CN113549565B - 一种具有降解苯系物能力的阴沟肠杆菌及其应用 - Google Patents
一种具有降解苯系物能力的阴沟肠杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有苯系物降解能力的阴沟肠杆菌及其在环境修复中的应用。本发明通过对从广东省广州市某垃圾填埋场垃圾渗滤液中进行筛选分离出了一种对苯系物具有良好降解能力的阴沟肠杆菌，并将此阴沟肠杆菌Enterobacter cloacae GDUTAN7，于2021年1月18日保藏于中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏编号为CCTCC NO：M 2021091；本发明所得到的阴沟肠杆菌Enterobacter cloacae GDUTAN7对环境中苯系物降解效果较好，可将其应用于降解环境中苯系物，实现环境修复的目的。
Description
技术领域
本发明属于微生物技术领域，特别涉及一种对苯系物具有降解能力的阴沟肠杆菌及其在环境修复和VOCs净化中的应用。
背景技术
苯系物污染主要存在于原油和石油产品生产加工行业中，在这些产品的加工和运输过程中会不可避免地排放和泄露环境中，并且伴随着降水和地表水等渗透到土壤中污染土壤和水体。同时，在油漆生产，塑料生产，印染和纺织加工等行业也会产生大量高浓度苯系物的废水和废气。由于苯系物具有强致癌、致畸和致突变性，难以降解，并且迁移能力强，引起了众多国家的高度重视，并被列为优先控制污染物。短期高剂量暴露会对中枢神经系统有麻醉，导致眼及上呼吸道出现明显的刺激症状，如眼结膜及咽充血，以及出现头晕、头痛、恶心、呕吐、胸闷、四肢无力、意识模糊等症状，长期接触会导致神经衰弱综合征等。
生物法是利用微生物的代谢活动降解环境中的苯系物，由于其投资成本小，无二次污染等优点得到人们的青睐。肠杆菌属的阴沟肠杆菌无芽孢、无荚膜、会形成大而湿润的黏液状菌落，是兼性厌氧的革兰阴性粗短杆菌，在人和动物的粪便、水、泥土、植物中均可检出，广泛存在于自然界中。已有研究证明，特定的阴沟肠杆菌菌种可被用于降解有机磷农药。但目前尚未有可用作苯系物降解的阴沟肠杆菌菌种的研究报道。
发明内容
本发明的首要目的在于提供一种具有苯系物降解能力的阴沟肠杆菌，本发明的目的通过下述技术方案实现：
阴沟肠杆菌(Enterobacter cloacae)GDUTAN7，于2021年1月18日保藏于中国典型培养物保藏中心(CCTCC)，地址：中国湖北省武汉市武汉大学，保藏编号为CCTCC NO：M2021091；
所述阴沟肠杆菌Enterobacter cloacae GDUTAN7具有如下的形态和生理生化特性：
a.根据常规的细菌生理生化鉴定方法和电子扫描显微镜观察，所述阴沟肠杆菌Enterobacter cloacae GDUTAN7为革兰氏阴性菌；在电子扫描显微镜下观察，其形态为杆状，菌体大小为0.1～0.2×0.3～0.5μm，无鞭毛；
b.阴沟肠杆菌Enterobacter cloacae GDUTAN7菌落的形态特性为：在LB固体培养基培养24h后，菌落形态为圆形，乳白色，半透明，菌落直径为0.5～1.5mm；
c.根据《伯杰氏细菌鉴定手册》第八版，对细菌生理生化指标进行检测，主要的生理生化特征如表1：
表1检测菌体的生理生化检测指标
注：反应状态分为阳性和阴性，阳性形状编码为“+”，阴性形状编码为“-”
d.所述对苯系物具有降解能力的阴沟肠杆菌Enterobacter cloacae GDUTAN7的16S rDNA序列如SEQ ID:No.1所示。
本发明的目的之二是提供一种具有降解苯系物能力的阴沟肠杆菌在环境修复中的应用。
优选地，所述阴沟肠杆菌Enterobacter cloacae GDUTAN7用于降解环境中的苯系物，所述环境包括大气、水体和土壤。
优选地，所述苯系物为间二甲苯。
本发明的目的之三是提供一种具有降解苯系物能力的阴沟肠杆菌在VOCs净化中的应用。
有益的技术效果:本发明中具有苯系物降解能力的阴沟肠杆菌Enterobactercloacae GDUTAN7能够应用于环境修复以及VOC净化治理，可降解环境中如大气、水体和土壤中的苯系物以及工业排放尾气中的苯系物，且其降解效果非常出色，降解率可高达99.8％以上。
附图说明
图1是本发明的阴沟肠杆菌Enterobacter cloacae GDUTAN7在电子扫描显微镜下的形态图一。
图2是本发明的阴沟肠杆菌Enterobacter cloacae GDUTAN7在电子扫描显微镜下的形态图二。
图3是本发明的阴沟肠杆菌Enterobacter cloacae GDUTAN7在电子扫描显微镜下的形态图三。
图4是本发明的阴沟肠杆菌Enterobacter cloacae GDUTAN7在电子扫描显微镜下的形态图四。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
1.实施例：
