CN114369542B - 一株烷烃降解菌及其应用 - Google Patents
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Abstract
本发明公开了一株烷烃降解菌及其应用。所述的烷烃降解菌Fast 30属于贝莱斯芽孢杆菌,保藏编号为CGMCC No:20883。本发明的贝莱斯芽孢杆菌Fast 30能以烷烃为唯一碳源进行生长繁殖,对烷烃具有良好的降解作用,能够在五天内对0.1%的石油实现43.2%的降解,可应用于对石油污染环境的修复与治理。并且贝莱斯芽孢杆菌Fast 30代谢产生的生物表面活性剂具有良好的乳化效果,能够增加油水的接触面积从而提高菌株对烷烃的利用率。
Description
技术领域
本发明属于环境污染物生物强化技术领域,涉及一株烷烃降解菌及其应用。
背景技术
石油作为工业生产和日常生活的主要能源,在对其的勘探、生产、精炼、运输和储存过程中经常发生意外泄漏,是造成水和土壤污染的主要原因。由于大多数动物和植物无法分解碳氢化合物,组织中逐渐积累的污染物会导致动植物的死亡或突变,所以碳氢化合物对土壤的污染会对当地生态系统造成严重的破坏。传统的土壤修复的技术包括机械、掩埋、蒸发、分散和洗涤。但是,这些技术不仅昂贵,会对环境造成二次污染并且不能使污染物完全降解。生物修复技术是指利用微生物的某些生命代谢活动来去除和降解环境中的某些污染物,生物修复技术成本低且不会对环境造成二次污染。天然微生物群体的生物降解是去除环境中石油和其他碳氢污染物的主要机制之一,并且比其他修复技术更经济。
环境中的碳氢化合物主要由细菌、酵母和真菌降解。据报道土壤真菌的降解效率为6%至82%,土壤细菌为0.13%至50%,海洋细菌为0.003%至100%。细菌是石油降解菌里面主要的一类微生物,有几种已知的细菌可以完全以碳氢化合物为碳源。如节杆菌,分枝杆菌,假单胞菌,鞘氨醇单胞菌和红球菌属。还有几种从被污染的河流中分离出的可以降解石油的细菌菌株,如荧光假单胞菌、铜绿假单胞菌、芽孢杆菌、产碱杆菌、不动杆菌和棒状杆菌等。
虽然能够利用烃类化合物的菌株已有很多报道,但是大多数菌株只能利用范围较窄的底物。例如朱杰等【朱杰,阮志勇,董卫卫,等.一株高效烷烃降解菌Acinetobactersp.LAM1007的分离鉴定及降解特性[J].微生物学通报,2017,044(007):1535-1546.】从大庆油田土壤中筛选的菌LAM1007仅对正十六烷降解效果较明显。并且大多数菌株对烷烃的耐受度较低。例如焦春燕等【焦春燕,聂麦茜.长链烷烃生长菌的筛选及其降解能力测定[J].环保科技,2011,17(001):6-9.】筛选的菌株S5对于低浓度的正十六烷(15mg/L)具有明显降解效果。
发明内容
本发明的目的是提供一种能够有效降解较高浓度的石油烃化合物的烷烃降解菌及其应用。
实现本发明目的的技术方案为:
本发明的烷烃降解菌经鉴定为贝莱斯芽孢杆菌(Bacillus velezensis),命名为Bacillus velezensis Fast 30。该菌株来源于石油污染场地的土壤,经人工富集、分离纯化得到。菌落呈乳白色,边缘整齐,扁平,不易挑取,为革兰氏阳性菌。菌株的16S rRNA的GenBank登录号为MT889689,于2020年10月13日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏,保藏编号为CGMCC No:20883,保藏地址为北京市朝阳区北辰西路1号院3号。
上述贝莱斯芽孢杆菌Fast 30的适宜培养基配方为:1g/L原油,磷酸二氢钾1~2.5g/L,磷酸氢二钾1~2g/L,氯化钠1~2g/L,氯化钙0.01~0.05g/L,硝酸铵1~3g/L,硫酸亚铁0.05~0.1g/L,硫酸镁0.01~0.2g/L,水。适宜培养pH为7~9,最适pH为7。适宜培养温度为30~40℃,最适培养温度为35℃。
本发明提供上述贝莱斯芽孢杆菌Fast 30在石油污染环境处理中的应用。本发明所述的石油污染环境为常规的石油污染环境,例如石油污染废水或石油污染土壤。
