CN104357035B - 防治高温水体中srb的生物菌剂及其抑制srb的方法 - Google Patents
防治高温水体中srb的生物菌剂及其抑制srb的方法 Download PDFInfo
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Abstract
本发明涉及油田采出液中硫酸盐还原菌抑制技术,特别涉及一种防治高温水体中SRB的生物菌剂及其抑制SRB的方法,其中防治油田高温水体环境中SRB的生物菌剂为JSHD‑4地芽孢杆菌,具有序列表SEQ ID NO:1 所示的序列,保藏于中国微生物菌种保藏管理委托员会普通微生物保藏中心,拉丁文学名为 Geobacillus toebii,保藏编号为CGMCC NO 7273 保藏日期为 2013年3月5日。所述JSHD‑4地芽孢杆菌通过微生物好氧发酵制得菌液浓度为1.0×106~1.3×1010cfu/mL的工业发酵液,耐受温度为50—95℃,最佳生长温度为60—70℃。采用本发明的JSHD‑4地芽孢杆菌配制的多功能生物菌剂投入井下后,在井下高温环境下使得硝酸盐还原菌群(DNB)迅速增生扩散,适合直接对井下高温环境的SRB进行在线处理。
Description
技术领域
本发明涉及油田水体中的硫酸盐还原菌抑制技术领域,特别涉及一种防治高温水体中SRB的生物菌剂及其抑制SRB的方法。
背景技术
油井腐蚀问题长期困扰着石油开采业,也是油井生产和科研人员一直关注的课题,至今没有得到较好的解决。大量研究表明,油井生物腐蚀主要是有害微生物硫酸盐还原菌(SRB)大量滋生,引起硫化物浓度增大造成的。目前,油井控制SRB和硫化物主要是采取化学和物理方法,存在成本高、易二次污染、形成耐药性等不足。
油田硫酸盐还原菌生物防治集输近年来逐渐兴起,该技术主要是利用生物竞争淘汰的方法,通过微生物种群的替代将有害的微生物问题变为有利因素;但是该技术主要研究对象为油田集输***的SRB治理,对于油井这种高温极端环境(50℃以上)的研究较少。
本发明中的多功能生物菌剂可以在50~95℃的油井高温环境下使用,使得硝酸盐还原菌群(DNB)迅速增生扩散,并在与SRB竞争生存空间和基质时,DNB优先选择使用油田***中的基质,有效阻止SRB获得所需要的营养物,从而控制SRB的代谢活性。
发明内容
本发明的目的是针对现有技术中SRB生物抑制技术的不足,通过向油田井下高温环境中注入嗜热多功能生物菌剂,有效的抑制高温环境中SRB的快速生长,消除已经产生的硫化物,同时刺激反硝化细菌的生长,改变水体微生物群落结构,从而达到防止生物腐蚀的目的。
本发明的目的之一是,提供一种防治油田高温水体环境中SRB的生物菌剂,所述生物菌剂为JSHD-4地芽孢杆菌,具有序列表SEQ ID NO:1 所示的序列,保藏于中国微生物菌种保藏管理委托员会普通微生物保藏中心,拉丁文学名为 Geobacillus toebii,保藏编号为CGMCC NO 7273 保藏日期为 2013年3月5日。
本发明中,所述JSHD-4地芽孢杆菌通过微生物好氧发酵制得菌液浓度为1.0×106~1.3×1010cfu/mL的工业发酵液,耐受温度为50—95℃,最佳生长温度为60—70℃。本发明中的JSHD-4地芽孢杆菌耐受温度高,可以适应中高温的井下环境中SRB的抑制,防止井下生物腐蚀。
本发明的另一个目的是,提供一种采用上述生物菌剂抑制油田高温水体环境中SRB的方法,按油田高温水体的量投加如下比例的多功能生物菌剂:JSHD-4地芽孢杆菌:通过微生物好氧发酵制得菌液浓度为1.0×106~1.3×1010cfu/mL的工业发酵液,所述工业发酵液的投加比例为油田高温水体体积0.5~1%;硝酸钠20~30 mg/L;亚硝酸钠50~150 mg/L;磷酸二氢钠5~10 mg/L
