CN117384794A - 拟无枝酸菌Amycolatopsis sp.MT3及其在降解卤代污染物中的应用 - Google Patents
拟无枝酸菌Amycolatopsis sp.MT3及其在降解卤代污染物中的应用 Download PDFInfo
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Abstract
本发明公开了一种拟无枝酸菌Amycolatopsis sp.MT3,于2023年3月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.47852。还公开了其在降解卤代污染物中的应用,以及利用磁纳米颗粒分离的方法。本发明筛选的菌株在好氧条件下能有效降解PCB77,还能有效降解PCB47、PCB110、BDE28、BDE47和BDE77。
Description
技术领域
本发明属于环境微生物工程技术领域,更具体的涉及一种拟无枝酸菌Amycolatopsis sp.MT3及其在降解卤代污染物中的应用。
背景技术
多氯联苯(Polychlorinated Biphenyls,PCBs)是典型的持久性有机污染物之一,曾作为变压器油、阻燃剂、导热剂、增塑剂等材料,广泛应用于化工领域。因氯原子取代位置与数量的不同,PCBs具有209种同系物,其中PCB77(3,3′,4,4′-Tentrachlorinatedbiphenyl)呈平面分子结构,难以微生物降解,在环境中半衰期长。同时,它也是毒性最强的一组PCBs组分,影响人体神经系统、免疫功能,并对生态系统造成危害。
针对环境PCBs污染,物理、化学和生物的修复方法均被采用,其中微生物修复技术因绿色、经济、可原位实施等优点具有很好的修复前景。目前已有研究报道从环境中分离出可好氧降解多氯联苯的微生物菌属,如Rhodococcus、Alcaligenes、Pseudomonas、Sphingomonas及Burkholderia等。但因多氯联苯降解菌资源不够丰富及外源微生物存活率较低,目前应用功能微生物修复多氯联苯污染场地的成功案例仍然较少。且污染场地中污染源分布复杂,往往伴随着多种污染物的交叉污染,如多种卤代污染物的共存,因此,从环境中筛选出更多的多氯联苯降解菌,并充分挖掘这些降解菌的降解谱,才有可能提高生物强化法的修复效果。
环境中微生物群落结构十分复杂,如何在复杂群落中定位到功能微生物仍存在较大的挑战。磁性纳米粒子分离技术在菌株分离方面具有重要价值,如在一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法(CN 110527640 A)中制备的纤维化磁性纳米粒子,成功应用于厌氧纤维降解菌的富集和分离;在一种利用磁性纳米颗粒技术分离废水中乙腈的原位降解菌的方法(CN 111041072 A)中利用磁性纳米颗粒促进了活性污泥中乙腈原位降解菌的富集,丰富了乙腈降解菌株的资料库。可见,磁性纳米技术介导分离技术是富集功能微生物的有效方法。
通过磁纳米粒子分离技术实现多氯联苯功能菌群的富集,在克服传统分离方法效率低、缺乏群体交互作用等弊端的同时,有助于我们进一步开展污染物微生物降解的研究,在难降解污染物的微生物修复方面具有重要的现实意义。
发明内容
1、发明目的。
本发明筛选出一株拟无枝酸菌Amycolatopsis sp.MT3,能有效降解卤代污染物。
2、本发明所采用的技术方案。
本发明的拟无枝酸菌Amycolatopsis sp.MT3,于2023年3月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其简称为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,邮编100101),分类命名为拟无枝酸菌(Amycolatopsis),保藏编号为CGMCC No.47852。
本发明的拟无枝酸菌Amycolatopsis sp.MT3菌株具有以下微生物学特征:
(1)菌落形态:白色至浅黄色,表面褶皱、边缘略带不平整
(2)菌体形态:放线状
(3)生理生化特征:革兰氏阳性
(4)培养特征:LB培养基,28℃
本发明的拟无枝酸菌Amycolatopsis sp.MT3可应用于降解卤代污染物,尤其是PCB77、PCB47、PCB110、BDE28、BDE 47或BDE77。
本发明还公开了上述的拟无枝酸菌Amycolatopsis sp.MT3的分离方法,其步骤包括:
(1)取氯代烃污染土壤,按一定固液比制备土壤悬液,首轮以PCB47为底物富集培养;
