Characterisation of a microbial community associated with a deep, coal seam methane reservoir in the Gippsland Basin, Australia

International Journal of Coal Geology 82 (2010) 232–239

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International Journal of Coal Geology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c o a l g e o

Characterisation of a microbial community associated with a deep, coal seam methane reservoir in the Gippsland Basin, Australia David J. Midgley a,⁎, Philip Hendry a, Kaydy L. Pinetown b, David Fuentes b, Se Gong b, Danielle L. Mitchell b, Mohinudeen Faiz c a b c

CSIRO Molecular and Health Technologies, P.O. Box 184, North Ryde, NSW, 1670, Australia CSIRO Petroleum, P.O. Box 136, North Ryde, NSW, 1670, Australia Origin, Exploration New Ventures, P.O. Box 148, Brisbane, Qld, 4001, Australia

a r t i c l e

i n f o

Article history: Received 2 July 2009 Received in revised form 21 January 2010 Accepted 21 January 2010 Available online 28 January 2010 Keywords: Bacteria Archaea Coal seam methane Coal bed methane Microbial community Methanogenesis

a b s t r a c t There is growing interest in optimising biogenic coal seam methane generation; however, relatively little is known about the microbiology of coal. To begin to address this deficiency, the biodiversity of a microbial community within a deep coal gas reservoir was investigated using the Amplified Ribosomal DNA Restriction Analyses (ARDRA) method. Additionally, a cultured subset of organisms from this community was examined for the ability to produce methane. ARDRA revealed that this community included both bacterial and archaeal lineages. The bacterial community was dominated by proteobacterial and Firmicutes taxa, though one actinobacterial taxa was also detected. This study is the first report of methanogenic archaea in an Australian coal seam gas reservoir. Cultures derived from the microbial community in the groundwater were able to produce methane from yeast extract and H2/CO2, but did not produce methane from coal. The ecological and physiological implications of these data are discussed. © 2010 Published by Elsevier B.V.

1. Introduction 1.1. Importance and background Rising extraction and remediation costs of coal production, along with climate change, have fuelled the global interest in alternative sources of energy. Worldwide, coal seam methane (CSM) reserves are large (1.4 × 1014 m3) and represent a significant energy resource (Dresselhaus and Thomas, 2001). Moreover, if coupled with CO2 capture and injection technologies, coal seam methane has the potential to be a comparatively clean source of energy (Damen et al., 2005). Methane associated with coal is known to have two origins. The first is thermogenic methanogenesis where methane is formed as a by-product of thermocatalytic reactions during the coalification process. The second, biogenic methane formation, results from microbial degradation of coal. In some coals, it has been suggested that biogenic methane generation may be a substantial, and importantly contemporary, source of CSM (Faiz and Hendry, 2006; Kotarba, 2001; Thielemann et al., 2004). Coals are made up of decomposed plant remains that initially form peats. After peatification, peat may be metamorphosed into coal of various ranks depending on its depth of burial and temperature.

⁎ Corresponding author. Tel.: +61 2 9490 5062. E-mail address: [email protected] (D.J. Midgley). 0166-5162/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.coal.2010.01.009

Coalification involves the loss of labile components leaving behind more resistant, typically aromatic compounds that are bonded into a complex, heterogeneous ultrastructure (Faison, 1993). Biogenic methane formation thus requires an array of catabolic enzymes, from numerous organisms, which act syntrophically to degrade coal. The initial stages of anaerobic coal degradation are presumably conducted by fermentative bacteria with methanogenic archaea performing the final conversions of CO2, H2, acetate, formate or other simple compounds to methane. 1.2. Microbial communities associated with coal There are relatively few studies of microbial communities associated with CSM reservoirs (Green et al., 2008; Li et al., 2008; Rogoff et al., 1962; Shimizu et al., 2007; Strąpoć et al., 2008). These studies suggest that coals within CSM reservoirs are generally colonised by an array of bacterial taxa that commonly include members of the Proteobacteria, most frequently from the families: Comamondaceae and Geobacteraceae, though other proteobacterial families are also reported (Li et al., 2008; Penner et al., 2008; Shimizu et al., 2007). In addition to Proteobacteria, numerous Firmicutes, mostly from the order Clostridiales have also been detected (Green et al., 2008; Shimizu et al., 2007; Strąpoć et al., 2008) along with a group of organisms from the Tenericutes family Acholeplasmataceae (Green et al., 2008). The Bacteroidetes also frequently feature in coal microbial assemblages, and while some taxa can be assigned to families such as the

