014626。
具体实施方式
下面通过具体实施例，对本发明的技术方案作进一步具体的说明，但是本发明并不限于这些实施例。
实施例1
本发明提供的Tzymx的分离筛选方法。
从滩涂围塘养殖沉积泥距地表下5～20cm处取表层泥土，作为菌源进行富集培养，将葡萄糖2g，胰蛋白胨4g，牛肉膏4g，MgSO40.8g，萘60mg、菲60mg、荧蒽60mg、芘60mg、苯并蒽60mg、苯并芘60mg；加入到400mL的蒸馏水中，滤膜除菌，在此培养基上培养二天。再配制模拟天然海水组成的培养基，将22.79g NaCl、11.18g MgCl2﹒6H2O、3.98g Na2SO4、1.46gCaCl2﹒2H2O、1.30g TAPSO、0.72g KCl、0.27g NH4Cl、89.00mg Na2HPO4﹒7H2O、83.00mg NaBr、31.00mg NaHCO3、27.00mg H3BO3、24.00mg SrCl2﹒6H2O、2.60mg NaF、2.00mg FeCl2﹒4H2O加入800mL蒸馏水中，再加入萘、菲、荧蒽、芘、苯并蒽、苯并芘的多环芳烃 化合物，加入蒸馏水定容至1000mL，上述模拟天然海水组成的培养基上述加入萘、菲、荧蒽、芘、苯并蒽、苯并芘的量要使配制好的培养液中萘、菲、荧蒽、芘、苯并蒽、苯并芘的最终浓度均为200mg/mL，将pH调至7.6，滤膜除菌，即得所需的培养液。取富集培养液在此培养基上培养三天，稀释涂布分离出一株菌命名为Tzymx，该菌于2014年12月4日保藏于“中国典型培养物保藏中心”其保藏号CCTCC NO:M 2014626。
Tzymx的16SrDNA基因的PCR扩增、测序由上海美吉生物公司完成。选择同源性大于97％的基因序列进行系统发育分析，表明菌株与Stenotrophomonas sp.A18同源性最高，结合菌株形态、菌落特征及生理生化特性，鉴定菌株属于寡养单胞菌属(Stenotrophomonassp.)。
实施例2
Tzymx降解洁净滩涂围塘养殖沉积泥中人工添加的多环芳烃。
选取添加芘、苯并蒽、苯并芘浓度各为200mg/kg的洁净滩涂围塘养殖沉积泥100g，Tzymx按2.0％(质量百分比)用量与泥土混合均匀，加水使含水率保持在15％，放入恒温振荡器中，控制培养温度在25℃的恒温条件下，振荡培养修复5天，即能实现对滩涂围塘养殖沉积泥进行修复，得到修复后的土壤，芘、苯并蒽、苯并芘的降解率均为70％以上。
实施例3
Tzymx降解受多环芳烃污染的滩涂围塘养殖沉积泥中的多环芳烃。
取浙江省台州市一滩涂围塘养殖沉积泥，HPLC分析测定主要多环芳烃污染物菲、萘、荧蒽、芘和PAHs总量分别为197.0、60.3、264.4、180.5、983.7ng/g，Tzymx按2.0％(质量百分比)用量 与泥土混合均匀，加水使含水率保持在15％，放入恒温振荡器中，控制培养温度在25℃的恒温条件下，振荡培养修复20天，测定残留菲、萘、荧蒽、芘和PAHs总量分别为69.4、19.2、150.0、99.6、486.1ng/g，降解率分别为66.4％、68.2％、43.3％、44.8％、50.6％。
利用本发明的方法对高盐条件下污染土壤进行修复，表明多环芳烃的修复效果好，对多环芳烃的降解效果明显，说明菌株耐盐并达到较好的多环芳烃降解的效果，与现有技术相比，提高了对多环芳烃污染环境的修复效果，更有利于实际中对海洋等高盐环境中多环芳烃的修复。
本发明中所描述的具体实施例仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (3)
1. 一株耐盐的多环芳烃降解菌，其特征在于，该菌株为寡养单胞菌(Stenotrophomonas sp.)Tzymx，在中国典型培养物保藏中心的保藏号为CCTCC NO:M2014626，所述多环芳烃为萘、菲、荧蒽、芘、苯并蒽、苯并芘。
2.根据权利要求1所述耐盐的多环芳烃降解菌在海洋多环芳烃污染环境的生物修复中的应用。
3. 利用权利要求1所述耐盐的多环芳烃降解菌进行污染环境生物修复的方法，其特征在于该方法由以下步骤组成：选取多环芳烃污染的滩涂围塘养殖沉积泥100g，寡养单胞菌(Stenotrophomonas sp.)Tzymx按质量百分比2.0％与泥土混合均匀，加水使泥土含水率保持在15%，放入恒温振荡器中，控制培养温度在25℃的恒温条件下，振荡培养修复5 天至20天。
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Application publication date: 20150902 Assignee: Taizhou Yongshang Trading Co.,Ltd. Assignor: TAIZHOU VOCATIONAL & TECHNICAL College Contract record no.: X2023980044891 Denomination of invention: A Salt-tolerant Polycyclic Aromatic Hydrocarbon Degrading Bacterium Tzymx and Its Application in Marine Polluted Environment Granted publication date: 20180109 License type: Common License Record date: 20231030 Application publication date: 20150902 Assignee: Taizhou Yier Technology Co.,Ltd. Assignor: TAIZHOU VOCATIONAL & TECHNICAL College Contract record no.: X2023980044675 Denomination of invention: A Salt-tolerant Polycyclic Aromatic Hydrocarbon Degrading Bacterium Tzymx and Its Application in Marine Polluted Environment Granted publication date: 20180109 License type: Common License Record date: 20231027 |