CN111271034A - 一种低级脂肪醇诱导提高生物煤层气产量的方法 - Google Patents
一种低级脂肪醇诱导提高生物煤层气产量的方法 Download PDFInfo
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Abstract
本发明属于煤层气增产技术领域,具体涉及一种低级脂肪醇诱导提高生物煤层气产量的方法。本发明通过采集煤层中产煤层气微生物菌群的异位富集培养,添加低级脂肪醇改变生物成气过程中厌氧发酵液中产甲烷古菌菌群,同时增加了煤层气产量。低级醇作为廉价的有机物,用于微生物生产煤层气具有增产效率高,产物无副作用的优点;特别是乙醇,更是促进生物煤层气生产的效应剂,而且具有绿色、环保的优点。
Description
技术领域
本发明属于煤层气增产技术领域,具体涉及一种低级脂肪醇诱导提高生物煤层气产量的方法。
背景技术
煤层气(CBM)是一种赋存在煤层中的甲烷气体,与燃烧煤相比,燃烧煤层气仅释放一半的CO2,CO和NOx的排放量减少80%。作为一种清洁能源,煤层气已经引起许多国家的关注和重视。我们国家由于天然气含量较少,因而作为天然气的替代能源,煤层气的开采显得尤为重要。过去的三十年累积的地质数据表明,生物成因气是煤层气的重要来源。从煤中生成二次生物成因气,通常发生在温度低于100℃的浅层,这是煤炭煤化后微生物群落活动的结果。但是,生物生成煤层气过程缓慢,导致其应用收到了制约。因而,如何提高微生物产煤层气的效率是目前需要解决的问题。
煤在某些条件下,可以在微生物的降解过程中产生新的甲烷。由于煤是一种非常复杂的杂环大分子,因此需要多种煤层微生物共同作用逐步降解,最后产生煤层气,基于此,目前普遍采用微生物厌氧降解煤的方法,结合产甲烷菌厌氧产甲烷的特性,以实现微生物增产煤层气。如CN201210035682.0公开了一种利用外源微生物增产煤层气的方法,具体为利用外源菌激活煤层中的本源菌,降解煤表面有机质生成甲烷;CN201610710769.1公开了一种利用本源菌提高煤层气产量的方法;CN201710721266.9公开了一种利用本源真菌提高生物煤层气产量的方法。
发明内容
针对上述问题本发明提供了一种低级脂肪醇诱导提高生物煤层气产量的方法。
为了达到上述目的,本发明采用了下列技术方案:
一种低级脂肪醇诱导提高生物煤层气产量的方法,包括以下步骤:
步骤1,采集多口煤层气井的煤粉和煤层出水样,充氮气以隔绝氧气并以铝箔纸包裹备用;
步骤2,将采集的煤粉和煤层出水样加入富集培养基中,驯化,培养,筛选单位质量煤粉产煤层气最高的菌群作为生物煤层气生产的菌源;
步骤3,将步骤2中筛选好的菌群以体积比为10%的比例加入900mL的产煤层气培养基中发酵,在发酵起始,或发酵液中产甲烷古菌含量降低时,加入0.1~2%的低级脂肪醇进行煤层气的生物生产;
步骤4,对采集的煤粉和煤层出水样和步骤2中的菌群,提取微生物DNA,以PCR-DGGE方法进行微生物菌群结构分析。
进一步,所述步骤1中富集培养基以5%的煤粉做唯一的碳源、0.2%的氮源、0.4%的磷和钾以及1%的微量元素溶液构成。
再进一步,所述煤粉为无烟煤、烟煤或褐煤中的任意一种;煤粉的颗粒度为60~160目,且需干燥6个月。
再进一步,所述氮源为有机氮源或无机氮源;
有机氮源为酵母粉、蛋白胨、大豆水解物或玉米浆干粉中的任意一种;
无机氮源为硫酸铵、氯化铵、硝酸铵或磷酸氢胺中的任意一种;
所述磷和钾为磷酸钾、磷酸二氢钾、磷酸氢二钾或硝酸钾中的任意一种。
再进一步,所述微量元素由氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO4 0.1g、NaCl 1.0g、COCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g组成。
进一步,所述步骤3中产煤层气培养基为:1L去离子水中加入酵母抽提物0.5g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g、半胱氨酸3g、0.2%刃天青2mL、微量元素溶液10mL,pH=7.0。
进一步,所述步骤3中产甲烷古菌为Methanosarcina、Methanobacterium或Methanoculleus。
进一步,所述步骤3中产甲烷古菌含量降低为在一个产期周期中,甲烷古菌的含量是先增加后降低,含量降低是与最高含量相比。
进一步,所述步骤3中低级脂肪醇为乙醇、丙醇或丁醇中的任意一种。
进一步,所述步骤4中PCR-DGGE方法为:电泳分析时,引入有物种标准的PCR-DGGE分析法。
与现有技术相比本发明具有以下优点:
煤地质微生物处在一种较为恶劣的环境下,呈现一种不活泼的状态,需要受到某些外界刺激,才能更好的进行代谢活动。与利用煤层本源菌直接产甲烷相比,通过对微生物刺激、增加煤溶解度和生物利用率等方面增产煤层气,特别是添加某些有机小分子物质刺激微生物复苏和生长,将会更有效地提高煤的降解率,促进生物甲烷的生成,显著提高生物煤层气的产量。
低级醇作为廉价的有机物,用于微生物生产煤层气具有增产效率高,产物无副作用的优点;特别是乙醇,更是促进生物煤层气生产的效应剂,而且具有绿色、环保的优点。
