CN107686849A - 一种促进油藏中石油烃厌氧降解产甲烷的方法 - Google Patents
一种促进油藏中石油烃厌氧降解产甲烷的方法 Download PDFInfo
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Abstract
本发明提供了一种促进油藏中石油烃厌氧降解产甲烷的方法，其包括如下步骤：(1)检测分析油藏中微生物群落结构，以判断其是否含有互营烃降解产甲烷菌系；(2)当油藏中含有互营烃降解产甲烷菌系时，向油藏中加入添加物A，然后封井，所述添加物A为氢营养型产甲烷古菌或含有氢营养型产甲烷古菌的混合菌群；当油藏中不含产甲烷互营烃降解菌系时，向油藏中加入添加物B，然后封井，所述添加物B为含有氢营养型产甲烷古菌与互营烃降解菌的混合菌。
Description
技术领域
本发明属于微生物产气技术领域，具体涉及一种促进油藏中石油烃厌氧降解产甲烷的方法。
背景技术
油藏是典型的极端环境，环境复杂，具有高温、高压、高矿化度、缺氧的特征。目前我国原油的平均采收率不到35％，很多长期注水开发和三次采油(化学驱、微生物驱、注气驱、热力采油等)的油藏，呈高含水、高采出度、低渗透率等特点，其开采效果和效益越来越低，如何提高低品位油藏的开采率，是石油开采的重大问题之一。
地下深层油藏经注水开发后，形成复杂的物生物区系。虽然采用注水开采原油，但是氧不大可能直接注入水进入地下深层油藏，油藏地层水中的溶解氧非常少，因此地下油藏主要是厌氧环境。好氧微生物难以正常生长繁殖，能够在油藏中生长代谢的微生物，只能是以硝酸盐、硫酸盐、三价铁、二氧化碳和有机酸等作为电子受体进行厌氧呼吸或发酵的发酵菌、硝酸盐还原菌、硫酸盐还原菌、铁还原菌和产甲烷菌(承磊,仇天雷,邓宇,等.油藏厌氧微生物研究进展[J].应用与环境生物学报,2006,12(5):740-744.)。这些油藏微生物的代谢活动能够改变油藏的地质化学环境，因此微生物采油技术应运而生。但自然条件下，油藏中营养物质匮乏，油藏本源微生物浓度低，其中发酵性细菌约为102-104个/ml，甲烷菌浓度约为1-102个/ml，且代谢活性低，难以有效的驱动原油开采(活化油藏中产甲烷菌转化二氧化碳生产甲烷的方法，申请号CN201310479743.7)。因此，外源添加营养物质和微生物制剂成为当前微生物采油技术的主流趋势。该技术通过微生物的代谢活动，降解原油，同时产生生物表面活性剂等代谢产物，从而提高原油采收率。影响微生物驱油效果的因素包括微生物的注入方式、营养物和氧的补充及油藏中微生物的发酵时间等。
基于好氧微生物采油的技术，无论是异源微生物，还是本源微生物，均会受限于环境氧浓度，这也严重限制了好氧微生物采油技术的应用。“一种微生物采油方法，申请号CN201010146472.X”需要对预培养的石油烃降解菌培养液进行纯氧曝气，再与石油烃降解菌菌液混合注入油藏。“一种微生物采油方法，申请号CN02151230.2”同样需要往油井中注入空气。随着氧气的消耗，注入的驱油微生物活性将大幅下降，极大的降低驱油效率。此外，微生物注入油藏后，随同注入的营养物质减少，微生物活性将大幅降低，不能长期连续有效的发挥微生物驱油作用。因此形成了一系列基于油藏营养元素匮乏的特点，建立一套补充油藏內源微生物营养物质从而激活微生物驱油的技术，例如“一种石油内源微生物激活体系及其筛选方法和应用，申请号CN201410405697.0”。以上可知，微生物采油技术的突破口在于，最大限度的利用油藏的地化环境，以及本源微生物的代谢活性。
针对地下油藏独特的缺氧环境，利用厌氧微生物的代谢活动驱油，为原油的开采提供了新思路。理论和实践已证明油藏中存在石油烃厌氧降解的生物学过程，并且石油烃可以被厌氧微生物降解产甲烷。因此，利用厌氧微生物的代谢作用，将油藏中难以开采的原油降解转化为甲烷，实现“油藏变气藏”，进行“气态”开采，将极大的提高油藏的开发利用率。
“油藏变气藏”需要在厌氧环境下多种微生物共同作用将石油烃降解为小分子有机物，再将这些小分子有机物转化为甲烷。这种互营过程，是限制石油烃转化甲烷速率的瓶颈。因为，标准状态下，没有氧气、硫酸盐、硝酸盐、铁(III)等外源电子受体时，厌氧微生物降解石油烃的吉布斯自由能大于零，是吸热反应，无法自发进行，例如标准状态下，正十六烷烃代谢产乙酸和CO2的吉布斯自由能分别为407.8KJ/mol和1230.5KJ/mol。处于厌氧生物链最末端的产甲烷古菌可以解除石油烃厌氧降解生物链的末端抑制，使得一系列的生化反应持续不断的进行。因此烃降解菌和产甲烷古菌偶联，通过产甲烷古菌消耗乙酸和/或H2，使乙酸和/或H2维持在极低浓度，即可保证烃降解菌持续不断的转化正十六烷烃(DolfingJ,Larter SR,Head IM(2008).Thermodynamic constraints on methanogenic crude oilbiodegradation.The ISMEjournal,2,442-452.)。
