耐低温产甲烷菌的分离及其产气活性的初探

在青藏高原地区的低温条件下通过滚管技术分离了一类耐低温产甲烷菌,并利用气相色谱法测定了产气活性。结果表明:这类甲烷菌最低产气温度为8℃,甲烷气体产生的高峰期在厌氧培养的第7 d;pH值与盐浓度对其产气活性均有影响,最佳条件为pH值7.0、盐度4%。     

不同水稻土甲烷氧化菌和产甲烷菌数量与甲烷排放量之间相关性的研究

对不施用任何肥料的浙江黄松土（发育于河流沉积物母质的水稻田土）、老黄筋泥田土（发育于第四纪红壤母质的水稻田土）、青紫泥田土（发育于滨海盐土母质的象山青紫泥水稻土）中甲烷氧化菌、产甲烷菌种群数量及甲烷排放量之间关系作了研究。结果表明,甲烷氧化菌种群数量范围在10^6-10^8cfu/g干土之间,其变化在2个数量级范围内,产甲烷细菌种群数量变化较大,其范围在10^3-10^7cfu/g干土之间,水稻田土壤的甲烷排放量受到甲烷氧化菌、产甲烷菌种群数量及其活性及土壤理化性状的影响,提出了当水稻田土壤的甲烷氧化菌种群数量在10^cfu/g干土、产甲烷菌种群数量在10^5cfu/g干土时,水稻田土壤几乎没有多余的CH4气体排放这一甲烷形成与甲烷氧化两类群微生物之间的数量关系。甲烷排量也与土壤砂粒（＜0．02mm的砂数）含量呈正相关性,土壤砂粒含量越高,其甲烷排放量亦高。     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性,而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法,包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR）,重点分析了分子检测中两类重要的标记基因,总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果,提出了我国在该领域今后应深入研究探讨的一些问题及建议。     

越冬沼气池中甲烷螺菌的分离及其系统分类学分析

通过改进的亨盖特(Hungate)厌氧技术,从越冬猪粪发酵沼气池中分离到1株产甲烷菌菌株SH01。该菌呈弯曲杆状,革兰氏阴性,有运动性,形成淡黄色菌落,能利用二氧化碳和甲酸钠作为碳源,但不能利用甲醇、乙酸钠和三甲胺。该菌最适生长pH为6.8～7.2,最适生长温度为35～40℃,最适Na 浓度低于0.1mol/L。通过生理、形态结构特征与16S rDNA序列的同源性分析,表明菌株SH01是甲烷螺菌属中的一个成员,为亨氏甲烷螺菌(Methanospirllum hungatei)。     

增温对冻土区泥炭沼泽土壤孔隙水甲烷关联微生物和溶解性有机碳的影响

多年冻土区泥炭沼泽土壤孔隙水甲烷关联微生物及底物的研究有助于深入理解气候变化背景下寒区湿地生态系统甲烷循环过程。选取大兴安岭连续多年冻土区柴桦-泥炭藓和狭叶杜香-泥炭藓两种典型植被群落泥炭沼泽,设置开顶箱(Open Top Chamber,OTC)增温实验。于生长季(6月、7月、8月和9月)采集土壤孔隙水样品,对比分析OTC内外土壤孔隙水中产甲烷菌数量、甲烷氧化菌数量及溶解性有机碳(Dissolved Organic Carbon,DOC)浓度的动态变化特征,并探究土壤孔隙水甲烷关联微生物与DOC浓度的关系。结果表明:增温提高了生长季大兴安岭多年冻土区土壤孔隙水中产甲烷菌数量和DOC浓度,而对甲烷氧化菌数量的影响因月份而异。生长季柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中产甲烷菌数量的平均增加幅度分别为54.52%和44.97%,DOC浓度的平均增加幅度分别为34.16%和28.33%。增温使得生长季柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中甲烷氧化菌平均数量分别降低了46.20%和31.42%。一元线性回归分析结果表明,土壤孔隙水中DOC浓度可分别解释柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中产甲烷菌数量变化的29.00%和24.10%(P<0.01),而对两种植被群落下土壤孔隙水中甲烷氧化菌数量的影响并不显著(P>0.05)。     

污泥厌氧消化产酸发酵过程中乙酸累积机制

[目的]研究污泥厌氧消化产挥发性脂肪酸(VFA)过程中的有机物碳流的转化机制,阐明乙酸累积机理。[方法]研究溴乙烷磺酸盐(BES)和氯仿(CHCl3)抑制模型下中间代谢产物和气体的累积,检测各产乙酸功能菌群数量,推断污泥产酸发酵过程中的有机物碳流方向和乙酸累积机理。[结果]BES模型乙酸浓度达27 mmol/L,fhs基因拷贝数比对照组高2-3倍,产氢产乙酸菌略有下降。CHCl3模型乙酸浓度达22 mmol/L,fhs基因拷贝数比BES组低一个数量级,产氢产乙酸菌下降明显。[结论]BES特异性较高,除产甲烷菌外对其他厌氧产酸细菌没有影响,乙酸浓度增加并且其主要来源于水解发酵产酸以及同型产乙酸过程。氯仿除抑制产甲烷菌外,对同型乙酸菌和产氢产乙酸菌也有强烈的抑制作用。     