D.J. Midgley et al. / International Journal of Coal Geology 82 (2010) 232–239

Porphyromonadaceae (Midgley DJ, unpublished data), many Bacteroidetes present come from as yet undescribed orders and families (Li et al., 2008; Shimizu et al., 2007; Strąpoć et al., 2008). In addition to bacterial taxa, coals are sometimes colonised by archaea. Generally, archaea in coal microbial assemblages are most commonly from the phylum Euryarchaeota (Green et al., 2008; Shimizu et al., 2007; Strąpoć et al., 2008), though Crenarchaeota have also been detected (Li et al., 2008; Shimizu et al., 2007). Like bacteria, the archaeal community diversity differs between sites. At one site in the Illinois Basin, for example, the family Methanocorpusculaceae were common (Strąpoć et al., 2008), while in the Powder River Basin, Methanosarcinaceae were abundant (Green et al., 2008). The most diverse archaeal community examined to date was detected in a deep coal associated aquifer in Japan where representatives of the Desulfurococcales, Methanobacteriales, Methanomicrobiales, Methanosarcinales, Thermoproteales orders and class Thermoplasmata were detected (Shimizu et al., 2007). 1.3. Geology of the sample site The Gippsland Basin is in southeast Victoria, Australia and is the largest known brown coal resource and petroleum province in the region. It is a passive margin basin formed during the Late Jurassic to Early Cretaceous break-up of Gondwana (Barton et al., 1995) and covers an area of ∼ 56,000 km2 (Holdgate et al., 2000). The major sedimentary sequences in the basin are the Strzelecki, Latrobe and Seaspray Groups as shown in the stratigraphic column proposed in (Gibson-Poole et al., 2008). Deposition of the Strzelecki Group began during the initial development of the basin and continued into the Middle Cretaceous. These sedimentary rocks include arkosic sandstones, mudstones and minor coal seams (Barton et al., 1995). Eocene to Oligocene sediments of the Latrobe Group were deposited under non-marine to fluvial-deltaic and marginal marine conditions, and consist of siliciclastic rocks, mudstones, carbonates and coals (Barton et al., 1995; Holdgate, 2005). The primary brown coal sequences, the Yallourn, Morwell and Traralgon Formations, are contained within the onshore sediments of the Latrobe Group (Barton et al., 1993). The Traralgon Formation comprises coarse-grained sandstones and conglomerates at the base, coals and shales in the middle, and, sandstones, shales, and minor coals near the top (Holdgate et al., 2000). The main coal seams of the Traralgon Formation are subdivided from the youngest, into the Traralgon 0 (T0), Traralgon 1 (T1) and Traralgon 2 (T2) seams, and the coals are ‘soft to hard brown coals’, classified as Lignite B within the American ASTM coal rank classification system (Holdgate et al., 2000). 1.4. Aims of the study Whilst some broad similarities between coal microbial communities are evident, it is also apparent that fine scale microbial community composition differs between sites. These differences may make optimising in situ methanogenesis challenging. Knowledge of the structure of coal microbial communities is thus critical in attempting to address this goal. The aim of the current study was therefore to examine the microbial community in a deep coal seam gas reservoir in the Gippsland Basin in southern Australia, and to examine the ability of a cultured microbial consortium from this community to degrade coal to methane. 2. Materials and methods

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as the upper seams of the Traralgon Formation (most likely T0) were intersected by the borehole (Fig. 1). The temperature of the water emerging from the pipe was ∼ 30 °C. One sample of approximately 600 ml of water was collected. The sample was collected after dewatering the hole to an approximate depth of 700 m. The water in this region was in contact with the formation as was evidenced by the congruence between the depth of the sample, the known depth of the coal seam and the presence of fine black material (assumed to be coal dust) in the collection (Fig. 1). The authors acknowledge both the potential for contamination along the borehole and from the lime- and mudstones that are directly above the coal formation, however, it is currently not possible to sample anaerobically at these depths without such limitations. The sample was collected in a sterile one litre bottle, immediately bubbled with high purity N2 (BOC, Australia), prior to the addition of 0.5 ml of both a 100 μM Na2S solution containing 0.1% resazurin, and 1.3 M cysteine HCl solution (all Sigma, Australia) to ensure the water remained oxygen-free. The sample was then transported to the laboratory at room temperature where they were transferred to an anaerobic glove box (Coy, Michigan, USA) with an atmosphere of 95% N2 and 5% H2 (BOC) prior to DNA extraction and culturing. Prior to culturing or DNA extraction, resazurin in the groundwater sample remained colourless indicating the oxygen-free status of the samples. The physicochemical characteristics of the groundwater (Table 1) were measured by Sydney Analytical Laboratories (Sydney, Australia). 2.2. Establishment of the methanogenic culture 2.2.1. Production of methane in mMSY medium Cultures were established in modified MSY (mMSY) liquid medium (Li et al., 2008) that contained (l− 1) 0.5 g yeast extract (Oxoid, Hampshire, UK); 0.4 g K2HPO4; 0.1 g NH4Cl; 0.1 g MgCl2.6H20; 1 ml of a 0.1% resazurin solution; 1 ml of SL-11 trace element solution (containing l− 1: 10 ml 25% HCl; 1.5 g FeCl2.4H2O; 0.1 g MnCl2.4H2O; 0.19 g CoCl2.6H2O; 70 mg ZnCl2; 36 mg NaMoO4.2H2O; 24 mg NiCl2. 2H2O; 10 mg AlKPO4; 6 mg H3BO3; 2 mg CuCl2.2H2O; 0.1 μg Na2SeO3) prior to autoclaving. After autoclaving the hot medium was transferred to the anaerobic glove box filled with a mixture of 95% N2 and 5% H2. After the medium had cooled to <50 °C, 1 ml of a filter sterile vitamin solution (containing l− 1: 10 mg pyridoxine HCl; 5 mg 4-aminobenzoic acid; 5 mg Ca pantothenate; 5 mg nicotinic acid; 5 mg riboflavin; 5 mg thiamine; 5 mg thioctic acid; 2 mg biotin; 2 mg folic acid and 0.1 mg B12), 1 ml of filter sterile 100 μM Na2S solution containing 0.1% resazurin and 0.5 ml of 1.3 M cysteine HCl solution were added, and the solution allowed to equilibrate for ∼2 h (all chemicals were from Sigma, except for K2HPO4, NH4Cl, MgCl2.6H20 supplied by Nuplex, New Zealand). The final medium pH was 6.8. Fifty milliliters of mMSY medium was then transferred asceptically to triplicate sterile, 120 ml serum vials (Crown Scientific, New South Wales, Australia) and inoculated with 1 ml of the BP sample under either a N2:H2 (19:1) or H2:CO2 (4:1) headspace gas mixture (BOC) to a final pressure of one atmosphere. The flasks were then sealed with butyl-rubber septa and aluminium crimps (Grace Davison Discovery Sciences, Illinois, USA) and removed from the anaerobic glove box. Cultures were inverted and incubated in the dark, shaking (50 rpm) at 30 °C. Culture vials were incubated in an inverted position to minimise loss of gases through the butyl-rubber septa. Gas chromatography (GC) measurements of methane were undertaken weekly for four weeks and the flasks re-gassed with either N2:H2 (19:1) or H2: CO2 (4:1) after sampling. Three flasks containing mMSY medium without inoculum were used as a negative control.