石油烃厌氧降解产甲烷过程是多种微生物互营代谢的结果。这类互营菌生长缓慢，致使石油烃互营降解产甲烷菌系建立需要数月甚至数年的时间(用于油藏残余油气化开采的菌群构建方法，申请号CN201110278644.3)。国内外研究表明，Syntrophaceae是石油烃厌氧降解产甲烷过程中的关键微生物(Gray ND,SherryA,Grant RJ,et al.(2011)Thequantitative significance of Syntrophaceae and syntrophic partnerships inmethanogenic degradation of crude oil alkanes.Environ Microbiol 13(11):2957-2975；Cheng L,Ding C,Li Q,He Q,Dai LR&Zhang H(2013)DNA-SIP reveals thatSyntrophaceae play an important role in methanogenic hexadecanedegradation.PLoS One 8(7):e66784)，但是互营微生物分离难度极大，迄今为止，尙未获得严格意义上的互营烃降解菌Syntrophaceae的纯培养物，不大可能利用分离纯培养的互营烷烃降解菌驱动原油转化为甲烷。但，地下油藏本身具有厌氧降解石油烃产甲烷的生物学过程，可以采用一定的手段，激活地下互营烃代谢产甲烷菌系，使其高效的利用石油烃产甲烷，实现“油藏变气藏”过程。因此，如何快速启动石油烃降解产甲烷过程并具有良好产甲烷速率的方法，是本领域面临的技术难题。
“一种调控石油烃厌氧生物降解产甲烷速率的方法，申请号201110278642.4”通过向油井中注入一定量的硫酸盐和磷酸盐，提高了石油烃厌氧转化甲烷的反应速率。但，硫酸盐的加入，会导致石油烃的代谢朝硫酸盐还原产硫化氢的方向进行，一方面大大降低石油烃产甲烷的转化率，另一方面产生的硫酸氢将会腐蚀采油管道及设备。“一种调控油藏微生物代谢产生物气的方法，申请号201410584212.9”通过油藏微生物检测及激活剂筛选和优化调控油藏产甲烷代谢活动，但添加硝酸盐，可能促使石油烃朝硝酸盐还原的方向发展，降低石油烃产甲烷的转化率，并且产甲烷菌激活剂的筛选需要3-6个月，时间周期长。随着微生物的代谢活动，油藏环境发生改变，最初筛选的产甲烷菌激活剂并不适合于动态变化的油藏环境。
发明内容
针对现有技术的缺点，本发明的目的在于提供一种促进油藏中石油烃厌氧降解产甲烷的方法，所述方法包括如下步骤：
(1)检测分析油藏中微生物群落结构，以判断其是否含有产甲烷互营烃降解菌系；
(2)当油藏中含有产甲烷互营烃降解菌系时，向油藏中加入添加物A，然后封井，所述添加物A为氢营养型产甲烷古菌或含有氢营养型产甲烷古菌的混合菌；当油藏中不含产甲烷互营烃降解菌系时，向油藏中加入添加物B，然后封井，所述添加物B为氢营养型产甲烷古菌与互营烃降解产甲烷菌系的混合菌。
所述产甲烷互营烃降解菌系指在厌氧条件下具有降解烃类化合物并产甲烷能力的微生物菌群，该菌群至少应该包括厌氧烃降解菌和产甲烷古菌两类微生物。
步骤(1)中，在检测分析油藏中微生物群落结构，以判断其是否含有产甲烷互营烃降解菌系时，所用方法包括：
(1)检测油藏中微生物的中间代谢产物；
(2)采用未培养技术分析油藏中的微生物群落结构；
(3)采用产甲烷互营烃降解菌系具有的功能基因分析油藏中的功能微生物；
(4)对油藏样品进行模拟培养，监测产甲烷趋势，分析其是否具有降解石油烃产甲烷潜力。
所述油藏中微生物的中间代谢产物包括烷基琥珀酸及其衍生物(如methylpentadecyl succinic acid(MPA)及其代谢产物4-methyloctadecanoic acid、4-methyloctadec-2,3-enoic acid；methyl tetradecyl succinic acid(MTA)及其代谢产物4-methylheptadecanoic acid、4-methylheptadec-2,3-enoic acid、2-methylpentadecanoic acid)、苯甲基琥珀酸及其衍生物、苯甲酸衍生物、芳香酸、脂肪酸中的至少一种。
所述未培养技术包括高通量测序、荧光原位杂交、克隆文库、T-RFLP、PCR-DGGE中的任意一种或多种。
所述油藏中微生物的功能基因包括烷基琥珀酸合成酶α亚基基因assA、苯甲基琥珀酸合成酶α亚基基因bssA、甲基辅酶M还原酶α亚基基因mcrA中的一种或多种。
所述assA的核苷酸序列包括如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ IDNO：4、SEQ ID NO：5所示的一种或多种。
优选的，所述assA的核苷酸序列为如SEQ ID NO：1和SEQ IDNO：2所示。
所述添加物A为氢营养型产甲烷古菌。添加量为102～109CFU/ml。
氢营养型产甲烷古菌与产甲烷互营烃降解菌系的混合菌中，氢营养型产甲烷古菌与互营烃降解菌系的菌数比为0.01～1000。
所述氢营养型产甲烷古菌包括Methanobacterium sp.CB12，Methanoculleusreceptaculi ZC-2、Methanothermobacter crinale Tm2中的至少一种。
对“油藏变气藏”的微生物采油技术而言，油藏中产甲烷古菌的生长繁殖可以促使发酵菌等微生物更好的生长繁殖，其代谢活动可改变油层中的微环境，实现微生物调剖、产酸产气从而提高采油率。
本发明发现，当向所述油藏中加入氢营养型产甲烷菌或氢营养型产甲烷古菌与产甲烷互营烃降解菌系的混合菌时，可以刺激油藏中互营烃降解菌的生长代谢，极好的利用了油藏及其本源微生物的环境条件和功能特性，充分的发挥了油藏中本源微生物驱油的性能，极有效的促进石油烃降解产甲烷过程。因此，本发明是一种科学高效的微生物驱油产气方法。
附图说明
图1为本发明实施例1涉及的油藏样品基因组、assA基因和mcrA基因PCR产物琼脂糖电泳图；
