CN113881594A - 一种优化的硫酸盐还原菌培养基及其应用 - Google Patents
一种优化的硫酸盐还原菌培养基及其应用 Download PDFInfo
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 30
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 15
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- KAEAMHPPLLJBKF-UHFFFAOYSA-N iron(3+) sulfide Chemical compound [S-2].[S-2].[S-2].[Fe+3].[Fe+3] KAEAMHPPLLJBKF-UHFFFAOYSA-N 0.000 description 1
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- 239000002341 toxic gas Substances 0.000 description 1
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Abstract
本发明公开了一种优化的硫酸盐还原菌培养基及其应用。培养基组成为:碳源、氮源、硫源、氯化钠、无水氯化钙、抗坏血酸、硫酸亚铁铵、保险粉、水余量;所述碳源组成为乳酸钠、葡萄糖、蔗糖或酒石酸钾钠的一种或二种以上的组合;所述氮源组成为:酵母浸粉、蛋白胨或氯化铵的一种或二种以上的组合;所述硫源组成为:无水硫酸钠、硫酸镁、硫代硫酸钠或单质硫的一种或二种以上的组合。本发明的硫酸盐还原菌培养基可满足油田环境中多种硫酸盐还原菌的营养需求,优化后的培养基有助于提高对油田硫酸盐还原菌活性检测及监测能力,有益于油田次生硫化氢问题防控及微生物腐蚀防控。
Description
技术领域
本发明涉及微生物数量监测、微生物检测及环境微生物学领域。具体涉及一种通过优化复配硫酸盐还原菌培养基中碳源、氮源和硫源营养成分,进而研发适用于油田采出水中硫酸盐还原菌的培养基。
背景技术
硫酸盐还原菌在生长繁殖的过程中会产生大量H2S,其是导致油藏酸败的主要原因,并给石油开采带来诸多危害:(1)硫化氢为一种无色有强烈臭鸡蛋气味的有毒气体,会对油田工人身体健康甚至生命产生危害。700ppm的H2S会使人出现中毒症状,甚至可能休克猝死。1000ppm的H2S被人不小心吸入,会导致人立刻死亡;(2)硫化氢为酸性气体,对采油管道具有较强的腐蚀性,严重时可能引起石油泄漏,污染环境,并造成经济损失。(3)硫酸盐还原菌的代谢产物H2S会增加原油和天然气的硫含量,降低原油和天然气的品质和油藏价值,同时使后续脱硫处理成本增加;(4)在化学驱油过程中,硫酸盐还原菌会引起聚合物溶液浓度降低,因而影响聚合物效果,导致驱油技术应用较差。
长期以来认为是油藏中硫酸盐还原菌利用含硫化合物进行生长繁殖并产生大量硫化氢过程,是导致油藏酸败的主要原因。但随着研究的不断深入,最新许多研究表明:1.油藏环境中还存在一些不能利用硫酸盐但可以产硫化氢的微生物。其可以利用单质硫、亚硫酸盐、硫代硫酸盐等一种或几种含硫底物进行生长繁殖并产生硫化氢;2.硫酸盐还原菌具有多样性,不同油田之间硫酸盐还原菌的种类存在差异性。
因此,亟待研发出针对各不同油田均有效满足多种硫酸盐还原菌生长、代谢、繁殖的培养基。进而能深入了解引发油田酸败的硫酸盐还原菌,并能有效检测油田环境中硫酸盐还原菌的数量和监测其数量变化,且找到油田酸败有效的控制方法。对于油田开发工业企业具有重要价值,也一直是油田企业所面临的一项重要挑战。
发明内容
本发明目的是提供一种适合油田环境中多种硫酸盐还原菌生长繁殖的培养基。