(2)培养一段时间后离心去除土壤大颗粒收获原始混合培养物,培养物在好氧环境中进行磁功能化,磁化菌群作为接种物以PCB77为底物进行新一轮培养;
(3)培养结束后,利用磁铁分离磁化和非磁化菌群,其中非磁化菌群即为PCB77降解菌群,经双层平板分离纯化得到单菌株Amycolatopsis sp.MT3。
优选的,步骤(1)中所述的固液比为1:5,底物PCB47浓度为5mg kg-1,富集周期为2个月。
优选的,步骤(2)中离心去除土壤大颗粒的离心条件为1400r,3min;所述磁功能化过程为:原始混合培养物与磁纳米粒子混合后于恒温培养箱中震荡1h(28℃,160r),其中,磁纳米颗粒为8g L-1Fe3O4纳米颗粒,添加量3%(v/v);底物PCB77浓度为5mg kg-1,磁化菌群接种量为20%(v/v),于恒温震荡培养箱中培养15天。
优选的,步骤(3)中所述的磁铁分离步骤为:磁铁置于培养瓶外壁一侧吸附磁化菌群20min,未被吸附的培养菌液为目标菌群;双层平板为下层为1.5%琼脂的MM固体培养基,上层为含有PCB77(20mg kg-1)为唯一碳源的MM固体培养基(1%琼脂)。
本发明涉及的磁纳米颗粒分离技术可以在复杂的原位环境下有效实现活性菌群的分离。磁性纳米颗粒非特异性的包裹在细胞表面,将细胞进行磁功能化。当加入某种特定的生长底物后,生长速度快的细胞能够随着反应的进行和细胞分裂逐渐丢失磁性,而生长速度慢的细胞则一直保有磁性,最后用磁铁定向吸附有磁纳米颗粒包裹的无代谢活性的微生物,分离上清液与沉淀,保留上清液即可获得具有特异性的功能菌群。经过平板加以分离纯化,得到降解菌株,并通过纯培养实验对其降解性能加以验证。
3、本发明所产生的技术效果。
(1)采用本发明提供的方法,利用磁纳米颗粒分离得到一株PCB77降解菌株MT3,经鉴定为Amycolatopsis属,目前尚未见此菌属用于PCB77降解的相关报道;
(2)本发明所得菌株MT3除PCB77外,对其他卤代污染物也具有良好的降解效果,包括PCB47、PCB110、BDE28、BDE47、BDE77;
(3)本发明提供的方法简便易行,同样适于分离其他难降解污染物的活性菌群。
附图说明
图1PCB降解菌MT3的系统发育树(a)和菌落细胞形态(b);
图1(a)中:Bootstrap次数设置为1 000,分支处数字表示Bootstrap支持率;括号内代表序列GenBank登录号。
图2菌株MT3对PCB77(a)和其他卤代污染物(b)的降解;
图2中:图中YE表示共代谢条件,具体为额外添加0.05%酵母抽提物。
具体实施方式
应该指出,以下具体说明都是示例性的,旨在对本发明提供进一步的说明。除非另有说明,本文所使用的所有科学和技术术语具有与本发明所属技术领域人员通常理解的相同含义。
下面结合具体实施例对本发明做进一步说明,但所举实施例不作为对本发明的限定。若为特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1:PCB降解菌的富集与分离
采集氯代烃污染场地土壤,采用PCB47作为底物富集PCB降解菌。具体实施如下:采用丙酮将PCB47引入250mL灭菌三角瓶(121℃,30min),待丙酮挥发后加入100mL MM培养基和20g土壤制备成含有5mg kg-1PCB47的土壤泥浆,三角瓶于28℃、160r/min培养2个月。富集结束后,取土壤悬液于1 400r/min离心3min,去除土壤大颗粒及杂质,得到初始富集培养物。将培养物进行磁化处理,以PCB77为底物进行培养,培养结束后进行磁性分离获得高效PCB77降解活性菌群。具体实施如下:向获得的富集培养物加入磁纳米颗粒在恒温震荡培养箱中培养1h对微生物进行磁化处理,取1mL磁化培养物接入5mL无机盐培养基中,PCB77(5mgkg-1)和联苯(0.1%)预先通过丙酮加入培养瓶中作为碳源底物,培养瓶置于28℃,160r的恒温震荡培养箱内培养15天,结束培养后在超净台用磁铁置于M组培养瓶外壁一侧吸附磁化菌群20min,未被吸附的培养菌液为目标菌液。取100μL目标菌液,涂布于PCB77双层板,于28℃培养箱中进行培养,待长出单菌落后,挑选单菌落在LB板上进行分离纯化。所得的单菌株在LB液体培养基中扩大培养并提取菌体DNA,采用通用引物27F(5′-AGTTTGATCMTGGCTCAG-3′)和1492R(5′-GGTTACCTTGTTACGACTT-3′)对细菌16S rRNA基因进行PCR,送公司(MajorBio,中国上海)进行测序,序列在NCBI数据库(blast.ncbi.nlm.nih.gov)中进行比对,采用MEGA5软件构建系统进化树,进行同源性分析。并在LB上反复划线纯化,挑取单菌落进行革兰氏染色,在显微镜下镜检,观察菌体形态。
