Microbial Diversity and Abundance in a ... - ACS Publications

Article pubs.acs.org/EF

Microbial Diversity and Abundance in a Representative SmallProduction Coal Mine of Central China Min Wei, Zhisheng Yu,* and Hongxun Zhang College of Resources and Environment, University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing 100049, People’s Republic of China ABSTRACT: Coal mine methane (CMM) is gaining global attention, and especially, its biogenic methane regeneration is of increasing interest in recent years. Microbial communities in coal and water in a representative small-production coal mine of central China were surveyed by cultivation-independent methods. A total of 22 330 and 53 622 high-quality sequences were obtained for archaea and bacteria, respectively, by 454 pyrosequencing. Diversity indices Chao1, ACE, and Shannon (H′) for microbes in mine water were higher than those in coal. The methane-producing archaea Methanosaeta and Methanosarcina were the dominated communities observed in coal and mine water, respectively. A variety of anaerobic fermentative bacteria were identified, and the most abundant genera in coal and mine water were Rheinheimera and Hydrogenophaga, respectively. On the basis of the results of real-time polymerase chain reaction (PCR), microbial abundance in coal was 3.29 × 105 and 8.56 × 104 cells g−1 for archaea and bacteria, respectively, while in mine water, it was 2.29 × 105 and 3.56 × 106 cells mL−1 for archaea and bacteria, respectively. Furthermore, the methanogens comprised 24.44 and 14.16% of total archaea in coal and mine water, respectively. The presence of methanogens and anaerobic fermentation bacteria may be responsible for the biogenic methane formation. This was the first study on microbial diversity and abundance in the small-production coal mine with lowconcentration methane based on 454 pyrosequencing and real-time PCR.

1. INTRODUCTION Coal mine methane (CMM) refers to the coal bed methane released during mining activities1 and is typically viewed in China as air mixed with low concentrations of methane from coal mines.2 For a long time, CMM has been considered to be harmful because of its impact on mining safety and the environment. The extremely important reason is that CMM is a main factor for coal mine explosions in underground coal mines. There have been thousands of fatalities because of coal mine explosions every year.3 The second reason is that methane is detrimental to global and local environments because of its greenhouse effect. Studies have demonstrated that methane is a major greenhouse gas preceded only by carbon dioxide, and it is 21 times more effective than carbon dioxide at being responsible for climate change.4 Underground coal mines are the largest source of CMM emissions in most countries. It is reported by the Global Methane Initiative that methane emissions from coal mines are estimated up to 584 million metric tons of carbon dioxide equivalent (MMTCO2E) in 2010, accounting for 8% of global methane emissions.5 By 2030, methane emissions are estimated as high as 8522 MMTCO2E without more effective abatement measures. Interestingly, more and more studies in recent years have indicated that CMM is not only a hazardous gas but also an energy source, which can be used as a renewable and clean energy in industrial applications. In fact, CMM emissions from both active and abandoned mines have become important energy sources for heat and power industries. For example, the growing use of CMM has significantly reduced the environmental impacts of coal power plants in Poland.6 CMM recovery and utilization have been developed in many Chinese coal mines.7 Methane from coal mines can be actually generated from thermogenic or biogenic © 2013 American Chemical Society

pathways. Thermogenic methane is formed during the coalification process, whereas biogenic methane is the result of coal degradation by microbes.8,9 Stable isotope ratios are routinely used to distinguish thermogenic and biogenic gases.10 It is worth noting that the mixed thermogenic and biogenic origin was typically observed in coal mines in Germany11 and coal seams in America,12 China,13 and Japan.14 However, various studies have suggested that biogenic methane comprises a significant portion in many coal beds, e.g., Powder River Basin15 and eastern Australian.16,17 Furthermore, biogenic methane has been generated by indigenous microbes inhabiting coal and produced water in both laboratory18−20 and in situ environments.12 Thus, it is vital to investigate indigenous microbial communities in coal seams, so that the in situ conditions can be modified to regenerate potential biogenic methane for the present and the future.21,22 In China, there are thousands of small-production coal mines. Their coal exploitation rate is only 30% by the survey of the China State Administration of Coal Mine Safety, and particularly, a lower rate is observed in some small coal mines. How to reuse the large amount of coal in abandoned mines has been a growing concern in recent years. To our knowledge, these small mines would be good potential resources for biogenic methane regeneration in the future. Previous studies have demonstrated the feasibility of biogenic methane regeneration from coal in abandoned coal mines.11 However, their indigenous microbial communities in coal mines are still unclear. Cultivation-independent methods, including 454 Received: March 28, 2013 Revised: June 15, 2013 Published: July 3, 2013 3821

dx.doi.org/10.1021/ef400529f | Energy Fuels 2013, 27, 3821−3829

Energy & Fuels

Article

Figure 1. Location of coal mines in Yichang, central China.

2. EXPERIMENTAL SECTION

pyrosequencing and real-time polymerase chain reaction (PCR), could provide such possibility to completely understand whole microbial communities in coal mines. The 454 pyrosequencing is a powerful tool in microbial investigation, which could provide comprehensive detection of an unseen majority of microbial communities.23 It has been applied to microbial investigation in a coal bed methane reservoir in eastern Ordos Basin of China.24 In addition, real-time PCR can provide sensitive and accurate quantification for microbes, and it has been successfully applied in numerous microbial studies, especially for the focus of coal seam microbes.25 In the present study, 454 pyrosequencing and real-time PCR were used to investigate the microbial community in coal and mine water in a small coal mine with low-concentration methane in central China. The main objective of this research was to understand the indigenous microbial diversity and evaluate whether the small coal mine has potential to produce biogenic methane in the future. To our knowledge, this is the first study on microbial community and abundance in coal and mine water, especially for a small-production coal mine using such cultivation-independent methods in China.