互花米草入侵对滩涂湿地甲烷排放的影响

在浙江省台州市附近滩涂湿地设置3个不同互花米草入侵密度梯度,即仅有本土植物样地、互花米草与本土植物混生样地和互花米草单优群落样地,研究互花米草入侵对滩涂湿地CH₄排放的影响.结果表明: 3个样地CH₄排放通量为0.68～5.88 mg·m^-2·h^-1,CH₄排放通量随着互花米草入侵梯度的增加而显著升高,互花米草单优群落样地CH₄排放通量分别为本土植物样地和混生样地的8.7和2.3倍.互花米草入侵显著提高了产甲烷菌数量、产甲烷潜力、甲烷氧化菌数量、甲烷氧化潜力、植物生物量、土壤有机碳含量和土壤pH,降低了土壤全氮含量.CH₄排放通量与土壤全氮呈显著负相关,与产甲烷菌数量、产甲烷潜力、甲烷氧化菌数量、甲烷氧化潜力、植物生物量和土壤pH呈显著正相关.互花米草的入侵提高了滩涂湿地植物群落生物量和土壤pH,促进了产甲烷菌数量和产甲烷潜力,从而提高了滩涂湿地的CH₄排放.     

一株高产细胞表面植酸酶酵母的实验研究

对一株高产细胞表面植酸酶酵母突变株WZ4菌的细胞植酸酶进行了研究。探讨了菌体生长与产酶的关系。结果表明:菌体在培养的前期植酸酶酶活很低,培养30h后酶活迅速增加,酶活在菌体生长的平衡期达到最大,该菌产植酸酶为非生长偶联型。在此基础了解WZ4菌细胞植酸酶的性质,实验表明:该酶的最适pH为5,最适温度为50℃,Km(以植酸钠为底物)为0666mmolL。通过磷对产酶影响因素的实验研究,初步得到WZ4菌产植酸酶受控于培养基中的磷浓度的结论,即最大产酶磷浓度为05mg100ml,当磷浓度大于10mg100ml时产酶被完全阻遏。     

内切中性纤维素酶EGⅡ的发酵条件优化

对毕赤酵母基因工程菌EIM-50-eg2产内切中性纤维素酶的主要影响因子进行研究,考察氮源、pH、温度、微量元素PTM1和甲醇浓度等对工程菌产酶的影响。单因素实验和正交实验结果表明,优化后的培养基组成及培养条件:磷酸氢二铵40 g/L,甲醇15 mL/L,硫酸镁10 g/L,磷酸二氢钾9 g/L,初始pH 6.0,培养温度28℃,PTM1添加量0.02%,甲醇诱导浓度1.5%。优化后内切葡聚糖酶活力可达4 158 U/(mL.min)是优化前1 449 U/(mL.min)的2.86倍。     

一株高温高效产氢细菌的分离及产氢特征研究

实验组采用非选择性培养、选择性培养、液体培养和固体培养一整套培养方法,结合平面厌氧操作技术与培养瓶厌氧技术,从混合产氢培养液中分离得到1株较好的产氢细菌。细菌需氧实验证明:此细菌为厌氧细菌;Gram实验和芽孢染色实验表明:此菌为革兰氏阴性杆菌,无芽孢。在不同的pH值、不同金属离子与温度下,于间歇式条件下测定其产氢量。实验结果表明:此菌株对pH值敏感,其最适产氢pH值为5.5,最适温度在45℃左右,在最适宜条件下产气量在1000 mL/(5 g葡萄糖),产气周期在15 d左右。金属离子影响实验表明:缺少铁和镁离子对此产气菌株的产气量有明显影响,此菌株对铜离子(0.1 g/L)无抗性。     

芦岭煤田微生物群落结构和生物成因气的产甲烷类型研究

【目的】揭示芦岭煤田微生物群落组成,并分析其潜在的产甲烷类型及产甲烷途径。【方法】采集芦岭煤田的煤层气样品和产出水样品,分别分析样品的地球化学性质特征;利用Illumina HiSeq高通量测序技术分析产出水中的微生物群落结构;采用添加不同底物的厌氧培养实验进一步证实芦岭煤田生物成因气的产甲烷类型。【结果】该地区煤层气为生物成因和热成因的混合成因气;古菌16S rRNA基因分析表明在产出水中含有乙酸营养型、氢营养型和甲基营养型的产甲烷菌。丰度较高的细菌具有降解煤中芳香族和纤维素衍生化合物的潜力。厌氧富集培养结果表明,添加乙酸盐、甲酸盐、H2+CO2为底物的矿井水样均有明显的甲烷产生。【结论】芦岭煤田具有丰富的生物多样性,该地区同时存在三种产甲烷类型。本研究为利用微生物技术提高煤层气的采收率,实现煤层气的可持续开采提供科学依据。     