作为本发明方法的进一步改进,所述JSHD-4地芽孢杆菌通过好氧发酵的工业发酵液的浓度为1.0~1.3×108cfu/mL,投加比例为油田高温水体体积0.8%;硝酸钠25 mg/L;亚硝酸钠75 mg/L;磷酸二氢钠8 mg/L。
作为本发明的再一步改进,本发明的方法中,向目标油井中投加所述多功能生物菌剂时,先将油井套管气压放至套压回零,JSHD-4地芽孢杆菌菌液直接采用冲击式投加加入油井套管内,硝酸钠、亚硝酸钠和磷酸二氢钠按比例称量后先溶解到25—30L的水中,然后采用冲击式投加加入油井套管内,最后再向油井套管内投加50L水。
本发明的有益效果是:本发明的JSHD-4地芽孢杆菌可以在50~95℃的油井高温环境下使用,菌种经生物好氧发酵可获得菌液浓度为1.0×106~1.3×1010cfu/mL芽孢的工业发酵液,采用JSHD-4地芽孢杆菌配制的多功能生物菌剂投入井下后,在井下高温环境下使得硝酸盐还原菌群(DNB)迅速增生扩散,可以有效的抑制高温环境中SRB的快速生长,消除已经产生的硫化物,同时刺激竞争微生物反硝化细菌的生长,改变水体微生物群落结构,其硫化物抑制率在93%以上,有效延长管道的使用寿命,改善水质,大大降低生产成本。
生物保藏
本发明所涉及的微生物已提交生物保藏:
具体实施方式
实施例1
本发明的JSHD-4地芽孢杆菌通过如下过程筛选:
(1)样品采集
在江苏油田采集7口油井的产出液,样品名称分别为:CH2-43、CH2-42、CH2-36、T83-1、T83-10、T83-7、H88-10。
(2)培养基配置
富集培养基由A溶液和B溶液混合组成,其中:
A溶液:无机盐K1 2.0 g,天冬酰胺1.0 g,蒸馏水500 ml,pH值7.0~7.2;
B溶液:柠檬酸钠8.5 g,乙酸钠2 g,葡萄糖2 g,KH2PO4 0.5 g,K2HPO4 0.5 g,MgSO4·7H2O 1.0 g,FeCl3·6H2O 0.05 g,CaCl2 0.1 g,yeast extract 0.5 g,蒸馏水500ml。pH值7.0~7.2。
NRB液体培养基:A液和B液单独121 ℃灭菌20 min,灭菌后等比例混合。
NRB固体培养基:A液和B液中各加入质量比为1.5% ~ 2%的琼脂,单独灭菌后等比例混合。
(3)富集培养
将步骤(1)中7口油井产出液样品等比例混合,然后加入NRB液体培养基,分别置于65 ℃、75 ℃(是否应改为70℃)培养箱中好氧培养;同时,以同样处理的采出液灭菌样品做为对照样。65 ℃条件下培养6d后以5%的接种量接种至相同的NRB液体培养基中进行转接培养,培养3 d后再次转接,转接培养3次,然后转接至70 ℃条件下进行驯化培养。培养后测定培养液中K1离子浓度见表1。
经过三次转接,65 ℃、70 ℃条件下培养溶液浑浊,而对照样培养液澄清,表明实验组培养液中微生物大量生长,同时,由表1培养液K1浓度测定表明,70 ℃驯化培养2 d,K1浓度由2.0 g/L降低至0.24 g/L,而65 ℃第三次转接培养液K1浓度测定为0,培养后溶液K1离子浓度大幅度降低说明微生物生长同时伴随K1离子大量消耗,表明实验组培养液含有硝酸盐还原菌,且在实验条件下生长旺盛。
(4)菌株分离纯化
将65 ℃三次转接、70 ℃驯化的样品涂布到NRB固体培养基平板培养,从所得培养平板上挑取单菌落于三角烧瓶中进行培养,培养3 d后置于4 ℃冰箱保存。
(5) 硝酸盐还原菌DNA提取与鉴定
(I)DNA提取
将分离纯化后的硝酸盐还原菌富集培养3天,用2 mL离心管在12000 × g条件下离心收集细胞,采用Axygen DNA提取试剂盒提取硝酸盐还原菌DNA,具体操作步骤如下:
1)在装有细胞的离心管中加入450 μL裂解液,旋涡振荡15 s,65 ℃水浴10 min;
2)继续加入400 μL蛋白质变性剂和1 mL 相分离液(4 ℃预冷),用力混合,12000× g离心2 min;
3)弃上相,保留相间沉淀和下相,加入1mL 4℃预冷的相分离液,混合,12000 × g离心2 min;
4)弃上相,将下相转移至滤器(滤器置于2 mL离心管中),12000 × g离心1 min;
5)弃滤器,在滤液中加入400 μL DNA结合液,混合均匀;
6)将制备管置于2 mL离心管中,将步骤(5)中的混合液移入制备管中,12000 × g离心1 min;
7)弃滤液,将制备管置回到原2 mL离心管中,加入500 μL冲洗液W1,12000 × g离心1 min;
8)弃滤液,将制备管置回到原2 mL离心管中,加入700 μL冲洗液W2,12000 × g离心1 min;
9)以同样的方法再用700 μL冲洗液W2洗涤一次;
10)弃滤液,将制备管置回到原2 mL离心管中,12000 × g离心1 min;
11)将制备管置于另一洁净的1.5 mL离心管中,在silica膜中央加100-200 μLEluent或去离子水,室温静置1 min,12000 × g离心1 min洗脱DNA,保存在-20 ℃下以备用。
(II)样品PCR扩增
将提取的JSHD04的DNA,采用16S rDNA基因通用引物1492R和27F进行PCR扩增。
1) 细菌16S rDNA PCR反应体系
PCR反应体系组成见表2。
2) 引物序列
2) 引物序列
1492R:5‘-GGTTACCTTGTTACGACTT-3’
27F:5‘>CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGAGAGTTTGATCTTGGCTCAG<3’
3) PCR反应程序
1.95 ℃ 3 min
2.94 ℃ 40 sec
3.52 ℃ 45 sec
4.Goto 2 35 times
5.72 ℃ 10 min
(III)测序结果
江苏油田产出液65 ℃富集样品分离出菌落(编号JSHD-4),挑取单菌落富集培养,培养细胞提取DNA,PCR扩增产物进行测序,结果如果如SEQ ID NO:1所示的序列。
经分析比对与经分析比对与G. toebii strain R-32639相似度为97%,为地芽孢杆菌的一个亚种。
实施例2
实验室体系条件下JSHD-4对SRB的抑制效果
按照表3改良SRB培养基配方,以无菌水为溶剂进行培养基配置,培养基经121 ℃灭菌20 min,冷却后每升培养基再分别加入微量元素溶液(组成如表4所示)1 mL、维生素溶液(组成如表5所示)1 mL和经紫外线灭菌的硫酸亚铁铵0.1g和L-半胱氨酸盐酸盐0.5 g;从固体斜面上用接种环挑取保存的SRB菌种(脱硫肠状菌)3~4环接种到SRB培养基中,在65℃下培养48小时,获得新鲜富集培养的SRB菌液。
同时将保存的JSHD-4在75 ℃下使用NRB液体培养基(同实例1中的NRB液体培养基)活化培养1 d,获得新鲜富集培养的JSHD-4菌液,然后按2%的接种比例将JSHD-4菌液和SRB菌液接种到改良SRB的培养基中(试验瓶为250ml的厌氧瓶),并加入10 mmol/L的硝酸钠在65℃下进行培养,同时以只接种SRB菌液和添加10 mmol/L的硝酸钠而不接种硝酸盐还原菌的为对照组,比较培养液变化,通过检测H2S的含量来表征多功能生物菌剂中的JSHD-4对SRB活性的抑制能力,检测结果如表6所示)。培养7 d以后,只加SRB和添加10 mmol/L的硝酸钠的对照组中的H2S含量很高,而在加入含JSHD-4的抑制体系中H2S浓度在第3 d和第7 d均保持很低,表明实验组中SRB的活性被明显抑制。