2.1. Sampling and physicochemical analyses The borehole from which the water samples were collected for the present study is located proximal to the current coastal margin, in the Sale–Yarram region. Interbedded limestones and mudstones, as well

2.2.2. Production of methane in carbon-free mMSY medium amended with gamma-sterile brown coal To determine methane generation from coal as the primary source of carbon, yeast extract, and cysteine HCl-free mMSY medium was

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Fig. 1. Schematic drawing of borehole showing sample locations and rock units that may have influenced water chemistry.

prepared and 50 ml aliquoted to 120 ml flasks as described above. To each flask 0.25 g of gamma sterilised Victorian (from the Port Phillip Basin, Bacchus Marsh region) brown coal was added (for coal characteristics see: Li et al., 2008). Flasks were sealed under a high purity N2 atmosphere (1 atm pressure), incubated, analysed by GC weekly for three weeks and re-gassed weekly, as described previously. 2.3. Gas sampling and chromotography Gas sampling was carried out inside the anaerobic glove box. Five ml gas samples were collected from sealed flasks via a gas-tight syringe. Samples were injected into a CP-3800 gas chromatograph

(GC) (Varian, Australia) equipped with a 2 m 1/8″ Haysep R 60/80 mesh packed column for the separation of hydrocarbons; this was connected in series by time switching to a 2 m 1/8″ Molsieve 5A 60/80 mesh packed column for the separation of permanent gases. Gases were detected using a two channel detector system combining a thermal conductivity detector and a flame ionisation detector. The electronic pressure control was set to 48 psi which equates to a column flow of 100 ml min− 1. After injection into the 250 μl sample loop, CO2 and C2–C6 hydrocarbons were separated on the Haysep R column. H2, O2/Ar, N2, methane and carbon monoxide were not retained and passed directly onto the Molsieve column where they were trapped and isolated at 1.7 min. At 5.5 min the contents of the Molsieve column

D.J. Midgley et al. / International Journal of Coal Geology 82 (2010) 232–239 Table 1 Physicochemical characteristics of the Basepipe (BP) sample.

pH EC Na K Ca Mg CaCO3 NH3 NO3 Total N PO4 Total P OCc a b c

BP (mg L− 1)

Uncertainty (%)

7.9a 2400.0b 480.0 48.0 9.0 4.4 520.0 38.0 <0.1 39.0 <0.1 0.37 235.0

6.2 6.1 6.7 10.4 8.3 7.4 12.4 17.3 19.7 14.2 15.7 13.2 5.8

pH units. Value is µS cm− 1. OC = organic carbon.

were put back in series with the Haysep R column together with the rest of the hydrocarbon gases. The temperature program had an initial temperature of 80 °C for 10 min followed by heating at 15 °C min− 1 to 200 °C (5 min hold). The GC was calibrated using a three point calibration using standard gas mixtures (BOC) with methane concentrations of 20.5 ppm, 2010 ppm, and 20,000 ppm. Sample methane concentration was calculated from the FID channel responses using the Varian Star software (vers. 6.20). 2.4. DNA extractions Five hundred milliliters of the sample was filtered through a Durapore® 0.22 μm filter (Millipore, Massachusetts, USA). The filter was subsequently divided into three sections, each of which was subjected to DNA extraction using the FastDNA® SPIN Kit for Soil (Bio101, California, USA) following the manufacturer's instructions.