为实现上述目的,本发明采用的技术方案为:一种优化的硫酸盐还原菌培养基,所述硫酸盐还原菌培养基组成为:碳源、氮源、硫源、氯化钠0.01~30g/L、无水氯化钙0.01~5g/L、抗坏血酸0.01~5g/L、硫酸亚铁铵0.01~5g/L、保险粉0.01~5g/L、水余量。
所述碳源组成为:乳酸钠0.1~30g/L、葡萄糖0.1~30g/L、蔗糖0.1~30g/L或酒石酸钾钠0.1~30g/L的一种或二种以上的组合。
所述氮源组成为:酵母浸粉0.01~15.0g/L、蛋白胨0.01~15.0g/L或氯化铵0.01~15.0g/L的一种或二种以上的组合。
所述硫源组成为:无水硫酸钠0.05~10g/L、硫酸镁0.1~20g/L、硫代硫酸钠0.01~10g/L或单质硫0.01~5g/L的一种或二种以上的组合。
优选的,上述的一种优化的硫酸盐还原菌培养基,所述碳源组成为:葡萄糖0.45~1.35g/L、蔗糖0.45~1.35g/L和酒石酸钾钠1.1~3.3g/L的组合。
更优选的,上述的一种优化的硫酸盐还原菌培养基,所述碳源组成为:葡萄糖0.90g/L、蔗糖0.90g/L、酒石酸钾钠2.2g/L。
优选的,上述的一种优化的硫酸盐还原菌培养基,所述氮源组成为:酵母浸粉0.25~0.75g/L、蛋白胨0.25~0.75g/L和氯化铵0.5~1.5g/L的组合。
更优选的,上述的一种优化的硫酸盐还原菌培养基,所述氮源组成为:酵母浸粉0.5g/L、蛋白胨0.5g/L、氯化铵1.0g/L。
优选的,上述的一种优化的硫酸盐还原菌培养基,所述硫源组成为:无水硫酸钠0.25~0.75g/L、硫酸镁0.7~2.1g/L、硫代硫酸钠0.1~0.3g/L、单质硫0.05~0.15g/L的组合。
更优选的,上述的一种优化的硫酸盐还原菌培养基,所述硫源组成为:所述硫源组成为:无水硫酸钠0.75g/L、硫酸镁2.1g/L、硫代硫酸钠0.3g/L、单质硫0.15g/L。
本发明提供的硫酸盐还原菌培养基在硫酸盐还原菌的富集培养、分离筛选和数量检测中的应用。
本发明的有益效果是:本发明的培养基可实现多种硫酸盐还原菌的富集培养、分离筛选和数量检测。本发明的培养基可满足不同油田环境中多种硫酸盐还原菌生长、代谢和繁殖。本发明的硫酸盐还原菌培养基可满足油田环境中多种硫酸盐还原菌的营养需求,优化后的培养基有助于提高对油田硫酸盐还原菌活性检测及监测能力,有益于油田次生硫化氢问题防控及微生物腐蚀防控。
具体实施方式
硫酸盐还原菌计数方法:用无菌注射器将待测定的水样进行梯度稀释逐级注入到硫酸盐还原菌培养基中,每组选三个平行样。如果测试瓶底部产生黑色硫化铁沉淀,并伴有硫化氢臭味产生,表明阳性反应,采用MPN法对被测试样的硫酸盐还原菌进行计数。
实施例1一种优化的硫酸盐还原菌培养基(一)碳源组成对培养基的影响
1、硫酸盐还原菌培养基的组成:
碳源、氮源、硫源、氯化钠10g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L、水余量。其中,
碳源组成如表1。
氮源组成为:酵母浸粉1.0g/L、氯化铵1.0g/L。
硫源组成为:无水硫酸钠0.5g/L、硫酸镁2.0g/L。
2、硫酸盐还原菌检测:
方法:测定西北某油田采出的水样中的硫酸盐还原菌。按1中不同碳源的硫酸盐还原菌培养基的组成,用严格厌氧的方法进行配制,使不同碳源的硫酸盐还原菌培养基的矿化度、pH和温度与实际采样的西北某油田采出水样的矿化度、pH和温度相同。将西北某油田采出的水样滴加于配置的不同碳源的硫酸盐还原菌培养基中,进行培养7天。采用MPN法对被测试样的硫酸盐还原菌进行计数,结果如表1。
表1:不同碳源的硫酸盐还原菌培养基中细菌量统计
碳源 | 细菌量(个/ml) | |
1 | 乳酸钠3.5g/L | 4.5×10<sup>3</sup> |
2 | 葡萄糖2.8g/L | 6.0×10<sup>3</sup> |