实施例3
首先将反硝化细菌JSHD-4通过常规微生物好氧发酵制备获得芽胞浓度为1.0×106cfu/mL工业发酵液,该工业发酵液可使用20L塑料桶储存,然后取硝酸钠、亚硝酸钠和磷酸二氢钠均为市售工业级产品,使用时按比例混合均匀,也可以依次加入目标油井采出液。
将本发明JSHD-4多功能生物菌剂按如下比例投入江苏油田王39井,处理前该井产出液的硫化物浓度为28mg/L。投加量为:JSHD-4工业发酵液(1.0×106cfu/mL),投加量为采出液含量的0.5%;硝酸钠:20mg/L;亚硝酸钠:50mg/L;磷酸二氢钠:5mg/L
向目标油井中投加上述多功能生物菌剂时,先将油井套管气压放至套压回零,JSHD-4工业发酵液直接采用冲击式投加加入油井套管内,硝酸钠、亚硝酸钠和磷酸二氢钠按比例称量后先溶解到25L的水中,然后采用冲击式投加加入油井套管内,最后再向油井套管内投加50L水。同时不定期检测硫化物含量,并适时补充添加多功能生物菌剂;以保持抑制效果。抑制效果如图1所示的硫化物变化曲线,60d抑制率达93.14%。
实施例4
与实施例3不同之处在于,实施油井为江苏油田韦2-53井,硫化物128.4mg/L,JSHD-4工业发酵液芽孢浓度为1.3×1010cfu/mL,投加量为油井采出液含量的1%;硝酸钠:30mg/L;亚硝酸钠:150mg/L;磷酸二氢钠:10mg/L。抑制效果如图1所示的硫化物变化曲线,60d抑制率达97.73%。
实施例5
与实施例3不同之处在于,实施油井为江苏油田黄88-39井,硫化物56mg/L,JSHD-4工业发酵液浓度为1.3×108cfu/mL,投加量为采出液含量的0.8%;硝酸钠:25mg/L;亚硝酸钠:75mg/L;磷酸二氢钠:8mg/L。抑制效果如图1所示的硫化物变化曲线,60d抑制率达94.93%。
序列表
<110>中国石油化工股份有限公司;中国石油化工股份有限公司江苏油田分公司
<120>防治高温水体中SRB的生物菌剂及其抑制SRB的方法
<160>1
<210>1
<211>1012
<212>DNA
<213>人工序列
<400>1
ATCATGCAAG TCGAGCGGAC CGAACGGAAG CTTGCTTCTG TTCGGTTAGC GGCGGACGGG 60
TGAGTAACAC GTGGGTAACC TGCCCGTAAG ACCGGGATAA CTCCGGGAAA CCGGGGCTAA 120
TACCGGATAA CACCGAAGAC CGCATGGTCT TTGGTTGAAA GGTGGCTTTT GCTACCACTT 180
ACGGATGGGC CCGCGGCGCA TTAGCTAGTT GGTGAGGTAA CGGCTCACCA AGGCGACGAT 240
GCGTAGCCGG CCTGAGAGGG TGACCGGCCA CACTGGGACT GAGACACGGC CCAGACTCCT 300
ACGGGAGGCA GCAGTAGGGA ATCTTCCGCA ATGGACGAAA GTCTGACGGA GCGACGCCGC 360
GTGAGCGAAG AAGGTCTTCG GATCGTAAAG CTCTGTTGTT AGGGAAGAAG AAGTACCGTT 420
CGAATAGGGC GGTACGGTGA CGGTACCTAA CGAGAAAGCC CCGGCTAACT ACGTGCCAGC 480
AGCCGCGGTA ATACGTAGGG GGCGAGCGTT GTCCGGAATT ATTGGGCGTA AAGCGCGCGC 540