图2为本发明实施例1油藏样品细菌和古菌的微生物群落结构情况；
图3为本发明实施例石油烃降解产甲烷模拟培养产甲烷趋势图；
图4和图5为本发明产气结果图。
具体实施方式
下面通过实施例对本发明进行具体描述，有必要在此指出的是以下实施例只是用于对本发明进行进一步的说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。
实施例1
从胜利油田采集油藏样品，采用bead-beating法提取样品总DNA，分别利用下述方法检测分析某油藏中的微生物群落结构，确认该油藏含有产甲烷互营烃降解菌系：
(1)油藏样品中互营烃降解产甲烷菌系功能基因检测
烃降解菌烷基琥珀酸合成酶α亚基和苯甲基琥珀酸合成酶α亚基基因assA和bssA基因的检测参考文献CallaghanAV,Davidova IA,Savage-AshlockK,Parisi VA,Gieg LM,Suflita JM,Kukor JJ&Wawrik B(2010)Diversity ofbenzyl-and alkylsuccinatesynthase genes in hydrocarbon-impacted environments and enrichmentcultures.Environ Sci Technol 44(19):7287-7294.其中PCR程序为：95℃预变性3min；95℃变性45s，55℃退火1min,72℃延伸2min，循环30-40次；72℃延伸10min。用1％琼脂糖电泳检测PCR产物。引物序列为：ass/bss F:5’-TTTGAGTGCATCCGCCAYGGICT-3’,ass/bss R:5’-TCGTCRTTGCCCCATTTIGGIGC-3’和/或bssA:1213F:5’-GACATGACCGAYGCCATYCT-3’，1987R:5’-TCRTCGTCRTTGCCCCAYTT-3’
产甲烷古菌甲基辅酶M还原酶α亚基基因mcrA的检测参考文献LutonPE,Wayne JM,Sharp RJ&Riley PW(2002)The mcrAgene as an alternative to 16S rRNAinthephylogenetic analysis ofmethanogen populations in landfill.Microbiology 148(Pt 11):3521-3530.其中mcrA基因引物为：mcr F:5′-GGTGGTGTMGGATTCACACARTAYGCWACAGC-3′，mcr R:5′-TTCATTGCRTAGTTWGGRTAGTT-3′.PCR程序：94℃预变性3min，94℃变性1min，65℃退火45s，72℃延伸45s，每个循环退火温度降低0.5℃，直至55℃，20个循环；然后94℃变性1min；55℃退火45s，72℃延伸45s，循环10次；72℃最终延伸10min.用1％琼脂糖电泳检测PCR产物。
经检测，PCR琼脂糖电泳中出现目标基因阳性条带，经DNA回收测序验证，阳性条带为烃降解菌assA基因和产甲烷古菌mcrA基因，可认为该油藏中含烃降解菌产甲烷菌系。
(2)油藏样品微生物群落结构分析
以总DNA为模板，对16S rRNA基因可变区进行PCR扩增，并进行高通量测序。分别选择细菌通用引物(B27F:5’-AGAGTTTGATCMTGGCTCAG-3，B533R:5’-TTACCGCGGCTGCTGGCAC-3’)和古菌通用引物(A344F:5’-ACGGGGYGCAGCAGGCGCGA-3’，A915R(5’-GTGCTCCCCCGCCAATTCCT-3’)来扩增细菌和古菌域微生物。其中细菌和古菌的正反向引物前面分别加上adaptorA和adaptorB，并在引物和adaptor之间连接一个8碱基片段(barcode)用来区分样品。利用Qiime等生物信息学方法对高通量测序数据进行OUT聚类，并进行微生物群落结构分析。如图2油藏样品中细菌和古菌的微生物群落结构所示，该油藏中含有烃降解菌Syntrophaceae和产甲烷菌Methanoculleus和Methanosaeta，可认为该油藏含有烃降解产甲烷菌系。
(3)石油烃降解产甲烷模拟培养
以油藏样品为接种源，石油烃为底物，厌氧培养，利用气相色谱连续监测培养物产甲烷趋势。检测发现，厌氧培养过程中该培养物有持续的甲烷产生，如图3所示，可判定该油藏样品含有烃降解产甲烷菌系，具有降解石油烃产甲烷的潜力。
实施例2
以实施例1中“石油烃降解产甲烷模拟培养”富集物为试验菌系，分别在55℃和45℃培养，构建石油烃菌系1和石油烃菌系2；氢营养型产甲烷菌Methanobacterium sp.CB 12为外源添加产甲烷菌，分析本发明所述方法对石油烃降解产甲烷趋势的作用。
(1)制备产甲烷菌无菌无氧培养基，55℃厌氧培养氢营养型产甲烷菌至对数生长期，厌氧条件下离心收集菌体，重悬至一定浓度，置于厌氧瓶中4℃保存待用。
(2)配置无机盐培养基，以10％的接种量接种石油烃菌系1或石油烃菌系2，实验组1加入十六烷烃、5×107个/ml产甲烷菌和10％的互营菌系，实验组2只加入十六烷烃和10％互营菌系，对照组1只接种10％的互营菌系，对照组2只接种10％的互营菌系和5×107个/ml产甲烷菌。