Microbial Methane Formation from Hard Coal and Timber in an Abandoned Coal Mine

About 7% of the global annual methane emissions originate from coal mining. Also, mine gas has come into focus of the power industry and is being used increasingly for heat and power production. In many coal deposits worldwide, stable carbon and hydrogen isotopic signatures of methane indicate a mixed thermogenic and biogenic origin. In this study, we have measured in an abandoned coal mine methane fluxes and isotopic signatures of methane and carbon dioxide, and collected samples for microbiological and phylogenetic investigations. Mine timber and hard coal showed an in-situ production of methane with isotopic signatures similar to those of the methane in the mine atmosphere. Enrichment cultures amended with mine timber or hard coal as sole carbon sources formed methane over a period of nine months. Predominantly, acetoclastic methanogenesis was stimulated in enrichments containing acetate or hydrogen/carbon dioxide. Molecular techniques revealed that the archaeal community in enrichment cultures and unamended samples was dominated by members of the Methanosarcinales. The combined geochemical and microbiological investigations identify microbial methanogenesis as a recent source of methane in abandoned coal mines.     

Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China

To identify the methanogenic pathways present in a deep coal bed methane (CBM) reservoir associated with Eastern Ordos Basin in China, a series of geochemical and microbiological studies was performed using gas and water samples produced from the Liulin CBM reservoir. The composition and stable isotopic ratios of CBM implied a mixed biogenic and thermogenic origin of the methane. Archaeal 16S rRNA gene analysis revealed the dominance of the methylotrophic methanogen Methanolobus in the water produced. The high potential of methane production by methylotrophic methanogens was found in the enrichments using the water samples amended with methanol and incubated at 25 and 35?°C. Methylotrophic methanogens were the dominant archaea in both enrichments as shown by polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE). Bacterial 16S rRNA gene analysis revealed that fermentative, sulfate-reducing, and nitrate-reducing bacteria inhabiting the water produced were a factor in coal biodegradation to fuel methanogens. These results suggested that past and ongoing biodegradation of coal by methylotrophic methanogens and syntrophic bacteria, as well as thermogenic CBM production, contributed to the Liulin CBM reserves associated with the Eastern Ordos Basin.     

Microbial production of biogas form organic wastes

Biogas production through bacterial digestion of water hyacinth, rice straw and sawdust inoculated with cow dung was investigated in a batch loading of laboratory digestors. It was found that the overall gas yield was 368.00 l from water hyacinth having 77.13% methane, 310.00 l from rice straw with 77.80% methane, and 144.80 l from sandust containing 62.28% methane. Along with the production of gas, the change in pH and microbial population were also monitored. The pH of the digestion mixture was initially low and found to be increasing as the digestion progressed. The total microbial population of bacteria, fungi, actinomycetes, cellulolytic organisms, and coliform bacteria were found to decrease with time.     

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

Methanogenesis and sulfate reduction in timber and drainage water from a gold mine

Biogenesis of methane in the heartwood of diseased trees has been shown, but never in timber in service. Studies were undertaken to establish whether methan‐ogens and sulfate‐reducers were present in wooden pit props and drainage water from underground sites in a gold mine. The predominant methanogen in the mine ecosystem was tentatively identified as Methanobacterium bryantii. The sulfate‐reducers comprised Desulfovibrio desulfuricans and Desulfotomaculum antarcticum. Most probable numbers (MPN) of bacteria indicated that 3.5 × 10⁵ methanogenic and 7.9 × 10³ sulfate‐reducing bacteria were present per milliliter of stagnant drainage water. MPN values per gram of timber were lower for methanogens but comparable for sulfate‐reducers. Laboratory model systems predicted a maximum rate of methan‐ogenesis of 2.3 mL methane/g wood per day; however, rates would never attain this value because of nutrient limitations and environmental restrictions. Analysis of gas samples extracted from sealed areas of the gold mine verified the presence of methane.     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

A contribution of hydrogenotrophic methanogenesis to the biogenic coal bed methane reserves of Southern Qinshui Basin,China

The activity of methanogens and related bacteria which inhabit the coal beds is essential for stimulating new biogenic coal bed methane (CBM) production from the coal matrix. In this study, the microbial community structure and methanogenesis were investigated in Southern Qinshui Basin in China, and the composition and stable isotopic ratios of CBM were also determined. Although geochemical analysis suggested a mainly thermogenic origin for CBM, the microbial community structure and activities strongly implied the presence of methanogens in situ. 454 pyrosequencing analysis combined with methyl coenzyme-M reductase (mcrA) gene clone library analysis revealed that the archaeal communities in the water samples from both coal seams were similar, with the dominance of hydrogenotrophic methanogen Methanobacterium. The activity and potential of these populations to produce methane were confirmed by the observation of methane production in enrichments supplemented with H₂ + CO₂ and formate, and the only archaea successfully propagated in the tested water samples was from the genus Methanobacterium. 454 pyrosequencing analysis also recovered the diverse bacterial communities in the water samples, which have the potential to play a role in the coal biodegradation fueling methanogens. These results suggest that the biogenic CBM was generated by coal degradation via the hydrogenotrophic methanogens and related bacteria, which also contribute to the huge CBM reserves in Southern Qinshui Basin, China.     

Thermus 2S from Thai hot springs: isolation and immobilization

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genusThermus, and is tentatively calledThermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum.