CN115058364B - 一株降解多环芳烃的微杆菌及其应用 - Google Patents
一株降解多环芳烃的微杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株降解多环芳烃的微杆菌及其应用，属于微生物技术领域。所述的菌株为微杆菌Z‑2，保藏编号为CGMCC No.24842。本发明的微杆菌Z‑2具有高效的多环芳烃降解能力，能够耐受高浓度多环芳烃，且降解周期短，适用于多环芳烃污染土壤和地下水的生物修复。
Description
技术领域
本发明属于微生物技术领域，涉及一株降解多环芳烃的微杆菌及其应用。
背景技术
近年来，随着石油行业的不断发展、农药的过度使用以及工业“三废”的大量排放，环境污染加剧，土壤质量也一直在下降，各种有毒物质及废弃污染物增多，这些有毒物质通过生物的生长过程进入到植物体内，再间接到人体，严重影响农业生产和人类健康。而其中，多环芳烃(PAHs)被认定为是影响人类健康的主要有机污染物。
多环芳烃是一类具有代表性的持久性有机污染物，在土壤中半衰期极长，难生物降解，且具有强烈的“三致”(致癌、致畸、致突变)毒性。现阶段，多环芳烃污染土壤的修复技术主要有物理法、化学法和生物法。物理法包括：热脱附、化学表面活性剂淋洗等，对于热脱附法，虽然原理简单、操作简便，但其耗能大、处理成本高；化学表面活性剂淋洗法操作简便、成本较低，但化学表面活性剂本身的残留会对土壤造成二次污染，残留药剂的回收也很麻烦。并且，这两种方法受限于土壤的渗透系数。化学法有氧化剂氧化法、以及以光催化和电化学为代表的高级氧化技术，氧化剂氧化法虽然高效稳定，但氧化剂的强氧化性会改变土壤的理化性质，其残留会造成二次污染；高级氧化技术受限于土壤性质，且成本较高。不同于物理方法和化学方法的局限性，包括植物修复、微生物修复在内的生物修复不仅成本低，且没有二次污染的问题，一直被认为是降解有机污染物的优先方法。然而，针对该类污染物，目前的降解大多依赖于土著菌，通过添加营养元素，构建生物刺激工艺进行降解，存在后期长、难以达标等问题。这与缺乏高效的降解菌株有关。大多数报道的微杆菌仅能耐受30mg/L以内的PAHs(例如：CGMCC No.18248、CGMCC No.3581等)，而且降解周期长达数月。
发明内容
本发明的目的是提供一种具有高效降解污染土壤中多环芳烃能力的微杆菌(Microbacterium sp.)，该菌株可应用于多环芳烃污染土壤的生物修复。
本发明的目的是通过以下技术方案来实现的：
本发明所述的降解多环芳烃的微杆菌，为微杆菌Z-2，已于2022年5月6日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏，保藏编号为CGMCC No.24842，保藏地址为中国北京市朝阳区北辰西路1号院3号。
本发明提供上述微杆菌的培养方法，具体如下：
将微杆菌Z-2接种至培养基中，37℃下摇床培养，所述的培养基的配方为：NaNO31～4g/L，MgSO4 0.2～0.4g/L，NaCl 1～3g/L，KCl 1～3g/L，CaCl2 0.04～0.06g/L，H3PO4 5～7ml/L，多环芳烃(碳源)100～400mg/L，pH＝7.0。
本发明还提供上述微杆菌在降解多环芳烃中的应用。
本发明中，所述的多环芳烃为菲、萘、蒽、茚、芴、苊、芘等。
与现有的降解多环芳烃的菌株相比，本发明的微杆菌Z-2具有高效的降解多环芳烃的能力，能够耐受高浓度多环芳烃，耐受菲的浓度可达400mg/L以上，耐受萘的浓度可达200mg/L以上，且降解周期短，适用于多环芳烃污染土壤和地下水的生物修复。
具体实施方式
下面结合具体实施例对本发明作进一步详述。
实施例1：微杆菌Z-2的分离筛选及鉴定
1、菌株富集
将2g取自被多环芳烃重度污染的土壤放入含60mL无菌蒸馏水的摇瓶中，至于37℃恒温摇床上培养15分钟，取2mL上清液放入60mL LB液体培养基进行富集培养12小时，获得可以在高浓度多环芳烃土壤中生存的菌株。LB液体培养基(g/L)的组成为：蛋白胨10，酵母提取物5，NaCl 10，pH＝7.0。
2、可降解PAHs菌株的筛选
1)以菲作为唯一碳源对菌株进行筛选：
