International Journal of Coal Geology 137 (2015) 66–76

Contents lists available at ScienceDirect

International Journal of Coal Geology

j ourna l homepage: www.e lsev ie r .com/ locate / i j coa lgeo

Temporal changes in microbial community composition during cultureenrichment experiments with Indonesian coals

Rita Susilawati a,b,⁎, Paul N. Evans c, Joan S. Esterle a, Steven J. Robbins c, Gene W. Tyson c,Suzanne D. Golding a, Tennille E. Mares a

a School of Earth Sciences, The University of Queensland, St Lucia, Queensland 4072, Australiab Geological Agency of Indonesia, Jl. Sukarno Hatta no 444, Bandung, West Java 40254, Indonesiac Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia

⁎ Corresponding author at: School of Earth Sciences, TLucia, Queensland 4072, Australia.

http://dx.doi.org/10.1016/j.coal.2014.10.0150166-5162/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 July 2014Received in revised form 30 October 2014Accepted 30 October 2014Available online 7 November 2014

Keywords:CoalMicrobial communityMethanogenesisCoalbed methane

Temporal changes in microbial community structures during methanogenesis were investigated in cultures ofSouth Sumatra Basin (SSB) coalbed methane (CBM) formation water (SSB5) grown on three coals of differentrank (Burung sub bituminous Rv 0.39%, Mangus sub bituminous Rv 0.5%, Mangus anthracite Rv 2.2%). Methaneproduction accelerated from day 6, peaked around day 17 and then levelled off around day 20. The initial bacte-rial community from the SSB formation water was predominantly Acetobacterium, Acidaminobacter, Bacteroidesand Pelobacter species, while the archaeal community consisted of Methanosaeta, Methanosarcina andMethanobacterium members. A general pattern was observed in all cultures with the three coals. Over time thebacterial members decreased in proportion whereas the archaeal component increased. The increase in the pro-portion of archaeal methanogens corresponded with an increase in methane production yield.Enrichment cultures produced similar communities when grown on coals from the same seam (Mangus subbituminous and Mangus anthracite), rather than from different seams of similar type (Burung sub bituminousand Mangus sub bituminous). Methanosaeta was the dominant methanogen species in the sub bituminousBurung coal culture, but was a lesser proportion in cultures of both Mangus sub bituminous and anthracitecoals where Methanosarcina species were a greater proportion. Interestingly, obligate hydrogenotrophicmethanogens from the genera ofMethanobacterium, which were present at low levels in culture enrichment ofall coal substrates, increased in proportion only in the absence of coal in the no-coal control enrichment cultures.These results suggest that the low rank Burung sub bituminous coal favours methane production by the obligateacetoclasticMethanosaetamembers while both Mangus coals also favour metabolically versatileMethanosarcinamembers, and the absence of coal favours hydrogenotrophicmethanogens. Despite the similarity of communitiesgrown on coals from the same seam, greater quantities of methane were generated from the lower rank coalswhen compared to higher rank coals.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Once considered to be an unconventional energy source, coalbedmethane (CBM) has become a significant energy resource in manycountries (Moore, 2012; Strąpoć et al., 2011). Over the last decade,the focus of CBM research has shifted from identifying and extractingthe resource to replenishing the methane by stimulating the nativemicrobial community to form methane in situ by degrading thedeep coal, effectively turning coal seams into real-time methanebioreactors. The potential for coal as a methane-forming bioreactorhas been assessed in several coal basins worldwide (Fry et al.,2009; Green et al., 2008; Guo et al., 2012; McIntosh et al., 2008;

he University of Queensland, St

Papendick et al., 2011; Penner et al., 2010; Singh et al., 2012;Strąpoć et al., 2008).

Methane in coal seams can be produced as a by-product of thethermal coalification process (thermogenic) and by methanogenicactivity (biogenic). Isotope analysis of the methane extracted fromsome CBM reservoirs has revealed biogenic origins, and have specu-lated that real time methane could be formed from the in situ coal inthose reservoirs (Butland and Moore, 2008; Faiz and Hendry, 2006;Flores et al., 2008; Golding et al., 2013; Hamilton et al., 2014;Kinnon et al., 2010; Mares and Moore, 2008; Strąpoć et al., 2007;Susilawati et al., 2013). To this end, several laboratory based studiesof the culture-dependant and independent methods have shown thepresence of active microbial consortia able to form methane fromcoal in a number of CBM reservoirs (Guo et al., 2012; McIntoshet al., 2008; Papendick et al., 2011; Penner et al., 2010; Singh et al.,2012; Strąpoć et al., 2008). These results suggest that the potential

67R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

exists to develop new and renewable biogenic gas resources fromdeep coal seams.

Despite microorganisms being present in coal and its associatedformation waters, the ability of the microbial community to decon-struct the coal is still poorly understood due to the heterogeneous-ly complex physical and chemical structures of the coal and to thelimited number of studies that identify coal degrading microbialspecies (Faison, 1991; Fakoussa and Hofrichter, 1999; Strąpoćet al., 2011). Consequently, the mechanism in which the microbialconsortium degrades the complex organic substrate into methanefrom coal remains unknown. A limited number of studies haveshown coal degradation begins with hydrolysis of polyaromatic,aromatic and other hydrocarbon substrates into compounds suchas volatile fatty acids (VFAs), alcohols and other organic acids,which in turn are formed into methanogenic substrates such as ac-etate, hydrogen/carbon dioxide and methanol (Jones et al., 2010;Strąpoć et al., 2011). Bacterial species from the genera Bacteroides,Pelobacter, Clostridium and Spirochaetes detected in coal and asso-ciated formation waters have been implicated in hydrolysis ofhydrocarbons (Aklujkar et al., 2012; Kudo et al., 1987; Murray,1986; Sträuber et al., 2012). However, despite being implicated inhydrocarbon degradation from CBM wells, those bacterial speciesare often found to be fermenters upon closer examination(Wawrik et al., 2014). In CBM reservoirs, those bacteria are typical-ly found associated with hydrogenotrophic (e.g.Methanobacteriumand Methanocorpusculum spp.), acetoclastic (e.g.Methanosaeta andMethanosarcina spp.) and methylotrophic methanogens (e.g.Methanolobus spp.) (Doerfert et al., 2009; Ghosh et al., 2014;Green et al., 2008; Midgley et al., 2010; Penner et al., 2010; Singhet al., 2012; Strąpoć et al., 2008; Tang et al., 2012).

Although broad similarities in the structure of the microbial consor-tia exist, each CBM reservoir site has a unique community structure thatdiffers with respect to its primary community members and theirdominance. Despite the broad understanding of the communities, onlya few studies have reported the influence of differing coal substrateson methane production from native coalbed microbial communities(Fallgren et al., 2013a; Jones et al., 2008; Susilawati et al., 2013). Tothis end, we investigated themicrobial communities present in a previ-ously unstudied South Sumatra Basin (SSB) CBM reservoir and the tem-poral changes in its microbial community structure during growth onthree native SSB coals of different rank.

2. Geology of the South Sumatra Basin

The geology of the SSB has been outlined by de Coster (1974),Darman and Sidi (2000) and Bishop (2001). The widespread accumula-tion of peatwas transformed into the coals of the SSB during the regres-sive phase of the Neogene depositional cycle (de Coster, 1974). A majorregressive sequence of Late Miocene–Pliocene sediments were deposit-ed as the Barisan mountains uplifted resulting in the deposition of theMuara Enim Formation, the main coal bearing unit in the SSB(Koesoemadinata, 2000). During the deposition of the Muara EnimFormation, the tectonic setting of the SSB produced a laterally extensivecoal in respect of thickness, mineral matter and split pattern, caused byexceptionally consistent subsidence coupled with the right type of cli-mate (Stalder, 1976). Coalification in the Muara Enim Formation on aregional scale was controlled predominantly by burial depth variationsas the geothermal gradientwas relatively constant throughout thebasin(Stalder, 1976). The higher gradient encountered in some areas resultsfrom the localised effects of igneous intrusions (Amijaya and Littke,2006; Moore et al., 2014; Susilawati and Ward, 2006).

Coal of theMuara Enim Formation has been exploited for decades atthe Bukit Assam field, where the threemain coal seams (Mangus, Subanand Petai) have an average aggregate thickness from30 to 40m. Severalminor seams (eg. Benuang and Burung), lenticular shales, sandstones,

carbonaceousmudstones, and tuffaceousmarker horizons are interbed-ded within these coals (Fig. 1).

3. Indonesian CBM

Indonesia is only just beginning to develop its CBM resources, andthe SSB has been highlighted as the most promising CBM basin inIndonesia (Fig. 1), estimated to hold around 183 Tcf of CBM resources(Stevens and Hadiyanto, 2004). The coal in the SSB ranges from indepth 300–1000 m and is mostly sub bituminous (Rv 0.4–0.5%) inrank with high vitrinite contents (N90%) and extremely low ash yields(b5%). Most Indonesian low rank coals are composed of hydrogen richorganic components (Davis et al., 2007) that are thought to makethem excellent substrates for biogenic methane generation.