2.1. Study Area and Sampling. Samples including coal and mine water were collected from a small-production coal mine in March 2012. The mine is located in Yichang of the Hubei province of central China (see Figure 1). Its annual coal production is about 30 000 tons per year, which is a representative small coal mine among the numerous small coal mines ranging from 20 000 to 90 000 tons of coal per year in Yichang. The depth for the samples was 70−80 m, with an in situ temperature of 15.50 °C. Coal seam (with the average thickness of 0.50 m) belonged to Jingmen-Dangyang Basin, and it was stable in geological structure. Jiuligang Formation was the coal-bearing strata and formed in upper Triassic. The methane (ventilation air methane) concentration in the sampling site was 0.25% (v/v) detected by a portable gas detector (GJC4/100, CCIEC, China Coal Industrial Equipment Corp., Ltd., China). For the sampling of coal, big-block coal (2−3 kg) was collected from the intact coal layer in the coal mining area using a sterile shovel and immediately put into sterilized glass bottles. To avoid sampling bias, at least five sample locations were covered. Mine water was drained by an uninterrupted drainage system. Water samples were pumped from the drainage pipes in the mining area without air and other contaminants. Three aseptic high-density polyethylene (HDPE) bottles of 10 L were full with mine water with no headspace, tightly sealed with butyl rubber stoppers, and then kept on ice in the field. 3822


Energy & Fuels

Article

The in situ temperature, air moisture, pH, conductivity, and dissolved oxygen were measured with a portable detector (HQ 40d, Hach, Loveland, CO) during sampling. Coal and water samples were immediately sent for analyses of chemical and physical properties. 2.2. DNA Extraction and Pyrosequencing of Archaea and Bacteria. The outer layer of about 2 cm from coal samples was removed, and the inner part of samples was stored in sterilized glass bottles flushed with N2 and further used for DNA extraction. Coal samples (50 g) were macerated in the 0.05 M phosphate buffer with 0.20% Tween 80 at pH 7.4 and then incubated at 37 °C at 150 rpm for 1 h. The samples were subsequently sonicated for 30 min with a 10 s interval after each 5 min sonication16 and centrifuged. The thallus obtained was collected to the Whatman filter (Whatman Japan KK, Tokyo, Japan). Genomic DNA was extracted from the filters using a FastDNA SPIN kit for soil (Bio101 Systems, Carlsbad, CA) according to the instructions of the manufacturer. Mine water (l L) was filtered through a 0.22 μm membrane filter. Genomic DNA of mine water was also extracted with the FastDNA SPIN kit. For pyrosequencing, 16S rRNA genes for archaea and bacteria were amplified with primer sets of AR-344F/915R26 and BAC-27F/533R,27 respectively. Nested PCR was performed for both archaea and bacteria in coal samples because the first PCR was typically not visible on the agarose gel.28 The primer sets of AR-109F/915R29 and BAC-27F/ 1492R30 were used for archaea and bacteria in the first amplification, respectively. The PCR was initiated at 95 °C for 4 min, followed by 20 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min, and extended at 72 °C for 10 min. The PCR products were purified with the DNA Purification Kit (Fermentas, Lithuania) and then sequenced using the pyrosequencing technology (454 Life Sciences, Roche Diagnostics, Indianapolis, IN). 2.3. Quantitative PCR of Archaea and Bacteria. Quantitative (real-time) PCR was carried out as described by Edwards et al.31 Plasmids from the bacterium of Escherichia coli DH5α containing the 16S rRNA gene of Methanosarcina spp. (KC215420) for archaea, Desulfosporosinus spp. (KC215425) for bacteria, and mcrA gene (the special gene of methyl coenzyme M reductase for methanogens) of Methanosarcina spp. (KC244184) for methanogens were respectively used to make standard curves. Plasmids were quantified on the basis of absorbance at 260 nm with a NanoDrop ND-1000 ultraviolet−visible (UV−vis) spectrophotometer (NanoDrop Technologies, Wilmington, DE) and diluted from 100−1010 copies uL−1. The copy numbers were calculated according to Zhang32 and cell numbers were estimated as the description of Beckmann.25 The detection limits for archaea, bacteria and methanogens were respectively 10−100 copies μL−1, 1− 10 copies μL−1 and 1−10 copies μL−1. The real-time PCR was performed on ABI 7300 sequence detection system (Applied Biosystems, Foster City, CA). The primer sets of AR-519F/915R,33 BAC-338F/518R,30 and MLf/r34 were used to determine the abundance of total archaea, bacteria, and methanogens, respectively, in the original samples. The final protocol contained 12.5 μL of 2× SYBR Green mix, 1 μL of each primer (10 nM), 1 μL of sample DNA, and double-distilled water (ddH2O) to a final volume of 25 μL. The PCR initiated at 95 °C for 10 min, then followed by 40 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 58 °C, and elongation for 30 s at 72 °C. Fluorescence signals were collected at 72 °C during the elongation step. Each DNA template was performed in triplicate.

Table 1. Properties of Coal Samples in the Coal Mine coal

yield (%)

proximate analyses (%, air-dried) moisture ash volatile matter fixed carbon ultimate analysis (vol %, dry and ash free) carbon hydrogen nitrogen sulfur oxygen petrographic analysis (vol %) vitrinite liptinite inertinite mineral matter reflectance, Ro,max (%) classification

3.70 39.20 13.90 43.20 89.80 3.40 1.80 2.60 2.40 69.80 10.70 0.10 19.40 2.6 ± 0.13 anthracite

(10.70%) was correlated to the methane content. In coal, there was a positive correlation between liptinite and methane contents in the laboratory.36 Coal macerals were related to the coalification level (coal rank). As the high-rank coal, anthracite had high reflectance (Ro,max of 2.60%). However, there was a negative correlation between the coal rank and maximum rates of methane generation in the laboratory.35 This may be one of the reasons for the low methane concentration in the coal mine. In addition, it was worth noting that the methane content would be gradually increased because of microbial biodegradation in the long run.11 3.2. Properties of Mine Water. Properties of mine water are listed in Table 2. Mine water was typically alkaline with pH 8.53. In comparison to the groundwater of coal seam from eastern Ordos Basin13 and northern Japan,14 the concentrations of SO42−, Ca2+, and Mg2+ were significantly higher, except for a relatively lower Cl− concentration. Dissolved oxygen (DO) in mine water was 9.22 mg/L, which was a suitable concentration Table 2. Physicochemical Properties of Mine Water in the Coal Minea

3. RESULTS AND DISCUSSION 3.1. Properties of Coal Samples. Coal from the mine contained a high proportion of carbon and a much lower proportion of hydrogen, oxygen, volatile matter, and moisture (Table 1), and it was therefore classified as anthracite.35 The ash yield and sulfur content were quite high, up to 39.20 and 2.60%. The coal macerals were composed of vitrinite, inertinite, liptinite, and mineral matter. Vitrinite contents (69.80%) in coal samples were quite high, whereas the inertinite content (0.10%) was significantly low. Typically, the liptinite content

water

content

SO42− (mg/L) Cl− (mg/L) NO3− (mg/L) NO2− (mg/L) NH4+ (mg/L) Na+ (mg/L) K+ (mg/L) Ca2+ (mg/L) Mg2+ (mg/L) DO (mg/L) conductivity (μs/cm) TOC (mg/L) total dissolved solid (mg/L) dissolved organic carbon (mg/L) dissolved inorganic carbon (mg/L)

35.40 3.39

0.27 34.60 2.10 53.30 28.70 9.22 604 442 136

The in situ water temperature was 17.70 °C. The detection limits for NO3−, NO2−, TOC, and dissolved organic carbon were 0.01, 0.01, 0.50, and 0.50 mg/L, respectively. a

3823


Energy & Fuels

Article

for organisms.37 Adequate DO was necessary for life in the water habitats, and the suggested DO for the organism was not lower than 5 mg/L.38 The high DO may result from the introduction of oxygen from meteoric water or human activities in the underground coal mine. It would decrease after coal mines were abandoned. The total organic carbon (TOC) in mine water was below the detection limit. TOC was considered to be an indicator for bacterial contamination.39,40 The low TOC value was consistent with the properties of coal because the high-rank coal had a low solubility in mine water. Therefore, it would take a long time for the obvious regeneration of biogenic methane after the coal could be degraded gradually by microbes. 3.3. Microbial Diversity and Richness by Pyrosequencing. To obtain comprehensive insight into microbial communities in the coal mine, 16S rRNA gene sequences of archaea and bacteria for coal and mine water were investigated by 454 pyrosequencing. Sequences were processed with chimeras removal, distance matrices calculation, cluster, and operational taxonomic units (OTUs, 97% similarity) generation in the software Mothur41 and compared to the Silva database (http://www.arb-silva.de/).42 Optimized 16S rRNA gene sequences were submitted to the GenBank database under the accession number SRA 065106. A total of 75 952 high-quality sequences were obtained by pyrosequencing with 22 330 and 53 622 sequences for archaea and bacteria, respectively. The average length of sequences for archaea and bacteria was 496 and 477 bp, respectively. Diversity indices Chao1, ACE, and Shannon (H′) and the coverage calculated at an evolutionary distance of 0.03 are listed in Table 3.

3.4. Microbial Community Determined by Pyrosequencing. 3.4.1. Archaeal Community. Archaea in coal were dominated by Methanosaeta (57.65%) and were followed by Halonotius (14.8%), Methanospirillum (6.26%), and Nitrososphaera (5.94%) (see Figure 2). The dominant Methanosaeta was also observed in coal in Ordos Basin.45 The genus of Methanosaeta was an obligate acetotrophic methanogen46 and also widely observed in natural wetlands47 and wastewater treatment plants.48 Hydrogenotrophic Methanospirillum detected in the present study was identified in coal and produced water in Powder River Basin.28 It was worth noting that Halobacteriaceae archaea were observed in coal. The genus of Halonotius was considered as extremely halophilic archaea,49,50 and its growth required at least 1.8 mol L−1 NaCl.51 Halonotius in coal might be related to the geological characteristic of the coal seam with an evolution from salinity limnetic facies to continental facies. Nitrososphaera was ammonia-oxidizing archaea,52 and it was also detected in coal samples.25 In contrast, archaea in mine water were significantly different and more complex than those in coal samples. Methanosarcina (70.94%) was the predominant archaea detected in mine water, which can use various substrates to generate methane.53 It was also observed in enrichment experiments of methanogens with acetate from abandoned coal mines in Germany11 and the Powder River Basin.18 In comparison to the methanogens in coal, more diverse methanogens were observed in mine water, including the acetotrophic Methanosarcina and Methanosaeta (0.46%), the hydrogenotrophic methanogens, including Methanosphaerula (2.33%), Methanobrevibacter (0.41%), and Methanoregula (0.16%), and the methylotrophic methanogens, e.g., Methanomethylovorans (1.91%),54 Methanolobus (0.55%), and Methanosphaera (0.3%), indicating diverse mathanogenesis pathways presented in mine water. In addition, a high proportion of ammonia-oxidizing archaea, including Nitrosoarchaeum (5.44%) and Nitrososphaera (0.61%), were detected, which might participate in the nitrogen cycle. 3.4.2. Bacterial Community. Bacteria in coal were dominated by Proteobacteria, comprising 80.96% of total bacteria. In fact, Proteobacteria were the most abundant bacteria in numerous coal bed methane reservoirs.21,55 Other abundant phyla were affiliated with Actinobacteria (5.94%), Bacteroidetes (5.03%), and Firmicutes (4.29%) (see Figure 3). The phyla of Actinobacteria, Bacteroidetes, and Firmicutes were also found in samples of coal and produced water.56 At the level of class, Gammaproteobacteria were the predominant bacteria in coal, followed by Betaproteobacteria, Actinobacteria, Bacilli, Sphingobacteria, Acidimicrobiia, Flavobacteria, and Alphaproteobacteria. The abundant OTUs affiliated with the closest genera are listed in Table 4. The predominant Gammaproteobacteria included the genera of Pseudomonas, Rheinheimera, and Acinetobacter. Furthermore, the most abundant Pseudomonas was reported to be capable of degrading hydrocarbon compounds,57 and it had previously been detected in coal beds in Sydney basin,58 western Canada,21 and Ordos Basin of China.45 Rheinheimera was observed for the first time in coal samples in the present study, which was typically found in a water habitat, e.g., marine,59 freshwater culture pond,60 and alkaline lake. 61 It was also possibly involved in oil degradation.62 Acinetobacter was also found in coal bed methane reservoirs in Ordos Basin in China24 and coal bed methane wells in south Texas,63 which could degrade aromatic compounds.64 Microthrix, the Actinobacteria bacteria, was a filamentous bacterium commonly found in sludge of waste-

Table 3. Diversity and Richness Indices of Archaea and Bacteria in Coal and Mine Watera reads

OTUs

coal water

14118 8212

413 372

coal water

27640 25982

1742 1925

ACE Archaea 491 372 Bacteria 2434 2859

Chao1

Shannon

coverage

486 601

4.09 3.54

0.99 0.99

2347 2852

4.70 5.89

0.98 0.97

a

The number of reads referred to the sequences passed quality controls. All of the diversity and richness indices were defined at an evolutionary distance of 0.03.

ACE for archaea in coal samples with the number of 491 was higher than that of 372 in water. However, ACE for bacteria in coal was estimated to be up to 2434. This was lower than that in mine water, with the number of 2859. Chao1 indices for archaea and bacteria in coal and mine water had the same trends. Shannon diversity indices indicated a high bacterial diversity (from 4.70 to 5.89), and they were comparable to other environmental samples, e.g., wastewater43 and soil.44 Furthermore, Shannon diversity indices in coal and mine water had the same trends as ACE. Coverage for archaeal sequences was 0.99, but it ranged from 0.97 to 0.98 for bacteria, which suggested a high sequencing depth. Such microbial diversity for both coal and mine water was much higher than other studies from coal and produced water,24 which may largely support the generation of biogenic methane in the small coal mine in the future. 3824


Energy & Fuels

Article

Figure 2. Archaeal community at the level of genus in coal and mine water. Uncultured and undefined archaea were classified as “others”.

water plants and could use lipids.65 Bacillus, which belonged to the Firmicutes bacteria, was helpful for coal desulfurization.66 The Bacteroidetes bacteria affiliated to Flavobacterium could play an important role in biological conversion of coal,67 which was typically found in soil and water.68 In mine water, Proteobacteria were predominant, accounting for almost 90% of the bacteria. Its dominant role was the same as mentioned above in coal. Such other phyla as Actinobacteria (5.08%), Bacteroidetes (2.02%), and Nitrospirae (2.29%) were abundant in mine water (see Figure 3). In comparison to the predominant Gammaproteobacteria observed in coal, Betaproteobacteria were the predominant class in mine water, followed by Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Nitrospira, Deltaproteobacteria, and Flavobacteria. The abundant OTUs affiliated with the closest genera are also listed in Table 4. The dominated Betaproteobacteria detected in the present study included the genera of Hydrogenophaga, Rhodocyclus, Thiobacillus, and Aquabacterium. Hydrogenophaga, the most abundant genus in mine water, was commonly found in a water habitat69 and was also detected in coal samples from an abandoned coal mine,70 which was reported to be an autotroph and capable of using hydrogen and carbon dioxide.71 Rhodocyclus was reported to be involved in phosphorus removal, which was most commonly found in wastewater treatment plants.72 However, it was reported little in coal and produced water. Thiobacillus was found in coal beds in western Canada.21 It could oxidize iron and inorganic sulfur compounds and produce sulfuric acid in acid mine drainage.73,74 Aquabacterium was the universal bacteria observed in water habitats,75,76 including coal-produced water in Ordos Basin in China.24 The Alphaproteobacteria detected in mine water were affiliated with the genera of Rhodobacter and Novosphingobium. Rhodobacter was described as a hydrogen-producing bacterium and was detected in the Illinois Basin, but its role in coal beds remains unknown.56 Novosphingobium was reported as a polycyclic-aromatic-hydrocarbon-degrading bacterium.77 It had been observed in coal seam groundwater in Japan14 and coalmining areas.78 Selected species of Arthrobacter from the phylum Actinobacteria, e.g., Arthrobacter oxidans B4, Arthrobacter sp. B1B, were capable of biodegrading benzo[a]pyrene79 and polychlorinated biphenyl.80 It was considered vitally important in biodegradation and solubilization of coal.81

3.5. Microbial Abundance Determined by Real-Time PCR. To better understand microbial potential for biogenic methane formation in such a small coal mine in the future, realtime PCR was performed to detect in situ microbial abundance in coal and mine water (Figure 4). In coal, archaea and bacteria were 3.29 × 105 and 8.56 × 104 cells g−1, respectively. In contrast, the archaea and bacteria in mine water were 2.29 × 105 and 3.56 × 106 cells mL−1, respectively. Total archaea in coal was higher than those in other abandoned coal mines,25,70 where archaea in coal was about 103−104 cells g−1. Bacteria in both coal and mine water were lower than those in the abandoned coal mines, with a varied abundance of 106−108 cells g−1.25 It was possible that bacterial abundance could gradually increase after coal mines were abandoned, which would contribute to the degradation of coal and generation of biogenic gas. For the detection of methanogens, the mcrA gene was targeted. Methanogens were 8.05 × 104 cells g−1, accounting for 24.44% of total archaea in coal, while in mine water, they were 3.24 × 104 cells mL−1, representing 14.16% of total archaea. These methanogens observed in the present study might be responsible for the generation of biogenic methane in the small-production coal mine in the present and the future. 3.6. Microbial Potential for Regeneration of Biogenic Methane. As previously mentioned, 454 pyrosequencing and real-time PCR demonstrated the presence of diverse bacteria involved in coal degradation and methanogens responsible for biogenic methane formation. In the present study, bacteria from the phyla of Acidobacteria, Bacteroidetes, Firmicutes, and Proteobacteria included a variety of hydrocarbon-metabolizing microbes, e.g., Acinetobacter, Flavobacterium, Hydrogenophaga, Novosphingobium, and Pseudomonas. These fermentative bacteria may play an important role in the initial decomposition of complex aromatic polymers and produce acetate, long-chain fatty acids, carbon dioxide, and hydrogen.35 The diverse methanogens, including Methanosarcina, Methanolobus, Methanobacteria, Methanocorpusculum, Methanosaeta, Methanococci, Methanoculleus, and Methanoregula, could convert carbon dioxide, hydrogen, acetate, formate, or other simple compounds to methane in the anaerobic coal seam. Both bacteria and methanogens detected in coal and mine water were typical 3825


Energy & Fuels

Article

Figure 3. Bacterial community at the level of (A) phylum and (B) class in coal and mine water. Bacteria of abundance less than 0.1% and undefined bacteria were classified as “others”, which included the phyla of Armatimonadetes (0.08 and 0.07%), candidate division BD1-5 (0.02 and 0.03%), BRC1 (0.01 and 0%), OP11 (0.02 and 0.01%), SR1 (0.07 and 0%), TM6 (0.03 and 0.02%), WS3 (0 and 0.01%), Chlorobi (0.05 and 0.01%), Deinococcus-Thermus (0.10 and 0.02%), Elusimicrobia (0.02 and 0%), Fibrobacteres (0.05 and 0%), Fusobacteria (0.03 and 0%), Gemmatimonadetes (0.09 and 0.02%), Lentisphaerae (0.01 and 0.02%), Spirochaetes (0.01 and 0.02%), and WCHB1-60 (0.03 and 0%).

microbial enrichment and isolation experiments, and in situ methane emission detection should be carried out for identifying the potential of biogenic methane regeneration in the following works.

microbes observed in the process of biogenic methane formation. Although it was reported that the deep buried coal was difficult for biodegradation in the confined environment,55 the enclosed environment might be destroyed by water recharge events.21 Nutrients and external microbes derived from meteoric water82 or groundwater would be introduced to the anaerobic coal seam, which could substantially activate indigenous microbial communities in the coal seam and eventually lead to the generation of secondary biogenic methane.83 However, more survey tracking ongoing time, e.g.,

4. CONCLUSION The indigenous microbial communities and their abundance in a small-production coal mine in central China were investigated by 454 pyrosequencing and real-time PCR. The following observations were found: (1) Pyrosequencing revealed a significantly high microbial diversity in coal and mine water 3826


Energy & Fuels

Article

Table 4. Dominant Genera of Archaea and Bacteria in Coal and Mine Watera archaea

a

bacteria

genus

coal

water

genus

coal

water

genus

coal

water

Nitrososphaera Nitrosoarchaeum Halonotius Methanobacterium Methanobrevibacter Methanolobus Methanomethylovorans Methanoregula Methanosaeta Methanosarcina Methanosphaera Methanosphaerula Methanospirillum Parvarchaeum Rice Cluster I Terrimonas Thermoplasma

5.94

0.61 5.44 0.01 0.20 0.41 0.55 1.91 0.16 0.46 70.94 0.30 2.33 2.42 0.01 0.02 0.01 0.11

Acidovorax Acinetobacter Aquabacterium Arthrobacter Bacillus Comamonas Delftia Ferribacterium Flavobacterium Hydrogenophaga Leeia Limnobacter Methylophilus Microthrix Nitrosomonas Nitrospira Nitrotoga

0.29 10.63 0.43 0.03 1.76 0.39 1.34 1.09 1.68 0.39

2.80 2.42 3.18 2.74

Novosphingobium Perlucidibaca Pseudomonas Ralstonia Rheinheimera Rhodobacter Rhodocyclus Sphingobium Thiobacillus Trichococcus

0.02 0.02 31.68 1.57 21.48 0.02

4.06 2.01 0.48 0.03 0.27 5.20 11.07 1.23 7.11

14.80

57.65

6.26

1.33 2.33 0.17 0.02 0.05

The abundance of genera listed in the table was more than 1%.

1.02 0.25 0.06 0.55 13.53 1.38 1.02 0.02

0.33 0.01 1.10

1.80 2.07 1.17

■

ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (21177153) and the National Science and Technology Major Project, China (2012ZX05060-005). We are very grateful to staff at the Production Safety Supervision Bureau in Hubei province for sample collection.

■

(1) Karacan, C. Ö .; Ruiz, F. A.; Cotè, M.; et al. Coal mine methane: A review of capture and utilization practices with benefits to mining safety and to greenhouse gas reduction. Int. J. Coal Geol. 2011, 86 (2), 121−156. (2) Yang, M. Climate change and energy policies, coal and coalmine methane in China. Energy Policy 2009, 37 (8), 2858−2869. (3) United Nations Economic Commission for Europe (UNECE). Best Practice Guidance for Effective Methane Drainage and Use in Coal Mines; United Nations Publications: Blue Ridge Summit, PA, 2010. (4) Warmuzinski, K. Harnessing methane emissions from coal mining. Process. Saf. Environ. 2008, 86 (5), 315−320. (5) Global Methane Initiative (GMI). Coal Mine Methane-Reducing Emissions, Advancing Recovery and Use Opportunity; GMI, 2012; http:// www.globalmethane.org/coal-mines/index.aspx. ́ (6) Sliwiń ska, A.; Czaplicka-Kolarz, K. Reducing life-cycle environmental impacts of coal-power by using coal-mine methane. Int. J. Energy Res. 2013, 37, 1044−1058. (7) Wang, L.; Cheng, Y.-P. Drainage and utilization of Chinese coal mine methane with a coal−methane co-exploitation model: Analysis and projections. Resour. Policy 2012, 37, 315−321. (8) Brown, A. Identification of source carbon for microbial methane in unconventional gas reservoirs. AAPG Bull. 2011, 95 (8), 1321− 1338. (9) Ni, Y.; Dai, J.; Zou, C.; et al. Geochemical characteristics of biogenic gases in China. Int. J. Coal Geol. 2013, 113, 76−87. (10) Strąpoć, D.; Schimmelmann, A.; Mastalerz, M. Carbon isotopic fractionation of CH4 and CO2 during canister desorption of coal. Org. Geochem. 2006, 37 (2), 152−164. (11) Krüger, M.; Beckmann, S.; Engelen, B.; et al. Microbial methane formation from hard coal and timber in an abandoned coal mine. Geomicrobiol. J. 2008, 25 (6), 315−321. (12) Wawrik, B.; Mendivelso, M.; Parisi, V. A.; et al. Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin. FEMS Microbiol. Ecol. 2012, 81 (1), 26−42.

Figure 4. Abundance of archaea, bacteria, and methanogens based on real-time PCR of 16S rRNA and mcrA genes. Microbial abundance in coal and mine water was indicated with cells g−1 and cells mL−1, respectively.

of the small coal mine. (2) Archaea in coal was dominated by Methanosaeta, whereas Methanosarcina was predominant in alkaline mine water. Rheinheimera and Hydrogenophaga were the predominant bacteria in coal and mine water, respectively. (3) Acetotrophic methanogens governed the archaeal communities in the coal mine. However, the presence of hydrogenotrophic and methylotrophic methanogens may suggest diverse mathanogenesis pathways in the coal mine. (4) The relatively high abundance of methanogens detected by real-time PCR together with hydrocarbon-metabolizing bacteria provided possibility for the potential biogenic methane regeneration from the small coal mine in the future.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-10-88256462. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 3827


Energy & Fuels

Article

(13) Guo, H.; Yu, Z.; Liu, R.; et al. Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China. Appl. Microbiol. Biotechnol. 2012, 96 (6), 1587−1597. (14) Shimizu, S.; Akiyama, M.; Naganuma, T.; et al. Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan. Geobiology 2007, 5 (4), 423−433. (15) Flores, R. M.; Rice, C. A.; Stricker, G. D.; et al. Methanogenic pathways of coal-bed gas in the Powder River Basin, United States: The geologic factor. Int. J. Coal Geol. 2008, 76 (1), 52−75. (16) Ahmed, M.; Smith, J. Biogenic methane generation in the degradation of eastern Australian Permian coals. Org. Geochem. 2001, 32 (6), 809−816. (17) Faiz, M.; Hendry, P. Significance of microbial activity in Australian coal bed methane reservoirsA review. Bull. Can. Pet. Geol. 2006, 54 (3), 261−272. (18) Green, M. S.; Flanegan, K. C.; Gilcrease, P. C. Characterization of a methanogenic consortium enriched from a coalbed methane well in the Powder River Basin, U.S.A. Int. J. Coal Geol. 2008, 76 (1−2), 34−45. (19) Harris, S. H.; Smith, R. L.; Barker, C. E. Microbial and chemical factors influencing methane production in laboratory incubations of low-rank subsurface coals. Int. J. Coal Geol. 2008, 76, 46−51. (20) Kimura, H.; Nashimoto, H.; Shimizu, M.; et al. Microbial methane production in deep aquifer associated with the accretionary prism in southwest Japan. ISME J. 2009, 4 (4), 531−541. (21) Penner, T. J.; Foght, J. M.; Budwill, K. Microbial diversity of western Canadian subsurface coal beds and methanogenic coal enrichment cultures. Int. J. Coal Geol. 2010, 82 (1−2), 81−93. (22) Ulrich, G.; Bower, S. Active methanogenesis and acetate utilization in Powder River Basin coals, United States. Int. J. Coal Geol. 2008, 76 (1−2), 25−33. (23) Logares, R.; Haverkamp, T. H. A.; Kumar, S.; et al. Environmental microbiology through the lens of high-throughput DNA sequencing: Synopsis of current platforms and bioinformatics approaches. J. Microbiol. Methods 2012, 91, 106−113. (24) Guo, H.; Liu, R.; Yu, Z.; et al. Pyrosequencing reveals the dominance of methylotrophic methanogenesis in a coal bed methane reservoir associated with eastern Ordos Basin in China. Int. J. Coal Geol. 2012, 93, 56−61. (25) Beckmann, S.; Krüger, M.; Engelen, B.; et al. Role of bacteria, archaea and fungi involved in methane release in abandoned coal mines. Geomicrobiol. J. 2011, 28 (4), 347−358. (26) Gantner, S.; Andersson, A. F.; Alonso-Sáez, L.; et al. Novel primers for 16S rRNA-based archaeal community analyses in environmental samples. J. Microbiol. Methods 2011, 84 (1), 12−18. (27) Liu, R.; Yu, Z.; Guo, H.; et al. Pyrosequencing analysis of eukaryotic and bacterial communities in faucet biofilms. Sci. Total Environ. 2012, 435−436, 124−131. (28) Klein, D. A.; Flores, R. M.; Venot, C.; et al. Molecular sequences derived from Paleocene Fort Union Formation coals vs. associated produced waters: Implications for CBM regeneration. Int. J. Coal Geol. 2008, 76 (1−2), 3−13. (29) Weiss, A.; Jérôme, V.; Burghardt, D.; et al. Investigation of factors influencing biogas production in a large-scale thermophilic municipal biogas plant. Appl. Microbiol. Biotechnol. 2009, 84 (5), 987− 1001. (30) Lane, D. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley and Sons: New York, 1991; pp 115−175. (31) Edwards, K., Logan, J., Saunders, N., Eds.; Real-Time PCR: An Essential Guide; Horizon Bioscience: Norfolk, U.K., 2004. (32) Zhang, G.; Tian, J.; Jiang, N.; et al. Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I. Environ. Microbiol. 2008, 10 (7), 1850−1860. (33) Liu, Y.; Beer, L. L.; Whitman, W. B. Methanogens: A window into ancient sulfur metabolism. Trends Microbiol. 2012, 20 (5), 251− 258.

(34) Juottonen, H.; Galand, P. E.; Yrjälä, K. Detection of methanogenic archaea in peat: Comparison of PCR primers targeting the mcrA gene. Res. Microbiol. 2006, 157 (10), 914−921. (35) Strąpoć, D.; Mastalerz, M.; Dawson, K.; et al. Biogeochemistry of microbial coalbed methane. Annu. Rev. Earth Planet. Sci. 2011, 39 (1), 617−656. (36) Scott, S.; Anderson, B.; Crosdale, P.; et al. Coal petrology and coal seam gas contents of the Walloon SubgroupSurat Basin, Queensland, Australia. Int. J. Coal Geol. 2007, 70 (1−3), 209−222. (37) Khanna, D. R. Microbial Ecology: A Study of River Ganga; Discovery Publishing House: Grand Rapids, MI, 2004. (38) Chin, D. A. Water-Quality Engineering in Natural Systems; John Wiley and Sons: New York, 2006. (39) Heinz, B.; Birk, S.; Liedl, R.; et al. Water quality deterioration at a karst spring (Gallusquelle, Germany) due to combined sewer overflow: Evidence of bacterial and micro-pollutant contamination. Environ. Geol. 2009, 57 (4), 797−808. (40) Zeri, C.; Kontoyiannis, H.; Giannakourou, A. Distribution, fluxes and bacterial consumption of total organic carbon in a populated Mediterranean Gulf. Cont. Shelf Res. 2009, 29 (7), 886−895. (41) Schloss, P. D.; Westcott, S. L.; Ryabin, T.; et al. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 2009, 75 (23), 7537−7541. (42) Pruesse, E.; Quast, C.; Knittel, K.; et al. SILVA: A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007, 35 (21), 7188−7196. (43) Roske, K.; Sachse, R.; Scheerer, C.; et al. Microbial diversity and composition of the sediment in the drinking water reservoir Saidenbach (Saxonia, Germany). Syst. Appl. Microbiol. 2012, 35 (1), 35−44. (44) Lin, Y.-T.; Whitman, W. B.; Coleman, D. C.; et al. Comparison of soil bacterial communities between coastal and inland forests in a subtropical area. Appl. Soil Ecol. 2012, 60, 49−55. (45) Tang, Y.-Q.; Ji, P.; Lai, G.-L.; et al. Diverse microbial community from the coalbeds of the Ordos Basin, China. Int. J. Coal Geol. 2012, 90−91, 21−33. (46) Ma, K.; Liu, X.; Dong, X. Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor. Int. J. Syst. Evol. Microbiol. 2006, 56 (1), 127−131. (47) Narihiro, T.; Hori, T.; Nagata, O.; et al. The Impact of aridification and vegetation type on changes in the community structure of methane-cycling microorganisms in Japanese wetland soils. Biosci., Biotechnol., Biochem. 2011, 75 (9), 1727−1734. (48) Steinberg, L. M.; Regan, J. M. mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Appl. Environ. Microbiol. 2009, 75 (13), 4435−4442. (49) Oren, A.; Arahal, D. R.; Ventosa, A. Emended descriptions of genera of the family Halobacteriaceae. Int. J. Syst. Evol. Microbiol. 2009, 59 (3), 637−642. (50) Robinson, J. L.; Pyzyna, B.; Atrasz, R. G.; et al. Growth kinetics of extremely halophilic Archaea (family Halobacteriaceae) as revealed by Arrhenius plots. J. Bacteriol. 2005, 187 (3), 923−929. (51) Horikoshi, K.; Grant, W. D. Extremophiles: Microbial Life in Extreme Environments; Wiley-Liss: New York: 1998. (52) Tourna, M.; Stieglmeier, M.; Spang, A.; et al. Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (20), 8420−8425. (53) Simankova, M. V.; Kotsyurbenko, O. R.; Lueders, T.; et al. Isolation and characterization of new strains of methanogens from cold terrestrial habitats. Syst. Appl. Microbiol. 2003, 26 (2), 312−318. (54) Jiang, B.; Parshina, S.; Van Doesburg, W.; et al. Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen from an anaerobic reactor fed with methanol. Int. J. Syst. Evol. Microbiol. 2005, 55 (6), 2465−2470. (55) Singh, D. N.; Kumar, A.; Sarbhai, M. P.; et al. Cultivationindependent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of 3828


Energy & Fuels

Article

coal into methane. Appl. Microbiol. Biotechnol. 2011, 93 (3), 1337− 1350. (56) Strapoc, D.; Picardal, F. W.; Turich, C.; et al. Methaneproducing microbial community in a coal bed of the Illinois Basin. Appl. Environ. Microbiol. 2008, 74 (8), 2424−2432. (57) Mikesell, M. D.; Kukor, J. J.; Olsen, R. H. Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer. Biodegradation 1993, 4 (4), 249−259. (58) Li, D.; Hendry, P.; Faiz, M. A survey of the microbial populations in some Australian coalbed methane reservoirs. Int. J. Coal Geol. 2008, 76 (1), 14−24. (59) Brettar, I.; Christen, R.; Höfle, M. G. Rheinheimera perlucida sp. nov., a marine bacterium of the Gammaproteobacteria isolated from surface water of the central Baltic Sea. Int. J. Syst. Evol. Microbiol. 2006, 56 (9), 2177−2183. (60) Chen, W. M.; Yang, S. H.; Young, C. C. Rheinheimera tilapiae sp. nov., isolated from a freshwater culture pond. Int. J. Syst. Evol. Microbiol. 2013, 63 (4), 1457−1463. (61) Liu, Y.; Jiang, J. T.; Xu, C. J.; et al. Rheinheimera longhuensis sp. nov., isolated from a slightly alkaline lake in northeast of China and emended description of genus Rheinheimera Brettar et al. 2002. Int. J. Syst. Evol. Microbiol. 2012, 62, 2927−2933. (62) Wang, J.; Ma, T.; Zhao, L.; et al. PCR−DGGE method for analyzing the bacterial community in a high temperature petroleum reservoir. World J. Microbiol. Biotechnol. 2008, 24 (9), 1981−1987. (63) Jones, E. J. P.; Voytek, M. A.; Corum, M. D.; et al. Stimulation of methane generation from nonproductive coal by addition of nutrients or a microbial consortium. Appl. Environ. Microbiol. 2010, 76 (21), 7013−7022. (64) Gerischer, U. Acinetobacter Molecular Microbiology; Horizon Scientific Press: Norfolk, U.K., 2008. (65) Nielsen, P. H.; Roslev, P.; Dueholm, T. E.; et al. Microthrix parvicella, a specialized lipid consumer in anaerobic−aerobic activated sludge plants. Water Sci. Technol. 2002, 46 (1−2), 73. (66) Graumann, P. Bacillus: Cellular and Molecular Biology; Caister Academic Press: Norfolk, U.K., 2007. (67) Meng, Q.; Wang, Y.; Zhou, A. Synergistic effect of photobiological co-conversion of Shenfu coal. J. China Univ. Min. Technol. (Engl. Ed.) 2011, 3, 438−442. (68) Otieno, P. O.; Lalah, J. O.; Virani, M.; et al. Soil and water contamination with carbofuran residues in agricultural farmlands in Kenya following the application of the technical formulation Furadan. J. Environ. Sci. Health, Part B 2010, 45 (2), 137−144. (69) Medihala, P.; Lawrence, J.; Swerhone, G.; et al. Transient response of microbial communities in a water well field to application of an impressed current. Water Res. 2013, 47 (2), 672−682. (70) Beckmann, S.; Lueders, T.; Kruger, M.; et al. Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines. Appl. Environ. Microbiol. 2011, 77 (11), 3749−3756. (71) Willems, A.; Busse, J.; Goor, M.; et al. Hydrogenophaga, a new genus of hydrogen-oxidizing bacteria that includes Hydrogenophaga flava comb. nov. (formerly Pseudomonas flava), Hydrogenophaga palleronii (formerly Pseudomonas palleronii), Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava and “Pseudomonas carboxydoflava”), and Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis). Int. J. Syst. Evol. Microbiol. 1989, 39 (3), 319−333. (72) Kong, Y.; Nielsen, J. L.; Nielsen, P. H. Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants. Appl. Environ. Microbiol. 2004, 70 (9), 5383−5390. (73) Takai, M.; Kamimura, K.; Sugio, T. A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1. Eur. J. Biochem. 2001, 268 (6), 1653−1658. (74) Peccia, J.; Marchand, E. A.; Silverstein, J.; et al. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium. Appl. Environ. Microbiol. 2000, 66 (7), 3065−3072.

(75) Chen, W. M.; Cho, N. T.; Yang, S. H.; et al. Aquabacterium limnoticum sp. nov., isolated from a freshwater spring. Int. J. Syst. Evol. Microbiol. 2012, 62 (3), 698−704. (76) Griebler, C.; Lueders, T. Microbial biodiversity in groundwater ecosystems. Freshwater Biol. 2008, 54 (4), 649−677. (77) Sohn, J. H.; Kwon, K. K.; Kang, J. H.; et al. Novosphingobium pentaromativorans sp. nov., a high-molecular-mass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment. Int. J. Syst. Evol. Microbiol. 2004, 54 (5), 1483−1487. (78) Smyk, D.; Mytilinaiou, M. G.; Rigopoulou, E. I.; et al. PBC triggers in water reservoirs, coal mining areas and waste disposal sites: From Newcastle to New York. Dis. Markers 2010, 29 (6), 337−344. (79) Peng, H.; Yin, H.; Deng, J.; et al. Biodegradation of benzo[a]pyrene by Arthrobacter oxydans B4. Pedosphere 2012, 22 (4), 554−561. (80) Gilbert, E. S.; Crowley, D. E. Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. strain B1B. Appl. Environ. Microbiol. 1997, 63 (5), 1933−1938. (81) Sekhohola, L. M.; Igbinigie, E. E.; Cowan, A. K. Biological degradation and solubilisation of coal. Biodegradation 2012, 1−14. (82) Flores, R. M. Microbes, methanogenesis, and microbial gas in coal. Int. J. Coal Geol. 2008, 76, 1−2. (83) Scott, A. R.; Kaiser, W. R.; Ayers, W. B., Jr. Thermogenic and secondary biogenic gases, San Juan Basin, Colorado and New MexicoImplications for coalbed gas producibility. AAPG Bull. 1994, 78 (8), 1186−1209.

3829


Microbial Diversity and Abundance in a ... - ACS Publications

Recommend Documents