此外,本发明还提供上述贝莱斯芽孢杆菌Fast 30生产的生物表面活性剂。
本发明的烷烃降解菌贝莱斯芽孢杆菌Fast 30,能以烷烃为唯一碳源进行生长繁殖,对烷烃具有良好的降解作用,对0.1%的原油的降解率可达43.2%。且对环境具有良好的适应性,在30~40℃之间降解率可以保持35%以上,pH 7~9之间可以保持30%以上的降解率,可应用于对石油污染环境的修复与治理。此外,贝莱斯芽孢杆菌Fast 30产生的表面活性剂具有良好的乳化效果,该表面活性剂能够增加油水的接触面积从而提高菌株对烷烃的利用率。
附图说明
图1是Bacillus velezensis Fast 30在固体培养基平板中生长的菌落图。
图2是Bacillus velezensis Fast 30的扫描电镜图。
具体实施方式
下面结合实施例和附图对本发明作进一步详述。
实施例1:贝莱斯芽孢杆菌(Bacillus velezensis)Fast 30的分离及鉴定
(1)菌株的分离
从被石油污染的地方采集土壤,取5g采集的土壤放入装有100ml液体LB培养基(培养基配方为:胰蛋白胨10g/L,酵母膏5g/L,氯化钠5g/L,调pH至7.0)的250ml的锥形瓶中,37℃、200r/min培养24h。静置1h后取5ml上清液转接至200ml含有500mg/L原油的无机盐培养基中(培养基配方为:磷酸二氢钾1~2.5g/L,磷酸氢二钾1~2g/L,氯化钠1~2g/L,氯化钙0.01~0.05g/L,硝酸铵1~3g/L,硫酸亚铁0.05~0.1g/L,硫酸镁0.01~0.2g/L)37℃、200r/min培养5天。5天后从富集培养物中取5ml进行梯度稀释。分别取10-4、10-5、10-6和10-7四个梯度的稀释液涂布于固体LB平板(培养基配方为:胰蛋白胨10g/L,酵母膏5g/L,NaCl5g/L,琼脂1.5g/L,调pH至7.0),37℃培养14h后挑取单菌落接种于3mL液体LB培养基。测其对石油的降解活性,对具有高降解活性的菌株进行分离纯化,获得菌株贝莱斯芽孢杆菌(Bacillus velezensis)Fast 30,将其进行-80℃低温保存。
(2)菌株的鉴定
对菌株Fast 30进行形态学、生理生化、16S rRNA基因序列的测定,将菌株的16SrRNA基因序列与GenBank数据库中的基因序列进行同源性比较并分析结果,从分子生物学水平上确定该菌的种属。
形态学特征:Fast 30菌落呈乳白色,边缘整齐,扁平,不易挑取,该菌株细胞呈杆状,尺寸为0.4-0.6μm×2.0-2.9μm。图1和图2分别为细菌Fast 30的平板菌落图和扫描电镜图。
生理生化特征:革兰氏阳性,H2O2酶阳性,淀粉水解阳性,油脂水解阳性,好氧,最适降解初始pH范围为6.5-8.0,最适降解温度为35℃。
分子生物学鉴定:以Fast 30菌株的核DNA为模板,以细菌扩增的通用引物进行PCR扩增,测定菌株的16S rRNA基因序列。将菌株的16S rRNA基因序列(序列表SEQ ID No.1)提交到GenBank数据库(GenBank登录号为MT889689)进行比对分析,结果表明,该菌株与Bacillus velezensis CR-502T的相似度为99.710%。
根据以上结果并结合其生理生化特性判定该菌株属于贝莱斯芽孢杆菌属(Bacillus velezensis),将其命名为Bacillus velezensis Fast 30。
实施例2:烷烃降解率的测定
按1%的比例将菌种接种到100ml含有1g/L原油的无机盐培养基中培养3天后向锥形瓶中加入50ml正己烷进行萃取,静置分层后取上层溶液,下层溶液重复上述步骤,萃取三次后将萃取液混合。取1ml混合后的萃取液稀释100倍,测量225nm处的紫外吸光值,计算降解率。
降解率%=(降解前吸光值-降解后吸光值)/降解前吸光值×100%
实施例3:pH对贝莱斯芽孢杆菌Fast 30降解性能的影响