To date, only two studies have reported the potential for biogenicCBM in Indonesia coal basins. Gas isotopic analysis and preliminaryenrichment work by Susilawati et al. (2013) identified a biogenic originfor CBM in the SSB, and highlighted the potential of its coal and forma-tion water for future natural gas regeneration. Additionally Fallgrenet al. (2013b), demonstrated methane production from an IndonesiaSouth Sumatra lignite and formation water for which there is no previ-ous history of gas production. To date, no study has reported themicro-bial methanogen community structure in SSB CBM reservoirs.

4. Materials and methods

4.1. Coal sampling and analysis

Collection of coal directly fromproducing CBM reservoirs was unfor-tunately not possible, at such, coal was sampled inMay 2012 at PT BukitAsam, a nearby coal mine in Tanjung Enim SSB. Three Bukit Asam coalsrepresenting different coal seams; Burung sub-bituminous (Burung SB),Mangus sub-bituminous (Mangus SB) and Mangus anthracite (MangusA) were used in this study (Fig. 1, Table 1). The rank of the twoManguscoals differed due to the effects of an igneous intrusion into the coalseam. Coal samples were collected from the inner side of a freshminingface after digging out about 10 cm of surface coal to minimize thechance of samples being oxidised. After coal lithotype description,fresh samples were wrapped with aluminium foil and plastic film,placed in a box and stored at 4 °C for one week before being shippedto Australia for culture enrichment purposes. In the laboratory, coalsamples were stored in an anaerobic chamber, to avoid further oxida-tion processes. Time from collection to anaerobic chamber storage wasless than 2 weeks.

The outer layer of the coal was pared away before the coal wascrushed in the anaerobic chamber using a mortar and pestle, milledusing an electric coffee grinder and sieved to collect the 220 to350 μm fractions. Subsequent petrographic analysis did not showany identifiable oxidation. The crushed coal was then split into twoparts using a riffle splitter, for chemical–physical analysis, and forenrichment studies (used as sole carbon and energy substrate). ALSLaboratory, Queensland, conducted coal chemical analyses (proxi-mate and ultimate) while coal physical analyses (rank and petro-graphic composition) were conducted using a Polarised Lightmicroscope Leica DM4500 P LED at the School of Earth Sciences,the University of Queensland, Australia.

4.2. Formation water collection

Formation water for microbial culturing was collected anoxicallyfrom a CBM pilot production well SSB5, located approximately300 km south of Palembang, South Sumatra, Indonesia, in May2012. The formation water came from a depth of 609 m where theSSB5 well intersects the Mangus seam. The formation water wasflushed for one minute through a Norprene® rubber tube connectedto the SSB5 well water line before the water was collected into

Fig. 1. Location of the study area: a)Water sampling (SSB5well: blue dot) and coal sampling (black dot); b) Coal stratigraphy of Bukit Asam area. Coal samples used in this study are fromBurung and Mangus seams. Stratigraphy of the coal seam is modified from Amijaya and Littke (2006). (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

68 R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

multiple 1 L Mason jars by overflowing the jar twice and sealing toexclude air. The Mason jars had been previously autoclaved andflushed with nitrogen gas and filled with 1 ml reduction solutionwhich consisted of 3 g sodium sulphide nonahydrate, 0.675 g sodiumhydroxide, 2.69 g L-cysteine hydrochloride and 20 μL resazurin dis-solved in 30 ml of anoxic deionized water. The formation water sam-ples were imported to Australia following Australian Quarantine andInspection Service (AQIS) guidelines under permit no IP13002699. Inthe laboratory, the formation water was stored in an anaerobicchamber (COY laboratory Products, MI, USA) with an atmosphereof 95% nitrogen and 5% hydrogen.

Awater sample from the SSB5 pilot well was also collected for anionand cation concentration analysis. The analysis was conducted atthe Centre of Groundwater Resource and Environmental Geology inBandung, Indonesia.

4.3. Enrichment culture experimental conditions

Experiments were designed to assess community changes duringthe process of biogenic methane formation in culture enrichments ofSSB5 formation water as inoculant and three SSB coals as carbon sub-strates. Basal coal-based medium, as described in Papendick et al.(2011) and Susilawati et al. (2013), was used for the microbial enrich-ment culture work and was prepared under anoxic conditions accord-ing to the methods outlined by these authors.

Culture tubes were prepared in 26 mL tubes (Bellco Glass) where0.25 g of coal substrate (b300 μm) was combined with 9 mL of basalmedium and capped with a butyl rubber stopper and then removedfrom the chamber. Outside the chamber, using the gasing manifoldand syringe, the headspace was vacuumed and flushed with nitrogen3 times to remove oxygen, then pressurized to 135 kPa with nitrogen

Table 1Formation water properties.

SSB5 water parameters Concentration

Temperature 32.0pH 6.6Salinity (ppm) 1350.0Conductivity (mS) 3.3CO3

2− (mg/L) ndHCO3

− (mg/L) 942.5SO4

2− (mg/L) 3.0Cl− (mg/L) 439.5Ca2+ (mg/L) 9.7Mg2+ (mg/L) 6.2Na+ (mg/L) 577.9K+ (mg/L) 126.1

Trace elementsCu (mg/L) 0.03Zn (mg/L) 0.32Ni (mg/L) ndCo (mg/L) 2.97Pb (mg/L) 0.00

69R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

before being autoclaved at 121 °C for 15 min. Culture tubes withoutadded coal (no-coal controls) were prepared as negative controls totest the ability of the microbial community to form methane fromcoal. Other tubes with both coal and 20 mM 2-bromoethanesulfonicacid (BESA) were also prepared as abiotic controls. Inside an anaerobicchamber and using a sterile 21-gauge syringe needle, 3 mL of formationwater was transferred into autoclaved culture tubes as the inoculum.Before inoculation, filter-sterilized vitamin solution (Tanner, 2002)was injected into all tubes (0.1 mL/tube) using a 1 mL sterile syringe.Cultures were then incubated at 37 °C until they reached the stationaryphase where cumulative methane concentrations no longer increased.

As with previous experiments using the SSB5 formation waterrevealed that the growth of the culture is in the range of 20–25 days(data not reported), eight replicate tubes for each coal enrichmentculture were prepared to capture the different growth phases (lag, log,peak and stationary). Changes in the relative abundance of themicrobi-al communities during growth on each of the coal enrichment cultureswere assessed using a molecular analysis method. For this purpose,one tube that produced the second highest methane yield from eachset of coal enrichment cultures was terminated every 2–3 days startingat day six. Terminated cultureswere transferred into sterile 15ml falcontubes and centrifuged at 3270 ×g for 30 min. The supernatant wasdiscarded, and the pellet frozen at −20 °C until required for DNAextraction.

4.4. Measurement of enrichment culture parameters and statisticalanalyses

Methane measurement was conducted every 2–3 days on all repli-cate tubes for each culture to generate methane production profiles.Headspace gas was drawn from culture tubes using an aseptic, anoxictechnique., Using a Hamilton 1710 gas tight syringe the gas thenmanu-ally injected into a Varian 9700 Gas Chromatograph equipped with a1177 column and a flame ionization detector (FID). Methane was cali-brated using a 1%, 4% and 10% CH4 standard (Scientific and TechnicalGases Ltd., UK).

Statistically significant differences of means in all culture treat-ments were evaluated by two-way analysis of variance (ANOVA)and post hoc multiple comparison t-tests using Holm–Sidak method,with alpha = 5.000% (Graph Pad Prism 6). For multiple comparisontests, each row was analysed individually, without assuming a con-sistent standard deviation (SD). The means of methane productionand standard deviation at each time point (lag, log, peak, stationary)were calculated and plotted as bar charts using Graph Pad Prism 6(Graph Pad Software, Inc.).

4.5. Growth curve and microbial community analysis

For the terminated enrichment culture tubes, the four thatcorresponded to the lag, log, peak and stationary phases were select-ed for DNA extraction and community profiling. For comparison, mi-crobial community analysis was also performed on initial formationwater (day 0) and no-coal control cultures (days 10 and 20). DNAwas extracted from the frozen enrichment culture cell pellets usinga modified phenol:chloroform:isoamyl alcohol method (Suzuki etal., 2001). For community profiling, approximately 20 ng of extract-ed DNA from enrichment cultures was used as a template for poly-merase chain reaction PCR. The 16S rRNA gene was PCR amplifiedusing the universal primers 926F and 1392R under the reaction con-ditions described inMondav andWoodcroft et al., (2014). Ampliconswere sequenced on the Roche 454 GS-FLX Titanium platform at theAustralian Centre for Ecogenomics, University of Queensland,Australia.