结果表明,将得到的单菌株定名为MT3,根据其16S rDNA序列结果发现菌株MT3为Amycolatopsis属,中文名为拟无枝酸菌。菌落为白色至浅黄色,革兰氏阳性,呈放线状。
实施例2降解菌对PCB77的降解
PCB降解菌MT3采用LB培养基于28℃、160r·min-1培养至对数生长期(OD600≈0.6),5 000×g离心10min收集菌体,MM培养基清洗菌体3轮后采用10%(v/v)比例进行接种。降解实验于20mL灭菌培养瓶(121℃,30min)进行,分别考察唯一碳源和共代谢条件下PCB77的降解,其中唯一碳源为5mg L-1PCB77,共代谢条件为额外添加0.05%酵母抽提物,有机污染物通过丙酮预先引入培养瓶,培养瓶于28℃、160r min-1培养9d。实验处理均为3个重复。培养结束后向瓶内加入两倍体积的正己烷提取残留污染物,吸取1mL有机相过0.22μm滤膜,采用气相色谱仪(GC,Agilent 7890A,岛津)测定残留污染物。
结果发现:比较了菌株MT3在唯一碳源和共代谢条件下对PCB77的降解能力。唯一碳源条件下,菌株MT3无PCB77降解能力(0%;图2a),酵母抽提物能够有效的促进菌株MT3对PCB77的降解(79.8%%;图2a)。
实施例3降解菌对其他卤代污染物的降解
测试菌株MT3在共代谢条件下对其他卤代污染物的降解,实验体系建立、培养条件、污染物提取及测定同实施案例2步骤。分别添加单个污染物作为微生物碳源:PCBs种类有PCB47和PCB110,使用浓度均为5mg L-1;PBDEs种类有BDE28、BDE47和BDE77,使用浓度均为0.1mg L-1,共代谢条件为额外添加0.05%酵母抽提物。培养瓶置于28℃、160r min-1的培养箱培养9d,实验处理包括空白均为3个重复。
结果发现:菌株MT3对供试污染物均有较好的降解表现,在单一PCBs作为碳源的条件下,MT3对PCB47和PCB110的降解率分别为15.9%和52.1%;在单一PBDEs的条件下,MT3对BDE28、BDE47和BDE77的降解率分别为6.42%、38.1%和37.1%(图2b)。
以上所述的实施例只是为更好地解释本发明而给出的优选实施方案,因此上述实施例并不用于限制本发明,在本发明的原则之内做的修改、改进和替换均应包含在本发明的保护范围之内。
Claims (7)
1.一种拟无枝酸菌Amycolatopsis sp.MT3,于2023年3月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.47852。
2.权利要求1所述的拟无枝酸菌Amycolatopsis sp.MT3在降解卤代污染物中的应用。
3.根据权利要求2所述的应用,其特征在于卤代污染物为:多氯联苯(包括PCB77、PCB47、PCB110)及多溴联苯醚(包括BDE28、BDE47、BDE77)。
4.权利要求1所述的拟无枝酸菌Amycolatopsis sp.MT3的分离方法,其特征在于其步骤包括:
(1)取氯代烃污染土壤,按一定固液比制备土壤悬液,首轮以PCB47为底物富集培养;
(2)培养一段时间后离心去除土壤大颗粒收获原始混合培养物,培养物在好氧环境中进行磁功能化,磁化菌群作为接种物以PCB77为底物进行新一轮培养;
(3)培养结束后,利用磁铁分离磁化和非磁化菌群,其中非磁化菌群即为PCB77降解菌群,经双层平板分离纯化得到单菌株Amycolatopsis sp.MT3。
5.根据权利要求4所述的拟无枝酸菌Amycolatopsis sp.MT3的分离方法,其特征在于步骤(1)中所述的固液比为1:5,底物PCB47浓度为5mg kg-1,富集周期为2个月。
6.根据权利要求4所述的拟无枝酸菌Amycolatopsis sp.MT3的分离方法,其特征在于步骤(2)中离心去除土壤大颗粒的离心条件为1400r,3min;所述磁功能化过程为:原始混合培养物与磁纳米粒子混合后于恒温培养箱中震荡1h(28℃,160r),其中,磁纳米颗粒为8gL-1Fe3O4纳米颗粒,添加量3%(v/v);底物PCB77浓度为5mg kg-1,磁化菌群接种量为20%(v/v),于恒温震荡培养箱中培养15天。
7.根据权利要求4所述的拟无枝酸菌Amycolatopsis sp.MT3的分离方法,其特征在于步骤(3)中所述的磁铁分离步骤为:磁铁置于培养瓶外壁一侧吸附磁化菌群20min,未被吸附的培养菌液为目标菌群;双层平板为下层为1.5%琼脂的MM固体培养基,上层为含有PCB77(20mg kg-1)为唯一碳源的MM固体培养基(1%琼脂)。
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