本发明中具有苯系物降解能力的阴沟肠杆菌Enterobacter cloacae GDUTAN7是在以苯系物为碳源和能源物质时，从广东省广州市某垃圾填埋场垃圾渗滤液中筛选分离、纯化而得。在无机盐培养基配方(K2HPO4·3H2O 1.2g/L,KH2PO4 1.2g/L,NH4Cl 0.4g/L,MgSO4·7H2O 0.2g/L,FeSO4·7H2O 0.01g/L，微量元素:CaCl2·2H2O 0.2g/L,MnSO4·4H2O0.2g/L,CuSO4·2H2O 0.01g/L,ZnSO4·7H2O 0.2g/L,CoCl2·6H2O 0.09g/L,Na2MoO4·2H2O0.12g/L,H3BO3 0.006g/L)添加一定量的苯系物筛选，驯化底物浓度依次为1mg/L、5mg/L、10mg/L、15mg/L、20mg/L。首先取10ml垃圾渗滤液加入含1mg/L苯系物的无机盐培养基，37℃驯化5天后，以10％接种量移入下一浓度中接着驯化，以此类推逐步提高浓度进行驯化。驯化结束后将终浓度的菌液稀释10-1～10-7倍，特别地，选取10-1、10-2、10-3、10-4、10-5、10-6、10-7等七个稀释度的稀释液，各取0.2ml上述稀释液分别均匀涂布在以苯系物为碳源的固体培养基中，挑选生长状态良好的单菌落富集培养后进行的降解实验，将具有降解效果最好的菌落进行保存并做菌种鉴定。鉴定结果如下：
(1)菌体的形态特性：
a.根据生理生化鉴定结果和电子显微镜观察，得出所筛选的阴沟肠杆菌Enterobacter cloacae GDUTAN7为革兰氏阴性菌；在电子显微镜下观察，其形态为杆状，菌体大小为0.1～0.2×0.3～0.5μm，无鞭毛，如图1-4所示；
b.在固体培养基(牛肉膏3.0g/L、蛋白胨10.0g/L、NaCl 5.0g/L)培养24h后菌落的形态特性：菌落形态为圆形，乳白色，半透明，菌落直径为0.5～1.5mm；
c.主要的生理生化特征如表2：
表2检测菌体的生理生化特征
注：反应状态分为阳性和阴性，阳性形状编码为“+”，阴性形状编码为“-”
(2)采用DNA提取试剂盒提取细菌总DNA。采用细菌的16SrDNA通用引物：
上游引物:27F(5'-AGTTTGATCMTGGCTCAG-3')
下游引物:1492R(5'-GGTTACCTTGTTACGACTT-3')
扩增其16S rDNA基因，测序结果详见SEQ ID:No.1。
将长为1372bp的16S rRNA基因序列与Genbank中已登录的基因序列进行比对分析发现菌种与Enterobacter cloacae strain OsEp_A&N_15A7最相似，相似度为99％。
综合上述的生理生化特性、16S rRNA基因序列结果，本发明所筛选的细菌应归属肠杆菌属，命名为Enterobacter cloacae GDUTAN7，于2021年1月18日保藏于中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏编号为CCTCC NO：M2021091。
2.以降解间二甲苯为例：
本发明筛选得到的阴沟肠杆菌Enterobacter cloacae GDUTAN7对间二甲苯具有较好的降解能力：
a.配置无机盐培养基：在顶空瓶中配制100mL无机盐培养液(K2HPO4·3H2O0.12g，KH2PO40.12g，NH4Cl 0.04g，MgSO4·7H2O0.02g，FeSO4·7H2O 0.001g)，100μl微量元素(CaCl2·2H2O 0.2g/L，MnSO4·4H2O 0.2g/L，CuSO4·2H2O 0.01g/L，ZnSO4·7H2O 0.2g/L，CoCl2·6H2O 0.09g/L，Na2MoO4·2H2O 0.12g/L，H3BO3 0.006g/L)，121℃高压灭菌30min。
b.首先将筛选的阴沟肠杆菌Enterobacter cloacae GDUTAN7在营养肉汤培养基(牛肉膏3.0g/L、蛋白胨10.0g/L、NaCl 5.0g/L)中富集增菌培养24h后，离心收集菌体，并用磷酸盐缓冲液洗涤三次，然后以10％接种量悬浮在上述装有100mL无机盐培养基的血清瓶中，然后加入一定量间二甲苯溶液使得其浓度分别为5mg/L、8mg/L、10mg/L、15mg/L、20mg/L、25mg/L加盖密封，在温度37℃、振动频率200r/min摇床下培养进行降解试验，定期取样测定气相中间二甲苯的浓度，并利用亨利定律来检测其在液相中的降解率。检测条件为：含氢焰离子化检测器(FID)GC9800气相色谱仪(AT.SE-54毛细管柱,30m×0.25mm×0.25μm)，检测器温度200℃，进样口气化室温度150℃，柱炉温度120℃。采用Agilent500μL气密针进样，进样量为500μL，不分流模式。结果如表3所示：
表3不同初始浓度的间二甲苯的降解率