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threshold of 95%. Assignment to domain, phylum, class, order and family level with 90% support or greater were considered to be acceptable, assignments with lower than 90% support were not reported (Wang et al., 2007). All sequences were submitted to the GenBank nucleotide database (GU060355–GU060372). It is noteworthy that the current study utilise the ARDRA technique to profile the microbial community. This method, like all molecular methods, has inherent biases in that: not all DNA is equally easily extracted from cells; all PCR primers have some bias towards certain phylogenetic groups and; the copy number of the 16S gene in bacteria and archaea varies between taxa from 1 to ∼20. Despite these limitations, ARDRA is a robust and widely used method for examining microbial communities. 2.6. Statistical analyses EstimateS version 8.0.0 (Colwell, 1997) was used to calculate Chao1 and Chao2 species richness estimates. The presence or absence of chimeric sequences was determined manually by alignment with closely allied sequences from GenBank. Methane generation data were tested for normality and equality of variance (Dytham, 2003). Significant differences between methane concentrations each week were determined using one-way ANOVA and where significant (P < 0.05) differences were observed Student–Newman–Keuls adjusted pairwise comparisons were conducted in SigmaPlot version 11.0. 3. Results 3.1. ARDRA Eighteen (18) unique RFLP-types were detected in the BP sample (Fig. 2, Table 2) from 95 clones examined. Chao 1 and Chao 2 diversity indices for the BP sample were 52 and 70, respectively, (with a lower and upper CI of 39-135). This suggested that significant diversity was

2.5. Amplified ribosomal DNA restriction analyses (ARDRA) Each sample was divided into three subsamples for DNA extraction. From each subsample a ∼1000 bp region of the 16S rDNA was amplified in reaction volumes containing 12.5 μl of GoTaq® Green Master Mix (Promega, Wisconsin, USA), 25 pmol (each) of universal 16S rDNA primers 533f (5′-GTGCCAGCMGCCGCGGTAA-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′), 1 μl of extracted DNA (∼10 ng) and filter sterile MilliQ® water (Millipore) to a total volume of 25 μl. Amplifications were performed in a MyCycler® Thermal Cycler (Bio-Rad Laboratories, Inc., California, USA) with a 4 min melt at 94 °C, preceding 25 cycles of 94 °C for 45 s, 53 °C for 45 s and 72 °C for 45 s, followed by an 8 min extension at 72 °C. The resulting amplicon was then diluted 1:10 prior to ligation and cloning using the StrataClone™ PCR Cloning Kit (Stratagene, California, USA). Ninety five clones (BP) were subjected to colony PCR using the protocol described above, however, the M13f (5′-GTAAAACGACGGCCAGT-3′), M13r (5′-GGAAACAGCTATGACCA-3′) primers were substituted for 533f/1492r. The resultant amplicon was then digested overnight with HaeIII (New England Biolabs, Massachusetts, USA) run on the MultiNA electrophoresis system (Shimadzu Biotech, Kyoto Prefecture, Japan) and sorted into RFLP-types. One representative clone was chosen at random for each RFLP-type and subjected to DNA sequencing by Macrogen Inc. (Seoul, South Korea). Sequences were aligned using Vector NTI (Invitrogen, California, USA) and then used as queries for BLASTn searches (http://blast.ncbi.nlm.nih.gov/) of the GenBank nucleotide database. Family affinity was determined using the Ribosomal Database Project (RDP) classifier, using a confidence

Fig. 2. Pie charts describing the 16S composition of the Basepipe (BP) sample n = 95. It is noteworthy that 16S composition is only representative of relative microbial community composition if all taxa have an equal number of copies of the 16S gene.

236 Table 2 The number of clones of each RFLP-type detected in the Basepipe (BP) sample, the closest two matches, and origin of these matches, from BLASTn queries between 16S rDNA sequences and the GenBank Nucleotide Database for each RFLPtype, and taxonomic affinity. Closest BLAST matches (all* 99–100%)

Origin of closest matches

Affinity

EF600586 Uncultured bacterium E2-22 AF453507 Uncultured bacterium pKD EU366499 Methanobacterium sp. TS2 DQ649335 Methanobacterium alcaliphilum DSM3387 AB294285 Uncultured bacterium YWB16 EF632886 Uncultured bacterium Par-s-66 DQ643978 Geosporobacter subterrenus VNs68* 98% EU443727 Thermotalea metallivorans B2-1 AY566219 Bacillus sp. BL8 EU816691 Geobacillus pallidus LK5 Identical matches (99% ID/100% Query) to many Shigella and Escherichia species making further identification difficult. EU491780 Uncultured bacterium EPR3967-O2-Bc31 DQ129577 Uncultured bacterium AKIW983 FJ984446 Ralstonia sp. BEB51 † FJ984435 Ralstonia solanacearum EU804113 Uncultured bacterium 6C232005 EU927145 Stenotrophomonas maltophilia G2 AJ302088 Halomonas sp. IB-I6 EU440679 Uncultured Halomonas sp. Plot17-H04 DQ643978 Geosporobacter subterrenus VNs68* 98% EU443727 Thermotalea metallivorans B2-1 EU234238 Uncultured bacterium C13 NR_025407 Desulfomicrobium norvegicum DSM 1741 FJ193019 Uncultured Delftia sp. GI5-002-F11 FJ192434 Uncultured Delftia sp. GI5-13-D07 AB237707 Uncultured bacterium HDBW-WB44 DQ394985 Uncultured bacterium VHS-B4-21 AY692042 Uncultured Desulfuromonas sp. M76 AB294283 Uncultured bacterium YWB14 AY362360 Desulfovibrio vulgaris I5 CP000527 Desulfovibrio vulgaris subsp. vulgaris DP4 DQ296472 Uncultured bacterium AR-89 AY214180 Uncultured bacterium clone ZZ12C4 DQ069225 Uncultured delta proteobacterium SRB9 CP001197 Desulfovibrio vulgaris str. ‘Miyazaki F’

Leaf litter Thermal spring Lake Tuosu deposit High pH anaerobic environments Deep coal seam groundwater Freshwater sediment Deep subsurface aquifer Great Artesian Basin (aquifer) Deep sea hydrothermal Sewerage – – Seafloor lava Urban aerosol Banana roots Banana roots Deep sea seawater Unknown Unknown Agricultural soil Deep subsurface aquifer Great Artesian Basin (aquifer) Production wastewater Oslo Harbour water Spacecraft assembly clean room Spacecraft assembly clean room Deep subsurface groundwater Harbour sediment Upflow anaerobic sludge blanket digester Deep coal seam groundwater Deep African gold mine Heavy metal-impacted lake sediment Quinoline degrading biofilm Benzene-contaminated groundwater Deep African gold mine Fresh water, Salt marsh, Soil

Bacteria; Proteobacteria; β proteobacteria; Burkholderiales; Burkholderiaceae Archaea; Euryarchaeota; Methanobacteria; Methanobacteriales; Methanobacteriaceae Bacteria Proteobacteria; δ proteobacteria; Desulfuromonales; Geobacteraceae Bacteria; Firmicutes; Clostridia; Clostridiales; Clostridiaceae Bacteria; Firmicutes; “Bacilli”; Bacillales; Bacillaceae; “Bacillaceae 1” Bacteria; Proteobacteria; γ proteobacteria Enterobacteriales; Enterobacteriaceae Bacteria; Actinobacteria; Actinobacteria; Actinomycetales; Micrococcineae; Micrococcaceae Bacteria; Proteobacteria; β proteobacteria; Burkholderiales; Burkholderiaceae Bacteria; Proteobacteria; γ proteobacteria; Xanthomonadales; Xanthomonadaceae Bacteria; Proteobacteria; γ proteobacteria; Oceanospirillales; Halomonadaceae Bacteria; Firmicutes; Clostridia; Clostridiales; Clostridiaceae Bacteria; Proteobacteria; δ proteobacteria; Desulfovibrionales; Desulfomicrobiaceae Bacteria; Proteobacteria; β proteobacteria; Burkholderiales; Comamonadaceae Bacteria; Firmicutes; Clostridia; Clostridiales

BP 2 (GU060356) BP 3 (GU060357) BP 4 (GU060358) BP 5 (GU060359) BP 6 (GU060360) BP 7 (GU060361) BP 8 (GU060362) BP 9 (GU060363) BP 10 (GU060364) BP 11 (GU060365) BP 12 (GU060366) BP 13 (GU060367) BP 14 (GU060368) BP 15 (GU060369) BP 16 (GU060370) BP 17 (GU060371) BP 18 (GU060372)

† These sequences were only partial reads ∼300–650 bp in length.

Bacteria Proteobacteria; δ proteobacteria; Desulfuromonales; Desulfuromonaceae Bacteria; Proteobacteria; δ proteobacteria; Desulfovibrionales; Desulfovibrionaceae Bacteria; Firmicutes; Clostridia; Clostridiales; Veillonellaceae Bacteria; Proteobacteria; δ proteobacteria; Desulfovibrionales; Desulfovibrionaceae

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RFLP accession no. BP 1 (GU060355)

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yet to be detected in the BP sample. The 16S clones from the BP sample were dominated by three RFLP-types. RFLP-type BP 4 represented >40% of clones examined, while two other RFLP-types (BP 3 and 15) were present at relative abundances > 10%.

and hydrogen, the quantity of methane produced did not differ from the negative control (data not shown).

3.2. DNA sequences from BP sample

This the first report of methanogenic Archaea in formation water associated with a Australian CSM reservoir. Methanogenic archaea have been previously observed in coal associated formation waters in Japan and USA (Green et al., 2008; Shimizu et al., 2007; Strąpoć et al., 2008) and the current study extends that range to include the Australian continent. The methanogen detected in this study was likely to be a Methanobacterium species. Assuming this methanogen is a Methanobacterium species, RFLP-type BP 2 is likely to reduce CO2 to methane using H2 as a source of electrons and protons. It is noteworthy that Li et al. (2008) did detect an Archaeologlobus species in a study on the microbial communities in Australian coal and coal formation waters. This taxon, however, produces very little methane and primarily uses sulphite, sulphate or thiosulfate as terminal electron acceptors (Garrity and Holt, 2001). This lack of genuine methanogens was intriguing as a geographically diverse range of both formation water and coal samples were examined. It seems likely that the differences in archaeal diversity discovered in Li et al. (2008) and the present study were due to differences in methods; firstly, Li et al. (2008) examined relatively few clones per sample, and these clones were chosen at random. Secondly, there may be bias in the A751F/UA1406R primer pair (Baker et al., 2003), used by Li et al. (2008), which may preferentially amplify the euryarchaeal classes Methanococci, Thermoplasmatales and Archaeoglobi to the detriment of other taxonomic groups. Data presented here demonstrate that bacterial communities in Australian coal formation waters are diverse. In a single site, three known bacterial phyla: Firmicutes, Proteobacteria and Actinobactera, were observed. This diversity was broadly similar to that observed by Li et al. (2008), where in numerous formation water samples bacteria from three phyla: Proteobacteria, Firmicutes and Bacteroidetes were observed. Li et al. (2008) also observed an actinobacterial taxon, however, this occurred in coal and not formation water. In the current study, proteobacterial lineages were abundant in the BP sample. It is noteworthy that the coal in this study was marineinfluenced and contained significant (0.5–4%) sulphur (Holdgate et al., 2000). This observation may explain the abundance of deltaproteobacterial RFLP-types, possibly involved in sulphur cycling, that occur in the BP sample. These Deltaproteobacteria were from the families Desulfuromonaceae, Geobacteraceae and Desulfovibrionaceae. Those RFLP-types with affinities to the Desulfuromonaceae (BP

All sequences examined from the BP sample were of microbial 16S rDNA sequence, chimeric sequences were not detected. RFLP-types from proteobacterial lineages (BP 1, 3, 6, 8, 9, 10, 12, 13, 15, 16, 17 and 19) were the most frequently observed. The majority of proteobacterial RFLP-types were from the deltaproteobacterial families Geobacteraceae (BP 3), Desulfomicrobiaceae (BP 12), Desulfuromonaceae (BP 15 and 16) and the Desulfovibrionaceae (BP 17 and 19), while members of the betaproteobacterial families Burkolderiaceae (BP1 and BP8) along with the Comamonadaceae (BP 13) were also observed. Three RFLP-types (BP 6, 9 and 10) with gammaproteobacterial affinities were also detected (Table 2); these three RFLP-types are likely members of the families Enterobacteriaceae, Xanthomonadaceae and Halomonadaceae, respectively (Table 2). In addition to the Proteobacteria, numerous Firmicutes RFLP-types were observed, these included the abundant RFLP-type BP 4 (Fig. 2). RFLP-type BP 4 is likely to be a member of the Clostridaceae family and its closest match was to Geosporobacter subterraneus (DQ643978) from a deep (800 m) subsurface aquifer (Klouche et al., 2007). Other Firmicutes were also observed including organisms with affinities to the Veillonellaceae (BP 18) and a putatively novel family (BP 14). In addition, one actinobacterial RFLP-type (BP 7) was also observed and was likely a member of the Actinomycetales family Micrococcaceae. In addition to bacterial sequences, one RFLP-type (BP 2) was detected from the domain Archaea (Table 2). This archaeal taxa was from the family Methanobacteriaceae and was likely a Methanobacterium species, whose closest relative seemed to be M. alcaliphilum. 3.3. Methanogenic activity of cultures Cultures derived from the BP samples in mMSY medium produced significant quantities of methane from the starting headspace gas mixtures and yeast extract (Fig. 3). In all treatments in mMSY medium, methane levels increased significantly (P < 0.05) after one week. The maximum weekly methane produced by the cultures were 42,000 ppm (±7000) (Fig. 3). Unsurprisingly, methanogenesis was greatest when the H2:CO2 (4:1) headspace gas mixture was applied (Fig. 3), indicating direct use of H2/CO2 by methanogens present. When Victorian brown coal was supplied as a sole source of carbon

4. Discussion

Fig. 3. Mean methane generation (± standard error) per week by the methanogenic cultures derived from the BP sample. Plots 1: mMSY medium under H2:CO2† (4:1) headspace gas mixture inoculated with BP groundwater. Plots 2: mMSY medium under N2:H2 (19:1) headspace gas mixture inoculated with the BP groundwater. Scales differ between plots. Within a plot different letters on columns indicate significant differences (P < 0.05) as determined by ANOVA and SNK-adjusted pairwise comparisons. The mean weekly quantity of methane in the negative controls was 105.7, 49.5, 347.6 and 396.9 PPM. †Significant reductions in pressure were observed in this treatment and thus reported concentrations of methane are overestimations of the concentration in this treatment.

