CN114644997B - 一种高效石油降解菌株及其应用 - Google Patents
一种高效石油降解菌株及其应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域,具体涉及一种高效石油降解菌株及其应用;该菌分类命名为神户肠杆菌(Enterobacter kobei)DH7,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.24280。所述菌株用于降解石油烃类污染物的用途,在7天内对油污中总石油烃的降解率达到了65%,14天降解率达到了84.6%;同时,菌株DH7还可应用于石油污染治理及生态修复、以及制备降解产品的用途;与现有原油降解菌相比,本发明提供的菌株对原油以及油污的降解效率高、培养简单、耐受性好,在微生物采油、含油废水处理、油污土壤的生态修复中具有良好的应用潜力。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种高效石油降解菌株及其应用。
背景技术
石油不仅是一种不可再生的商品,更是国家生存和发展不可或缺的战略资源,对保障国家经济和社会发展以及国防安全有着不可估量的作用。但在海上或陆上生产、运输、储存、加工或使用石油时,可能会意外或操作性地产生石油污染。石油泄漏对陆地和海洋生态系统造成了极大的危害。石油含有成千上万种复杂的不同化合物,这些化合物基本上分为环烷烃、直链烷烃、芳香烃、树脂和沥青质,以及表征较差的化合物。这些化合物大部分是稳定的、有毒的和致癌的,威胁着人类的生命和健康。当它们意外释放到环境中,它们严重破坏了周围的生态系统,造成严重污染时,所有生物群落都会收到影响,严重威胁着人类的健康和土壤生态系统的安全。对石油污染的环境修复迫在眉睫,对石油污染修复的技术也成为了当今的研究热点。
由于石油污染物对人体及生态系统造成影响的严重性,研究人员开发了一系列的修复处理技术,几种物理化学方法如焚烧法,泡沫浮选,溶剂萃取、高温分解、超声法生,通过添加表面活性剂/生物表面活性剂/碱/表面活性剂进行化学处理,这些都已应用于处理石油污染物,但实施这些方法会产生一些问题,涉及高化学品消耗、产生二次污染物、不环保,还需要进行前处理和后处理。典型的修复方法中,生物修复具有成本低、对环境友好、耗时少等优点,是一种可行的石油污染土壤修复方法。
能够降解石油烃类的原生微生物在环境中无处不在。研究发现,200多种微生物有降解石油污染物的能力,包括藻类、真菌、细菌,具有修复环境污染物的能力,并且降解微生物能以石油作为碳源、能源的唯一来源。微生物降解是从污染环境中去除石油污染物的主要机制。在利用石油的各种微生物中,细菌被认为是最活跃和最重要的微生物,同时进行高效降解方面发挥着重要作用。迄今为止,已鉴定出许多可降解碳氢化合物的细菌种类。例如,芽孢杆菌属、短小芽孢杆菌属、假单胞菌属、铜绿假单胞菌属、红球菌属和克雷伯菌属等。
在石油污染修复中,细菌的研究必不可少的,通过人工培养筛选、分离出的菌株,大多存在降解效率低、降解周期长的问题。通过向油污土壤中添加氮和磷来刺激微生物,可以加速细胞生长,从而提高对石油污染物的降解速率。从另一个角度来看,通过接种特殊微生物群进行生物强化是一种有希望的低成本生物修复各种含油污染物的策略。然而,由于缺乏适应性,将外来细菌添加到新环境中并不十分有利。因此筛选分离的代谢优越、高效降解的石油菌株对于作为生物增强剂的应用以及对微生物修复土壤的前景意义重大。
发明内容
本发明的第一个目的是克服现有技术存在的石油降解菌株降解效率不高、不足的问题,提供一种高效石油降解菌株,所述菌株为神户肠杆菌(Enterobacter kobei)DH7,于2022年01月10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,邮编:100101,保藏中心登记入册编号:CGMCC NO.24280;所述菌株的16S rRNA基因序列如SEQ ID NO.1所示。
本发明的第二个目的在于所述神户肠杆菌(Enterobacter kobei)DH7,用于石油污染治理及生态修复的用途;
本发明的第三个目的在于所述神户肠杆菌(Enterobacter kobei)DH7,用于降解石油烃类污染物的用途;
本发明的第四个目的在于制备一种降解石油烃类污染物的产品,其所述活性成分为石油降解菌株DH7;所述产品包括液体或固体的生物制剂。
本发明具体应用于废机油中总石油烃的降解,包括以下步骤:
将含有废机油的基础无机盐培养基pH调节为6.5-7.8,按体积百分比10%-40%的接种量添加OD600值为1-2的神户肠杆菌(Enterobacter kobei)DH7,于28-37℃条件下进行振荡反应,反应后即可实现废机油中总石油烃的降解。
优选的,所述含有废机油的基础无机盐培养基中废机油与基础无机盐培养基的体积比为2:100。
优选的,所述振荡反应时的振荡速度为160~200r/min,振荡反应的时间为7d-14d。
优选的,所述的基础无机盐的组分为(g/L),以1L计:硫酸镁0.02g,氯化钙0.002g,磷酸二氢钾0.1g,磷酸氢二钾0.1g,硫酸铵0.1g,氯化铁0.005g,蒸馏水定容至1L;pH=7.0~7.4,121℃灭菌20min。
