菌种类型及EDTA二钠用量对生物甲烷生成的影响

采用平板分离和培养瓶厌氧技术,结合PCR扩增技术,以沙曲矿煤为发酵基底,中泰矿业和沙曲矿井水及白腐菌为菌种来源,进行产甲烷菌培养实验,并添加不同浓度梯度的EDTA二钠,进行产甲烷菌培养实验,测定实验结束后的产甲烷量。实验证明:中泰矿业矿井水中有产甲烷菌的存在,而沙曲矿井水在含反硝化菌和pH偏高的情况下,抑制了产甲烷菌的生长。同等条件下沙曲矿批次实验中没有甲烷气体的产生,而中泰矿业的有气体产出,适宜浓度的EDTA二钠浓度会显著提高产气量,EDTA二钠用量为1.0 g/L时,中泰矿井水CH4产率达到最大,为10.0 mL/g煤,反应后溶液的pH值明显增高。     

Ultrastructure of methanotrophic yeasts.

The cellular structure of two yeast strains capable of growth on methane was investigated by electron microscopy. Microbodies were observed in cells of Sporobolomyces roseus strain Y and Rhodotorula glutinis strain CY when grown on methane but rarely when grown on glucose. The size of the microbodies and the number observed per cell in a thin section did not increase with culture age. No crystalline organization was observed within these organelles. Similar microbodies were also observed in cells of R. glutinis CY grown on hexadecane. The plasma membranes of both methane and hexadecane-grown cells exhibited increased invagination compared to that of glucose-grown cells. Catalase activity was detected in the microbodies of alkane-grown cells by using 3,3'-diaminobenzidine as a cytochemical stain. The data presented suggest that microbodies, and the catalase contained within them, play a role in eucaryotic methane metabolism.     

Assessment of bioavailability of soil-sorbed atrazine

Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact. Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine. Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp. strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp. strain M91-3) were used in the bioavailability assays. Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO(2) production over time. Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients. Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite. The desorption profiles were well described by the three-site desorption model. In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine. However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed. Even the assumption of instantaneous desorption could not account for the elevated rates. A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations. Characteristics of high sorbed-phase concentration, chemotaxis, and attachment of cells to soil particles seem to contribute to the bioavailability of soil-sorbed atrazine.     

一株甲烷利用菌的分离及其在甲烷气体测定中的应用

从山西太原晋阳湖水样中分离得到一株能以甲烷为唯一碳源生长的菌株ME16.气相色谱分析表明ME16菌株能利用甲烷.ME16菌株的16S rDNA 序列与铜绿假单胞菌(Pseudomonasaeruginosa,ATCC 10145,AF094713)相似性为99%.该菌株最适培养条件为30℃、2%接种量、25%甲烷含量和培养基pH为6.0.用电化学法研究了ME16固定化细胞体系中不同含量甲烷对溶氧的响应时间以及溶氧变化与甲烷含量的关系.结果表明,加入固定化细胞后.溶氧变化在100s内达到平衡,溶氧消耗量与通入甲烷气体含量在0～16%呈线性关系,相关系数为0.9954.对样品气体8次测量,RSD为3.34%,表明该反应体系重现性良好,为该菌株进一步研究甲烷传感器奠定基础.     

Assessment of Bioavailability of Soil-Sorbed Atrazine

Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact. Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine. Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp. strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp. strain M91-3) were used in the bioavailability assays. Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO₂ production over time. Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients. Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite. The desorption profiles were well described by the three-site desorption model. In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine. However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed. Even the assumption of instantaneous desorption could not account for the elevated rates. A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations. Characteristics of high sorbed-phase concentration, chemotaxis, and attachment of cells to soil particles seem to contribute to the bioavailability of soil-sorbed atrazine.     

In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in association with an archaea methanogen is stimulated by a probiotic strain of Saccharomyces cerevisiae.

The effects of a live strain of Saccharomyces cerevisiae on hydrogen utilization and acetate and methane production by two hydrogenotrophic ruminal microorganisms, an acetogenic bacterial strain and an archaea methanogen, were investigated. The addition of yeast cells enhanced by more than fivefold the hydrogenotrophic metabolism of the acetogenic strain and its acetate production. In the absence of yeasts, and in a coculture of the acetogen and the methanogen, hydrogen was principally used for methane synthesis, but the presence of live yeast cells stimulated the utilization of hydrogen by the acetogenic strain and enhanced acetogenesis.     

Development of a biofilter for the removal of methane from coal mine ventilation atmospheres

Several strains of methane-oxidizing bacteria were isolated and studied to determine their physiological suitability for removal of methane in coal mine atmospheres. One strain, Methylomonas fodinarum ACM 3268, was selected as the most suitable culture for use in the development of a continuous biofilter to be used as a ventilation air scrubber. The experimental biofilter utilising a biofilm of M. fodinarum was shown to reduce methane levels substantially provided the residence times were sufficiently long. In the range 0.25–1.0% methane in air, commonly experienced in coal mine atmospheres, more than 70% of the methane was removed with a residence time of 15 min, with a 90% reduction at 20 min. Even at a residence time of 5 min approximately 20% of the methane in air was removed. Equal quantities of O₂ are consumed during the bacterial oxidation of methane and 1% methane is converted to 0.7% CO₂. Scale-up and alternative biofilter packings are likely to reduce the residence times in the biofilter.     

