微生物技术治理煤层瓦斯理论及应用研究
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摘要
随着这些年我国采掘速度加快，采深加大，导致地应力不断增加，煤层透气性系数不断降低，瓦斯抽采、排放越来越困难，以至于更容易出现抽采“空白带”，在单位时间内瓦斯排放呈现更加不充分等问题，加之抽放时间长、效率低，浪费大量人力物力而效果并不明显，严重制约着煤矿的安全生产。尤其是重庆地区，近年来瓦斯含量和压力不断增加，突出事故的规模不断加大，原来以瓦斯为主的突出逐渐转变为煤与瓦斯共同、大规模突出，矿井现有以预抽为主要手段的措施到目前已出现抽放难。研究表明瓦斯突出呈现这些特征的根本原因是渗透率极低。通过对其煤体孔和裂隙的测试分析得知，重庆地区煤层小孔多，大孔少；总孔体积大，有效连通孔体积小，这有利于瓦斯储集而不利于其流动。传统对瓦斯主要采取避、驱、排等策略的瓦斯治理方法，希望通过瓦斯抽排实现安全采煤的目标，但对于上述情况其效果并不理想。同时，矿井低浓度瓦斯提纯技术成本也相当高昂，所需要耗费的能量及成本可能是提纯之后瓦斯所产生效益所无法补偿的，目前国内外大部分地区都将低浓度煤矿瓦斯直接排放进入大气中，对生态环境造成恶劣影响。
在此背景下，本文从微生物的角度出发，跳出传统的瓦斯治理思路，研究微生物方法处理甲烷的作用机理及工程应用，利用甲烷氧化菌处理甲烷，解决煤矿瓦斯抽采难问题，消除煤矿预抽后的低浓度或残余瓦斯，力争避免矿井瓦斯爆炸、窒息等事故的发生，减少甲烷气体的直接排放，降低甲烷对臭氧层的破坏。
本文研究了甲烷氧化菌的筛选、分离纯化、鉴定、基因解析及大规模培养的最适合生长条件，并对其氧化甲烷的性能进行了验证，最后用该菌进行了地面可行性实验及井下现场试验，结果表明微生物能够降解煤层瓦斯。主要结论如下：
（1）分析了甲烷氧化菌的多样性及其氧化甲烷的机理，并分别阐述了其氧化过程中四个主要特征酶的特点及作用。
（2）通过采集五种土样并在NMS培养基中培养观察，发现水稻田的淹水环境中甲烷氧化菌降解甲烷效率最高，氧化效果最好。经分离纯化后得到菌落M02-019。
（3）通过革兰氏染色观察、16S rDNA的PCR扩增及其序列分析等实验，确定菌株M02-019与Methylophilus位于同一分支，相似度较高，故M02-019属于Methylophilus属，即嗜甲基菌属。
（4）设计了阴性对照实验、物质守恒实验及小型放大实验，三个实验均显示了该菌具有降解甲烷的活性。
（5）对M02-019菌株大规模培养的生长条件进行了研究，实验结果表明：该菌株的生长周期为12天，从接种的第三天开始进入对数生长期；甲烷和甲醇同时作为碳源时菌株生长最好，且不易染菌；菌株在硝基氮和少量的氨基氮同时存在时长势较好；最适生长温度为30℃，最适pH值为6.5；Fe~(2+)浓度为0.4mg/L，Cu~(2+)浓度为0.03mg/L。
（6）对重庆地区煤层的特征进行具体分析，包括煤层突出的宏、微观特征及煤质与孔隙特征。在对其煤化学成分、煤体孔径结构、孔隙形状、煤体渗透性做数据测试后，分析认为煤体存在压敏效应和水敏效应，通过分析煤层孔和裂隙的“双敏效应”与低渗透的关系，提出微生物技术，即在水力扩孔的基础上，将注水改为注入甲烷氧化菌溶液，不断氧化甲烷、降低瓦斯浓度，既能有效卸除应力，又能缓解了水敏效应带来的孔道阻塞问题。为了解煤层注入微生物溶液的可行性，实施了煤层注液研究，结果表明注液量理论上随注液压力及时间的递增而增加，但考虑到煤层底板被压穿后底板泥岩遇水后泥浆化反而会造成孔道堵塞，故采用微生物技术注液时不宜选择太大的注液压力，而宜采用中低压长时间注液，以保证溶液较大程度的渗入煤体。
（7）在井下工程试验前先用HCA-1型高压容量法瓦斯吸附成套装置等在地面做了注水和注微生物培养液对比可行性试验，证明了微生物培养液处理矿井瓦斯具有其可行性，可进一步在井下进行微生物处理瓦斯实验。通过井下实验观测与分析，甲烷氧化菌能够不同程度地降低两个实验地点的瓦斯动力现象、回风瓦斯浓度、吨煤瓦斯含量、煤层瓦斯压力和钻屑瓦斯解析指标K1值。两个实验地点喷孔距离明显减小，回风瓦斯浓度分别降低了22.54%和77.23%，吨煤瓦斯含量分别降低了39.67%和13.45%，平均瓦斯压力降幅约76%和18%，钻屑瓦斯解析指标K_1值平均降幅分别为62.8%和26.88%。
（8）无论是地面可行性试验还是井下现场实验，其效果均较为明显，表明微生物方法处理煤层瓦斯是可行的，表明该方法具有应用于工业的潜在价值。
As the depth of mine excavation goes further, the ground stress is continuouslyincreasing but the permeability coefficient of coal seam reduce. It leads to the difficultyof gas extraction or emission. More time, manpower or resources are needed todischarge gas and the effect is not very obvious.Therefore, mining conditions are moreand more complicated and the coal and gas outburst accident become more and morefrequent. Especially Chongqing area, the gas content and pressure is on the increasethese years, the outburst scale continuously aggravate and coal and gas extensiveoutburst replace the former basically gas outburst. The major means of pre-draining ishard to meet the current state, the long-term drainage remains below standards. The rootof these phenomena is the low permeability.Though tests of coal hole and fracture, thereare mainly micropore compared with macropore in Chongqing area. Total volume of thepores is large while the volume of interconnected pores is small, and the coals wouldmanifest pressure and water sensibility, all these are positive for storage of gas butnegative for moving of gas.Traditional methods of gas control mainly which base onavoiding, driving, elimination are not very ideal for realizing the goal of safe mining.Meanwhile, the cost of gas purification for low concentration gas is so high that theproduce benefit after purification is far below the cost of energy and fund demanded. Somost of the mines at home and abroad discharge the low concentration coal mine gasinto the atmosphere directly, that would cause bad influence on the ecologicalenvironment.