优选的,所述LB培养基(g/L),以1L计:蛋白胨10.0g、酵母浸粉5.0g、NaCl 10.0g,其余为蒸馏水,pH=7.0-7.4,121℃灭菌20min。
优选的,所述固体LB培养基(g/L)以1L计::蛋白胨10.0g、酵母浸粉5.0g、NaCl10.0g、琼脂15.0g、蒸馏水定容至1L,pH=7.0-7.4,121℃灭菌20min。
所述神户肠杆菌(Enterobacter kobei)DH7的16S rRNA序列,SEQ ID NO.1:
GCAATGGCGGCGGCCTACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGAWGNCCANCCACNCTGGAANCTGAGACACGNTCCAGACTCNTACGGNAGNCAGNCAGTGGGNANTATNGCACAWTGGGCGCANGCNTGATGCAGCNATGCCGCGTGTATGAAGAAGNCCTTCGGNTNGTAAAGTACTTTCAGCGGGGAGGAARGKKKTGTGGTTAATAACCACAGCAATTGACGTTACCNGCAGAAGAAGCACCGGCTAACTCCNGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTNCCCGCANCGAGCGCAACCNTTATCCTTTGKTGCNAGCGNTCNGGCCGGGANCTCAANGNAGACTGSCAGTGATAANCTGRAGKANGKTGGNNATGACGTCMAGNTCATCATGGMCCTTANCGANGTAGGGCWACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGATGCTGC
本发明相对于现有技术具有如下的优点及效果:
(1)本发明从中国江苏省连云港市废机油污染土壤中筛选到具有降解废机油能力的神户肠杆菌(Enterobacter kobei)DH7,能够在高浓度的废机油环境下生长。
(2)本发明的神户肠杆菌(Enterobacter kobei)DH7能够降解总石油烃,降解率可达65%-83%。
(3)本发明的神户肠杆菌(Enterobacter kobei)DH7降解废机油中的总石油烃相对于物理化学法成本低,且无二次污染。
附图说明
图1为石油降解菌株DH7的系统发育分析树。
图2为废机油中总石油烃的最大吸收波长图。
图3为神户肠杆菌(Enterobacter kobei)DH7降解7d后的残余废机油的GC-MS图。
图4为未添加任何菌株降解7d后的残余废机油的GC-MS图。
图5为石油降解菌株DH7在降解过程中的生长曲线图。
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。
针对江苏省连云港市桃林镇的一个循环经济产业园周围潜在的石油烃污染土样,从中筛选出一株高效石油降解菌株,并进一步研究其降解能力,以其应用于修复石油污染土壤。
实施例1:菌株筛选与鉴定
本发明的石油降解菌株分离筛选自连云港东海县桃林镇的污染土壤。筛选鉴定方法如下:
(1)取样:土壤样品从江苏省连云港市桃林镇的一个循环经济产业园石油污染土壤中进行采集,运回实验室,4℃保存。
(2)菌株的分离筛选与驯化:先取5g的石油污染的土壤样品,加入到50mL己灭菌的基础无机盐培养基中,在37℃、180r/min下振荡培养3d,取出,待明显分层后取上层菌液2mL转移至已灭菌的50mL基础无机盐培养基中,并加入1mL废机油(已无菌过滤)作为碳源,在37℃、180r/min进行振荡培养7d。7d后将静置后的上层菌液采用平板划线分离法接种于固体LB培养基上,至于恒温培养中37℃倒置培养12h,待菌落长出后挑取单菌落,并以同样的方法接种至固体LB培养基。重复上述划线分离过程,直至形成形态单一的纯化菌落,将单菌落在LB培养基中扩繁,并甘油管藏并保存于-80℃,备用。使用前将保存的菌株在LB液体培养基中活化。
(3)鉴定:使用Tiangen细菌基因组DNA提取试剂盒(天根生化科技有限公司)对分离纯化的石油降解菌株DH7进行基因组DNA提取。以石油降解菌株DH7的DNA为此模板并采用27F,1492R引物扩增16S rRNA片段。PCR反应参数为:94℃预变性5min,94℃变形1min,54℃退火1min,72℃延伸2min,共30个循环;最后72℃延伸2min再降温4℃即可。PCR产物由上海生工生物工程公司测序,测得的序列在Genbank中与已知16S rRNA进行Blast,确定其为肠杆菌属(Enterobacter),并用MEGA—X计算出序列的系统进化距离,构建系统发育树。综合分子生物学及生化鉴定结果,并构建系统发育树如图1所示,确定菌株的种属关系。
由双数方法鉴定所得菌株命名为神户肠杆菌(Enterobacter kobei)DH7,保藏于中国普通微生物菌种保藏中心,保藏编号CGMCC NO.24280,保藏日期为2022年01月10日,该菌株的存活经由保藏中心于保藏日检测为存活状态。
