CN110669700B - 一株高效石油烃降解菌pa16_9及其筛选方法与应用 - Google Patents
一株高效石油烃降解菌pa16_9及其筛选方法与应用 Download PDFInfo
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Abstract
一株高效石油烃降解菌PA16_9及其筛选方法与应用，涉及海洋石油烃降解菌。一株高效石油烃降解菌为泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的菌落呈圆形，米黄色半透明，表面光滑湿润，边缘规则，无晕环，中央微凸起，直径2～4mm；一株泰坦尼克号盐单胞菌可在厌氧条件降解有机化合物中的应用。经鉴定一株高效石油烃降解菌(Halomonas titanicae)PA16‑9在32℃下，厌氧避光静置培养，能降解初浓度约为3.867g/L的正十六烷，3个月后测得降解率为76.7％～86.5％，厌氧条件下芘的降解率达到58％，好氧条件下降解率可达74.5％。
Description
技术领域
本发明涉及海洋石油烃降解菌，尤其是涉及一株高效石油烃降解菌PA16-9及其筛选方法与应用。
背景技术
随着我国社会经济的不断发展，对石油的需求和依赖日益增加，从而引导了石油相关产业的持续繁荣，却也造成了目前日趋严重的环境污染问题。受限于目前技术流程与管理理念方面的不完善，含有大量烃成分的石油产品直接或间接(天然渗漏、意外溢出以及精炼石油产品的广泛使用)流入土壤、海洋等生态圈(El-Sheshtawy,Khalil,Ahmed,&Abdallah,2014)，引起一系列严重且持续的污染问题。饱和烃(烷烃)是石油的主要成分(Tissot&Welte,1984)，泄露的烃类物质灌注入土壤的空隙中，影响土壤的通透性，破坏原油的土壤水相、气相和固相结构，影响土壤中微生物的生长，阻碍土壤中植物根系的呼吸及水分养料的吸收，甚至使植物根系腐烂坏死，严重危害植物的生长，降低该区域的土地耕种价值。同时，土壤中的石油污染物亦常伴随水的流动而发生迁徙，导致污染区域不断扩大。另一方面，石油中的有害物质，尤其是多环芳烃类物质具有致癌、致畸、致突变等作用，可以通过食物链进入生物体乃至人体中，直接危害人体健康。
目前，常用的石油烃污染修复方法主要有：物理修复、化学修复和生物修复(Guarino,Spada,&Sciarrillo,2017)。相比于化学、物理等修复方法，生物修复对人和环境造成的影响小，操作简便，成本较低等优势(Cappello et al.,2015)，生物修复费用仅为传统物理、化学修复的30％～50％(Bragg,Prince,Harner,&Atlas,1994)。因此，生物修复以其投入小，无二次污染的优势被视为最有前途的环境治理手段(Head&Swannell,1999；Juhasz&Naidu,2000)。
发明内容
本发明的第一目的在于提供一株高效石油烃降解菌(Halomonas titanicae)HTPA16_9。
本发明的第二目的在于提供一株高效石油烃降解菌(Halomonas titanicae)HTPA16_9的筛选方法。
本发明的第三目的在于提供一株高效石油烃降解菌(Halomonas titanicae)HTPA16_9 16S rDNA核苷酸序列。
本发明的第四目的在于提供一株高效石油烃降解菌(Halomonas titanicae)HTPA16_9可用于盐碱性土壤深层较为缺氧区域烃污染的治理，以及含盐丰富、含氧量较低的烃污染海域中应用。
所述一株高效石油烃降解菌为泰坦尼克号盐单胞菌(Halomonas titanicae)HTPA16_9的菌落呈圆形，米黄色半透明，表面光滑湿润，边缘规则，无晕环，中央微凸起，直径2～4mm；所述泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9已于2019年10月22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，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，保藏中心登记入册编号：CGMCC No.18722。
所述泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9筛选自太平洋深海沉积物，样品编号45II-KW1-S37-BC29，距沉积物表层22～26cm，大洋45航次第2航段KW1海区，W 154°14.9909′，N 9°29.9927′，沉积物为黄褐色，无味，弱黏性，表层呈半流动状，手搓略有粉砂感，向下稠度增加，10cm一下富含黄白色团块混杂。
所述泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的筛选方法包括以下步骤：
1)取156mL厌氧瓶，洗净后加入100mL基础盐培养基，通N2除去残余空间的氧气，补加半胱氨酸(-HCl)去除盐溶液中残余的氧气，胶塞封口加铝盖，121℃高温灭菌20min，待体系冷却至室温后，在超净工作台补加微量元素溶液、维生素溶液、磷酸盐溶液、亚硒酸盐-钨酸盐溶液和碳源等；