AGGCGGTCC CTTAAGTCTGA TGTGAAAGCC CACGGCTCAA CCGTGGAGGG TCATTGGAAA 600
CTGGGGGACT TGAGTGCAGA AGAGGAGAGC GGAATTCCAC GTGTAGCGGT GAAATGCGTA 660
GAGATGTGGA GGAACACCAG TGGCGAAGGC GGCTCTCTGG TCTGTAACTG ACGCTGAGGC 720
GCGAAAGCGT GGGGGAGCAA ACAGGATTAG ATACCCTGGT AGTCCACGCC GTAAACGATG 780
AGTGCTAAGT GTTAGAGGGG TTTTCCCTTT AGTGCTGTAG CTAACGCGTT AAGCACTCCG 840
CCTGGGGAGT ACGGCGCATG CTGAACTCAA GAATTGACGG GGCCCGCACA AGCGTGAGCA 900
TGTGGTTTAA TTCGAGCACG CGAGACCTTA CAGTCTGACA TCCCTGACAC CTGGAAATGC 960
GTTCCCCCTC GGGGGACAGG TGACAGGGGG GCATGGTGTC GTCACACTCG TG 1012
<210>2
<211>19
<212>DNA
<213>人工序列
<400>2
GGTTACCTTGTTACGACTT
<210>3
<211>60
<212>DNA
<213>人工序列
<400>3
CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGAGAGTTTGATCTTGGCTCAG
Claims (5)
1.一种防治高温水体中SRB的生物菌剂,其特征在于,所述生物菌剂为JSHD-4地芽孢杆菌,具有序列表SEQ ID NO:1 所示的序列,保藏于中国微生物菌种保藏管理委托员会普通微生物保藏中心,拉丁文学名为 Geobacillus toebii,保藏编号为CGMCC NO 7273 保藏日期为 2013年3月5日。
2.权利要求1所述的防治高温水体中SRB的生物菌剂,其特征在于,所述JSHD-4地芽孢杆菌通过微生物好氧发酵制得菌液浓度为1.0×106~1.3×1010cfu/mL的工业发酵液,耐受温度为50—95℃,最佳生长温度为60—70℃。
3.一种权利要求2所述的生物菌剂抑制高温水体中SRB的方法,其特征在于,按油田高温水体的量投加如下比例的多功能生物菌剂:所述JSHD-4地芽孢杆菌通过好氧发酵的工业发酵液的投加比例为油田高温水体体积0.5~1%;硝酸钠20~30 mg/L;亚硝酸钠50~150mg/L;磷酸二氢钠5~10 mg/L。
4.根据权利要求3所述的生物菌剂抑制高温水体中SRB的方法,其特征在于,按油田高温水体的量投加如下比例的多功能生物菌剂:所述JSHD-4地芽孢杆菌通过好氧发酵的工业发酵液的浓度为1.0×108~1.3×108cfu/mL,投加比例为油田高温水体体积0.8%;硝酸钠25mg/L;亚硝酸钠75 mg/L;磷酸二氢钠8 mg/L。
5.根据权利要求3或4所述的生物菌剂抑制高温水体中SRB的方法,其特征在于,向目标油井中投加所述多功能生物菌剂时,先将油井套管气压放至套压回零,JSHD-4地芽孢杆菌菌液直接采用冲击式投加加入油井套管内,硝酸钠、亚硝酸钠和磷酸二氢钠按比例称量后先溶解到25—30L的水中,然后采用冲击式投加加入油井套管内,最后再向油井套管内投加50L水。
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