取2mL的菌株富集液，加入到60mL含200mg/L菲的无机盐培养基中，然后在37℃恒温摇床进行培养。无机盐培养配方为：NaNO3 4g/L，MgSO4 0.2g/L，NaCl 1g/L，KCl 1g/L，CaCl2 0.04g/L，H3PO4 5ml/L，菲200m g/L，pH＝7.0。
培养72小时后，将培养液转接入含有400mg/L菲的4-2无机盐培养基中筛选培养，培养3天后，利用稀释涂布平板法筛选单菌落，在37℃的培养箱中培养10小时，得到40个单菌落，筛选培养基为LB固体培养基(g/L)的组成为：蛋白胨10，酵母提取物5，NaCl 10，琼脂粉15，pH＝7.0。挑选生长旺盛的单菌落，在相同的培养条件下反复进行筛选培养，由此可以获得纯种菌株。
2)以菲作为唯一碳源对菌株的活力进行检测：
将上述获得的纯种菌株在60mL LB液体培养基中进行富集培养后，取2mL扩大培养后的液体放入60mL以200mg/L菲为唯一碳源的无机盐液体培养基中，在37℃恒温摇床进行培养。无机盐液体培养基配方为：NaNO3 4g/L，MgSO4 0.2g/L，NaCl 1g/L，KCl 1g/L，CaCl20.04g/L，H3PO4 5ml/L，菲100～400mg/L，pH＝7.0。
培养72小时后，对培养基内液体进行pH测定和菌浓的测定，发现pH有明显变化，菌浓从OD600＝0.305增长到OD600＝0.344，证明菌株可以摄取利用菲，因此有降解菲的能力。
3)菌株的驯化
为了提高纯种菌株降解多环芳烃和耐受高浓度多环芳烃的能力，进行了如下驯化。将分离获得的菌株接入LB培养基中富集，并以2％体积比连续接种于以400mg/L多环芳烃为唯一碳源的培养基中，在37℃的温度下培养4天，然后转接进去新鲜培养基。培养基的组成为：NaNO3 4g/L，MgSO4 0.2g/L，NaCl 1g/L，KCl 1g/L，CaCl2 0.04g/L，H3PO4 5ml/L，菲400mg/L，pH＝7.0。经过30天左右的驯化，其对400mg/L的菲降解效率从23％上升至58％。
3、菌株的分类学鉴定
驯化得到的菌株，革兰氏染色阴性，形态呈杆状、单个、无芽孢。
采用分子生物学的方法对驯化得到的菌株进行鉴定，测得其16S rRNA序列(SEQID No.1)，在GenBank核酸数据库中进行比对，通过序列比对确认为微杆菌，命名为微杆菌(Microbacterium)Z-2，并于2022年05月06日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏，保藏编号为CGMCC No.24842。
实施例2：微杆菌Z-2降解多环芳烃能力的测定
将分离的微杆菌Z-2接种至不同菲(100、200、300、400mg/L)和萘(30、50、100、200mg/L)含量的无机盐培养基中，置于37℃摇床中培养5天，然后分析培养基中残留的菲和萘含量，并计算菲和萘的降解率。无机盐培养基的组成为：NaNO3 4g/L，MgSO4 0.2g/L，NaCl1g/L，KCl 1g/L，CaCl2 0.04g/L，H3PO45 ml/L，pH＝7.0。
由上表可以看出，微杆菌Z-2可高效地降解菲和萘，且耐受这两种多环芳烃的能力较强，降解周期短。
以上所述，仅为本发明专利的较佳示例而已，并非用于限定本发明专利的保护范围。除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变化形成的技术方案，均落在本发明要求的保护范围。
序列表
<110> 南京理工大学
<120> 一株降解多环芳烃的微杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
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<213> 微杆菌(Microbacterium)
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gttccccccc tttcgacggc tccctccaca agggttaggc caccggcttc aggtgttacc 60
gactttcatg acttgacggg cggtgtgtac aagacccggg aacgtattca ccgcagcgtt 120