From the view of microorganisms, this paper jumps out of the traditional gasmanagement ideas and studys on making use of microbial technology to deal with coalseam gas. By the using of methane oxidation bacteria, the gas could be controlledsimplely and safely, and the coat will be low,too. This technology will reduce the directdischarging of gas and the amount of accidents of gas explosion or asphyxia.
This paper studys the enrichment, isolation, identification, gene analysis andsuitable growth conditions of methanotrophs, and its property of the oxidation ofmethane are verified through laboratory experiences. Then the feasibility test on theground and field experiment underground were conducted. The results of theexperiments showed that degrading coal seam gas by methanotrophs was rational.
(1) Analyzed the diversity and mechanism of methane oxidation of methanotrophs, and introduced the characteristics and functions of the four main enzymes.
(2) Collected five soil samples and observed in NMS culture medium, and foundthat methanotrophs collected form the water environment of paddy field was of thehighest efficiency of methane degradation and enrichmen. The highestone kind ofmethanotrophs was named M02-019.
(3) The gram observation,16S rDNA of PCR amplification and sequence analysisexperiments showed M02belonged to the fungus of degradation or decomposition ofmethane, and made sure that the strain M02-019is in the same branch ofMethylophilus.
(4) Negative control experiment, experiment of conservation of mass and smallamplification experiment were conducted, and the results showed that M02was of havehigh activity of degrading methane.
(5) Studied the growth conditions of mass culture. The experimental resultsshowed that the growth cycle for this bacterial strain was12days, and its growthentered the logarithmic phase from the third day after inoculation. Using methane andmethanol together as the carbon source of strain growth was best, and it was not easy todye bacteria. It would grow better when using nitrocellulose nitrogen and a smallamount of amino nitrogen together as the nitrogen source of strain. The optimumgrowth temperature for M02-019was30℃and optimum PH was6.5, Fe~(2+)concentration was0.4mg/L and Cu~(2+)concentration was0.03mg/L.
(6) Analyzed the main reason for the gas outburst of the coal seam, and thechemical composition, aperture structure, pore configuration and the permeability of thecoal mass were tested. The results showed that both pressure-sensitive effect and watersensitivity effect exist in the coal seam. After analyzing the relationship between the lowpermeability and them, the methods were put forward in order to increase thepermeability. The study of injecting fluid into the coal seam indicated that the way ofinjection was feasible and the performance of injection depended on the pressure andtime of injection, as well as the wettability of coal seam.