The resulting sequence reads were demultiplexed and chimericsequences removed using the UCHIME software (Edgar et al., 2011),while homopolymer errors were corrected using Acacia (Bragg et al.,2012). Using QIIME scripts sequences were clustered into operationaltaxonomic units (OTUs) at a 97% cut-off. A representative sequencewas then randomly selected within each cluster which is then given ataxonomic affiliation by performing a BLAST search against theGreengenes 16S rRNA gene database (De Santis et al., 2006). The resultsin the form of the abundance of different OTUs and their taxonomicassignments in each sample are tabulated. To avoid misleading statisti-cal comparisons that could result from different sequencing depths andcomparisons of diversity with the bias of uneven sampling effort(a higher number of low-abundance OTUs will be recovered by chancewith increasing sequencing effort), the number of readswas normalisedto 1150, to match the sample with the lowest number of sequences.Measures of microbial diversity (Shannon index), species communityrichness (Chao 1) and composition skew (Evenness) were estimatedusing the programme EstimateS (Colwell, 2013).

5. Results

5.1. Water chemistry

The SSB5 formation water is sodium bicarbonate-chloride type, andhas very low concentration of sulphate and low concentration ofcalcium and magnesium (Table 1). These signatures are typical of for-mation water associated with CBM (Papendick et al., 2011; Taulis andMilke, 2007; Van Voast, 2003). The dissolution of carbonate mineralssuch as calcium carbonate and magnesium carbonate may contributeto the calcium,magnesium and bicarbonate levels in thewater. Howev-er, significantly high bicarbonate alkalinities of SSB5 water may alsoindicate that the source of dissolved inorganic carbon is from otherthan carbonate dissolution. The same phenomenon was also noticedin the Forest City Basin (McIntosh et al., 2008). It has been suggestedthat a high concentration of bicarbonate in the water indicates themicrobial decomposition of reduced carbon stored in organic matter.The SSB5 water has higher total dissolved solid as shown by higherconcentration of sodium and chloride. Sulphate in SSB5 water wasonly detected in very low concentration and that may not favour thegrowth of bacterial sulphate reducers.

5.2. Coal characterisation

Petrographic analysis (Table 2) showed that the Burung SB coal wasthe lowest rank (Rv = 0.39%) followed by the Mangus SB (Rv = 0.5%)and the Mangus A (Rv = 2.2%). Huminite in Burung SB and MangusSB coals or vitrinite in Mangus A coal was the major maceral groupspresent in coals and accounted for 81–97% of maceral composition.Liptinite group content is 6–7%, and inertinite group content varies

Table 2Petrographic and chemical composition of coal samples used in this study. Bases are asdetermined (a.d.b.) and dry ash free (d.a.f.).

Analysis Burung Sb Mangus Sb Mangus A

Proximate % (a.d.b.)Moisture 16.8 8.0 1.2Ash 2.2 0.7 1.2Volatile matter 38.1 42.5 14.7Fixed carbon 42.9 48.8 82.9Total sulphur 0.8 0.3 1.1

Ultimate % (d.a.f.)Carbon 76.2 78.2 91.0Hydrogen 5.1 5.3 3.8Nitrogen 1.7 1.2 1.6Sulphur 1.1 0.4 1.1Oxygen (by difference) 15.9 15.1 2.6

Petrography (%)Rv (Rank) 0.39 0.50 2.20Maceral compositionHuminite/Vitrinite

Textinite 6 3Ulminite 23 22Attrinite 24 20Densinite 17 27Corpohuminite 8 4Gelinite 4 5Total 82 81 97

InertiniteFusinite 5 3Semifusinite 1 1Funginite 2 5Inertodetrinite 2 3 2Macrinite 0 0Total 10 12 2

LiptiniteSporinite 1 1Cutinite 1 1Resinite 1 0Liptodetrinite 2 2Suberinite 1 2Exsudatinite 1 0 0Total 7 6 0

Mineral matter 1 1 1

Fig. 2. Plot of average methane production from Burung (Brg), Mangus Sub Bituminous(MSb), Mangus Anthracite (Ma) and No coal-control (Ncc) cultures. Error bars representstandard deviation for replicates tubes.

Table 3Analysis of Variance (ANOVA) results of methane productions in SSB5 cultureexperiments.

ANOVA table SS DF MS F (DFn, DFd) P value

Interaction 1315 9 146 F (9, 62) = 2.4 P = 0.0203Time 46199 3 15400 F (3, 62) = 254.4 P b 0.0001Treatments 5659 3 1886 F (3, 62) = 31.2 P b 0.0001Residual 3753 62 61

SS = sum of square, DF = degree of freedom, MS = mean of square, DFn = degrees offreedom numerator. Dfd = degrees of freedom denominator. DFn = a − 1, DFd = N − a,a = number of treatments, N = the total number of measurement.

70 R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

from 2–12% (Table 2). All three coals had very low mineral matter(≤1%; corroborated by low ash yields b10%), which is typical forIndonesian coal. The anthracite by comparison had much lower mois-ture and higher reflectance and as a result, higher vitrinite content asthe liptinite group macerals were devolatilised.

5.3. Methane production from coal enrichment cultures

Negligible amounts of methane produced from BESA treated abioticcontrol tubes (data were not presented) suggested that little methanein cultures was from methane adsorbed in the substrate coal. Overall,methane from each of the enrichment cultures increased slowly untilday 6. After day 6, methane production increased significantly and con-tinued until around day 17 when the peak of methane yield wasachieved (Fig. 2). After day 10, methane production rate started to de-cline and was confirmed by day 20 when no increases in methanewere seen for all coal types and no-coal controls (Fig. 2).

Using two-factor Anova, the difference between the methaneproduction yields from the four culture treatments (three cultureswith coal substrate and 1 control) over time was analysed. The Anovatest produced P values less than 5% for the three parameters beingmeasured (interaction, row and column factor) (Table 3) suggestingthat methane production in this experiment was affected by both timeand substrate. Although the mean methane production and maximummethane production in all coal cultureswere higher than no coal controlculture, post hocmultiple comparison test showed that only the Burungcultures consistently showed statistically significant differences in their

methane concentration compared to the no-coal control cultureswhereas no significant differences were observed in the mean betweenboth Mangus cultures and no-coal control cultures (Table 4). At peakproduction (day 17), the methane produced by the Burung cultureswas also significantly higher than other coal treated cultures (Fig. 2,Table 4). This result suggests there is some ability of the SSB5 microbialcommunity to metabolise the coal and indicates the influence of coalsubstrate to methanogenesis in SSB5 cultures. Even though differencesin methane concentration among the 3 coal cultures were not all statis-tically significant according to the ANOVA test, the mean of methaneproduction data indicated declining methane production yields withincreased rank (Fig. 2). Burung SB coal culture has the highest methaneproduction yield, followed by the Mangus SB and Mangus A coal as theleast (Fig. 2). However, the rank trend observed in the maximum totalproduction rate is not as evident in the net rate (treatment–control)(Fig. 3).

It has been reported that headspace gas composition and mediabicarbonate concentrations can have a significant effect on gas andmethane production in culturing experiments (Lin et al., 2013; Patraand Yu, 2014). The concentration of bicarbonate in our media wasshown to have no deleterious effect on coal to methane rate and yields(Papendick et al., 2011). Bicarbonate in the inoculum may provideexcess CO2, which in the presence of H2 rich substrates may stimulatemethanogenesis. The headspace gas containing H2 can provide hydro-gen needed by hydrogenotrophic methanogens to reduce CO2 to pro-duce methane (Patra and Yu, 2014). Although we use H2N2 as a gas

Table 4Post hoc multiple comparison tests of methane production in SSB5 culture experiments.

Time(days)

Post hoc multiple comparison test (Holm Sidak method)

Brg vsMSb

Brg vsMA

Brg vsNcc

MSB vsMA

MSB vsNcc

MA vsNcc

MSB vsNcc

MA vsNcc

6 b 0.001 b 0.001 b 0.001 0.150 0.370 0.630 0.370 0.63010 0.020 0.240 0.001 0.600 0.039 0.080 0.039 0.08017 0.006 0.005 0.002 0.400 0.037 0.181 0.037 0.18120 0.024 0.010 0.012 0.347 0.088 0.402 0.088 0.402

Brg = Burung, MSb = Mangus Sub-bituminous, Ma = Mangus Anthracite, Ncc = no-coal control.

71R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

mix in our anaerobic vessel, the presence of this gas in our culture tubeswas replaced by N2, as such the influence of additional H2 to the meth-ane production in our experiment can be eliminated. Nitrogen can be alimiting factor (in the hydrolysis phase) but also as a source for fermen-tation for subsequent methane production by methanogenic archaea(Wagner et al., 2012). We did not test the effect of N2 gas on our meth-ane production. However, since all cultures, including the no-coal con-trol, were treated similarly with 135 kPa N2, the presence of this gasin our culture headspace will not affect treatment comparisons.