3 | 蔗糖2.67g/L | 4.5×10<sup>3</sup> |
4 | 酒石酸钾钠6.6g/L | 6.0×10<sup>3</sup> |
5 | 乳酸钠1.2g/L+葡萄糖0.90g/L+蔗糖0.90g/L | 4.5×10<sup>3</sup> |
6 | 乳酸钠1.2g/L+葡萄糖0.90g/L+酒石酸钾钠2.2g/L | 6.0×10<sup>3</sup> |
7 | 乳酸钠1.2g/L+蔗糖0.90g/L+酒石酸钾钠2.2g/L | 6.0×10<sup>3</sup> |
8 | 葡萄糖0.90g/L+蔗糖0.90g/L+酒石酸钾钠2.2g/L | 7.5×10<sup>3</sup> |
由表1可见,第8组碳源组成,富集培养的细菌数量最多,因此,本发明优选碳源组成为:葡萄糖+蔗糖+酒石酸钾钠。
(二)氮源组成对培养基的影响
1、硫酸盐还原菌培养基的组成:
碳源、氮源、硫源、氯化钠10g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L、水余量。其中,
碳源组成为:葡萄糖0.90g/L+蔗糖0.90g/L+酒石酸钾钠2.2g/L。
氮源组成如表2。
硫源组成为:无水硫酸钠0.5g/L+硫酸镁2.0g/L。
2、硫酸盐还原菌检测:
方法:测定西北某油田采出的水样中的硫酸盐还原菌。按1中不同氮源的硫酸盐还原菌培养基的组成,用严格厌氧的方法进行配制,使不同氮源的硫酸盐还原菌培养基的矿化度、pH和温度与实际采样的西北某油田采出水样的矿化度、pH和温度相同。将西北某油田采出的水样滴加于配置的不同氮源的硫酸盐还原菌培养基中,进行培养7天。采用MPN法对被测试样的硫酸盐还原菌进行计数,结果如表2。
表2:不同氮源的硫酸盐还原菌培养基中细菌量统计
氮源 | 细菌量(个/ml) | |
1 | 酵母浸粉1.0g/L+氯化铵1.0g/L | 7.5×10<sup>3</sup> |
2 | 蛋白胨1.0g/L+氯化铵1.0g/L | 9.5×10<sup>3</sup> |
3 | 酵母浸粉0.5g/L+蛋白胨0.5g/L+氯化铵1.0g/L | 1.15×10<sup>4</sup> |
由表2可见,第3组氮源组成,富集培养的细菌数量最多,因此,本发明优选氮源组成为:酵母浸粉+蛋白胨+氯化铵。
(三)硫源组成对培养基的影响
1、硫酸盐还原菌培养基的组成:
碳源、氮源、硫源、氯化钠10g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L、水余量。其中,
碳源组成为:葡萄糖0.90g/L+蔗糖0.90g/L+酒石酸钾钠2.2g/L。
氮源组成为:酵母浸粉0.5g/L+蛋白胨0.5g/L+氯化铵1.0g/L。
硫源组成如表3。
2、硫酸盐还原菌检测:
方法:测定西北某油田采出的水样中的硫酸盐还原菌。按1中不同硫源的硫酸盐还原菌培养基的组成,用严格厌氧的方法进行配制,使不同硫源的硫酸盐还原菌培养基的矿化度、pH和温度与实际采样的西北某油田采出水样的矿化度、pH和温度相同。将西北某油田采出的水样滴加于配置的不同硫源的硫酸盐还原菌培养基中,进行培养7天。采用MPN法对被测试样的硫酸盐还原菌进行计数,结果如表3。
表3:不同硫源的硫酸盐还原菌培养基中细菌量统计
由表3可见,第4组硫源组成,富集培养的细菌数量最多,因此,本发明优选硫源组成为:无水硫酸钠+硫酸镁+硫代硫酸钠+单质硫。
(四)以优选的碳源组成、氮源组成和硫源组成进行正交试验
碳源组成、氮源组成和硫源组成的三因素三水平设计如表4,培养基的其余组成为氯化钠10g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L。
表4三因素三水平L9(34)正交试验表