gctgatctgc gattactagc gactccgact tcatgaggtc gagttgcaga cctcaatccg 180
aactgggacc ggctttttgg gattcgctcc acctcacggt attgcagccc tttgtaccgg 240
ccattgtagc atgcgtgaag cccaagacat aaggggcatg atgatttgac gtcatcccca 300
ccttcctccg agttgacccc ggcagtatcc catgagttcc caccataacg tgctggcaac 360
atagaacgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420
gacaaccatg caccacctgt tcacgagtgt ccaaagagtt gaccatttct ggcccgttct 480
cgtgtatgtc aagccttggt aaggttcttc gcgttgcatc gaattaatcc gcatgctccg 540
ccgcttgtgc gggtccccgt caattccttt gagttttagc cttgcggccg tactccccag 600
gcggggaact taatgcgtta gctgcgtcac ggaatccgtg gaaaggaccc cacaactagt 660
tcccaacgtt tacggggtgg actaccaggg tatctaagcc tgtttgctcc ccaccctttc 720
gctcctcagc gtcagttacg gcccagagat ctgccttcgc catcggtgtt cctcctgata 780
tctgcgcatt ccaccgctac accaggaatt ccaatctccc ctaccgcact ctagtctgcc 840
cgtacccact gcaggcccga ggttgagcct cgggatttca cagcagacgc gacagaccgc 900
ctacgagctc tttacgccca ataattccgg ataacgcttg cgccctacgt attac 955
Claims (4)
1. 降解多环芳烃的微杆菌（Microbacterium sp.），为微杆菌Z-2，保藏编号为CGMCCNo. 24842。
2.根据权利要求1所述的微杆菌的培养方法，其特征在于，具体如下：
将微杆菌Z-2接种至培养基中，37℃下摇床培养，所述的培养基的配方为：NaNO31~4 g/L，MgSO4 0.2~0.4 g/L，NaCl 1~3 g/L，KCl 1~3 g/L，CaCl2 0.04~0.06 g/L，H3PO4 5~7 ml/L，多环芳烃100~400 mg/L，pH=7.0。
3.根据权利要求1所述的微杆菌在降解多环芳烃菲或萘中的应用。
4.根据权利要求1所述的微杆菌在生物修复多环芳烃菲或萘污染土壤或地下水中的应用。
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| KR20010073276A (ko) * | 2000-01-13 | 2001-08-01 | 안태영 | 다환식 탄화수소 오염 토양의 생물정화를 위한 고분자다환식 탄화수소 분해 미생물, 그의 제조 방법 및 분해미생물을 포함하는 유류 분해 조성물 |
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| CN112481149A (zh) * | 2019-10-29 | 2021-03-12 | 北京博诚立新环境科技股份有限公司 | 一种降解高环多环芳烃的菌株及其污染修复应用 |
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| KR20010073276A (ko) * | 2000-01-13 | 2001-08-01 | 안태영 | 다환식 탄화수소 오염 토양의 생물정화를 위한 고분자다환식 탄화수소 분해 미생물, 그의 제조 방법 및 분해미생물을 포함하는 유류 분해 조성물 |
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