在步骤1)中，所述基础盐培养基的组分可为：氯化铵0.33g/L，硝酸钠0.88g/L，氯化钾0.5g/L，氯化钙(2H2O)0.5g/L，氯化镁(6H2O)3g/L，氯化钠22g/L，无水硫酸钠3g/L，PIPESbuffer5g/L，刃天青(1g/L)1mL/L，pH调至6.8。所述维生素溶液的组分可为：盐酸吡哆醇10mg/L，盐酸硫胺素5mg/L，核黄素5mg/L，盐酸5mg/L，钙D-(+)-泛酸盐5mg/L，对氨基苯甲酸5mg/L，硫辛酸5mg/L，生物素2mg/L，叶酸2mg/L，维生素B12 0.1mg/L，过滤除菌。所述微量元素溶液的组分可为：先将1.5g次氮基三乙酸加入约500mL水中，用KOH调节pH至6.5溶解，硫酸镁(7H2O)3g，硫酸锰(H2O)0.5g，氯化钠1g，硫酸铁(7H2O)0.1g，氯化钴(6H2O)0.1g，无水氯化钙0.1g，氯化锌(7H2O)0.1g，硫酸铜(5H2O)0.01g，硫酸铝钾(12H2O)0.01g，硼酸0.01g，钼酸钠(2H2O)0.01g，用蒸馏水将体积调至1L，过滤除菌。所述磷酸盐溶液的组分可为：KH2PO45g溶解在100mL蒸馏水中，121℃高压灭菌20min。所述亚硒酸盐-钨酸盐溶液的组分可为：氢氧化钠0.5g/L，亚硒酸钠(5H2O)3mg/L，钨酸钠(2H2O)4mg/L，过滤除菌；所述碳源可为混合芳烃或烷烃。
2)取出太平洋深海沉积物，按比例以无菌基础盐培养基溶解太平洋深海沉积物，形成浊液，通过注射器注入密封的厌氧培养基中，室温下避光静置培养两个月；
在步骤2)中，所述厌氧培养基的组成比例可为：1mL微量元素溶液、1mL维生素溶液、1mL亚硒酸盐-钨酸盐溶液、8mL磷酸盐溶液以及0.2％的碳源添加至1L基础盐培养基中。
3)取样涂布在正十六烷与十二烷为唯一碳源的厌氧固体平板上，置于厌氧操作箱中室温培养三周后，形成肉眼可见的菌落，经HLB固体平板反复分离纯化，得一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9。
在步骤3)中，所述厌氧固体平板可在基础盐培养基的基础上添加1.5％琼脂；所述HLB固体平板的组成可为：氯化钠30g/L、酵母提取物5g/L、蛋白胨10g/L、琼脂15g/L。
本发明提取一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的基因组DNA并以此为模板采用通用27F，1492R引物扩增16S rDNA片段，并在EZBiocloud上选取高相似度序列，用MEGA-X计算出序列的系统进化距离，构建系统发育树。
在平板上挑取单菌落接种于30mL体系的厌氧培养基中，正十六烷为唯一碳源，初始添加量为0.07734g，恒温33℃且避光静置，厌氧培养3月后，测残余正十六烷量。计算降解率约为76.7％～86.5％。
所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的16S rDNA核苷酸序列为：
TGGTTACCTTGTTACGACTTCACCCCAGTGATGAAGCACACCGTGGTGATCGCCCTCTTGCGTTAGGCTAACCACTTCTGGTGCAGTCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGACCGGCTTTAAGGGATTCGCTGACTCTCGCGAGCTCGCAGCCCTTTGTACCGGCCATTGTAGCACGTGTGTAGCCCTACCCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCGACATTACTCGCTGGCAAATAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTTACAGTTCCCGAAGGCACACCAGAATCTCTTCCGGCTTCTGTAGATGTCAAGGGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTATCGCGTTAACTTCGCCACAAAGTGCTCTAGGCACCCAACGGCTGGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCACCTCAGTGTCAGTGTCAGTCCAGAAGGCCGCCTTCGCCACTGGTATTCCTCCCGATCTCTACGCATTTCACCGCTACACCGGGAATTCTACCTTCCTCTCCTGCACTCTAGCCTGACAGTTCCGGATGCCGTTCCCAGGTTGAGCCCGGGGCTTTCACAACCGGCTTATCAAGCCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTGATGTCTTTCCTACCGGGTATTAACCGATAGGCGTTCTTCCTCGCTGAAAGTGCTTTACAACCCGAGGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTCGGGCCGTGTCTCAGTCCCGATGTGGCTGATCATCCTCTCAGACCAGCTACGGATCGTTGCCTTGGTAAGCCATTACCTTACCAACTAGCTAATCCGACATAGGCTCATCCAATAGCGGGAGCCGAAGCCCCCTTTCTCCCGTAGGACGTATGCGGTATTAGCCTGGGTTTCCCCAGGTTATCCCCCACTATCGGGCAGATTCCTATGCATTACTCACCCGTCCGCCGCTCGTCAGCGGGTAGCAAGCTAGATCTGTTACCGCTCGACTTGCATGTGTTAGGCCTGCGGCGCCGCGTTCAATCTGAGCCAGGATCAAACTCTAA。
所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9可在厌氧条件降解有机化合物中的应用。所述有机化合物可为石油烃类化合物，所述石油烃类化合物可为中链与长链烷烃、单环与多环芳烃、沥青、卤代烃等烃类有机物。
经鉴定一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9在32℃下，厌氧避光静置培养，能降解初浓度约为3.867g/L的正十六烷，3个月后测得降解率为76.7％～86.5％，厌氧条件下芘的降解率达到58％，好氧条件下降解率可达74.5％。因此，所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9可应用于被烃污染的高盐土壤或海域等较极端环境的生态修复以及参与污水处理等过程。
附图说明
图1为本发明所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的多环芳烃降解率测定。
图2为本发明所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的烷烃降解率测定。
图3为本发明所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的菌落宏观形态图。
图4为本发明所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9在透射电镜下的形态。
图5为本发明所述一株泰坦尼克号盐单胞菌(Halomonas titanicae)HT PA16_9的系统发育分析树。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
实施例1：菌株形态特征
将单菌落划线接种至HLB固体平板中，将平板倒置于恒温培养箱内，32℃培养48h，菌落呈圆形，米黄色半透明，表面光滑湿润，边缘规则，无晕环，中央微凸起，直径2～4mm，见图3。取初生菌体制片，进行透射电镜观察，见图4，菌株为革兰氏阴性菌，兼性好氧，呈杆状，长度为1.6～2μm，外围有菌毛，主要依赖鞭毛进行运动。
实施例2：菌株的筛选和鉴定
(1)太平洋深海沉积物样品取自大洋45航次第二航段KW1海区(样品编号45II-KW1-S37-BC29，距沉积物表层22～26cm，W 154°14.9909′，N 9°29.9927′)，沉积物为黄褐色，无味，弱黏性，表层呈半流动状，手搓略有粉砂感，向下稠度增加，10cm以下富含黄白色团块混杂其中。沉积物样品在无菌收集后，经低温保存后运送至实验室进行下一阶段研究工作。
(2)取156mL厌氧瓶，洗净后加入100mL基础盐培养基，通N2除去残余空间的氧气，补加半胱氨酸(-HCl)去除盐溶液中残余的氧气。胶塞封口加铝盖，121℃高温灭菌20min，待体系冷却至室温后，在超净工作台补加微量元素溶液、维生素溶液、磷酸盐溶液和碳源(混合芳烃及烷烃)等。取出前述沉积物样品，按比例以无菌基础盐培养基溶解沉积物样品，形成浊液，通过注射器注入密封的厌氧培养基中。室温下避光静置培养两月，取样涂布在正十六烷与十二烷为唯一碳源的固体平板上，置于厌氧操作箱中室温培养3周后，形成肉眼可见的菌落，经反复平板分离纯化，得到纯净的泰坦尼克号盐单胞菌(Halomonas titanicae)HTPA16_9。
提取该菌的基因组DNA并以此为模板采用通用27F，1492R引物扩增16SrDNA片段，并在EZBiocloud(https://www.ezbiocloud.net/)上选取高相似度序列，用MEGA-X计算出序列的系统进化距离，构建系统发育树(NJ)，参见图5。
实施例3：菌株的部分烃降解能力测定
(1)配制厌氧液体培养基，碳源浓度为0.33％(正十六烷，约为0.07734g)，培养体系为30mL，从HLB固体平板刮取适量菌体溶于液体培养基，涡旋混匀，接种到厌氧培养基中，接种量为1％，三组实验组避光32℃静置培养。
(2)培养3个月后打开瓶口，采用正己烷反复萃取培养基中的正十六烷(共萃取四次)，合并萃取液，N2吹后保证有0.5ml(约10滴)正十六烷防止氧化。通过GC-MS(气相色谱-质谱联用仪)得到样品的质量谱图，测得正十六烷残余量，得到降解率为76.7％～86.5％，芘的厌氧降解率达58％，好氧条件下达74.5％，参见图1和2。
序列表
<110> 自然资源部第三海洋研究所；中国大洋矿产资源研究开发协会（中国大洋事务管理局）
<120> 一株高效石油烃降解菌PA16-9及其筛选方法与应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1498
<212> DNA/RNA
<213> 泰坦尼克盐单胞菌(Halomonas titanicae)
<400> 1
tggttacctt gttacgactt caccccagtg atgaagcaca ccgtggtgat cgccctcttg 60
cgttaggcta accacttctg gtgcagtcca ctcccatggt gtgacgggcg gtgtgtacaa 120
ggcccgggaa cgtattcacc gtgacattct gattcacgat tactagcgat tccgacttca 180
cggagtcgag ttgcagactc cgatccggac tgagaccggc tttaagggat tcgctgactc 240
tcgcgagctc gcagcccttt gtaccggcca ttgtagcacg tgtgtagccc tacccgtaag 300
ggccatgatg acttgacgtc gtccccacct tcctccggtt tgtcaccggc agtctcctta 360
gagttcccga cattactcgc tggcaaataa ggacaagggt tgcgctcgtt acgggactta 420
acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctt acagttcccg 480
aaggcacacc agaatctctt ccggcttctg tagatgtcaa gggtaggtaa ggttcttcgc 540
gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcatttga 600
gttttaacct tgcggccgta ctccccaggc ggtcgactta tcgcgttaac ttcgccacaa 660
agtgctctag gcacccaacg gctggtcgac atcgtttacg gcgtggacta ccagggtatc 720
taatcctgtt tgctacccac gctttcgcac ctcagtgtca gtgtcagtcc agaaggccgc 780
cttcgccact ggtattcctc ccgatctcta cgcatttcac cgctacaccg ggaattctac 840
cttcctctcc tgcactctag cctgacagtt ccggatgccg ttcccaggtt gagcccgggg 900
ctttcacaac cggcttatca agccacctac gcgcgcttta cgcccagtaa ttccgattaa 960
cgcttgcacc ctccgtatta ccgcggctgc tggcacggag ttagccggtg cttcttctgc 1020
gagtgatgtc tttcctaccg ggtattaacc gataggcgtt cttcctcgct gaaagtgctt 1080
tacaacccga gggccttctt cacacacgcg gcatggctgg atcagggttg cccccattgt 1140
ccaatattcc ccactgctgc ctcccgtagg agttcgggcc gtgtctcagt cccgatgtgg 1200
ctgatcatcc tctcagacca gctacggatc gttgccttgg taagccatta ccttaccaac 1260
tagctaatcc gacataggct catccaatag cgggagccga agcccccttt ctcccgtagg 1320
acgtatgcgg tattagcctg ggtttcccca ggttatcccc cactatcggg cagattccta 1380
tgcattactc acccgtccgc cgctcgtcag cgggtagcaa gctagatctg ttaccgctcg 1440
acttgcatgt gttaggcctg cggcgccgcg ttcaatctga gccaggatca aactctaa 1498
Claims (2)
1.一株泰坦尼克号盐单胞菌（Halomonas titanicae）HT PA16_9，已于2019年10月22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，保藏中心登记入册编号：CGMCCNo. 18722。
2.如权利要求1所述一株泰坦尼克号盐单胞菌（Halomonas titanicae）HT PA16_9在厌氧条件降解有机化合物中的应用；
所述有机化合物为正十六烷和芘。
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