(7) Before field experiment underground, the feasibility test on the ground wasconducted through HCA-1type device of high pressure capacity method for gasadsorption. By the comparison of the effect of injecting water and microbial culturesolution, the feasibility of gas control by microbial culture solution was testified, andfurther experiment underground could be conducted. Through the observation andanalysis of underground experiment, the methanotrophs remarkably weakened the dynamic phenomenon of gas and reduced the gas concentration of return air, gas content,gas pressure and the index of K1of the two experimental sites in different degree. Thegas concentration of return air of the two experimental sites respectively reduced by22.54%and77.23%, the gas content reduced by39.67%and13.45%, the gas pressurereduced by76%and18%, and the index of K1reduced by62.8%and26.88%.
(8) Whether the feasibility test on the ground or field experiment underground, theeffect were obvious. Meantime, this microbiological method is simple and practicablefor gas control; therefore this technique is of the great industrial value.
在此背景下，本文从微生物的角度出发，跳出传统的瓦斯治理思路，研究微生物方法处理甲烷的作用机理及工程应用，利用甲烷氧化菌处理甲烷，解决煤矿瓦斯抽采难问题，消除煤矿预抽后的低浓度或残余瓦斯，力争避免矿井瓦斯爆炸、窒息等事故的发生，减少甲烷气体的直接排放，降低甲烷对臭氧层的破坏。
本文研究了甲烷氧化菌的筛选、分离纯化、鉴定、基因解析及大规模培养的最适合生长条件，并对其氧化甲烷的性能进行了验证，最后用该菌进行了地面可行性实验及井下现场试验，结果表明微生物能够降解煤层瓦斯。主要结论如下：
（1）分析了甲烷氧化菌的多样性及其氧化甲烷的机理，并分别阐述了其氧化过程中四个主要特征酶的特点及作用。
（2）通过采集五种土样并在NMS培养基中培养观察，发现水稻田的淹水环境中甲烷氧化菌降解甲烷效率最高，氧化效果最好。经分离纯化后得到菌落M02-019。
（3）通过革兰氏染色观察、16S rDNA的PCR扩增及其序列分析等实验，确定菌株M02-019与Methylophilus位于同一分支，相似度较高，故M02-019属于Methylophilus属，即嗜甲基菌属。
（4）设计了阴性对照实验、物质守恒实验及小型放大实验，三个实验均显示了该菌具有降解甲烷的活性。
（5）对M02-019菌株大规模培养的生长条件进行了研究，实验结果表明：该菌株的生长周期为12天，从接种的第三天开始进入对数生长期；甲烷和甲醇同时作为碳源时菌株生长最好，且不易染菌；菌株在硝基氮和少量的氨基氮同时存在时长势较好；最适生长温度为30℃，最适pH值为6.5；Fe~(2+)浓度为0.4mg/L，Cu~(2+)浓度为0.03mg/L。
（6）对重庆地区煤层的特征进行具体分析，包括煤层突出的宏、微观特征及煤质与孔隙特征。在对其煤化学成分、煤体孔径结构、孔隙形状、煤体渗透性做数据测试后，分析认为煤体存在压敏效应和水敏效应，通过分析煤层孔和裂隙的“双敏效应”与低渗透的关系，提出微生物技术，即在水力扩孔的基础上，将注水改为注入甲烷氧化菌溶液，不断氧化甲烷、降低瓦斯浓度，既能有效卸除应力，又能缓解了水敏效应带来的孔道阻塞问题。为了解煤层注入微生物溶液的可行性，实施了煤层注液研究，结果表明注液量理论上随注液压力及时间的递增而增加，但考虑到煤层底板被压穿后底板泥岩遇水后泥浆化反而会造成孔道堵塞，故采用微生物技术注液时不宜选择太大的注液压力，而宜采用中低压长时间注液，以保证溶液较大程度的渗入煤体。
（7）在井下工程试验前先用HCA-1型高压容量法瓦斯吸附成套装置等在地面做了注水和注微生物培养液对比可行性试验，证明了微生物培养液处理矿井瓦斯具有其可行性，可进一步在井下进行微生物处理瓦斯实验。通过井下实验观测与分析，甲烷氧化菌能够不同程度地降低两个实验地点的瓦斯动力现象、回风瓦斯浓度、吨煤瓦斯含量、煤层瓦斯压力和钻屑瓦斯解析指标K1值。两个实验地点喷孔距离明显减小，回风瓦斯浓度分别降低了22.54%和77.23%，吨煤瓦斯含量分别降低了39.67%和13.45%，平均瓦斯压力降幅约76%和18%，钻屑瓦斯解析指标K_1值平均降幅分别为62.8%和26.88%。
（8）无论是地面可行性试验还是井下现场实验，其效果均较为明显，表明微生物方法处理煤层瓦斯是可行的，表明该方法具有应用于工业的潜在价值。
As the depth of mine excavation goes further, the ground stress is continuouslyincreasing but the permeability coefficient of coal seam reduce. It leads to the difficultyof gas extraction or emission. More time, manpower or resources are needed todischarge gas and the effect is not very obvious.Therefore, mining conditions are moreand more complicated and the coal and gas outburst accident become more and morefrequent. Especially Chongqing area, the gas content and pressure is on the increasethese years, the outburst scale continuously aggravate and coal and gas extensiveoutburst replace the former basically gas outburst. The major means of pre-draining ishard to meet the current state, the long-term drainage remains below standards. The rootof these phenomena is the low permeability.Though tests of coal hole and fracture, thereare mainly micropore compared with macropore in Chongqing area. Total volume of thepores is large while the volume of interconnected pores is small, and the coals wouldmanifest pressure and water sensibility, all these are positive for storage of gas butnegative for moving of gas.Traditional methods of gas control mainly which base onavoiding, driving, elimination are not very ideal for realizing the goal of safe mining.Meanwhile, the cost of gas purification for low concentration gas is so high that theproduce benefit after purification is far below the cost of energy and fund demanded. Somost of the mines at home and abroad discharge the low concentration coal mine gasinto the atmosphere directly, that would cause bad influence on the ecologicalenvironment.