从表3中可以看出，本发明得到阴沟肠杆菌Enterobacter cloacae GDUTAN7在48h对间二甲苯的降解能力可达99.8％以上；对苯系物具有良好的降解效果，可以应用于气体，水体，土壤的苯系物污染的修复及工业排放的VOC净化治理中。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，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，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 广州紫科环保科技股份有限公司
<120> 一种具有降解苯系物能力的阴沟肠杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1372
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgccctcccg aaggttaagc tacctacttc ttttgcaacc cactcccatg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgtagcatt ctgatctacg attactagcg 120
attccgactt catggagtcg agttgcagac tccaatccgg actacgacgc actttatgag 180
gtccgcttgc tctcgcgagg tcgcttctct ttgtatgcgc cattgtagca cgtgtgtagc 240
cctggtcgta agggccatga tgacttgacg tcatccccac cttcctccag tttatcactg 300
gcagtctcct ttgagttccc ggcctaaccg ctggcaacaa aggataaggg ttgcgctcgt 360
tgcgggactt aacccaacat ttcacaacac gagctgacga cagccatgca gcacctgtct 420
cacagttccc gaaggcacca atccatctct ggaaagttct gtggatgtca agaccaggta 480
aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgattt aacgcgttag 600
ctccggaagc cacgcctcaa gggcacaacc tccaaatcga catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgca cctgagcgtc agtctttgtc 720
cagggggccg ccttcgccac cggtattcct ccagatctct acgcatttca ccgctacacc 780
tggaattcta cccccctcta caagactcca gcctgccagt ttcgaatgca gttcccaggt 840
tgagcccggg gatttcacat ccgacttgac agaccgcctg cgtgcgcttt acgcccagta 900
attccgatta acgcttgcac cctccgtatt accgcggctg ctggcacgga gttagccggt 960
gcttcttctg cgggtaacgt caattgctgt ggttattaac cacaacacct tcctccccgc 1020
tgaaagtact ttacaacccg aaggccttct tcatacacgc ggcatggctg catcaggctt 1080
gcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1140
ttccagtgtg gctggtcatc ctctcagacc agctagggat cgtcgcctag gtgagccgtt 1200
accccaccta ctagctaatc ccatctgggc acatctgatg gcaagaggcc cgaaggtccc 1260
cctctttggt cttgcgacgt tatgcggtat tagctaccgt ttccagtagt tatccccctc 1320
catcaggcag tttcccagac attactcacc cgtccgccac tcgtcacccg ag 1372
Claims (3)
1.一种具有降解苯系物能力的阴沟肠杆菌（Enterobacter cloacae），其特征在于：该菌株名称为阴沟肠杆菌（Enterobacter cloacae）GDUTAN7，保藏在中国典型培养物保藏中心，保藏地址为湖北武汉市武汉大学；保藏编号为CCTCC NO：M 2021091。
2.根据权利要求1所述的具有降解苯系物能力的阴沟肠杆菌在环境修复中的应用，所述苯系物为间二甲苯。
3.根据权利要求2所述的具有降解苯系物能力的阴沟肠杆菌在环境修复中的应用，其特征在于：所述阴沟肠杆菌（Enterobacter cloacae）GDUTAN7用于降解环境中的苯系物，所述环境包括大气、水体和土壤。
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