(3)55℃或45℃厌氧静置培养，连续监测CH4变化趋势。如图4和图5所示，本发明所述方法的培养组与对照组相比具有较高的产甲烷速率和甲烷气量。其中石油烃菌系1试验组，添加甲烷菌的实验组1最大甲烷比产气率是未加甲烷菌的实验组2的1.91倍，长链烷烃转化率(57.85±7.58％)是未加甲烷菌的实验组2转化率(13.41±1.85％)的4.32倍。石油烃菌系2试验组，添加甲烷菌的实验组1最大甲烷比产气率是未加甲烷菌的实验组2的1.41倍，长链烷烃转化率(45.46±5.23％)是未加甲烷菌的实验组2转化率(10.37±0.5％)的4.38倍。
序列表
<110> 农业部沼气科学研究所
<120> 一种促进油藏中石油烃厌氧降解产甲烷的方法
<130> 2017
<160> 5
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ttagcccagt ccctggatgg tccgctggat gatgttgtcc tgggtcttcc ggctgatgtc 60
cacgaagtgg gcgctgtagc cggccacgcg gacaatcagc tcctggtact tctccggctc 120
ccgctgggcc gctcttaagg tctggtcgtc gaccatgttg aactgcacgt ggtcgagtcc 180
taaatcggcc caggtcttta tgtaactctt ccagagctgg tacccctttt cgccctgcag 240
ctgggtgggg gagagacgct ggttcaagag gaaggcccgc ccgtcgctga tgtggttgat 300
cttggcgcag gagcgcaaga cggccgtggg acctttcttg tccaggccgg ggccggggga 360
gatgccgccg tcgtagaggg cgtcgcccag cctccgcccg ttgggcaaag cgccgacgac 420
gttggcgtag tggatgttcc cgctgacgtt ttccggcagg acgggccagg ggttgccgga 480
agggcacctt atctcccagg aatgtttggc gatgtccctt aagcagcgga ccatgatctg 540
gtccacgtag tcgtcgtcgt taccccactt gggcgcgttt ttgacgaagt ccgctgtcga 600
cgttttccac ccaggtaaac caggtgatcc aggcgttccc ccgctcccct tcggggctgg 660
tcgcgtcgat gcccagttcc accgctcttt ccgtaatagc cgaaaggtag gggcggccgt 720
tgaattccgg agcctttgcc cggcccaggt tgacggtgcg gacgaggatg tccatcagcc 780
acttcatctg tttcacccag gcggcgtaga gctcctcgaa gtccttgaac ttccgggcgt 840
cccccgtctg cggccccatc tgcatcttga ccacccggtc gtagccgttg aacagggtgt 900
actcgatgat cttggcgcta ttggcggtcg ccgtggccat ccggtagggc tgggcgccgt 960
gtttggtggt cggggagggg gacatgcagg cctggtgcac ccacgtgcgg gcttcttcca 1020
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gcgggtgcca gcggaaggca aaggtggggt tggatacccg caccagacgg gccgccttca 1200
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gccaggcggt tcacttttaa ttcgtaaaca gttcactttt aaaaaattcc taacagtttt 1080
ttcaacaaat cgataaataa ttttgatcaa aatatacatt cttattgccc tcagatttta 1140
tttgagataa atatatcaat atattgaaca aatcctaatc tgaccgtcgg aggtggagga 1200
tggctattga atttaaagca tcaaagggtc tggctcaaca aatagctgag tatctcggct 1260
ttcgtatcat ccacatggag ttgaagccag gcgaacgact ccttgaggca aaactcgctg 1320
ccgagatggg tgtaagccga gcccctttgc gtgaagcatt gcgtatcctg gaaatgaatg 1380
gccttgtaga gtttgtaccc aggcgtggag tgagggtcag cgagatgacg gagaaaacta 1440
ttctggcaat taacgatgtg gttaaagaag taataggtct gcttgcacag cgtggtgtag 1500
aaaatgcaaa cgcagaagat tatgaaataa tcactcagga agggagagcg ttgcaggaat 1560
atgccgaagc agaagatatt tcggggtacc ttaatgctac actggagttt atccgttctg 1620