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15 and 16) were presumably, like their cultured relatives, strict anaerobes that most commonly use sulphur or ferric iron as a terminal electron acceptor. Desulfuromonaceae are not commonly detected in coals or coal formation waters, though they were previously detected in a deep CSM reservoir in Japan (Shimizu et al., 2007). All cultured members of the Geobacteraceae exclusively use ferric iron as a terminal electron acceptor and RFLP-type BP 3 probably behaves in a similar fashion in situ. Geobacteraceae are relatively common coal associated microbes and have been previously detected in Australia, Japan and North America (Li et al., 2008; Penner et al., 2008; Shimizu et al., 2007), their occurrence in the present study further extends their ubiquity in these environments. The families Desulfovibrionaceae are similar, being most commonly involved in sulphate reduction to sulphide or H2S, or rarely, fermentative in their metabolism (Kuever et al., 2001; Kuever et al., 2005). Interestingly, some fermentative Desulfovibrio species produce H2 and may be involved in syntrophic relationships with other bacteria (Richardson et al., 2002) and related RFLP-types in the present study may be involved in similar roles in coal degradative pathways. In addition to Deltaproteobacteria, a small number of Betaproteobacteria were also detected in the present study. Previous studies have detected a range of Betaproteobacteria in coal or coal associated water. Most commonly, these Betaproteobacteria were from the family Comamonadaceae (Li et al., 2008; Penner et al., 2008; Shimizu et al., 2007). The detection of comamonads in the present study, in both the MP and BP samples, further supports the contention that this family is ubiquitous in coal associated formation water. Despite their abundance, and the readily culturable nature of some comamonads (Wen et al., 1999), those that occur in coal appear to be recalcitrant to pure culture and do not appear to be recovered in isolated enrichment cultures from coal formation water (Li et al., 2008; Strąpoć et al., 2008). Unusually, the BP sample in the present study also included members of the family Burkolderiaceae (BP 1 and BP8); this family has not been previously reported from coals or coal formation water. Based on their 16S sequences, these two RFLP-types may be Ralstonia species. If this classification is correct, these RFLP-types were unusual as most cultured members of this genus are aerobes (Yabuuchi et al., 2001) although, one species, Ralstonia eutropha, can grow anaerobically, oxidising H2 using NO− 3 as a terminal electron acceptor (Schwartz and Friedrich, 2001). Ralstonia species have also been implicated in the degradation of phenolic compounds (Agarry et al., 2008), and BP 1 and BP 8 in the present study may have similar catabolic potential. Numerous gammaproteobacterial lineages were detected in the current study from the families: Enterobacteriaceae, Halomonadaceae and Xanthomonadaceae. Enterobacteriaceae species closely related to the genera Shigella and Escherichia are commonly found in coal and coal associated formation waters (Li et al., 2008; Penner et al., 2008). Many Enterobacteriaceae species are facultative anaerobes, though in these environments they are likely to be fermentative in their metabolism. One Enterobacteriaceae RFLP-type was detected in the current study and occurred in both the MP and BP samples. This RFLPtype may be involved in the fermentation of simple compounds to H2 and CO2. Halomonads are also relatively common in the coal environment, having been detected in coal or coal associated formation water in Australia (Li et al., 2008. and the present study) and in North America (Penner et al., 2008). Like Enterobacteriaceae species, some halomonads are fermentative, while others reduce NO− 3 to NO− 2 . The roles of halomonads in coal degradative pathways are unknown, though they may contribute to H2 production via fermentation. Unusually, one RFLP-type in the current study was from the family Xanthomondaceae. Members of this family have not been previously observed in coal or formation water and for the most part are regarded as strict aerobes. The means by which this taxon survives in this environment are unclear; however, the family may be more physiologically diverse than has been reported to date.

Along with Proteobacteria, a range of Firmicutes were observed in BP sample from the families (or candidate groupings sensu Ludwig et al., 2008); Bacillaceae 1, Clostridaceae (sensu stricta) and Veillonellaceae. In addition, several RFLP-types, likely to be novel at the level of family (BP 14), could not be assigned to known taxonomic lineages. As a phylum, Firmicutes are common members of coal or coal formation water communities and have been detected in Australia, Japan, Canada and the USA previously (Green et al., 2008; Li et al., 2008; Penner et al., 2008; Shimizu et al., 2007; Strąpoć et al., 2008). In Australia, numerous clostridial families including Clostridiaceae, Eubacteriaceae, Peptostreptococcaceae, “Incertae Sedis XI” and “Incertae Sedis XII” along with the Bacillales grouping “Bacillus j” were detected by Li and co-workers in 2008. Similarly, most of these families also occurred in a deep coal seam aquifer in northern Japan where several other families, the Peptococcaceae, Ruminococcaceae, Syntrophomonadaceae and Veillonellaceae, were also detected (Shimizu et al., 2007). Intriguingly, the Firmicute diversity seems much lower in the North American coals, for example in the Powder River Basin only Clostridaceae and “Incertae Sedis XII” were observed (Green et al., 2008). Similarly, only one unknown Firmicutes could be detected from the Illinois Basin in the USA (Strąpoć et al., 2008). The current study confirms the ubiquity of the Bacillaceae 1, Clostridiaceae and Veillonellaceae in these environments. In addition to the Proteobacteria and Firmicutes the Actinobacteria were present at much lower abundance, being represented by just a single clone in this sample. Actinobacterial taxa have been reported previously in the coal and coal associated formation water in Canada and Australia (Li et al., 2008; Penner et al., 2008). To date, all actinobacterial taxa in coal belong to the suborder Micrococcineae in the order Actinomycetales, and those identified in the current study are also part of this group, though there relatedness to each other remains unknown. Some related Micrococcineae are able to grow anaerobically reducing selenate to elemental selenium (von Wintzingerode et al., 2001) and Micrococcinaeae in coal microbial assemblages may also be involved in reduction of metals or metalloids. It is noteworthy, that ARDRA does not distinguish active members of the microbial community from inactive or dormant cells. The community structure presented here may therefore not necessarily be indicative of the structure and activity present in situ. Other techniques, such as stable isotope probing, combined with ARDRA may better inform future studies of these environments, though there are technical challenges with manipulating the environment at 700 m subsurface. Furthermore, our rarefraction analyses indicated that only a relatively small portion of the BP sample was observed in this study. While this indicates significant diversity is yet to be characterised, it also demonstrates that coal seam methane reservoirs are highly diverse microbial communities. Interestingly the cultures derived from the BP water sample readily produced methane from yeast extract but were unable to produce methane on brown coal as a sole source of carbon and hydrogen. This presumably reflects either specificity between the coal and its associated microbial community, or the absence of some key member of the community in culture required to degrade coal or a deficiency in the medium used in the present study. Specificity between coal derived microbial consortia and their source of coal has been demonstrated to effect methanogenesis in laboratory experiments (Midgley DJ, unpublished data) and may be the cause of the failure of this consortium to produce methane on a foreign, gammasterile coal obtained from a site ∼ 250 km distant. Based on the N2:H2 (19:1) gas mixture provided to the culture, approximately 25 000 ppm of methane could be produced if all of the 5% hydrogen was converted to methane. It is noteworthy, however, that the maximum weekly methane concentration produced exceeded this figure by 17,000 ppm. This indicates that at least some yeast extract, and possibly cysteine, is being fermented and the products of this fermentation converted to methane via methanogensis.