取五个250ml锥形瓶分别加入100ml无机盐培养基,分别调pH到5、6、7、8、9,加入1g/L的原油,按1%接种贝莱斯芽孢杆菌Fast 30,35℃、200r/min培养5天后分别测其降解率(方法同实施例2)。结果见表1,发现pH 7-9之间可以保持30%以上的降解率,pH为7时降解率最高,可达40.2%,且酸性环境比碱性环境对降解率的影响更大。
表1:pH对菌株降解性能的影响
pH | 降解率% |
5 | 15.8 |
6 | 26.4 |
7 | 40.2 |
8 | 36.4 |
9 | 30.7 |
实施例4:温度对贝莱斯芽孢杆菌Fast 30降解性能的影响
100ml无机盐培养基中加入1g/L的原油,按1%接种贝莱斯芽孢杆菌Fast 30,分别在20、25、30、35、40℃条件下培养,5天后测定225nm处紫外吸光值,计算降解率(方法同实施例2)。结果见表2,结果表明最适降解温度在35℃左右,降解率可达41.5%。温度低于35℃时,随温度升高降解率逐渐增高,当温度高于35℃时,温度越高降解率越低。
表2:温度对菌株降解性能的影响
实施例5:表面活性剂的提取及其性能测定
将1%的贝莱斯芽孢杆菌Fast 30接种于1L的液体LB培养基中,35℃、200r/min培养3天。离心收集上清,用滤膜过滤后用盐酸将上清液pH调至2.0,4℃静置过夜。离心收集沉淀,将沉淀用酸化的无菌水清洗过后用无菌去离子水溶解,再用等体积的二氯甲烷/甲醇溶液萃取,分离有机相并蒸干,得到黄色表面活性剂粗制品。表面活性剂的产量可达560mg/L。
将表面活性剂粗制品制成400mg/L的水溶液,将4mL生物表面活性剂水溶液及4mL液体石蜡分别加入刻度试管中,40℃水浴10min,用涡旋仪剧烈振荡10min,静置24h后,观察乳化层高度,并测量乳化层及混合液体高度,用H(H=乳化层高度/液体总高度×100%)表示测试样的乳化活性。其乳化指数可达38%。
序列表
<110> 南京理工大学
<120> 一株烷烃降解菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1421
<212> DNA
<213> 贝莱斯芽孢杆菌(Bacillus velezensis)
<400> 1
tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420
ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200
aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260
cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380
ccgaagtcgg tgaggtaacc tttaggagcc agccgccgaa g 1421
Claims (6)
1. 烷烃降解菌,为贝莱斯芽孢杆菌(Bacillusvelezensis) Fast 30,保藏编号为CGMCC No:20883。
2. 根据权利要求1所述的烷烃降解菌的培养方法,其特征在于,适宜培养基配方为:1g/L原油,磷酸二氢钾1~2.5 g/L,磷酸氢二钾1~2 g/L,氯化钠1~2 g/L,氯化钙0.01~0.05g/L,硝酸铵1~3 g/L,硫酸亚铁0.05~0.1 g/L,硫酸镁0.01~0.2 g/L,水;适宜培养pH 为7~9;适宜培养温度为30~40℃。
3.根据权利要求2所述的烷烃降解菌的培养方法,其特征在于,最适培养pH为7,最适培养温度为35℃。
4.根据权利要求1所述的烷烃降解菌在石油污染环境修复中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的石油污染环境为石油污染废水或石油污染土壤。
6.根据权利要求1所述的烷烃降解菌在生产生物表面活性剂中的应用。
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