cctgtaaagc cactaagaat tctttcctgg agaaggttgt tctctacttc tggcctgtag 1680
ccagccgaat tcaatatgtt tcacgtatct ccaaaaaaga agaattaaaa aagaacgtga 1740
aatacttcga gcgtgcttta aaatacctta atgatgggaa tgctgaaatg acagcccaag 1800
aaatgagaaa cctggtcgaa aatgaagcaa agaatgccat ccaatatgtg cgtgaaaaag 1860
gctatgccaa atagtattca taccattcac tcttcatatc agaagaaact atacggaaat 1920
aagtattatc gggttgttaa ctcgttgttc tttcaaacaa gttctcgttt tgtagtgcgg 1980
cttccttcta attacttaca ggttcatttt gagagagtgt aagaacaagt ggattcgggc 2040
acaagtctgc tcccagccgg gaaattcatc aaacctgcgg cgggctgaca atgccagcgc 2100
cagaagaata tccctatttc gacacaaatg acgaagttgg ctttccaagg cagtcgaatc 2160
acctggtttg accaataaac catttttatt atcttgaata atttcattcc cacctcctgc 2220
ggaagaggca atgggcacaa caccataagc catcgcttcc aggtaaacaa ttccgaaccc 2280
ttcataaaat gaaggcatca ccaaaacatg actgttttgt aaaagcgaaa caatgttgga 2340
atcaggtgtt tctcccagga aaattacccg gctggaaa 2378
<210> 3
<211> 1028
<212> DNA
<213> M82_109
<400> 3
cgcattacta aggaagaggc tttggatctg gtcggtgagt tcctgatcag agctgcagaa 60
gtttcccagt acaaacccaa atgggttcgg gaaggattgc agggtatcga gggaacctgg 120
gtctggacct tgggcggggt caatcagcac ggcgacgatg cctgcaacgg tatgacgatt 180
gctctgctgc aggccgcccg tctggtgaga gtggccaacc cgactttcgc tttccgttgg 240
catcctaaag tcaaggaaga agttctgcgc gaatgtttcg agtgcatccg tcagggtctg 300
ggttatccct ccatgcgcaa cgatcctctt ctgattgcca attccatgta ttggcacgga 360
catcccattg aagaggctcg tacctgggtg catcaggcct gcatgtcacc gtgtcccacg 420
accaagaaag gtttccagcc gatgcgcatg gccaatgcca cggccaactg cgccaagatc 480
atagagtacg tctttaccag cggatttgat cccatagtca acatgcagat cggagcggaa 540
acgccggatg cggcgacctt caccgatttc gagcaggtat acgatgcctg ggttacgcag 600
atgaagacga ttttctccgt catcgtccgt gcggttaatg ccgccagaac aatggctccg 660
gacatgactc ctcggccgtt cttatcggct atttccgagc gctcggtaga aagcggtctt 720
gatgttttta cgccttcaat cagccgcggt aactcctgga tcacggcatt tacctgggtg 780
gaaaacgccg acagtctggc cgccatcaag aaactggtct ttgaagaaaa gaaatatacc 840
atggccgagc tcaagaaggc tctggccgat gattggcagg gtcatgaaga gatgcgtctg 900
gacttcgtga aaaatgcgcc caagtggggc aatgatgacg actacgtaga tagcatcatg 960
ctccgatgcc tgcgcgattg cgcgaaattt tcacacgagc tgaaggatcc gatgggcaac 1020
aactggcc 1028
<210> 4
<211> 2505
<212> DNA
<213> Sulfate-reducing bacterium AK-01 alkylsuccinate synthase gene
<400> 4
atggaaatgg tagcagaacc agcacaggac cagtctgtcc aagaactgga agacaaacag 60
gaatggtggt gggtcgctga aaaaaagcgt tccaaacgtc tggattactt aagaaaatct 120