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5. Conclusions ARDRA revealed that that the microbial communities in the BP sample included numerous bacterial taxa and a methanogenic archaeal lineage. Like previously described coal seam methane reservoirs, the bacterial communities were dominated by proteobacterial and Firmicutes taxa, though one less common phylum, the Actinobacteria was also detected. Prior to this study, no methanogenic archaea had been reported from Australian CSM reservoirs. In the present study a methanogen, most likely a Methanobacterium species, was observed. Cultures derived from the groundwater sample produced significant quantities of methane from yeast extract, indicating the presence of at least one methanogenic archaeal taxon and syntrophic production of H2, CO2, acetate or some other simple substrate for methanogenesis by bacteria in these cultures. These cultured consortia were unable to produce methane using gamma-sterile brown coal as a sole source of carbon and hydrogen and this may indicate some specificity between coal and isolated coal-degrading consortia. If locally sourced microbial communities are required to gasify coals of various compositions, then improved understanding of microbial ecology and microbial physiology of coal associated microbes is critical to the goal of microbially enhanced coal seam methane production. Acknowledgments The authors thank Mr. Lance Smith of Sydney Analytical Laboratories for his assistance in chemically analysing the water samples. We also thank Drs. Stephen Sestak for technical assistance with gas equipment and Drs. Dongmei Li and Neil Sherwood for critical comments on the manuscript. References Agarry, S.E., Durojaiye, A.O., Solomon, B.O., 2008. Microbial degradation of phenols: a review. International Journal of Environment and Pollution 32, 12–28. Baker, G.C., Smith, J.J., Cowan, D.A., 2003. Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods 55, 541–555. Barton, C.M., Gloe, C.S., Holdgate, G.R., 1993. Latrobe Valley, Victoria, Australia: a world class brown coal deposit. International Journal of Coal Geology 23, 193–213. Barton, C.M., Bolger, P.F., Holdgate, G.R., Thompson, B.R., Webster, R.L., 1995. In: Ward, C.R., Harrington, H.J., Mallett, C.W., Beeston, J.W. (Eds.), Gippsland Basin. In Geology of Australian Coal Basins. Geological Society of Australia Incorporated Coal Group Special Publication, pp. 541–549. Colwell, R.K., 1997. EstimateS: statistical estimation of species richness and shared species from samples. Version 8.0.0. User's Guide and application published at http://purl.oclc.org/estimates, 1997. Damen, K., Faaij, A., van Bergen, F., Gale, J., Lysen, E., 2005. Identification of early opportunities for CO2 sequestration—worldwide screening for CO2-EOR and CO2-ECBM projects. Energy 30, 1931–1952. Dresselhaus, M.S., Thomas, I.L., 2001. Alternative energy technologies. Nature 414, 332–337. Dytham, C., 2003. Choosing and Using Statistics: A Biologist's Guide, 2nd edn. Wiley Blackwell, London. Faison, B.D., 1993. In: Crawford, D.L. (Ed.), The Chemistry of Low Rank Coal and Its Relationship to the Biochemical Mechanisms of Coal Biotransformation. In Microbial Transformations of Low Rank Coals. CRC Press, Boca Raton, pp. 2–24. Faiz, M., Hendry, P., 2006. Significance of microbial activity in Australian coal bed methane reservoirs — a review. Bulletin of Canadian Petroleum Geology 54, 261–272.

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