Important Role of Fungi in the Production of Secondary

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Important role of fungi in the production of secondary biogenic coalbed methane in China’s Southern Qinshui Basin Hongguang Guo, Jinlong Zhang, Qing Han, Zaixing Huang, Michael Allan Urynowicz, and Fei Wang Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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Important role of fungi in the production of secondary biogenic coalbed methane in China’s Southern Qinshui Basin Hongguang Guo

a,b*

, Jinlong Zhang a, Qing Han a, Zaixing Huang b, Michael A

Urynowicz b, Fei Wang a a

College of Mining Technology, Taiyuan University of Technology, 79 Yingze West

Road, Taiyuan 030024, PR China b

Center for Biogenic Natural Gas Research, Department of Civil and Architectural

Engineering, University of Wyoming, Laramie, Wyoming 82071, USA

Abstract It is commonly accepted that biogenic coalbed methane (CBM) is formed by anaerobic bacteria and methanogens via coal biodegradation. While the syntrophic cooperation between fungi and methanogens has been well established in the production of methane from rumen, little is known about the role that fungi play in the formation of biogenic CBM. Miseq sequencing and mcrA gene library was employed to investigate the fungal, archaeal, and bacterial communities in produced water from Qinshui Basin, a major site for CBM exploitation in China. The syntrophic relationship between fungi degrading coal and methanogens producing methane was also investigated. A diversity of fungal communities was found in produced water from different coal seams with the dominance of Ascomycota and Basidiomycota. Hydrogenotrophic methanogens, Methanobacterium were also found to be predominant in produced water as revealed by Miseq sequencing and mcrA gene 1

ACS Paragon Plus Environment

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library analysis. Bacterial communities with potential to degrade coal were also recovered in produced water. Large yields of methane were produced in incubations with produced water and coal. Incubations that included antibiotics achieved 62.24% to 97.53% of the methane production as compared to the incubations without antibiotics. These results confirmed that most of the biogenic gas was produced by hydrogenotrophic methanogens and demonstrated the important role that fungi play in the biodegradation of coal. Keywords

Secondary

biogenic

coalbed

methane,

hydrogenotrophic methanogenesis, Qinshui Coal Basin

2


fungi,

methanogens,

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1. Introduction Secondary biogenic coal bed methane (CBM) has become a topic of academia and industry interest in recent years. It has been recognized as an important contributor to CBM reserves all over the world, regardless of coal rank, region or formation history 1-3

. In some basins it has even been identified as the primary source of CBM 4.

Secondary biogenic CBM is believed to be generated by coal biodegradation via anaerobic bacteria and methanogenic archaea following the coalification process. Microbially enhanced CBM (MeCoM) has been impressed as a low-cost and environmentally friendly alternative for improving CBM yield through the introduction of microorganisms, nutrition and/or other stimuli 5. Coal is a complex and heterogeneous compound which makes it difficult to degrade naturally. However, the pathway of coal biodegradation can still be understood using the classic steps associated with anaerobic digestion. First, coal is hydrolyzed into soluble coal-derived compounds that are further degraded into simple organic acids, carbon dioxide and hydrogen, which are the primary substrates for methanogenesis 4. Understanding the key functions of these microorganisms is essential for determining the mechanisms of coal biodegradation in the interest of promoting MeCBM development. For years, scientists have investigated the microbial communities in produced water and coal samples from various CBM fields and coal seams using both traditional sequencing and next generation sequencing methods. The 16S rRNA gene clone

3


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library was first used to investigate coal seam bacterial and archaeal communities in northern Japan where Methanoculleus, Methanolobus and syntrophic bacteria were detected 6. Similar methods were later used to investigate the microbial communities in produced water and coal obtained from the Sydney, Surat, Phillip and Gippsland Basins in Australia

7, 8

, and the Power River and Illinois Basins in the USA 9, 10, the

Waikato coalfield in New Zealand

11

, and the Alberta Basin in Canada

12

, and the

Ordos Basin in China 13, 14. In these studies, methanogens that are capable of utilizing three methanogenesis pathways were detected, suggesting the presence of secondary biogenic CBM. However, as a result of the intrinsic shortages of cloning and sequencing throughput, results obtained by traditional clone and sequencing methods often underestimate the real microbial diversity

15

. Next generation sequencing

methods are now beginning to be introduced so as to provide additional information regarding the microbial communities related to the production of secondary biogenic CBM. In previous work, 454 pyrosequencing was used to analyze produced water, coal, and CBM gas from the Ordos Basin as well as produced water from Qinshui Basin to reveal the existence of methanogens and fermentative bacteria16-18. Methanogens and bacteria were also found in the coal and mine water from Yichang, China, using the 454 pyrosequencing approach

19

. In the USA, pyrosequencing results showed that

Methanolobus and β-Proteobacteria were the main components of microbial communities

within the Powder River Basin 20, and Methanolobus, Methanosarcina

and Firmicutes, Bacteroides dominated microbial communities in produced water in 4


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the Cook Inlet Basin 21. Previous studies have focused almost exclusively on bacterial and archaeal communities with few studies reporting on fungi relating to aerobic 22

communities

or fungi following incubation without clear composition of fungal

communities 23. There is a great deal of evidence showing that fungi can degrade all kinds of large and complex organic compounds including synthetic dyes, lignin, and polycyclic aromatic 24-26

hydrocarbons (PAHs)

. Lignin derived compounds and PAHs are some of the

main components of coal. Moreover, several fungi have been shown to degrade coal directly

27, 28

. Although it is still unknown whether anaerobic fungi exist in situ,

anaerobic fungi have been shown to degrade an array of plant monosaccharides including fructose, glucose, xylose, cellobiose and gentiobiose

29

. In addition, the

symbiosis between anaerobic fungi and methanogens has already been well established in bovine rumen

30

with the enriched co-cultures of anaerobic fungi and

methanogens from rumen digesta being shown to metabolize lignocellulose (barley straw) into methane

31

. These studies strongly suggested that anaerobic fungi could

also degrade coal, thereby providing substrates for methanogenesis. In this paper, produced water and coal samples were obtained from Qinshui Basin in China, where the existence of secondary biogenic CBM have previously been demonstrated

17

. This is the first study to investigate the presence of fungal

communities at this site. Fungal communities in produced water from three different targeted coal seams were evaluated using Miseq sequencing. The archaeal, bacterial communities in produced water were also investigated as well as the potential for 5


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producing methane from coal using fungi and methanogens compared with fungi, bacteria and methanogens by adding antibiotics to restrict bacteria. 2. Material and methods 2.1 Sample collection The sampling sites were located in the southern Qinshui Basin (see the location map in Guo, Yu, Thompson and Zhang

17

). The stratigraphic column of coal-bearing

sequence in Qinshui Basin is shown in Figure 1. Produced water samples were collected from active CBM wells from Sihe and Yuecheng coal mines. These wells were selected to insure that the targeted coal seams included all coal seams currently producing CBM in the southern Qinshui Basin, including coal seam 3, coal seam 9, and coal seam 15. Unfortunately, it was impossible to distinguish produced water samples from one coal seam to another. Produced water was sampled from nine wells. These wells target different coal seams. For examples, in the Sihe district SH-3 represents 3 wells targeting coal seam 3, SH-3915 are 3 wells for coal seams 3, 9 and 15 while YC-915 represents 3 wells in the Yuecheng district targeting coal seams 9 and 15 (see the schematic diagram in Figure 1). All the CBM wells were drilled several years ago. Prior to sampling, produced water was pumped continuously from each well for a period of more than one month. Produced water samples were collected in sterile 2 L bottles with 20 mL of water containing 0.1 % resazurin, 1.25 % cysteine, and 1.25 % Na2S. After sampling, the bottles were tightly sealed and transported to the laboratory on ice. The anthracite 6


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sample was obtained from coal seam 3 in Sihe coal mine, and coal cores were resampled in laboratory as substrate for cultivation. 2.2 Physicochemical properties analysis Three composite produced water samples were collected. Each sample consists of water from wells targeting the same coal seam(s) with equal volume (3 wells from SH-3, 3 from YC-915 and 3 from SH-3915). The concentrations of major cations (K+, Na+, Ca2+, Mg2+) were measured utilizing an inductively coupled plasma optical emission spectrometer (ICP-OES; Spectro Analytical Instruments, Kleve, Germany). Major anions (Cl-, SO42-, NO2-, NO3-) were analyzed using an ion chromatography (Metrohm Ltd, Herisau, Switzerland). The concentration of ammonium (NH4+) was analyzed utilizing a colorimetric method with mercuric iodide and potassium iodide according to Chinese standard methods (GB 5749-2006). Total organic carbon (TOC) concentrations were measured with a TOC analyzer (Shimadzu TOC analyzer). 2.3 DNA extraction and Miseq sequencing Each 1 L composite water sample was filtered with a 0.22 µm membrane filter (Millipore, USA) to collect the microorganisms. The filters were then stored at -20°C until extraction. The genome DNA of each filter was extracted using the UltraClean™ Soil DNA Isolation Kit (Mobio, USA) according to manufacturer’s instructions. The DNA was quantified using a NanodropTM 1000 Spectrophotometer (Nanodrop, Wilmington, DE, USA), and confirmed by agarose gel electrophoresis. The DNA obtained from the procedure was stored at -20 °C until further process. 7


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The primer pairs 344F-915R

32, 33

, 338F-806R

Page 8 of 43

34

, and ITS1F-ITS2R

35

were used to

amplify the archaeal 16S rRNA genes, bacterial 16S rRNA genes, and fungal ITS genes. Each forward primer contained a unique barcode to distinguish the samples. All the PCR reactions were performed as follows: 94 °C for 4 min; 30 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and, 72 °C for 10 min. Each sample was amplified in triplicate, and then pooled and purified using the AxyPrepDNA gel recovery kit (Axygen Biosciences, USA). Sequencing was performed using an Illumina Miseq platform. 2.4 Miseq data analysis After sequencing was completed, all sequence reads were quality checked using Trimmomatic soft

36

according to the following thresholds: length >50bp; no

ambiguous base in the entire sequence; and, no mismatches in the primer sequence. And any poor quality reads and primer dimers were removed. The rest of the sequences were assigned to operational taxonomic units (OTUs) with a cutoff of 97% identity using Usearch v 7.1 (http://drive5.com/uparse/). A representative sequence from each OTU was selected and assigned a taxonomic rank. 16S rRNA gene sequences were compared against the Silva database 37 and fungal ITS gene sequences were compared against the Unite fungal database

38

. Mothur v.1.30.1

39

was used to

calculate diversity and richness indices under the same sampling depth (Chao1, Shannon, and Coverage) and perform rarefaction analysis. 2.5 mcrA gene clone library analysis

8


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mcrA genes were amplified using primer pair ML-F and ML-R 40. The PCR reactions were the same as those performed to amplify 16S rRNA. The cloning, transformation, and sequencing of mcrA genes were performed as described by Guo, Yu, Thompson and Zhang

17

. PCR products of each sample were cloned and transformed into

competent E. coli DH5α cells using pGEM-T Easy Vector (Promega Corporation, Madison, WI). The positive recombinant clones were checked by PCR using M13 primers sequenced on an Applied Biosystems 3730xl (Applied Biosytems, Darmstadt, Germany). The sequences obtained were assigned to OTUs with a 3 % distance level using Mothur software

39

. Representative sequences for each OTU were compared

with the NCBI nucleotide sequence database (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were aligned with Clustal X

41

and a phylogenetic tree was constructed

with MEGA 4 software using the neighbor-joining method and bootstrap resampling analysis for 1,000 repeats 42. 2.6 Incubations Incubations with coal as the sole carbon resource were performed to determine the ability of the microbial communities, especially fungal communities in the produced water samples, to degrade coal thereby facilitating methanogenesis. The anaerobic medium included (1 L) 100 mL basic medium, 30 mL trace metals solution, 30 mL vitamin solution, 10 mL Fe(NH4)2(SO4)2 (6%), 10 mL cysteine (15%) - Na2S (15%), 10 mL HEPES buffer (1 M, pH 7.5), and 1 mL resazurin (1%) 14, 17. The basic medium included KCl (3.35 g/L), MgCl2·2H2O (27.5 g/L), MgSO4·7H2O (34.5 g/L), NH4Cl (2.5 g/L), CaCl2·2H2O (1.4 g/L), K2HPO4 (1.4 g/L), NaCl (110 g/L), yeast 9


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extract (10 g/L). The trace mineral solution contained FeCl2·4H2O (1500 mg/L), ZnCl2 (70 mg/L), MnCl2·4H2O (100 mg/L), CuCl2 (2 mg/L), CoCl2·6H2O (190 mg/L), AlK(SO4)2 (10 mg/L), NiCl2·6H2O (24 mg/L), NaMoO4 (6 mg/L), H3BO3 (36 mg/L), and 0.25 % HCl. The vitamin solution contained biotin (2 mg/L), folic acid (2 mg/L), pyroxidine HCl (10 mg/L), thiamine HCl (5 mg/L), riboflavin (5 mg/L), nicotinic acid (5 mg/L), lipoic acid (5 mg/L), paminobenzoic acid (5 mg/L), and vitamin B12 (0.1 mg/L). The filters containing the microorganisms were placed in autoclaved 100 mL serum bottles with an anaerobic medium (30 mL), supplemented with 1 g coal in each bottle. The bottles, which also contained 0.1 mM ampicillin and 0.2 mM streptomycin, were designed to restrict the activities of bacteria and determine the methane production by fungi and methanogens. The bottles without antibiotics were set as the positive control and the bottles without a filter or coal were set as the negative control. Bottle headspace was filled with N2 gas at 1 atm. All the treatments were performed in triplicate and incubated without shaking at 35 °C. The concentration of methane (CH4) in the headspace was measured using a gas chromatograph (GC-7890A, Agilent Technologies) equipped with a thermal conductivity detector. 2.7 Nucleotide sequence accession number The 16S rRNA gene and ITS gene sequences derived from Miseq sequencing were deposited in the NCBI Sequence Read Archive with the accession number SRP074542. The mcrA gene sequences derived from clone library have been deposited in GenBank with the accession no. KX234740 to KX234797. 10


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3. Results and Discussion 3.1 physicochemical properties of produced water The physicochemical properties of the three samples were similar (Table 1) and consistent with previous observations 17. The concentration of Na+ was relatively high, ranging from 13.43 to 18.01 mmol/L, while the concentrations of other cations, including K+, Ca2+, and Mg2+ were all below 0.3 mmol/L The concentration of Clranged from 1.36 mmol/L to 1.53 mmol/L, and SO42- ranged from 0.15 to 0.32 mmol/L. Each of the samples contained similar amounts of NH4+ (0.03 mmol/L, 0.04 mmol/L and 0.03 mmol/L respectively) and the NO2- and NO3- concentrations were 0.22 × 10-3 mmol/L and 0.48 × 10-3 mmol/L for each of the samples. TOC was relatively low with respect to the general range for CBM produced water at 10% of sequence reads included Tremellomycetes (38.06%), Dothideomycetes (20.05%) and Eurotiomycetes (14.35%) in SH-3; Eurotiomycetes (48.53%), Dothideomycetes (18.12%) and Microbotryomycetes (14.94%) in YC-915; Dothideomycetes (28.08%), Sordariomycetes (25.89%) and Agricomycetes (16.32%) in SH-3915. The phylogenic compositions of fungal communities at the genetic level was shown in Table 3. A large number of fugal genera were found in the produced water samples. There were 11 genera containing >5% of sequence reads, such as Cryptococcus (31.65%), Malassezia (8.36%) and Aspergillus (6.42%) in SH-3, Penicillium (42.29%), Rhodotorula (14.94%), Phoma (7.28%), and Cryptococcus (5.08%) in YC-915, Phoma (16.12%), Mortierella (7.21%), Nectria (5.06%) and Pseudallescheria (5.02%) in SH-3915. Most fungi were reported to degrade organic compounds with large molecular weight. For example, Cryptococcus spp. is known as a crude oil degrader producing lipid 44. Aspergillus spp. is often used to pretreat olive mill wastewater to facilitate anaerobic methanogenesis 45. It can also degrade plant-derived carbohydrates 46 and rice straw 47. Rhodotorula spp. anaerobically grows and degrades aromatic compounds such as dimethyl phthalate esters 48 and ethyl carbamate 49. Lignin degrading Agaricomycetes spp.

50

, cellulose and wood degrading Penicillium spp.

51, 52

13


were also found. Some

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fungi were shown to be either anaerobic or facultatively anaerobic. Schizophyllum spp. was reported to anaerobically convert polysaccharides or wood chips to ethanol 53 and capable of growth at oxygen tensions below 0.2%

54

. Mortierella spp., Acremonium

spp., Fusarium spp., Trichoderma spp., and Aspergillus spp. were uncovered by Kurakov and co-workers as facultatively anaerobic mycelial fungi in soil 55. As coal is a complex compound with the dominance of aromatic compounds and lignite derived constituents, these fungi are likely to function on the coal directly, producing soluble coal-derived compounds. The abilities of fungi in produced water to degrade coal facilitating methanogenesis were further determined by anaerobic cultivation with coal as the primary substrates (See section 3.6). 3.4 Archaeal communities revealed by Miseq sequencing and mcrA gene library Figure 4a showed the composition of archaeal communities at genetic level as revealed by Miseq sequencing. Methanobacteriaceae was the most abundant archaeal family, comprising 96.05% of sequence reads in SH-3, 93.37% in YC-915, and 96.73% in

SH-3915.

Only

the

genus

Methanobacterium

and

unclassified

Methanobacteriaceae were detected in this family with 95.07% of SH-3, 83.34% of YC-915, and 1.35% of SH-3915 classified into the genus Methanobacterium. GOM_Arc_I group made up 0.99% of sequence reads in SH-3, and 0.07% in SH-3915, which was also found to be dominant in CBM gas 18. Methanolobus made up 0.1% of sequence reads in YC-915. The other genera detected, including Methanocella,

Methanomethylovorans,

Methanoregula,

Methanospirillum

Methanothermobacter, accounted < 0.1% of the sequence reads in each sample. 14


and

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Figure 5 showed the phylogenic tree of the representative mcrA gene sequences from each OTU and related sequences. In the mcrA gene clone library, a total of 14 positive clones in SH-3 were sequenced and clustered into 2 OTUs (SH3-OTU1 and SH3-OTU2), 15 sequences in YC-915 were clustered into 5 OTUs (YC-915-OTU1 to YC-915-OTU5), and 29 sequences in SH-3915 were clustered into 4 OTUs (SH-3915-OTU1 to SH-3915-OTU4). Except for SH-3915-OTU4 with 1 sequence, all sequences were clustered into one genus Methanobacterium and four groups. SH-3-OTU1, SH-3-OTU2, YC-915-OTU1, YC-915-OTU3, YC-915-OTU4, and SH-3915-OTU2 were clustered into one group, which was closely related to the mcrA gene from the cultured organisms Methanobacterium sp. GH and Methanobacterium alcaliphilum. YC-915-OTU2, SH-3915-OTU1, and SH-3915-OTU3 were clustered into one group, which showed the highest similarity to the mcrA gene from cultured organism Methanobacterium sp. TS-2. The mcrA gene sequences and 16S rRNA gene sequences also showed close identity to those of Methanobacterium sp. GH and Methanobacterium sp. TS-2

17

. YC-915-OTU5 was closely related to the mcrA gene

from cultured organism Methanobacterium sp. NBRC 105039. SH-3915-OTU4 showed the closest similarity to the mcrA gene from Methanospirillum lacunae. Combining the results of archaeal Miseq sequencing and mcrA gene library analysis, hydrogenotrophic methanogens, mainly belonged to Methanobacterium, were proved to be dominant in all three different coal seams, which was consistent with our previous work

17

. Although most of the Miseq sequencing reads in sample SH-3915

were classified to unclassified Methanobacteriaceae, nearly all the sequences were 15


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clustered into the genus Methanobacterium using mcrA gene library analysis. And the other methanogenic genera detected in produced water were mostly hydrogenotrophic, such as Methanocella, Methanoregula, Methanospirillum and Methanothermobacter. The

others

were

methylotrophic

Methanomethylovorans.

These

methanogens,

results

e.g.,

suggested

that

Methanolobus

and

hydrogenotrophic

methanogenesis was the predominant pathway to generate biogenic CBM in Qinshui Basin. 3.5 Bacterial communities in produced water The phylogenetic composition of bacterial communities at the phylum level was shown in Figure 4b. Most of the bacterial sequences belonged to Proteobacteria, consisting of 95.70%, 84.17%, and 96.09% of sequence reads in SH-3, YC-915, and SH-3915, respectively. This is a quite common observation in CBM fields 4. The other phyla, which contained slightly > 1% sequence reads in each sample, included Bacteroidetes (2.86%, 1.35%, and 1.19% in SH-3, YC-915, and SH-3915), Actinobacteria (respectively 8.53% and 2.25% in YC-915 and SH-3915), Firmicutes (1.10% in SH-3), and Candidatus Saccharibacteria (5.08% in YC-915). The composition of bacterial communities at the genetic level was shown in Figure 6. The major genera in SH-3 were Rhizobium (46.81%), Aquabacterium (11.75%), and Acidovorax (8.16%). Members of the genera Devosia (39.68%), Rhizobium (12.47%), Methylotenera (9.40%), Brevundimonas (9.12%), and Hydrogenophaga (5.38%) were dominant in sample YC-915 and the main populations in SH-3915 were Hydrogenophaga spp. (33.20%), Thiovirga spp. (8.29%), and Methylotenera spp. 16


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(5.79%). The detected bacterial phyla were all commonly found in CBM fields as reported by previous works 4, such as Proteobacteria, Firmicutes, Bacteroidetes, Actinobateria, and so on (Figure 4b). Most of the dominant genera, such as Rhizobium, Aquabacterium,

Devosia,

Hydrogenophaga,

Thiovirga,

Acidovorax

and

Methylotenera, were also detected in our previous study, the study site of which was about 24 km away 17. Devosia spp., Brevundimonas spp., and Acinetobacter spp. have recently been isolated from coal samples

56

. These bacteria would function as

fermenters, sulfate-reducers or nitrate-reducers in coal biodegradation. 3.6 Incubations with antibiotics control Incubations were carried out to investigate the potential of fungi to degrade coal, resulting in enhanced methanogenesis. Ampicillin and streptomycin were used to inhibit bacteria which made it possible to determine the role of fungi in coal biodegradation. They are widely and successfully used for eliminaiton of bacteria in cell culture and mixed cultivation. They have been reported not to be inhibitory to several methanogens 57, 58. Ampicillin was also used to inhibit bacteria in a mix culture of bacteria and methanogen resulting in no debromination activity

59

. Thus, the

addition of ampicillin and streptomycin would inhibit bacteria but less effect methanogensis in methanogenic cultivation with coal as the main substrate. Inhibition of antibiotics on bacteria was confirmed by PCR assay. The results showed no PCR product in the treatments with antibiaotics using the same bacterial primer as Miseq

17


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sequencing after cultivation (data not shown). In this study, anaerobic medium including mineral elements, vitamin, and reducing agent was employed to determine the potential of microbial communities in produed water to biodegrade coal and produce methane. The pressure of cultivations was set to 1 atm. Coal samples were pulverized into 125 µm. Similar cultivation conditions have been used in our previous works and other studies 10, 14, 17. Comparing with coal seams where nutritions are limited, pressure is higher, and pulverized coal is barely existed, the cultivations are in much more perfect condition which would facilitate the growth of microorganisms and the production of methane by biodegrading coal. And some microbes would not be cultivated due to that lots of microorganisms still can not be enriched or cultivated using culture-dependent methods nowadays. Further studies on the formation of biogenic methane under in situ conditions would be performed to confirm the methane production by coal biodegradation and investigate the influence of cultivation conditions on methane production and microbial communities. Methane production was observed in the incubtaions supplemented with coal and produced water, regardless of the addition of antibiotics (Figure 7). This suggests that methanogens, bacteria, and fungi were anaerobically active in produced water and participated in coal biodegradation. The highest methane production occurred in the positive control without antibiotics. While methane was barely detectable in the negative control (only coal or water), all three samples treated with antibiotics showed surperisely high amounts of methane produced after being incubated for 150 days. SH-3 produced the highest yield of methane (223.66 µmol/g coal) on the 75th day of 18


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incubation without antibiotics and 139.20 µmol/g coal on the 125th day of incubation with antibiotics. YC-915 produced the highest yield of methane (265.25 µmol/g coal) on the 115th day of incubation in the treatments without antibiotics while 244.45 µmol/g coal was produced with antibiotics. For sample SH-3915, the highest methane yield was 295.75 µmol/g coal in the 75th day of incubation with antibiotics and 288.44 µmol/g coal in 115th day of incubation without antibiotics. 4. Conclusions In this study, we employed Miseq sequencing, mcrA gene library and anaerobic cultivation to analyze the microbial communities in produced water from Qinshui Basin, determine their abilities to degrade coal, and more importantly, to determine whether fungi survived in targeted coal seams and capability of sytrophically degrading coal with methanogens. The detection of high diversity of fungi in produced water and the observation of high methane yields produced in co-cultures of fungi and methanogens with coal as the main substrates strongly suggested that active fungi existed in situ and play an important role in biodegrading coal to fuel methanogens. It was further proved that hydrogenotrophic methanogenesis would be the main pathway to produce biogenic CBM in the sampling site based on the dominance of hydrogenotrophic methanogens, Methanobacterium, in produced water, the abundant of anaerobic bacterial and fungal communities in produced water and their remarkable activities to degrade coal. It is well known that coal degrading microorganisms can produce secondary biogenic

19


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Page 20 of 43

CBM and that it can be enhanced by stimulating microbial activity in situ. However, coal is a complex heterogeneous compound dominated by aromatic and lignin-derived macromolecules that are hard for microorganisms to degrade. Consequently, it is thought that upstream microbial degradation processes including coal hydrolysis and fermentation limit methane yield

60-62

. For the last few years, researchers been

investigating coal seam bacterial communities and their ability to ferment coal in the hope of revealing the mechanism(s) of coal degradation 4, 63. However, little effort has been expended to investigate anaerobic fungal communities within these subsurface environments and how they may contribute to coal degradation and other important microbial processes. Even though numerous studies have shown that fungi are capable of degrading relatively recalcitrant organic compounds

64

, anaerobic fungi, mostly

isolated from bovine rumen, have been found to degrade cellulose, straw long ago 65. This is the first report detailed the communities and metabolisms of fungi in coal seams. Nearly all the previously reported anaerobic fungi were gut fungi, particularly rumen fungi, while only a few literatures detected anaerobic fungi in other habitats such as landfill

66

. In herbivores, there is a complex syntrophic association of

anaerobic fungi with methanogens. Not only anaerobic fungi facilitate methanogens by producing H2, CO2, formate and acetate as the primary microbes to colonize and degrade ingested plant biomass, hydrogen transfer also changes fungal catabolic pathways from more oxidized end products to more reduced products

67, 68

. To date,

only one anaerobic fungal phylum, Neocallimastigomycota containing eight genera is currently described. In this study, we did not detect any genera in this phylum from 20


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produced water using the specific primer sets MN100F/MNGM2 (data not shown) which have been successfully used in 454 pyrosequencing of Neocallimastigomycota in 30 different herbivore species

69

. Using universal fungal primer pairs ITS1/ITS2,

some facultative anaerobic fungi were found, e.g. Rhodotorula spp., Mortierella spp., Acremonium spp., Fusarium spp., Trichoderma spp., Aspergillus spp., and Schizophyllum spp.. These fungi are likely to degrade coal, especially the plant-derived, aromatic, and lignin-like compounds in coal, to produce soluble coal-derived compounds and small molecular, thus facilitating methanogenesis. And the activities of fungi to syntrophically produce methane with methanogens by coal biodegradation were further determined by cultivations. High methane yields were observed in the incubations of produced water and coal with antibiotics. Methane yields by fungi and methanogens represented 62.24%, 92.16% and 97.53% when compared with methane produced by bacteria, fungi and methanogens in samples SH-3, YC-915 and SH-3915, respectively. These results suggested that anaerobic fungi can survive in groundwater in coal seams and involve in biodegrading coal and producing methane syntrophically with methanogens.

Acknowledgments This work was supported by the National Natural Science Foundation of China (51404163), Natural Science Foundation of Shanxi (2014021036-2) and Coal seam gas Joint Foundation of Shanxi (2014012006).

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Table 1. Physicochemical parameters of the produced water samples.

SH-3 YC-915 SH-3915

K+ (mmol/L)

0.02

0.04

0.04

Na+ (mmol/L)

18.01

13.43

15.31

Ca2+ (mmol/L)

0.12

0.26

0.22

Mg2+ (mmol/L)

0.01

0.05

0.05

Cl- (mmol/L)

1.36

1.50

1.53

SO42- (mmol/L)

0.32

0.15

0.26

NH4+ (mmol/L)

0.03

0.04

0.03

NO2- (10-3 mmol/L)

0.22

0.22

0.22

NO3- (10-3 mmol/L)

0.48

0.48

0.48

TOC (mmol/L)

0.14

0.06

0.14

32


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Table 2. Summary of archaeal, bacterial and fungal Miseq sequencing reads, operational taxonomic units (OTUs), and diversity estimators of produced water samples SH-3, YC-915, SH-3915 at 0.97 similarity. Sample

Reads

OTUs

Coverage

SH-3

33,028

26

0.999

YC-915

28,903

58

0.999

SH-3915

31,747

59

0.999

SH-3

33,166

104

0.999

YC-915

24,374

88

0.999

SH-3915

22,927

105

0.999

SH-3

39,323

84

0.999

YC-915

44,156

89

0.999

SH-3915

38,755

116

0.999

Chao1

Shannon

29

0.28

(27, 44) a

(0.27,0.29) a

58

0.8

(58, 62) a

(0.79,0.82) a

61

0.35

(59, 75) a

(0.33,0.36) a

133

2.24

(113, 192) a

(2.23,2.26) a

109

2.38

(95, 153) a

(2.36,2.4) a

128

3.1

(113, 173) a

(3.08,3.12) a

84

3.41

(84, 84) a

(3.39,3.42) a

89

2.52

(89, 95) a

(2.5,2.54) a

116

3.99

(116, 122) a

(3.98,4.01) a

Archaea

Bacteria

Fungi

a

The confidence intervals for Chao1 and Shannon estimators

33


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Table 3. Distribution of fungal communities in three produced water samples (SH-3, YC-915, and SH-3915) at the genetic level. Only genus containing >1% of sequence reads are shown.

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Titles and legends to figures Figure 1. Stratigraphic column of coal-bearing sequence modified from Xu, et al. 70 and schematic diagram of sampled wells targeted different coal seams in Qinshui Basin. Figure 2. Rarefaction curves of archaeal (a), bacterial (b) and fungal (c) community richness estimates of samples SH-3 (black square), YC-915 (red circle) and SH-3915 (blue triangle) at 0.97 similarity. Figure 3. Venn diagram showing the distribution of fungal and bacterial OTUs between samples SH-3, YC-915, and SH-3915. Figure 4. Phylogenetic composition of microbial communities in the three produced water samples (SH-3, YC-915, and SH-3915) based on the Miseq sequencing data. Archaeal community populations at genus level (a), bacterial community populations at phylum level (b), and fungal community populations at class level (c) are shown. Taxonomy was assigned respectively by comparing against the Silva database and Unite fungal database. Figure 5. Phylogenetic tree of representative mcrA gene sequences from each OTU obtained from produced water samples SH-3, YC-915, and SH-3915 and their related sequences. The number of sequences in the each OTU are also showed. The phylogenetic tree was constructed with the MEGA 4 software using the neighbor-joining method. Bootstrap support values (1,000 replicates) above 50 % are shown at the nodes. The scale bar represents 5 % estimated sequence divergence. 35


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Figure 6. Composition of bacterial communities in the produced water samples (SH-3, YC-915, and SH-3915) at the genus level. Only genera containing >1% of sequence reads are shown. Figure 7. Methane production from produced water sample SH-3 (a), YC-915 (b), and SH-3915 (c) with coal as the primary carbon resource. Produced water was incubated with coal (squares) as positive control. Antibiotics (ampicillin and streptomycin) were added to determine the fungal activities (diamond). The incubations contained only water (up triangles) or coal (down triangles) were set as negative controls.

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Figure 1. 320x180mm (300 x 300 DPI)


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Figure 2. 139x107mm (300 x 300 DPI)


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Figure 3. 260x90mm (300 x 300 DPI)


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Figure 4. 105x139mm (300 x 300 DPI)


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Figure 5. 127x78mm (300 x 300 DPI)


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Figure 6. 233x138mm (300 x 300 DPI)


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Figure 7. 288x551mm (300 x 300 DPI)


Important Role of Fungi in the Production of Secondary

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