5.4. Microbial community

5.4.1. Microbial diversityAt the 97% cut-off score for an operational taxonomic unit (OTU), a

total of 46 bacterial and10 archaeal generawere observed in the SSB5mi-crobial communities grown on the Burung SB, Mangus SB and Mangus Acoals. Those OTUs over 1% are shown in Tables 5 and 6. The microbialcommunity of the Burung SB enrichment cultures had both the highestbacterial and archaeal diversities among the coal enrichment cultures(Tables 7 and 8), and produced the greatest quantity of methane(Fig. 2). For all three coals rank enrichment cultures, the bacterial diversi-ty and community skew, measured by evenness, decreased. Archaeal di-versity and skew remained static across all time points (Tables 7 and 8).

The SSB water bacterial sequences were dominated by members ofthe Pelobacter and Acetobacterium genera across all cultures and timepoints, while other species belonging to the orders Bacteroidetes, Spiro-chaetes, Synergistetes and Tenericutes were consistently present at lowlevels (Table 5). For the archaeal component of the communities, allenrichment cultures were dominated by acetoclastic methanogens

Fig. 3.Maximummethane production rate in SSB5 cultures, plotted as total rate (rate fromtreatment tube where source of carbon came from both coal substrate and native fine coalparticle or dissolved constituent present in inoculum) and net rate (rate from treatmenttubes subtracted by rate of no-coal control where coal is a major carbon substrate).

from the genera of Methanosaeta (Fig. 4 and Table 6). Lower levels ofMethanosarcina, Methanobacterium, Methanospirillum and CandidatusMethanoregula were also detected across all enrichment cultureswhereas members belonging to Methanofollis, Methanomethylovoransand Thermoplasmata were only found at very low abundance in mostcultures (b1%) and are grouped in Table 6 as “other archaea”.

5.4.2. Temporal changes in community structureThe initial formation water contained a community composed of

14% bacteria and 86% archaea. Culturing successfully enriched thebacterial communities, and by day six (the beginning of log phase),the relative abundance of bacteria had almost doubled. At day six, thebacterial population was at its highest proportion in all three cultures(bacteria 43–48% and archaea 52–57%) and the community structurewas similar between all three cultures, owing to the same inoculumsource. Over time the relative abundance of the bacterial populationtended to decline, dropping from 43–48% at day 6 to 15–29% by day20. In contrast, in the no-coal controls, during the log phase (day 10)bacteria occupied 17%, but increased to 28% at stationary phase (day20), whereas archaea decreased from 83% at day 10 to 71% at day 20.

In the Burung SB culture, the proportion of Acetobacterium andPelobacterwere similarwith both tending to decreasewith time,where-as in the Mangus SB, Mangus A and no-coal control cultures, Pelobacteris more dominant than Acetobacterium. The Burung SB culture had thehighest Bacteriodales population. Molecular analysis showed significantvariations in methanogen communities, with shifts in the dominance ofdifferent groups (Fig. 4). It appeared that in the Burung SB culture, mostof the methane production was associated with growth of the obligateacetoclastic methanogen, Methanosaeta. At day six, in both Mangus SBand Mangus A, the acetoclastic (Methanosaeta) and hydrogenotrophicmethanogens (Methanoregula and Methanospirillum) were present inalmost the same proportion but then decreased towards the end ofgrowth. In contrast, despite its low proportion at the early stage ofgrowth, Methanosarcina was present in significant proportions at thestationary phase. In the no-coal control culture, Methanosaeta was thedominantmethanogen until day 10. However, at the end of experimentMethanosarcina and Methanobacteria were found to be equivalent inabundance. The Methanobacteriales population was only observed inthe early growth stage of Burung SB culture and at the late stage ofno-coal control culture. Its closest match was to the Methanobacteriumformicicum type strain.

6. Discussion

6.1. Methane production

Although the differences are not considered significant by theANOVA tests, the total and net methane production from the experi-ments increased consistently over time, with some variation attribut-able to the rank of the coal substrates. The small differences betweenmethane yield from coal tubes and no-coal controls in our experimentwere likely to be a result of low concentration of critical coal degradingbacterial consortium in the initial formation water inoculum, as suchonly a small amount of coal organic material can be biodegraded.More-over, during storage, it is likely that a key nutrient needed for the rapidgrowth of the microbial community available in the initial formationwater becomes depleted, or toxic by-products of microbial metabolismwere accumulated, which further influenced the growth of microbialcommunities capable of converting coal to methane. Additionally, itseems that many organic constituents are also available as substratesin the initial formation water, likely from continued microbial metabo-lism. This product alongwith fine coal particles carried over in thewatercan provide substrates for metabolism in coal and no-coal enrichmentcultures.

The variation in methane production among experimental coal andno-coal control replicate tubes may relate to the concentration of

Table 5The dominant bacterial taxa detected in SSB5 cultures and the closest matches at 99% similarity to known bacteria in GeneBank database.

No Closest BLAST sequence match Origin of closest match Taxonomic String

1 Bacteroidetes bacterium AY548787 Metal-containing wastewater Bacteria; Bacteroidetes; Bacteroidia; Bacteroidales; unclassified_Bacteroidales2 Geovibrio ferrireducens X95744 Surface sediment of hydrocarbon-

contaminated ditchBacteria; Deferribacteres; Deferribacterales; Deferribacteraceae; Geovibrio.

3 Acetobacterium carbinolicumAB546236

Oil storage tank Bacteria; Firmicutes; Clostridia; Clostridiales; Eubacteriaceae; Acetobacterium.

4 Acidaminobacter hydrogenoformansAF016691

Black mud Bacteria; Firmicutes; Clostridia; Clostridiales; Clostridiales Family XII. Incertae Sedis;Acidaminobacter.

5 Azospira sp. R-25019 AM084030 Municipal wastewater treatment plant Bacteria; Proteobacteria; Betaproteobacteria; Rhodocyclales; Rhodocyclaceae;Azospira.

6 Uncultured bacterium GQ181535 Purified terephthalic acid (PTA) wastewater Bacteria;Proteobacteria; Deltaproteobacteria; Desulfovibrionales;Desulfovibrionaceae; Desulvofibrio.

7 Desulfomicrobium sp. AY570692 Production water of oil reservoir Bacteria; Proteobacteria; Deltaproteobacteria; Desulfovibrionales;Desulfomicrobiaceae; Desulfomicrobium.

8 Uncultured bacterium AB701661 Natural gas field Bacteria; Proteobacteria; Deltaproteobacteria; Desulfuromonadales; Pelobacteraceae;Pelobacter

9 Geobacter pelophilus U96918 Freshwater enrichment culture Bacteria; Proteobacteria; Deltaproteobacteria; Desulfuromonadales; Geobacteraceae;Geobacter.

10 Uncultured bacterium GQ181438 Purified terephthalic acid (PTA) wastewater Bacteria; Spirochaetes; Spirochaetales; Spirochaetaceae; Treponema.

72 R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

microbial methanogen consortium member present in each 3 ml of theinoculum added to these experimental systems. Each tube likelycontained slightly varying concentrations of the critical microbialmethanogen consortium member, which then led to differences in themethane yield. It can be assumed that the tube with the highest meth-ane production contains the highest concentration of critical consor-tium members. The fact that differences showed a consistent changewith rank could be coincidental, or reflect the influence of coal on theprocess.

Our data suggests that the source of carbon for methane productionin all SSB5 cultures was from both the additional coal substrate and theinoculum (Fig. 3). The greatest influence of coal substrate occurred fromday 6 to 10. In general, total production rate was higher than net pro-duction rate, indicating that the organic precursor in the inoculumwas easier to biodegrade than the additional coal substrate. This is notsurprising since the consortia may well have adapted to the nativesource of carbon present in the water.

6.2. Microbial methanogen community structure

In the future, CBM resources may become an important energy sup-plement to meet some of the global demand for energy resources. Re-plenishment of current and future CBM resources by in situmethanogenesis is one way to increase the productive lifetime ofthese fields but is dependent on the ability of coal to be degraded bymi-crobial communities. To this end, we tested the ability of Indonesiancoals to provide substrates for a native microbial community from anIndonesian CBM well. The results of our experiments suggest thatthese coals can provide carbon and energy source for a microbial con-sortium. Lower rank coals produced slightly more methane than thehigher rank coal in the experiments. Whereas cultures grown on coals

Table 6The dominant archaeal taxa detected in SSB5 cultures and the closest matches at 99% similarit

No Closest BLAST sequence match Origin of closest match T

1 Methanobacterium formicicumAF169245

Culture collection, Institute ofMicrobiology CAS China

AM

2 Uncultured Methanospirillaceaearchaeon AB236073

River sediment AM

3 Methanoregula formicicaAB479390

Granular sludge AM

4 Uncultured archaeonJN397698

River bank sediment AM

5 Methanosarcina mazeiiDQ987528

Isolated strain from lake sediment AM

of similar rank and type produced different community structures,coal of the same seam with different rank revealed similarity in theircommunity structures, suggesting that community structure could bedependent on coal source.