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.     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal

The use of hexamethyldisilazane (HMDS) as a drying agent was investigated in the specimen preparation for scanning electron microscopy (SEM) imaging of bacterial surface colonization on sub-bituminous coal. The ability of microbes to biofragment, ferment and generate methane from coal has sparked interest in the initial attachment and colonization of coal surfaces. HMDS represents an attractive alternative to critical point drying (CPD) in the imaging of cells on coal, negating the need for expensive equipment. Coal is easily fragmented into sub-micron particles, which can be problematic in critical point drying procedures. In this study, both individual and aggregated cells appeared well shaped with minimal occurrence of flattened cells, signifying the suitability of HMDS in cell attachment studies on sub-bituminous coal. In the absence of glucose, microcolonies of short and long cells showed similar positive results using this method. EPS shrinkage found in microcolonies was inevitable, though this enabled observation of points of attachment between cells and with coal, which would be less effective if the EPS was intact. Overall the use of HMDS drying is preferred over the more commonly used CPD method as it is safer, cheaper and more practical.     

Methane and trichloroethylene oxidation by an estuarine methanotroph, Methylobacter sp. strain BB5.1.

An estuarine methanotroph was isolated from sediment enrichments and designated Methylobacter sp. strain BB5.1. In cells grown on medium with added copper, oxidation of methane and trichloroethylene occurred with similar Ks values, but the Vmax for trichloroethylene oxidation was only 0.1% of the methane oxidation Vmax. Cells grown on low-copper medium did not oxidize trichloroethylene and showed a variable rate of methane oxidation.     

Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

Biogenic Methane Cycling in a Laboratory Model of an Abandoned Bituminous Coal Mine

Microbial communities in decommissioned coal mines have the potential to promote methane generation. Here, two 1 m x 10 cm diameter column bioreactors designed to mimic an abandoned coal mine were monitored for a year, with zones of methanogenesis in the bottom, saturated waters and aerobic coal degradation and methane oxidation at the top. The resilience of aerobic methanotrophs to survive periods with low methane and oxygen conditions suggests methanotrophs may be useful in decreasing atmospheric methane fugitive emissions from decommissioned mines. When biogenic methane production from coal did occur, the rate was slow, ≤ 0.073 nmol CH₄/g coal/day.     

Nutrient and acetate amendment leads to acetoclastic methane production and microbial community change in a non‐producing Australian coal well

Coal mining is responsible for 11% of total anthropogenic methane emission thereby contributing considerably to climate change. Attempts to harvest coalbed methane for energy production are challenged by relatively low methane concentrations. In this study, we investigated whether nutrient and acetate amendment of a non‐producing sub‐bituminous coal well could transform the system to a methane source. We tracked cell counts, methane production, acetate concentration and geochemical parameters for 25 months in one amended and one unamended coal well in Australia. Additionally, the microbial community was analysed with 16S rRNA gene amplicon sequencing at 17 and 25 months after amendment and complemented by metagenome sequencing at 25 months. We found that cell numbers increased rapidly from 3.0 × 10⁴ cells ml^?1 to 9.9 × 10⁷ in the first 7 months after amendment. However, acetate depletion with concomitant methane production started only after 12–19 months. The microbial community was dominated by complex organic compound degraders (Anaerolineaceae, Rhodocyclaceae and Geobacter spp.), acetoclastic methanogens (Methanothrix spp.) and fungi (Agaricomycetes). Even though the microbial community had the functional potential to convert coal to methane, we observed no indication that coal was actually converted within the time frame of the study. Our results suggest that even though nutrient and acetate amendment stimulated relevant microbial species, it is not a sustainable way to transform non‐producing coal wells into bioenergy factories.     

Bacterial exopolymer and PHB production in fluctuating ground-water habitats

Abstract The physiological response of a strain of ground-water bacteria to short-term fluctuations in the quality of percolating water was followed in a continuous-flow water-saturated soil column. The cells reduced their production of uronic acids-characterized extracellular polysaccharides by more than 50% when pure ground water was enriched with phosphate and glucose but resumed it gradually as the poor growth conditions returned. Bacteria that were sorbed to the mineral particles in the soil column produced more exopolymers-about six times more in pure ground water-than free-living bacteria, but the composition of the polymer was the same. Glucose, mannose and galacturonic acid accounted for about 90% of the carbohydrate. The cells stored about five times more poly-β-hydroxybutyric acid (PHB) in pure than in enriched ground water, and sorbed cells had five times higher concentrations than free-living cells. The quality variations of the ground water were also reflected in the population size of bacteria, which differed by two to four orders of magnitude between poor and enriched conditions.     

Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2. Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis. The ability to metabolize formate at high rates was observed only in cells previously grown on formate. Two such strains were distinguished: strain F and strain HF. Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2. Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate. Initial adaptation of strain H to growth on formate was preceded by a long lag. The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings. Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium. High levels of formate dehydrogenase were also constitutive in strain F. Only low formate dehydrogenase activities were observed in strain H. High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2. In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase.     

微生物增产煤层气技术研究进展

微生物增产煤层气技术能够产生新的煤层气,有效缓解能源紧张问题,是煤层气开采、增产的研究热点。微生物增产煤层气技术的核心机理是煤的厌氧生物降解产甲烷。国内外研究学者通过实验室模拟研究了本源和外源微生物的降解煤产甲烷能力及影响因素。部分煤层气公司富集或分离获得了高效产甲烷菌群,并研究了微生物增产煤层气技术的现场实施方法。开发煤的预处理技术、构建新型高效菌群等可进一步提高微生物的增产煤层气能力,促进微生物增产煤层气技术的应用。     

On Comparison Between Fungal and Bacterial Pretreatment of Coal for Enhanced Biogenic Methane Generation

Owing to better understanding of subsurface geochemical carbon recycling and real-time active methanogenesis in major coal basins around the globe, substantial share of subsurface methane generation is attributed to biogenic origin. Since coal, being complex geopolymer, does not appear to be a favorable microbial substrate, enhancement in biogenic methane yield depends on its degradation into simpler organic substrates. This review puts forward a comparative analysis of fungal and bacterial pretreatment for determining the extent of facilitation in initial degradation of coal, which is still rate limiting step in overall conversion of coal into methane. Primarily, the initial fungal degradation of coal differs from bacterial pretreatment of coal in terms of the nature of released organics. On the basis of previous reports, fungal pretreatment of coal yields, majorly, polyaromatic hydrocarbons, however, bacterial pretreatment results in the generation of mixed organics pool of aromatics and aliphatics. The presence of aliphatics may be prospected for achieving greater conversion rates of coal conversion into methane. Considering the criticality of preliminary degradation of coal and associated issues, the fate of commercial biogenic methane generation would be dictated by the factors pertaining to geological considerations and reservoir geology, chemistry of coal and associated water tables, geomicrobial considerations and economic viability.     

Thermophilic methane production and oxidation in compost

Methane cycling within compost heaps has not yet been investigated in detail. We show that thermophilic methane oxidation occurred after a lag phase of up to one day in 4-week old, 8-week old and mature (>10-week old) compost material. The potential rate of methane oxidation was between 2.6 and 4.1 micromol CH4(gdw)(-1)h(-1). Profiles of methane concentrations within heaps of different ages indicated that 46-98% of the methane produced was oxidised by methanotrophic bacteria. The population size of thermophilic methanotrophs was estimated at 10(9) cells (gdw)(-1), based on methane oxidation rates. A methanotroph (strain KTM-1) was isolated from the highest positive step of a serial dilution series. This strain belonged to the genus Methylocaldum, which contains thermotolerant and thermophilic methanotrophs. The closest relative organism on the basis of 16S rRNA gene sequence identity was M. szegediense (>99%), a species originally isolated from hot springs. The temperature optimum (45-55 degrees C) for methane oxidation within the compost material was identical to that of strain KTM-1, suggesting that this strain was well adapted to the conditions in the compost material. The temperatures measured in the upper layer (0-40 cm) of the compost heaps were also in this range, so we assume that these organisms are capable of effectively reducing the potential methane emissions from compost.     

Characterization of a methane‐utilizing strain and its application for monitoring methane

Aims: To explore new resources of methane‐utilizing micro‐organism and develop a microbial biosensing system for monitoring methane released from natural and semi‐natural ecosystems. Methods and Results: A methane (CH₄)‐utilizing bacterial strain was isolated from paddy soil using CH₄ as the sole carbon source and identified as Klebsiella sp. ME17 by phenotyping and 16S rDNA sequence analysis. The efficiency of CH₄ utilization of strain ME17 was 83·2% by gas chromatography analysis. A microbial biosensing system for CH₄ detection was developed by combining immobilized cells of strain ME17 with a dissolved oxygen sensor. It was found that response time of the system to CH₄ was <90s. The dissolved O₂ consumption increased with increasing CH₄ from 0% to 16·0% (v/v) demonstrating a positive linear relationship with a low detection limit of 0·2% (v/v). The relative standard deviation is 3·48%. Conclusions: Klebsiella sp. ME17 isolate is capable of utilizing CH₄. The microbial biosensing system of strain ME17 has been successfully applied to measure standard CH₄ sample with satisfactory results. Significance and Impact of the Study: This study suggests that certain strains of Klebsiella genus are capable of utilizing CH₄. Our proposed method appears very attractive for CH₄ measurement in coal mine.