实施例2:降解能力测试
测试方法如下:
将保存的石油降解菌株DH7,以0.5%的接种量接种至200mL LB培养基活化12h,将活化好的菌液,3000r/10min得到菌泥,并用基础无机盐培养基以同样的转速清洗2次;将清洗好的菌泥转移至200mL的石油培养基中,放入37℃、180r/min的恒温摇床中进行培养14天,得到降解后的溶液。
上述石油培养基组分为(g/L):基础无机盐培养机中按体积比添加2%废机油作为石油降解菌株的筛选以及降解用液体培养基,121℃灭菌20min。
将降解后的溶液倒入250mL的分液漏斗﹐加50mL石油醚(30-60℃沸程)冲洗摇瓶后一并倒入分液漏斗中,加盖充分振摇2min,并注意放气,静置分层2min,上层为萃取出的有机溶剂,下层为菌液;将下层菌液放回摇瓶中。用上面放有5mm厚无水硫酸钠的漏斗过滤上层萃取出的有机溶剂,滤液置于250mL容量瓶中。再将下层菌液倒入分液漏斗﹐用10mL石油醚(30-60℃沸程)冲洗摇瓶。再次加入50mL石油醚(30-60℃沸程)重复萃取3次,合并4次上层萃取出的有机溶剂于250mL容量瓶中,以石油醚(30-60℃沸程)定容,在最大吸收波长处,以石油醚作为参比,测定吸光度,确定得到的菌株降解后残余废机油含量。计算公式如下:降解效率%=(A-B)/A×100%
公式中:A为对照组:未添加任何菌株的石油培养基中残余废机油中的含量,B为实验组:含有菌株DH7在石油培养基中降解后残余废机油含量。
经测试计算,本发明的石油降解菌DH7在7d内对废机油中的总石油烃既有降解效率达到65%,14d降解效率达到了84.6%。
确定废机油中石油烃的最大吸收波长:将废机油用石油醚(30-60℃沸程)配置成2%体积浓度的溶液,测定不同稀释倍数的溶液在200nm-300nm的吸收波长,如图2所示,确定最大吸收波长283nm。
将神户肠杆菌(Enterobacter kobei)DH7降解7天后的产于废机油的GC-MS分析结果可以看出(如表1、表2),原始的废机油主要为己烷烃或其同分异构体、庚烷烃或其同分异构体、3-戊醛、甲基环己烷。神户肠杆菌(Enterobacter kobei)DH7微生物降解后的废机油中含有己烷烃、庚烷烃、3-戊醛,说明废机油在微生物作用下发生了分子结构的变化,大分子结构的污染物质在微生物的作用下被氧化成小分子结构,进而被降解。同时微生物降解废机油中的庚烷烃或其同分异构体,2.37min以后的时间内都没有被检测出来,说明神户肠杆菌(Enterobacter kobei)DH7对庚烷烃或其同分异构体具有明显的降解能力,且该菌株对废机油中的甲基环己烷的降解能力较强,降解率达100%。
从图3、图4的GC-MS分析图对比看出,神户肠杆菌(Enterobacter kobei)DH7对废机油中总石油烃具有较强的降解能力。
在降解过程中也测定不同时间段的神户肠杆菌(Enterobacter kobei)DH7的生物活性OD600。图5中对照为:对照为:未添加任何菌株的石油培养基在600nm下吸光度值;菌株7为:添加神户肠杆菌(Enterobacter kobei)DH7的石油培养基在600nm下吸光度值。如图5所示,很明显看出神户肠杆菌(Enterobacter kobei)DH7在降解废机油中总石油烃时保持较高的活性。神户肠杆菌(Enterobacter kobei)DH7降解7天后,残余废机油中仍有部分石油烃类物质残留,可以延长增加微生物降解时间和改变微生物降解条件进一步降解。
表1神户神户肠杆菌(Enterobacter kobei)DH7降解7d后的残余废机油的GC-MS结果
表2对照组(未添加任何菌株)降解7d后的残余废机油的GC-MS结果
说明:以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案;因此,尽管本说明书参照上述的实施例对本发明已进行了详细的说明,但是本领域的普通技术人员应当理解,仍然可以对本发明进行修改或等同替换;而一切不脱离本发明的精神和范围的技术方案及其改进,均应涵盖在本发明的权利要求范围内。
序列表
<110> 江苏大学
<120> 一种高效石油降解菌株及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1450
<212> DNA
<213> 神户肠杆菌(Enterobacter kobei)
<400> 1
gcaatggcgg cggcctacac atgcaagtcg aacggtagca cagagagctt gctctcgggt 60
gacgagtggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac 120
tggaaacggt agctaatacc gcataacgtc gcaagaccaa agagggggac cttcgggcct 180
cttgccatca gatgtgccca gatgggatta gctagtaggt ggggtaacgg ctcacctagg 240
cgacgatccc tagctggtct gagaggawgn ccanccacnc tggaanctga gacacgntcc 300
agactcntac ggnagncagn cagtgggnan tatngcacaw tgggcgcang cntgatgcag 360
cnatgccgcg tgtatgaaga agnccttcgg ntngtaaagt actttcagcg gggaggaarg 420
kkktgtggtt aataaccaca gcaattgacg ttaccngcag aagaagcacc ggctaactcc 480
ngtgccagca gccgcggtaa tacggagggt gcaagcgtta atcggaatta ctgggcgtaa 540
agcgcacgca ggcggtctgt caagtcggat gtgaaatccc cgggctcaac ctgggaactg 600
cattcgaaac tggcaggcta gagtcttgta gaggggggta gaattccagg tgtagcggtg 660
aaatgcgtag agatctggag gaataccggt ggcgaaggcg gccccctgga caaagactga 720
cgctcaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780
aaacgatgtc gacttggagg ttgtgccctt gaggcgtggc ttccggagct aacgcgttaa 840
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 900
cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta cctactcttg 960
acatccagag aactttccag agatggattg gtgccttcgg aactctgaga caggtgctgc 1020
atggctgtcg tcagctcgtg ttgtgaaatg ttgggttaag tncccgcanc gagcgcaacc 1080
nttatccttt gktgcnagcg ntcnggccgg ganctcaang nagactgsca gtgataanct 1140
gragkangkt ggnnatgacg tcmagntcat catggmcctt ancgangtag ggcwacacac 1200
gtgctacaat ggcgcataca aagagaagcg acctcgcgag agcaagcgga cctcataaag 1260
tgcgtcgtag tccggattgg agtctgcaac tcgactccat gaagtcggaa tcgctagtaa 1320
tcgtagatca gaatgctacg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccatgggagt gggttgcaaa agaagtaggt agcttaacct tcgggagggc gcttaccact 1440
ttgatgctgc 1450
Claims (10)
1.一种高效石油降解菌株,其特征在于,所述菌株为神户肠杆菌(Enterobacterkobei)DH7,于2022年01月10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.24280;所述菌株的16S rRNA基因序列如SEQ ID NO.1所示。
2.根据权利要求1所述的高效石油降解菌株的用途,其特征在于,用于降解石油烃类污染物的用途。
3.根据权利要求2所述的用途,其特征在于,具体应用于废机油中总石油烃的降解。
4.根据权利要求3所述的用途,其特征在于,包括以下步骤:将含有废机油的基础无机盐培养基pH调节为6.5-7.8,按体积百分比10%-40%的接种量添加OD600值为1-2的神户肠杆菌DH7,于28-37℃条件下进行振荡反应,反应后即可实现废机油中总石油烃的降解。
5.根据权利要求4所述的用途,其特征在于,所述含有废机油的基础无机盐培养基中废机油与基础无机盐培养基的体积比为2:100。
6.根据权利要求4所述的用途,其特征在于,所述振荡反应时的振荡速度为150-200r/min,振荡反应的时间为7d-14d。
7.根据权利要求4或5所述的用途,其特征在于,所述的基础无机盐的组分为,以1L计:硫酸镁0.02g,氯化钙0.002g,磷酸二氢钾0.1g,磷酸氢二钾0.1g,硫酸铵0.1g,氯化铁0.005g,其余为蒸馏水,pH=7.0-7.4,121℃灭菌20min。
8.根据权利要求1所述的石油降解菌株DH7应用于石油污染治理及生态修复的用途。
9.一种降解石油烃类污染物的产品,其特征在于,所述产品的活性成分为权利要求1所述的石油降解菌株DH7。
10.根据权利要求9所述一种降解石油烃类污染物的产品,其特征在于,所述产品包括液体或固体的生物制剂。
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