附图说明
图1本发明实验条件下乙醇诱导煤层微生物产煤层气的情况图。
具体实施方式
实施例1
在厌氧手套箱中,将50mL煤层产出水样和2g煤样加入到500mL厌氧瓶中,加入200mL的灭菌富集培养基,再加入终浓度为0.04%的除氧L-半胱氨酸及20g褐煤煤粉,轻轻混匀,迅速密封厌氧瓶,并于25℃条件下静置培养。期间每周用无菌注射器从厌氧瓶瓶顶插入进行气体的自动收集,收集到的气体取0.5mL进行气相色谱分析,并计算甲烷生成的相对含量。(富集培养筛选菌群)
富集培养基:
1L去离子水中加入酵母抽提物2g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl20.4g、半胱氨酸3g、刃天青(0.2%)2mL、微量元素溶液10mL,pH=7.0,维生素溶液5ml。
微量元素溶液配方为:氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO4 0.1g、NaCl 1.0g、CoCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g。
1L维生素溶液包括:生物素2mg,叶酸2mg,B6 10mg,B2 5mg,B1 5mg,烟酸5mg,B120.1mg,硫辛酸5mg,对氨基苯甲酸5mg。
实施例2
向1L厌氧瓶中加入25g无烟煤煤粉、350mL产煤层气培养基及0.408mg刃天青,1×105Pa灭菌30min。在厌氧手套箱内向1L厌氧瓶中加入800μL无菌的20%L-半胱氨酸及乙醇(浓度分别为0,0.5%,1%或2%),然后向厌氧瓶中持续通入高纯N2,至培养基颜色近无色。在厌氧手套箱中,在上述1L厌氧瓶中(含无烟煤)接入实施例1培养液50mL。密封各产气实验用厌氧瓶,于厌氧箱中静置培养1~3个月,期间每周从厌氧瓶瓶顶插入无菌注射器进行气体的自动收集,收集到的气体取0.5mL进行气相色谱分析,并计算甲烷生成的相对含量。同时取5~10mL培养液进行菌群定性和定量分析。如图1乙醇诱导煤层微生物产煤层气的情况图所示。
产煤层气培养基:
1L去离子水中加入酵母抽提物0.5g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl1.8g、MgCl20.4g、半胱氨酸3g、刃天青(0.2%)2mL、微量元素溶液10mL,pH=7.0。
微量元素溶液配方为:氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO4 0.1g、NaCl 1.0g、CoCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g。
实施例3
向1L厌氧瓶中加入20g褐煤煤粉、350mL产煤层气培养基及0.408mg刃天青,1×105Pa灭菌30min。在厌氧手套箱内向1L厌氧瓶中加入800μL无菌的20%L-半胱氨酸及丙醇(浓度分别为0,0.5%,1%或1.5%),然后向厌氧瓶中持续通入高纯N2,至培养基颜色近无色。在厌氧手套箱中,在上述1L厌氧瓶中(含褐煤)接入实施例1培养液50mL。密封各产气实验用厌氧瓶,于厌氧箱中静置培养1~3个月,期间每周从厌氧瓶瓶顶插入无菌注射器进行气体的自动收集,收集到的气体取0.5mL进行气相色谱分析,并计算甲烷生成的相对含量。同时取5~10mL培养液进行菌群定性和定量分析。
产煤层气培养基:
1L去离子水中加入酵母抽提物0.5g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl1.8g、MgCl20.4g、半胱氨酸3g、刃天青(0.2%)2mL、微量元素溶液10mL,pH=7.0。
微量元素溶液配方为:氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO4 0.1g、NaCl 1.0g、CoCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g。
实施例4
向1L厌氧瓶中加入25g烟煤煤粉、350mL产煤层气培养基及0.408mg刃天青,1×105Pa灭菌30min。在厌氧手套箱内向1L厌氧瓶中加入800μL无菌的20%L-半胱氨酸及丁醇(浓度分别为0,0.5%,1%或1.5%),然后向厌氧瓶中持续通入高纯N2,至培养基颜色近无色。在厌氧手套箱中,在上述1L厌氧瓶中(含烟煤)接入实施例1培养液50mL。密封各产气实验用厌氧瓶,于厌氧箱中静置培养1~3个月,期间每周从厌氧瓶瓶顶插入无菌注射器进行气体的自动收集,收集到的气体取0.5mL进行气相色谱分析,并计算甲烷生成的相对含量。同时取5~10mL培养液进行菌群定性和定量分析。
产煤层气培养基:
1L去离子水中加入酵母抽提物0.5g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl1.8g、MgCl20.4g、半胱氨酸3g、刃天青(0.2%)2mL、微量元素溶液10mL,pH=7.0。