After an initial lag phase, typical of lag phases observed by other re-searchers for microbial consortia grown on coal (Green et al., 2008;Papendick et al., 2011), the microbial population was consistentamong all enrichments grown on coal and consisted predominantly ofthe obligate acetoclastic Methanosaeta methanogen species. However,due to the time taken to start enrichment experiments, it is likely thata key nutrient needed to drive the metabolism of the microbial consor-tia might have forced the community to this structure. As little methanewas formed during the lag phase, theMethanosaetamembers are poten-tially significant constituents of the original formation water inoculum,which is consistent with other studies that show Methanosaeta speciesas a large proportion of indigenous communities (Jones et al., 2010;Wawrik et al., 2012). The predominant bacterial members of the com-munity during this lag phase were Acidaminobacter, Acetobacteriumand Pelobacter. The members of these bacteria are known to be able tohydrolyze and ferment aromatic compounds and hydrocarbons(Kreikenbohm and Pfennig, 1985; Schink, 2006a; Sträuber et al., 2012)and have previously been identified in studies of coal associated enrich-ment cultures across several CBM reservoirs (Barnhart et al., 2013;Dawson et al., 2012; Green et al., 2008; Jones et al., 2010; Penneret al., 2010; Strąpoć et al., 2011; Wawrik et al., 2012). It is likely thatthese bacteria were the primary degraders of coal constituents in ourcultures. Moreover, the involvement of Acetobacterium in the utilizationof substrates produced by coal degradation has been suggested byBarnhart et al. (2013). Consistent with this result, our experimentshowed that Acetobacterium species were always of lesser proportionsof the no-coal community over the time series compared to coal

y to known bacteria in GeneBank database.

axonomic String

rchaea; Euryarchaeota; Methanobacteria; Methanobacteriales; Methanobacteriaceae;ethanobacterium.rchaea;Euryarchaeota; Methanomicrobia; Methanomicrobiales; Methanospirillaceae;ethanospirillum.rchaea; Euryarchaeota; Methanomicrobia; Methanomicrobiales;ethanomicrobiales_incertae_sedis; Methanoregula.rchaea; Euryarchaeota; Methanomicrobia; Methanosarcinales; Methanosaetaceae;ethanosaeta.rchaea; Euryarchaeota; Methanomicrobia; Methanosarcinales; Methanosarcinaceae;ethanosarcina.

Table 7Bacterial diversity in SSB5 cultures during different stages of growth.

Inoculum (rawformation water andno-coal control)

Burung sub-bituminous Mangus sub-bituminous Mangus anthracite

Micobial taxa RFW C10 C20 6 10 17 20 6 10 17 20 6 10 17 20

Bacteroideteso_Bacteroidales 3.2 1.6 0.4 0.9 0.6 0.3 0.8 0.7 0.7 0.9 1.0 0.7 0.5 0.4 0.4FirmicutesAcidaminobacter 0.0 0.2 0.0 1.4 0.6 0.1 0.0 1.9 1.0 0.0 0.1 2.1 1.0 1.0 2.4Acetobacterium 2.7 6.0 0.1 19.6 6.4 4.1 5.0 11.8 4.2 4.1 6.7 14.1 8.2 6.3 7.7Proteobacteriaf_Comamonadaceae 0.3 0.1 0.0 0.0 0.1 0.1 0.2 0.1 0.0 2.3 0.0 0.1 0.1 0.1 0.0Desulfomicrobium 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.2 0.0 1.2 0.2 0.0 0.4 0.6 0.1Desulfovibrio 0.0 0.5 0.4 0.5 0.9 0.3 0.7 0.6 0.2 1.0 0.7 0.3 0.5 0.3 0.5f_Geobacteraceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 1.7 0.0 0.0 0.0 0.0 0.0f_Pelobacteraceae 2.4 5.3 23.3 16.9 6.0 3.9 5.1 29.1 11.7 25.7 15.9 30.0 22.8 21.2 12.6SpirochaetesTreponema 0.6 0.2 1.0 0.7 0.4 0.7 1.5 0.6 0.3 0.2 0.9 0.3 0.5 0.9 0.3All Synergistetes 1.4 0.3 0.8 0.3 0.1 0.2 0.2 0.5 0.3 0.0 0.7 0.2 0.4 0.3 0.1All Tenericutes 1.0 0.3 0.0 0.0 0.0 0.2 0.2 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1Other Bacteria 2.8 2.7 2.3 2.8 1.0 1.5 1.6 0.8 0.8 2.1 2.3 0.4 2.0 1.5 0.7Total read abundance of Bacteria taxa (% ofnormalised OTU read)

14.4 17.3 28.3 43.2 16.1 11.5 15.3 46.3 19.4 39.2 28.5 48.2 36.5 32.6 24.9

Species richness (Chao 1) 33.9 22.5 20.5 33.1 14.5 18.48 21.1 34.9 35.9 19.9 18.9 38.9 21.5 15.0 23.9(24. 5, (20.4, (18.4, (27.1, (13.2, (16.4, (19.3, (20.9, (26.7, (15.8, (14.7, (23.1, (15.3, (15.67, (21.5,75.9) 35.2) 34.9) 58.3) 28.0) 32.9) 32.3) 98.1) 78.1) 47.1) 46.0) 104.2) 56.4) 16.2) 38.9)

Evenness 0.8 0.7 0.4 0.5 0.6 0.7 0.7 0.4 0.5 0.5 0.5 0.4 0.5 0.5 0.5Shannon Diversity Index 2.4

(0.08)2.0(0.07)

1.0(0.03)

1.5(0.03)

1.6(0.04)

1.8(0.08)

1.9(0.09)

1.1(0.02)

1.4(0.04)

1.3(0.02)

1.4(0.06)

1.2(0.02)

1.4(0.04)

1.4(0.02)

1.6(0.04)

OTU = Operational Taxonomy Unit; RFW = Raw Formation Water; C10 = control at day 10, C20 = control at day 20. p = phylum, c = class, o = ordo, f = family.Evenness calculated as H/ln(S), H = Shannon Diversity, S = Chao 1. For Chao 1, values between brackets represent lower bound and upper bound.For Shannon Diversity Index, values between brackets are standard deviation. Only taxa that have read abundance ≥1% from total are plotted in this table.Taxa that have read abundance b1% are grouped together. Dominant taxa are highlighted.

73R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

containing cultures, suggesting that Acetobacterium species likelymetabolise substrates directly from the coal, even though the exactmechanisms remain unknown.

After the lag phase, methane production increased rapidly andreached maximum production rate by day 11 in all enrichmentcultures. During this time the relative proportions of the microbialcommunity measured at days 10 and 17 had changed from day 6,with an increased archaeal and corresponding decreased bacterialfraction. This result suggested that methanogenesis was occurring

Table 8Archaeal diversity in SSB5 cultures during different stages of growth.

Inoculum (rawformation water andno-coal control)

Burung sub-bitu

Micobial taxa RFW C10 C20 6 10

MethanobacterialesMethanobacterium 0.1 0.0 36.6 9.3 0.0MethanomicrobialesCandidatus Methanoregula 23.5 9.0 0.0 12.3 11.7Methanospirillum 8.9 2.4 0.0 2.0 3.7MethanosarcinalesMethanosaeta 50.7 65 16.4 26.4 61.2Methanosarcina 1.3 6.0 18.2 6.7 7.0Other Archaea 1.4 0.3 0 0.5 0.6Total read abundance of Archaea taxa (% ofnormalised OTU read)

85.9 82.7 71.2 57.2 84.2

Species richness (Chao 1) 8.0 7.0 7.0 8.0 10.5(8.0, (7.0, (6.1, (8.2, (9.2,8.9) 8.0) 19.7) 9.1) 24.1)

Evenness 0.5 0.4 0.6 0.7 0.4Shannon Diversity Index 1.1

(0.00)0.8(0.00)

1.1(0.00)

1.4(0.01)

0.9(0.00)

OTU = Operational Taxonomy Unit; RFW = Raw Formation Water; C10 = control at day 10,Evenness calculated as H/ln(S), H = Shannon Diversity, S = Chao 1. For Chao 1, values betweFor Shannon Diversity Index, values between brackets are standard deviation. Only taxa that hTaxa that have read abundance b1% are grouped together. Dominant taxa are highlighted.