以表4设计的三因素三水平L9(34)进行正交试验,从试验结果得知,用A2B2C3培养基通过MPN法测得西北某油田硫酸盐还原菌细菌量最大,硫酸盐还原菌细菌量达4.5×105个/ml。因此本发明优化的硫酸盐还原菌培养基组成为:葡萄糖0.90g/L、蔗糖0.90g/L、酒石酸钾钠2.2g/L、酵母浸粉0.5g/L、蛋白胨0.5g/L、氯化铵1.0g/L、无水硫酸钠0.75g/L、硫酸镁2.1g/L、硫代硫酸钠0.3g/L、单质硫0.15g/L、氯化钠10g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L。
(五)对比例
采用常规培养基:乳酸钠2.5g/L、酵母浸粉1.0g/L、氯化铵1.0g/L、无水硫酸钠0.5g/L、硫酸镁2.0g/L、无水氯化钙0.1g/L、抗坏血酸0.1g/L、硫酸亚铁铵0.1g/L、保险粉0.1g/L。对西北某油田硫酸盐还原菌进行检测,通过MPN法测硫酸盐还原菌细菌量仅为4.5×103个/ml。
可见,本发明的硫酸盐还原菌培养基更能有效检测油田环境中硫酸盐还原菌的数量。适应用硫酸盐还原菌的富集培养、分离筛选、数量检测等应用,对于油田开发工业企业具有重要价值。
Claims (8)
1.一种优化的硫酸盐还原菌培养基,其特征在于,所述硫酸盐还原菌培养基组成为:碳源、氮源、硫源、氯化钠0.01~30g/L、无水氯化钙0.01~5g/L、抗坏血酸0.01~5g/L、硫酸亚铁铵0.01~5g/L、保险粉0.01~5g/L、水余量;
所述碳源组成为:乳酸钠0.1~30g/L、葡萄糖0.1~30g/L、蔗糖0.1~30g/L和酒石酸钾钠0.1~30g/L的一种或二种以上的组合;
所述氮源组成为:酵母浸粉0.01~15.0g/L、蛋白胨0.01~15.0g/L和氯化铵0.01~15.0g/L的一种或二种以上的组合;
所述硫源组成为:无水硫酸钠0.05~10g/L、硫酸镁0.1~20g/L、硫代硫酸钠0.01~10g/L和单质硫0.01~5g/L的一种或二种以上的组合。
2.根据权利要求1所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述碳源组成为:葡萄糖0.45~1.35g/L、蔗糖0.45~1.35g/L和酒石酸钾钠1.1~3.3g/L的组合。
3.根据权利要求2所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述碳源组成为:葡萄糖0.90g/L、蔗糖0.90g/L、酒石酸钾钠2.2g/L。
4.根据权利要求1所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述氮源组成为:酵母浸粉0.25~0.75g/L、蛋白胨0.25~0.75g/L和氯化铵0.5~1.5g/L的组合。
5.根据权利要求4所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述氮源组成为:酵母浸粉0.5g/L、蛋白胨0.5g/L、氯化铵1.0g/L。
6.根据权利要求1所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述硫源组成为:无水硫酸钠0.25~0.75g/L、硫酸镁0.7~2.1g/L、硫代硫酸钠0.1~0.3g/L、单质硫0.05~0.15g/L的组合。
7.根据权利要求6所述的一种优化的硫酸盐还原菌培养基,其特征在于,所述硫源组成为:无水硫酸钠0.75g/L、硫酸镁2.1g/L、硫代硫酸钠0.3g/L、单质硫0.15g/L。
8.权利要求1-7任意一项所述的硫酸盐还原菌培养基在硫酸盐还原菌的富集培养、分离筛选和数量检测中的应用。
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