atctggaaaa aaggcgcttt ggggggcaat tacgccccgg gaattaagct ggatttggaa 180
tgcgcgaccc tttttacgga catgtggaaa ttctggaaat acgacccaat catgatgcgc 240
agagccaagg ccatcgccca tgtcctggac aaaaaaacga tcttcatcac cgaccacgcc 300
caattggtgg gctacttcgg cagcctgccc aacaccatca tgtggcgtgt ggacggcgcg 360
tccatggtca acgaggaagc ctacaacgaa ccgggcatca tgcccgagcc cgaaaatgaa 420
tccctgcaaa aagtggcgga gctcaatgac tattgggcgg gccagacagc cgtggacaag 480
gtggcccgca tcctggaccc cgaagacgcg gtgaaattcc tatccggcgc cattggctgg 540
ggagcgcctt cctccgccta cggatactcc ggcaaggatt atgagtacct gtttgcggga 600
aggcggggat ttgaagacat tatcgaggaa atcaacgcag ccattgaaaa ggccgaggat 660
aaaaccgtgg gcgttcccgg ccctgaaatc ctggatatct acgacaggct tcagaactgg 720
gacgccatga tcctggtcct ggaggcgggc atccgtcacg ccaagcggta tgcaaggctg 780
gcgagaacca tggccgaaaa catggagacg gatgaaaaac ggcgggaaga actgcttaaa 840
atcgccgaaa cctgcgaaag agtgcctgcc agggcgccca ggaatctcca ggaatccctg 900
cagtacgacc acttcatcca gatattcgcc agaacggaag cccacgaagg ggcctggccg 960
gcaaggcccg actattatca cgggccctat tacgacaagg acgtgaacgt cgacaaaacc 1020
ctcaccaagg aagacgcatt ggacctggtg ggcgagttca tgattcgggc ttacgaggtg 1080
ggcggcttcg ccccccggtg ggcgcgggaa ggccttcaag gcattaccgg cacctgggtc 1140
tggaccctgg gaggcgtgaa caaggacggc tcggacgcgt gcaacgatct gaccgtcgcc 1200
tttctccagg cggcccgcct ggtcagggtg tccaatccca cctttgggtt ccggtggcat 1260
cccaaagtca aggacgaagt cctgcgcgaa tgctttgagt gcatccgcca cggcctgggg 1320
tatccgtcca tgcgcaacga ccccttgctt atccagaacg ccatgcactg gcacggccat 1380
cctctggaag aggcgcgcac ctgggtgcat caggcctgca tgagcccctg ccccaccacc 1440
aagcacgggg tgcagcccat gcgcatggcc tcggccaccg ccaactgcgc caagatggtg 1500
gaatacgccc tgcacaacgg ctatgaccac gtggtgggca tgcaaatggg gcctgaaacc 1560
ggggacgccg ccaagttcca tgattttgag gaccttttcc aggcctgggt caaacagatg 1620
gaatggctca ccagcctcct ggtccgcacc gtcaacctgg gcagatacaa ggaccccgag 1680
tttttcggca ggccttttct ctccggcatg agcgaacgca gtgtggaatc cggtctggac 1740
gtggtcagcc cggtggggga tcgcggcaac tgctgggtca cggcattcac ctgggtggag 1800
aacatcgact ccctggccgc ggtcaaaaag ctggtttttg acgataaaaa atacaccatg 1860
gagcagttgc tcaccgccct caaggccaat tgggacggct acgaggaaat gcgcctggac 1920
ttcgtcaaca acgctcccaa atggggcaat gacgacgatt acgtggacga catcatgctt 1980
cgctgcctcc gggagaccgc caggcacagc cgcgtcatga aatgcccctc gggcaacagt 2040
tggcccatcc tgcccgaaaa cgtctccggc aatattcatt acgcctccat cgtgggcgca 2100