from hydrolysis and fermentation of end products produced by thebacterial component. As the proportion of the obligate acetoclasticMethanosaeta had increased, such as in the Burung microcosm, it islikely that it syntrophically consumes the acetate being producedby the Pelobacter, Acetobacterium and Acidaminobacter members,which are known acetogens (Beckmann et al., 2011; Green et al.,2008; Schink, 1997; Schink, 2006a,b; Stams, 1994; Stams andHansen, 1984). It has been reported that acetate production caneventually inhibit the growth of Acetobacterium, but in the presence

minous Mangus sub-bituminous Mangus anthracite

17 20 6 10 17 20 6 10 17 20

0.1 0.1 0.2 0.0 0.0 0.1 0.2 0.0 0.0 0.2

7.8 7.2 17.9 14.6 8.4 11.3 21.1 6.8 7.8 9.83.1 1.5 4.3 4.2 8.4 3.2 4.5 2.6 1.3 3.2

70.8 71.1 29.2 48.1 43.6 22.9 21.1 38.7 33.8 26.05.9 4.3 1.8 13.6 0.5 33.6 4.2 14.9 24.4 35.30.8 0.6 0.8 0.1 0.2 0.6 1.1 0.7 0.3 0.588.5 84.8 54.2 80.6 61.1 71.7 52.2 63.7 67.6 75.0

7.0 11.0 8.0 8.0 11.0 7.0 8.0 6.0 6.0 7.0(7.0, (8.4, (8.0, (8.4, (8.4, (7.0, (8.1, (6.0, (6.1, (7.1,8.1) 32.9) 9.4) 9.1) 32.9) 8.4) 9.1) 7.4) 7.2) 8.1)0.4 0.3 0.6 0.5 0.5 0.6 0.5 0.6 0.5 0.60.7(0.00)

0.6(0.00)

1.3(0.01)

1.1(0.00)

1.1(0.00)

1.2(0.00)

1.1(0.01)

1.1(0.01)

0.8(0.00)

1.2(0.02)

C20 = control at day 20. p = phylum, c = class, o = order, f = family.en brackets represent lower bound and upper bound.ave read abundance ≥1% from total are plotted in this table.

a)

c)

b)

d)

Fig. 4. Bar charts depicting microbial methanogen community dynamics and diversity at phylum level in SSB5 cultures during different stages of growth; a) Burung, b) Mangus Sub bitu-minous, c) Mangus Anthracite, d) Inoculum (initial formation water at time 0 and no-coal controls at day 10 and 20). Microbial communities presented in the graphs are the ones thatoccupy at least 1% of the total microbial community in any stage of growth. Bacterial community displayed as area with solid colour while methanogen community displayed as areawith pattern.

74 R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

of Methanosaeta, acetate is utilised to produce methane and car-bon dioxide (Bainotti et al., 1997; Winter and Knoll, 1989).Acetobacterium would likely compete with hydrogenotrophicmethanogens for hydrogen when grown on coal which may explainwhy the proportion of hydrogenotrophic methanogens onlyincreased in the no-coal controls culture in the near absence ofAcetobacterium. Based on the ability of Methanosaeta to outcompeteMethanosarcina species for acetate (Liu and Whitman, 2008), thedominance of the Methanosaeta members over Methanosarcina spe-cies suggests that in our experiment acetate was only present atlow concentrations. Furthermore, Methanosarcina members ob-served in our study were closely related to Methanosarcina mazeii,a versatile strain that is able to metabolise H2–CO2, acetate, methyl-amines and methanol (Hovey et al., 2005; Jäger et al., 2009), there-fore an increase in the proportion of Methanosarcina species likelyaccounted for increases in methane concentration and could be dueto these members being able to metabolise methylated compoundssuch as methylamines, methyl sulphide and methanol when pro-duced towards the end of fermentation (Allison and Macfarlane,1989).

During the times between 10 and 17 days it became apparent thatthe communities on the different coals diverged. Themicrobial commu-nity grown with the Mangus SB coal was more similar to the Mangus Acoal, than to the Burung SB coal. Pelobacter species weremore dominantin both Mangus cultures than in Burung SB culture. In the Mangus coalcommunities,Methanosarcina became a significant proportion while inthe Burung enrichment it was present in low proportion. The increaseof Methanosarcina in both Mangus cultures is concomitant with thedecrease of bothMethanosaeta and Methanoregula. We postulated thatboth Mangus coal substrates reduced the particular contribution of theobligate acetoclastic methanogen Methanosaeta and the obligatehydrogenotrophic methanogen Methanoregula to methane production.On the contrary both coal substrates increased the relative contribu-tion of Methanosarcina to methane production in the cultures.

Methanosarcina has versatile methanogenic pathways, thus dependingon the environment condition these microbes may have advantage toproduce methane via an effective pathway and outcompete the othermethanogen genera. In the end, it appeared that Burung coals led tothe growth ofMethanosaetawhile both of the Mangus coals stimulatedthe growth of Methanosarcina. Except that the coals come from thesame seam, it is not clearwhat factors are responsible for the similaritiesin themicrobial communities of cultures onMangus SB and A coals. Thetwo coals have different chemical and petrographic properties due tothe significant differences in rank, but formerly shared the same originand may have some characteristics that force the communities toreach a similar population structure. Although the communities on theMangus SB was similar to Mangus A coals, maximum methane pro-duced from the Mangus SB was higher than Mangus A, suggesting thatthe coal rank has an effect in the ability of the community to generatemethane. Overall, there also appeared to be an inverse correlationbetweenmaximummethane yield and coal rank which is in agreementwith previous studies (Scott, 1999; Strąpoć et al., 2011). The lower rankcoal Burung SB culture is likely to be less recalcitrant and is easier to bebiodegraded than higher rank Mangus anthracite coal. It has beenreported that an increasing recalcitrant material has an inhibitory effecton methanogenesis (Minderlein and Blodau, 2010). It is likely that thecoal maturity and the organofacies of the source material govern thedominant extractable compounds and their concentrations in the coalsamples (Glombitza et al., 2009).

By day 20, methane production had ceased, and differences betweenthe methane concentrations of the microbial consortia when grown onthe three coal substrates were still observed when compared to the nocoal control, although these differences were not always significant.Interestingly, in the no-coal control enrichment cultures, the proportionof the acetoclasticMethanosaeta species decreased significantly where-as the proportion of the obligate hydrogenotrophic methanogensMethanobacterium increased which suggested that hydrogen/carbondioxide and not acetate is a more important intermediate in microbial

Fig. 5. Cross plot of methanogen abundance against methane yield in SSB5 cultures. Levelof confidence (P = 0.05) plot as dot line.

75R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

metabolism in cultures without a coal substrate. Overtime we notedthat in all cultures there was a tendency that the bacterial membersdecreased in proportion whereas the archaeal component increasedand the increase in methanogen population corresponded with anincrease in methane production yield (Fig. 5).

7. Conclusion

In this study, we show that an Indonesian CBM well produced anactive methane-forming microbial community, which adds to theknown basins where microbial communities can form methane fromcoal. The diversity of the community derived from the Indonesian coalformation water was similar to previous studies that have enriched formicrobial communities from coal basins worldwide. Further, to enrich-ment culture study and microbial community profiling, we show com-munity structure changes over time, which exhibited more similaritywhen grownon coal from the same seambut of different ranks (MangusSB and Mangus A), than to coal from similar type (Mangus SB andBurung SB). It is noteworthy that the enrichment culture grown onthe lowest rank coal produced the greatest quantity ofmethane, where-as the highest rank coal produced the least methane per gram of coal.Overall, our results indicate a potential relationship between coal typeand rank, microbial community composition and methane production.Further work should focus on factors that may enhance or limit coalmethanogenesis. We also highlight the need for more detailed studieson coals of different types and ranks and their potential for microbesto form methane.

Acknowledgement

The Australia Awards Scholarship (2010) provided funding for thisresearch. The TSOP Spackman award (2011) was used to fund part ofthe laboratory consumables and gas isotope analyses for this study. PTMedco CBM Sekayu and PT Bukit Asam kindly allowed site and sampleaccess. The authors thank Gordon Southam for the constructive reviewof this manuscript, also Soleh Basuki Rahmat, Eko Budi Tjahyono andErlina Lena Ria from the Geological Agency of Indonesia for the fieldassistance.

References

Aklujkar, M., Haveman, S.A., DiDonato Jr., R., Chertkov, O., Han, C.S., Land, M.L.,Brown, P., Lovley, D.R., 2012. The genome of Pelobacter carbinolicus reveals sur-prising metabolic capabilities and physiological features. BMC Genomics 13, 690.

Allison, C., Macfarlane, G.T., 1989. Influence of pH, nutrient availability, and growth rateon amine production by Bacteroides fragilis and Clostridium perfringens. Appl. Environ.Microbiol. 55, 2894–2898.

Amijaya, H., Littke, R., 2006. Properties of thermally metamorphosed coal from TanjungEnim Area, South Sumatra Basin, Indonesia with special reference to the coalificationpath of macerals. Int. J. Coal Geol. 66, 271–295.

Bainotti, A.E., Futagami, K., Nakashimada, Y., Chang, Y.J., Nagai, S., Nishio, N., 1997. pHAuxostat continuous culture of Acetobacteriumsp. coupled with removal of acetateby Methanosaeta concilii. Biotechnol. Lett. 19, 989–993.

Barnhart, E.P., De León, K.B., Ramsay, B.D., Cunningham, A.B., Fields, M.W., 2013. Investi-gation of coal-associated bacterial and archaeal populations from a diffusive microbi-al sampler (DMS). Int. J. Coal Geol. 115, 64–70.

Beckmann, S., Lueders, T., Kruger, M., Netzer, F.v, Engelen, B., Cypionka, H., 2011.Acetogens and acetoclastic Methanosarcinales govern methane formation in aban-doned coal mines. Appl. Environ. Microbiol. 77, 3749–3756.

Bishop, M.G., 2001. South Sumatra Basin Province, Indonesia: the Lahat/Talang Akar-Cenozoic total petroleum system. Open File Report 99-50S. US Geological Survey,p. 15.

Bragg, L., Stone, G., Imelfort, M., Hugenholtz, P., Tyson, G.W., 2012. Fast, accurateerror-correction of amplicon pyrosequences using Acacia. Nat. Methods 9,425.

Butland, C.I., Moore, T.A., 2008. Secondary biogenic coal seam gas reservoirs inNew Zealand: a preliminary assessment of gas contents. Int. J. Coal Geol. 76,151–165.

Colwell, R.K., 2013. EstimateS: Statistical estimation of species richness and shared speciesfrom samples. Version 9. User's Guide and application published at: http://purl.oclc.org/estimates.

Darman, H., Sidi, F.H., 2000. An outline of the Geology of Indonesia. In: Darman, H., Sidi,F.H. (Eds.), Indonesian Association of Geologists, Jakarta, p. 192.

Davis, R.C., Noon, S.W., Harrington, J., 2007. The petroleum potential of Tertiary coals fromWestern Indonesia: relationship to mire type and sequence stratigraphic setting. Int.J. Coal Geol. 70, 35–52.

Dawson, K.S., Strapoc, D., Huizinga, B., Lidstrom, U., Ashby, M., Macaladya, J.L., 2012.Quantitative fluorescence in situ hybridization analysis of microbial consortiafrom a biogenic gas field in Alaska's Cook Inlet Basin. Appl. Environ. Microbiol.78, 3599–3605.

de Coster, G.L., 1974. The geology of the Central and South Sumatra Basins. 3th AnnualConvention Indonesian Petroleum Association Jakarta, pp. 77–110.

De Santis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., Huber, T., Dalevi,D., Hu, P., Andersen, G.L., 2006. Greengenes, a Chimera-Checked 16S rRNA Gene Da-tabase and Workbench Compatible with ARB. Applied and environmental microbiol-ogy 72, 5069–5072.

Doerfert, S.N., Reichlen, M., Iyer, P., Wang, M., Ferry, J.G., 2009. Methanolobus zinderi sp.nov., a methylotrophic methanogen isolated from a deep subsurface coal seam. Int.J. Syst. Evol. Microbiol. 59, 1064–1069.

Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensi-tivity and speed of chimera detection. Bioinformatics 27, 2194–2200.

Faison, B.D., 1991. Biological coal conversions. Crit. Rev. Biotechnol. 11, 347–366.Faiz, M., Hendry, P., 2006. Significance ofmicrobial activity in Australian coal bedmethane

reservoirs — a review. Bull. Can. Petrol. Geol. 54, 261–272.Fakoussa, R.M., Hofrichter, M., 1999. Biotechnology and microbiology of coal degradation.

Appl. Microbiol. Biotechnol. 52, 25–40.Fallgren, P.H., Jin, S., Zeng, C., Ren, Z., Lu, A., Colberg, P.J.S., 2013a. Comparison of

coal rank for enhanced biogenic natural gas production. Int. J. Coal Geol. 115,92–96.

Fallgren, P.H., Zeng, C., Ren, Z., Lu, A., Ren, S., Jin, S., 2013b. Feasibility of microbial produc-tion of new natural gas from non-gas-producing lignite. Int. J. Coal Geol. 115, 79–84.

Flores, R.M., Rice, C.A., Stricker, G.D., Warden, A., Ellis, M.S., 2008. Methanogenic pathwaysof coal-bed gas in the Powder River Basin, United States: the geologic factor. Int. J.Coal Geol. 76, 52–75.

Fry, J.C., Horsfield, B., Sykes, R., Cragg, B.A., Heywood, C., Kim, G.T., Mangelsdorf, K.,Mildenhall, D.C., Rinna, J., Vieth, A., Zink, K.-G., Sass, H., Weightman, A.J., Parkes, R.J.,2009. prokaryotic populations and activities in an interbedded coal deposit, includinga previously deeply buried section (1.6–2.3 km) above ∼150 Ma basement rock.Geomicrobiol J. 26, 163–178.

Ghosh, S., Jha, P., Vidyarthi, A.S., 2014. Unraveling themicrobial interactions in coal organ-ic fermentation for generation of methane — a classical to metagenomic approach.Int. J. Coal Geol. 125, 36–44.

Glombitza, C., Mangelsdorf, K., Horsfield, B., 2009. Maturation related changes in the dis-tribution of ester bound fatty acids and alcohols in a coal series from the New ZealandCoal Band covering diagenetic to catagenetic coalification levels. Org. Geochem. 40,1063–1073.

Golding, S.D., Boreham, C.J., Esterle, J.S., 2013. Stable isotope geochemistry of coal bed andshale gas and related production waters: a review. Int. J. Coal Geol. 120, 24–40.

Green, M.S., Flanegan, K.C., Gilcrease, P.C., 2008. Characterization of a methanogenic con-sortium enriched from a coalbed methane well in the Powder River Basin, U.S.A. Int.J. Coal Geol. 76, 34–45.

Guo, H., Liu, R., Yu, Z., Zhang, H., Yun, J., Li, Y., Liu, X., Pan, J., 2012. Pyrosequencing revealsthe dominance of methylotrophic methanogenesis in a coal bed methane reservoirassociated with Eastern Ordos Basin in China. Int. J. Coal Geol. 93, 56–61.

Hamilton, S.K., Golding, S.D., Baublys, K.A., Esterle, J.S., 2014. Stable isotopic and molecularcomposition of desorbed coal seam gases from the Walloon Subgroup, eastern SuratBasin, Australia. Int. J. Coal Geol. 122, 21–36.

Hovey, R., Lentes, S., Ehrenreich, A., Salmon, K., Saba, K., Gottschalk, G., Gunsalus, R.P.,Deppenmeier, U., 2005. DNAmicroarray analysis ofMethanosarcina mazei Go1 revealsadaptation to different methanogenic substrates. Mol. Gen. Genomics. 273, 225–239.

76 R. Susilawati et al. / International Journal of Coal Geology 137 (2015) 66–76

Jäger, D., Sharma, C.M., Thomsen, J., Ehlers, C., Vogel, J., Schmitz, R.A., 2009. Deep sequenc-ing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogenavailability. Proc. Natl. Acad. Sci. 106, 21878–21882.

Jones, E.J.P., Voytek, M.A., Warwick, P.D., Corum, M.D., Cohn, A., Bunnell, J.E., Clark, A.C.,Orem,W.H., 2008. Bioassay for estimating the biogenic methane-generating potentialof coal samples. Int. J. Coal Geol. 76, 138–150.

Jones, E.J.P., Voytek, M.A., Corum, M.D., Orem, W.H., 2010. Stimulation of methane gener-ation from nonproductive coal by addition of nutrients or a microbial consortium.Appl. Environ. Microbiol. 76, 7013–7022.

Kinnon, E.C.P., Golding, S.D., Boreham, C.J., Baublys, K.A., Esterle, J.S., 2010. Stable isotopeandwater quality analysis of coal bedmethane production waters and gases from theBowen Basin, Australia. Int. J. Coal Geol. 82, 219–231.

Koesoemadinata, R.P., 2000. Tectono-stratigraphic framework of Tertiary coal deposits ofIndonesia, Southeast Asia Coal Geology Conference. Directorate General of Geologyand Mineral Resources of Indonesia, Bandung Indonesia, pp. 8–16.

Kreikenbohm, R., Pfennig, N., 1985. Anaerobic degradation of 3,4,5-trimethoxybenzoateby a defined mixed culture of Acetobacterium woodii, Pelobacter acidigallici, andDesulfobacter postgatei. FEMS Microbiol. Lett. 31, 29–38.

Kudo, H., Cheng, K.-J., Costerton, J.W., 1987. Interactions between Treponema bryantii andcellulolytic bacteria in the in vitro degradation of straw cellulose. Can. J. Microbiol. 33,244–248.

Lin, Y., Lü, F., Shao, L., He, P., 2013. Influence of bicarbonate buffer on the methanogeneticpathway during thermophilic anaerobic digestion. Bioresour. Technol. 137, 245–253.

Liu, Y., Whitman, W.B., 2008. Metabolic, phylogenetic, and ecological diversity of themethanogenic Archaea. Ann. N. Y. Acad. Sci. 1125, 171–189.

Mares, T.E., Moore, T.A., 2008. The influence of macroscopic texture on biogenically-derived coalbed methane, Huntly coalfield, New Zealand. Int. J. Coal Geol. 76,175–185.

McIntosh, J., Martini, A., Petsch, S., Huang, R., Nüsslein, K., 2008. Biogeochemistry of theForest City Basin coalbed methane play. Int. J. Coal Geol. 76, 111–118.

Midgley, D.J., Hendry, P., Pinetown, K.L., Fuentes, D., Gong, S., Mitchell, D.L., Faiz, M., 2010.Characterisation of a microbial community associated with a deep, coal seam meth-ane reservoir in the Gippsland Basin, Australia. Int. J. Coal Geol. 82, 232–239.

Minderlein, S., Blodau, C., 2010. Humic-rich peat extracts inhibit sulfate reduction,methanogenesis, and anaerobic respiration but not acetogenesis in peat soils of atemperate bog. Soil Biol. Biochem. 42, 2078–2086.

Mondav, R., Woodcroft, B.J., Kim, E.H., McCalley, C.K., Hodgkins, S.B., Crill, P.M., Chanton, J.,Hurst, G.B., VerBerkmoes, N.C., Saleska, S.R., Hugenholtz, P., Rich, V.I., Tyson, G.W.,2014. Discovery of a novel methanogen prevalent in thawing permafrost. Nat.Commun. 5, 3212.

Moore, T.A., 2012. Coalbed methane: a review. Int. J. Coal Geol. 101, 36–81.Moore, T.A., Bowe, M., Nas, C., 2014. High heat flow effects on a coalbed methane reser-

voir, East Kalimantan (Borneo), Indonesia. Int. J. Coal Geol. 131, 7–31.Murray, W.D., 1986. Cellulose hydrolysis by Bacteroides cellulosolvens. Biomass 10, 47–57.Papendick, S.L., Downs, K.R., Vo, K.D., Hamilton, S.K., Dawson, G.K.W., Golding, S.D.,

Gilcrease, P.C., 2011. Biogenic methane potential for Surat Basin, Queensland coalseams. Int. J. Coal Geol. 88, 123–134.

Patra, A.K., Yu, Z., 2014. Effects of gas composition in headspace and bicarbonate concen-trations in media on gas and methane production, degradability, and rumen fermen-tation using in vitro gas production techniques. J. Dairy Sci. 96, 4592–4600.

Penner, T.J., Foght, J.M., Budwill, K., 2010. Microbial diversity of western Canadian subsur-face coal beds and methanogenic coal enrichment cultures. Int. J. Coal Geol. 82,81–93.

Schink, B., 1997. Energetics of syntrophic cooperation in methanogenic degradation.Microbiol. Mol. Biol. Rev. 61, 262–280.

Schink, B., 2006a. The genus Pelobacter. In: Dworkin, M., Falkow, S., Rosenberg, E.,Schleifer, K.-H., Stackebrandt, E. (Eds.), The Prokaryotes. Springer, New York,pp. 5–11.

Schink, B., 2006b. Syntrophic associations in methanogenic degradation. In: Overmann, J.(Ed.), Molecular Basis of Symbiosis. Springer, Berlin Heidelberg.

Scott, A.C., 1999. Improving coal gas recovery with microbially enhanced coalbedmethane. In: Mastalerz, M., Glikson, M., Golding, S.D. (Eds.), Coalbed Methane: Scien-tific, Environmental and Economic Evaluation. Kluwer, Dordrecht, pp. 89–110.

Singh, D.N., Kumar, A., Sarbhai, M.P., Tripathi, A.K., 2012. Cultivation-independent analysisof archaeal and bacterial communities of the formation water in an Indian coal bed toenhance biotransformation of coal into methane. Appl. Microbiol. Biotechnol. 93,1337–1350.

Stalder, P., 1976. A Review of The South Sumatra Coal Basin. Shell Minjbuow N.V., Jakarta,p. 17.

Stams, A.M., 1994. Metabolic interactions between anaerobic bacteria in methanogenicenvironments. Antonie Van Leeuwenhoek 66, 271–294.

Stams, A.J.M., Hansen, T.A., 1984. Fermentation of glutamate and other compounds byAcidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolatedfrom black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria. Arch. Microbiol. 137, 329–337.

Stevens, S.H., Hadiyanto, 2004. Indonesia: coalbed methane indicators and basin evalua-tion. SPE Asia Pacific Oil and Gas Conference and Exhibition. Society of PetroleumEngineers, Perth Australia.

Strąpoć, D., Mastalerz, M., Eble, C., Schimmelmann, A., 2007. Characterization of the originof coalbed gases in southeastern Illinois Basin by compound-specific carbon andhydrogen stable isotope ratios. Org. Geochem. 38, 267–287.

Strąpoć, D., Picardal, F.W., Turich, C., Schaperdoth, I., Macalady, J.L., Lipp, J.S., Lin, Y.S.,Ertefai, T.F., Schubotz, F., Hinrichs, K.U., Mastalerz, M., Schimmelmann, A., 2008.Methane-producing microbial community in a coal bed of the Illinois basin. Appl.Environ. Microbiol. 74, 2424–2432.

Strąpoć, D., Mastalerz, M., Dawson, K., Macalady, J., Callaghan, A.V., Wawrik, B., Turich, C.,Ashby, M., 2011. Biogeochemistry of microbial coal-bed methane. Annu. Rev. EarthPlanet. Sci. 39, 617–656.

Sträuber, H., Schröder, M., Kleinsteuber, S., 2012. Metabolic and microbial communitydynamics during the hydrolytic and acidogenic fermentation in a leach-bed process.Energ. Sustain. Soc. 2, 1–10.

Susilawati, R., Ward, C.R., 2006. Metamorphism of mineral matter in coal from the BukitAsam deposit, south Sumatra, Indonesia. Int. J. Coal Geol. 68, 171–195.

Susilawati, R., Papendick, S.L., Gilcrease, P.C., Esterle, J.S., Golding, S.D., Mares, T.E., 2013.Preliminary investigation of biogenic gas production in Indonesian low rank coalsand implications for a renewable energy source. J. Asian Earth Sci. 77, 234–242.

Suzuki, M.T., Béjà, O., Taylor, L.T., DeLong, E.F., 2001. Phylogenetic analysis of ribosomalRNA operons from uncultivated coastal marine bacterioplankton. Environ. Microbiol.3, 323–331.

Tanner, R.S., 2002. Cultivation of bacteria and fungi, In: Hurst, C.J., Crawford, R.L., Knudsen,G.R., McInerney, M.J., Stetzenbach, L.D. (Eds.), Manual of Environmental Microbiolo-gy, 2nd ed. ASM Press Washington, D.C., pp. 62–70.

Tang, Y.-Q., Ji, P., Lai, G.-L., Chi, C.-Q., Liu, Z.-S., Wu, X.-L., 2012. Diverse microbial commu-nity from the coalbeds of the Ordos Basin, China. Int. J. Coal Geol. 90–91, 21–33.

Taulis, M., Milke, M., 2007. Coal seam gas water from Maramarua. N. Z. Charact.Comp. U. S. Analog. J. Hydrol. 46, 1–17 (New Zealand).

Van Voast, W.A., 2003. Geochemical signature of formation waters associated withcoalbed methane. AAPG Bull. 87, 667–676.

Wagner, A.O., Hohlbrugger, P., Lins, P., Illmer, P., 2012. Effects of different nitrogen sourceson the biogas production — a lab-scale investigation. Microbiol. Res. 167, 630–636.

Wawrik, B., Mendivelso, M., Parisi, V.A., Suflita, J.M., Davidova, I.A., Marks, C.R., VanNostrand, J.D., Liang, Y., Zhou, J., Huizinga, B.J., Strąpoć, D., Callaghan, A.V., 2012.Field and laboratory studies on the bioconversion of coal to methane in the SanJuan Basin. FEMS Microbiol. Ecol. 81, 26–42.

Wawrik, C.B., Callaghan, A.V., Stamps, B.W., Wawrik, B., 2014. Genome sequence ofYoungiibacter fragilis, the type strain of the genus Youngiibacter. Genome announc. 2.

Winter, J., Knoll, G., 1989. Methanogens—synthrophic dependence on fermentative andacetogenic bacteria in different ecosystems. Adv. Space Res. 9, 107–116.