微量元素溶液配方为:氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO4 0.1g、NaCl 1.0g、CoCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g。
实施例5
细菌和古菌的16s rRNA基因PCR扩增选用引物分别为BAC-338F(加GC夹)/BAC-518R和AR-344F(加GC夹)/AR-519R。
PCR反应体系:95℃预变性5min,95℃30s,55℃30s,72℃30s,30个循环,72℃10min,10℃保存。
细菌和古菌的DGGE分析条件相同。DGGE凝胶为浓度是10%(v/v)的聚丙烯酰胺胶,胶的变性梯度范围是40%~60%。
电泳条件是在恒温60℃、电压85V下运行13h。电泳结束后,取下凝胶,在4S redplus nucleic acid stain中染色15min,然后放入纯水中,脱色摇床脱色10min。凝胶影像分析系统下观察并拍照,得到DGGE图谱。DGGE图谱结果分析采用Quantity One软件。
物种定性分析:样品经PCR扩增后,产物与本发明的煤层微生物物种DGGEMarker(CN 2017103488119.0,CN201710349248.2)同时上样于聚丙烯酰胺变性胶,电泳后成像,与Marker处于同一位置的样品DNA片段代表一种与Marker相同的种属。
上述内容对实施例做了详细的说明,但本发明不受上述实施方式和实施例的限制,在不脱离本发明宗旨的前提下,在本领域技术人员所具备的知识范围内还可以对其进行各种变化和改进,这些变化和改进均落入本发明要保护的范围之内。
Claims (10)
1.一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:包括以下步骤:
步骤1,采集多口煤层气井的煤粉和煤层出水样,充氮气以隔绝氧气并以铝箔纸包裹备用;
步骤2,将采集的煤粉和煤层出水样加入富集培养基中,驯化,培养,筛选单位质量煤粉产煤层气最高的菌群作为生物煤层气生产的菌源;
步骤3,将步骤2中筛选好的菌群以体积比为10%的比例加入900mL的产煤层气培养基中发酵,在发酵起始,或发酵液中产甲烷古菌含量降低时,加入0.1~2%的低级脂肪醇进行煤层气的生物生产;
步骤4,对采集的煤粉和煤层出水样和步骤2中的菌群,提取微生物DNA,以PCR-DGGE方法进行微生物菌群结构分析。
2.根据权利要求1所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述步骤1中富集培养基以5%的煤粉做唯一的碳源、0.2%的氮源、0.4%的磷和钾以及1%的微量元素溶液构成。
3.根据权利要求2所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述煤粉为无烟煤、烟煤或褐煤中的任意一种;煤粉的颗粒度为60~160目,且需干燥6个月。
4.根据权利要求2所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述氮源为有机氮源或无机氮源;
有机氮源为酵母粉、蛋白胨、大豆水解物或玉米浆干粉中的任意一种;
无机氮源为硫酸铵、氯化铵、硝酸铵或磷酸氢胺中的任意一种;
所述磷和钾为磷酸钾、磷酸二氢钾、磷酸氢二钾或硝酸钾中的任意一种。
5.根据权利要求2所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述微量元素由氮三乙酸1.5g、CaCl2 0.1g、MgSO4·7H2O 3.0g、H3BO3 0.05g、FeSO40.1g、NaCl 1.0g、CoCl2 0.1g、MnSO4 0.5g、ZnSO4 0.1g、NaMO4 0.05g、A1K(SO4)2 0.01g、NiCl2 0.1g、CuSO4 0.01g组成。
6.根据权利要求1所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述步骤3中产煤层气培养基为:1L去离子水中加入酵母抽提物0.5g、K2HPO4 2.9g、KH2PO4 1.5g、NH4Cl 1.8g、MgCl2 0.4g、半胱氨酸3g、0.2%刃天青2mL、微量元素溶液10mL,pH=7.0。
7.根据权利要求1所述的一种补料发酵提高生物煤层气产量的方法,其特征在于:所述步骤3中产甲烷古菌为Methanosarcina、Methanobacterium或Methanoculleus。
8.根据权利要求1所述的一种补料发酵提高生物煤层气产量的方法,其特征在于:所述步骤3中产甲烷古菌含量降低为在一个产期周期中,甲烷古菌的含量是先增加后降低,含量降低是与最高含量相比。
9.根据权利要求1所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述步骤3中低级脂肪醇为乙醇、丙醇或丁醇中的任意一种。
10.根据权利要求1所述的一种低级脂肪醇诱导提高生物煤层气产量的方法,其特征在于:所述步骤4中PCR-DGGE方法为:电泳分析时,引入有物种标准的PCR-DGGE分析法。
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