ctgcccaacg ggcgccggtg cggggacgcc ctgtacgacg gcggcatttc ccccggccct 2160
ggcctggaca aaaaaggccc ctcggccgtt ttgaaatcct gcggcaaaat cgaccacgtt 2220
tccgacggcc gggcgttcct gctgaaccag cgtctttccc ccacccaatt ggccggggaa 2280
aaaggctatc agctttggaa agcctatatc cgcacctggg ccgacctggg gctggaccac 2340
gtccagttca atatggtttc ggacgaaacc ctgcgggccg ctcaaaagga cccggagaaa 2400
tactccgagg tcatcgtccg ggtggccgga tacagcgccc actttgtgga tatttcccgc 2460
aaaacccagg acaacatcat ccaaagaacc gtccagggca tctaa 2505
<210> 5
<211> 2502
<212> DNA
<213> Sulfate-reducing bacterium AK-01 alkylsuccinate synthase (assA2)gene
<400> 5
atgactgcag aacccgcaaa gttggatgta agtctccaag aacacgaaga gaatcaggaa 60
tggtggtgga tcgcggaaaa aaagcgctcc aaaaggctgg actatttaag gaaggccgtg 120
tggaaaaaag gggctttggg cggcaactac gcccctggaa tcaaagtgga cctggaaggt 180
cccaagctgt ttacggacat gtggaatttt tggaaattcg atcccattat catgcgcagg 240
gccaaagccc tggcccatgt ttttgacaac atttccattt ttataacgga ccattctcaa 300
atcgttggat attggggcag cgccccccat accatcagtt ggcgcgtgga cggcgcgtcc 360
atcgtgaatg aagagctgta caacgaaccc ggcatcatgc ccgagcctga agaggaatcc 420
ctccgcaagg tggccgagat caacgactac tgggccggcc agaccgccgt ggataaagtg 480
gcccgcattc tggacccgga agacgcggtg aagttccttt ccggcgccat cggatggggc 540
gcccccactt cggcttacgg ctattcgggc aagaattacg aatattacct taagggcgag 600
cgcgggtttg aagacatcat cgccgatatt gaagatcaca tcgccgaagc cgaagaaaaa 660
accatcggca cccccggccc ggacatcctg cccatttatg atcgcatcca gaactgggag 720
gccatgatca ccgtgctgga agcggctatc cgttttgcca aacgctacgc gcgcctggcc 780
aggaccatgg ccgagcacct ggaaaccgat gagaagcgca aggaagaact tttgcggatc 840
gccgaaacct gcgagcgggt tccggccaag gcgccccgga atctccagga atccttccag 900
atggacatgc tgatccagac catgtgccgt tttgaagcca gcgaaggcgc ctggcccgcc 960
cggcccgact attaccacgg ccccttttac gaaaaagacg ttctccaaga taaaaccctg 1020
accgaagaag aagccaccga cctcatcggc gagttcatga tccgggctta cgaagtgggc 1080
ggcttcggcc cccgttgggc ccgtgaaggc atgcagggca tcacaggcac ctgggtgtgg 1140
accatcggcg gcgtcaagcc cgacggatcg gacgcatgca acgccctgac caccgccttc 1200
ttgcgcaccg cgcgcctgat ccgggtgtcc aatcccacct tcgccttcag gtggcatccc 1260
aaggtctccg acgaagtcat gcgggagtgc tttgagtgca tccgccacgg cctgggctac 1320
ccctctttcc gccatgatcc cattctggtg gccaattgca tgaactggca cggccatccg 1380
gtggaggaag cccgcacctg ggtgcatcag gcttgcatga gcccgtgccc caccaccaaa 1440
aatggcgttc agcctttccg catggcgtcg gccacagcca attgcgccaa aatggtggaa 1500