From the view of microorganisms, this paper jumps out of the traditional gasmanagement ideas and studys on making use of microbial technology to deal with coalseam gas. By the using of methane oxidation bacteria, the gas could be controlledsimplely and safely, and the coat will be low,too. This technology will reduce the directdischarging of gas and the amount of accidents of gas explosion or asphyxia.
This paper studys the enrichment, isolation, identification, gene analysis andsuitable growth conditions of methanotrophs, and its property of the oxidation ofmethane are verified through laboratory experiences. Then the feasibility test on theground and field experiment underground were conducted. The results of theexperiments showed that degrading coal seam gas by methanotrophs was rational.
(1) Analyzed the diversity and mechanism of methane oxidation of methanotrophs, and introduced the characteristics and functions of the four main enzymes.
(2) Collected five soil samples and observed in NMS culture medium, and foundthat methanotrophs collected form the water environment of paddy field was of thehighest efficiency of methane degradation and enrichmen. The highestone kind ofmethanotrophs was named M02-019.
(3) The gram observation,16S rDNA of PCR amplification and sequence analysisexperiments showed M02belonged to the fungus of degradation or decomposition ofmethane, and made sure that the strain M02-019is in the same branch ofMethylophilus.
(4) Negative control experiment, experiment of conservation of mass and smallamplification experiment were conducted, and the results showed that M02was of havehigh activity of degrading methane.
(5) Studied the growth conditions of mass culture. The experimental resultsshowed that the growth cycle for this bacterial strain was12days, and its growthentered the logarithmic phase from the third day after inoculation. Using methane andmethanol together as the carbon source of strain growth was best, and it was not easy todye bacteria. It would grow better when using nitrocellulose nitrogen and a smallamount of amino nitrogen together as the nitrogen source of strain. The optimumgrowth temperature for M02-019was30℃and optimum PH was6.5, Fe~(2+)concentration was0.4mg/L and Cu~(2+)concentration was0.03mg/L.
(6) Analyzed the main reason for the gas outburst of the coal seam, and thechemical composition, aperture structure, pore configuration and the permeability of thecoal mass were tested. The results showed that both pressure-sensitive effect and watersensitivity effect exist in the coal seam. After analyzing the relationship between the lowpermeability and them, the methods were put forward in order to increase thepermeability. The study of injecting fluid into the coal seam indicated that the way ofinjection was feasible and the performance of injection depended on the pressure andtime of injection, as well as the wettability of coal seam.
(7) Before field experiment underground, the feasibility test on the ground wasconducted through HCA-1type device of high pressure capacity method for gasadsorption. By the comparison of the effect of injecting water and microbial culturesolution, the feasibility of gas control by microbial culture solution was testified, andfurther experiment underground could be conducted. Through the observation andanalysis of underground experiment, the methanotrophs remarkably weakened the dynamic phenomenon of gas and reduced the gas concentration of return air, gas content,gas pressure and the index of K1of the two experimental sites in different degree. Thegas concentration of return air of the two experimental sites respectively reduced by22.54%and77.23%, the gas content reduced by39.67%and13.45%, the gas pressurereduced by76%and18%, and the index of K1reduced by62.8%and26.88%.
(8) Whether the feasibility test on the ground or field experiment underground, theeffect were obvious. Meantime, this microbiological method is simple and practicablefor gas control; therefore this technique is of the great industrial value.
引文
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