tacgctttgc acaacggcta cgaccacgtg gtgggcatgc aaatggggcc ccagaccggc 1560
gacgcccgca cctttacgga ctttgagcag cttttcgacg cctggaccag gcagatgcag 1620
tggctcctga gccttctggt ccgcacggtc aacctgggca ggtataagga cgccgagttt 1680
tacggcaggc ctctcctctc cggcatcacc gaacgggccg ttgaaagggg cattgatgcg 1740
gtcaatccgg aaggcgagcg cggcaattgc tggatcaccg gctttacctg ggtggaaaac 1800
gccgactccc tggccgccgt caaaaagctg gtgttcgacg ataaaaaata caccatggat 1860
cagttgatca cagccctgga atccaactgg gacggctacg agcaaatgcg cctggatttc 1920
gtcaacaaag cgcccaaatg gggcaacgac gacgactatg tggacgacat catgctccgg 1980
tgcctgcgca ccctggccaa gcacagccgg gtcatgcgct gcacctccaa caatacctgg 2040
cccatttcgc cccagaacgt ttccggcaac atccattact cctccgtagt gggcgccctg 2100
cccaacggca gaaggctggg cgacgccctg tacgacggcg gcatctcccc cggaccgggc 2160
ctggataaaa aaggccccac ggccgtgttg aagtcctgcg gcaagatcga ccacgtgggc 2220
gacggcaggg cctttttact gaaccagcgc ctttccccca cccagatggc cggtgagaaa 2280
ggctatcaac tctggcgcgc ctacatgcgc acctgggccg acctgggcct ggaccacatc 2340
caattcaaca tggtctcgga caaaaccctt cgggccgccc agaaagaccc ggaaaaatac 2400
caggaagtga tcgtgcgggt ggcgggctac agcgcccact ttgtggatat ctcccgcaag 2460
acccaggaca acatcatcca gaggacggtc cagggcattt aa 2502
Claims (10)
1.一种促进油藏中石油烃厌氧降解产甲烷的方法，其特征在于，所述方法包括如下步骤：
(1)检测分析油藏中微生物群落结构，以判断其是否含有产甲烷互营烃降解菌系；
(2)当油藏中含有产甲烷互营烃降解菌系时，向油藏中加入添加物A，然后封井，所述添加物A为氢营养型产甲烷古菌或含有氢营养型产甲烷古菌的混合菌群；当油藏中不含产甲烷互营烃降解菌系时，向油藏中加入添加物B，然后封井，所述添加物B为氢营养型产甲烷古菌与互营烃降解菌系的混合菌。
2.根据权利要求1所述的方法，其特征在于，步骤(1)中，在检测分析油藏中微生物群落结构，以判断其是否含有产甲烷互营烃降解菌系时，所用方法包括：
(1)检测油藏采出水、原油或油泥中的中间代谢产物；
(2)采用未培养技术分析油藏中的微生物群落结构；
(3)采用产甲烷互营烃降解菌系具有的功能基因分析油藏中的功能微生物；
(4)对油藏采出水、原油或油泥样品进行模拟培养，监测产甲烷趋势，分析其是否具有降解石油烃产甲烷潜力。
3.根据权利要求2所述的方法，其特征在于，所述油藏中微生物的中间代谢产物包括烷基琥珀酸及其衍生物、苯甲基琥珀酸及其衍生物、苯甲酸衍生物、芳香酸、脂肪酸中的至少一种。
4.根据权利要求2所述的方法，其特征在于，所述未培养技术包括高通量测序、荧光原位杂交、克隆文库、T-RFLP、PCR-DGGE中的任意一种或多种。
5.根据权利要求2所述的方法，其特征在于，所述功能基因包括烷基琥珀酸合成酶α亚基基因assA、苯甲基琥珀酸合成酶α亚基基因bssA、甲基辅酶M还原酶α亚基基因mcrA中的一种或多种。
6.根据权利要求5所述的方法，其特征在于，所述assA的核苷酸序列包括如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5所示的一种或多种。
7.根据权利要求6所述的方法，其特征在于，所述assA的核苷酸序列为如SEQ ID NO：1和SEQ ID NO：2所示。
8.根据权利要求1所述的方法，其特征在于，所述添加物A为氢营养型产甲烷古菌；优选的，所述添加物A的添加量为102～109CFU/ml。
9.根据权利要求1所述的方法，其特征在于，氢营养型产甲烷古菌与产甲烷互营烃降解菌系的混合菌中，氢营养型产甲烷古菌与产甲烷互营烃降解菌系的菌数比为0.01～1000。
10.根据权利要求1所述的方法，其特征在于，所述氢营养型产甲烷古菌包括但不限于Methanobacterium sp.CB12，Methanoculleus receptaculi ZC-2、Methanothermobactercrinale TM2的至少一种。
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