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

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

PII: S0166-5162(14)00225-0DOI: doi: 10.1016/j.coal.2014.10.015Reference: COGEL 2394

To appear in: International Journal of Coal Geology

Received date: 24 July 2014Revised date: 30 October 2014Accepted date: 30 October 2014

Please cite this article as: Susilawati, Rita, Evans, Paul N., Esterle, Joan S., Robbins,Steven J., Tyson, Gene W., Golding, Suzanne D., Mares, Tennille E., Temporal changesin microbial community composition during culture enrichment experiments with Indone-sian Coals, International Journal of Coal Geology (2014), doi: 10.1016/j.coal.2014.10.015

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TITLE:

Temporal changes in microbial community composition during culture enrichment experiments

with Indonesian Coals

CONTRIBUTORS:

Rita Susilawatia,b,*, Paul N. Evansc, Joan S. Esterlea, Steven J. Robbinsc, Gene W. Tysonc, Suzanne D.

Goldinga, Tennille E. Maresa

AFFILIATIONS:

a School of Earth Sciences, The University of Queensland, St Lucia, Queensland 4072 Australia;

b Geological Agency of Indonesia, Jl. Sukarno Hatta no 444, Bandung, West Java 40254, Indonesia;

c Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences,

The University of Queensland, St Lucia, Queensland 4072, Australia.

*Corresponding author

ABSTRACT

Temporal changes in microbial community structures during methanogenesis were

investigated in cultures of South Sumatra Basin (SSB) coalbed methane (CBM) formation water

(SSB5) grown on three coals of different rank (Burung sub bituminous Rv 0.39%, Mangus sub

bituminous Rv 0.5%, Mangus anthracite Rv 2.2%). Methane production accelerated from day 6,

peaked around day 17 and then levelled off around day 20. The initial bacterial community from the

SSB formation water was predominantly Acetobacterium, Acidaminobacter, Bacteriodes and

Pelobacter species, while the archaeal community consisted of Methanosaeta, Methanosarcina and

Methanobacterium members. A general pattern was observed in all cultures with the three coals.

Over time the bacterial members decreased in proportion whereas the archaeal component

increased. The increase in the proportion of archaeal methanogens corresponded with an increase

in methane production yield.

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Enrichment cultures produced similar communities when grown on coals from the same

seam (Mangus sub bituminous and Mangus anthracite), rather than from different seams of similar

type (Burung sub bituminous and Mangus sub bituminous). Methanosaeta was the dominant

methanogen species in the sub bituminous Burung coal culture, but was a lesser proportion in

cultures of both Mangus sub bituminous and anthracite coals where Methanosarcina species were a

greater proportion. Interestingly, obligate hydrogenotrophic methanogens from the genera of

Methanobacterium, which were present at low levels in culture enrichment of all 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

obligate acetoclastic Methanosaeta members while both Mangus coals also favour metabolically

versatile Methanosarcina members, and the absence of coal favours hydrogenotrophic

methanogens. Despite the similarity of communities grown on coals from the same seam, greater

quantities of methane were generated from the lower rank coals when compared to higher rank

coals.

Keywords: coal; microbial community; methanogenesis; coal bed methane

1. Introduction

Once considered to be an unconventional energy source, coal bed methane (CBM) has

become a significant energy resource in many countries (Moore, 2012; Strąpod et al., 2011). Over

the last decade, the focus of CBM research has shifted from identifying and extracting the resource

to replenishing the methane by stimulating the native microbial community to form methane in situ

by degrading the deep coal, effectively turning coal seams into real-time methane bioreactors. The

potential for coal as a methane-forming bioreactor has been assessed in several coal basins

worldwide (Fry et al., 2009; Green et al., 2008; Guo et al., 2012; McIntosh et al., 2008; Papendick et

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

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Methane in coal seams can be produced as a by-product of the thermal coalification process

(thermogenic) and by methanogenic activity (biogenic). Isotope analysis of the methane extracted

from some CBM reservoirs has revealed biogenic origins, and have speculated that real time

methane could be formed from the in situ coal in those 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; Strąpod et al., 2007; Susilawati et al., 2013). To this end, several laboratory

based studies of the culture-dependant and independent methods have shown the presence of

active microbial consortia able to form methane from coal in a number of CBM reservoirs (Guo et al.,

2012; McIntosh et al., 2008; Papendick et al., 2011; Penner et al., 2010; Singh et al., 2012; Strąpod et

al., 2008). These results suggest the potential exists to develop new and renewable biogenic gas

resources from deep coal seams.

Despite microorganisms being present in coal and its associated formation waters, the

ability of the microbial community to deconstruct the coal is still poorly understood due to the

heterogeneously complex physical and chemical structure of the coal and to the limited number of

studies that identify coal degrading microbial species (Faison, 1991; Fakoussa and Hofrichter, 1999;

Strąpod et al., 2011). Consequently, the mechanism in which the microbial consortium degrades the

complex organic substrate into methane from coal remains unknown. A limited number of studies

have shown coal degradation begins with hydrolysis of polyaromatic, aromatic and other

hydrocarbon substrates into compounds such as volatile fatty acids (VFAs), alcohols and other

organic acids, which in turn are formed into methanogenic substrates such as acetate,

hydrogen/carbon dioxide and methanol (Jones et al., 2010; Strąpod et al., 2011). Bacterial species

from the genera Bacteroides, Pelobacter, Clostridium and Spirochaetes detected in coal and

associated formation waters have been implicated in hydrolysis of hydrocarbons (Aklujkar et al.,

2012; Kudo et al., 1987; Murray, 1986; Sträuber et al., 2012). However, despite being implicated in

hydrocarbon degradation from CBM wells, those bacterial species are often found to be fermenters

upon closer examination (Wawrik et al., 2014). In CBM reservoirs, those bacteria are typically found

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associated with hydrogenotrophic (e.g. Methanobacterium and Methanocorpusculum spp.),

acetoclastic (e.g. Methanosaeta and Methanosarcina 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; Singh et al., 2012; Strąpod et al., 2008; Tang et al., 2012).

Although broad similarities in the structure of the microbial consortia exist, each CBM

reservoir site has a unique community structure that differs with respect to its primary community

members and their dominance. Despite the broad understanding of the communities, only a few

studies have reported the influence of differing coal substrates on methane production from native

coalbed microbial communities (Fallgren et al., 2013a; Jones et al., 2008; Susilawati et al., 2013). To

this end, we investigated the microbial communities present in a previously unstudied South

Sumatra Basin (SSB) CBM reservoir and the temporal changes in its microbial community structure

during growth on three 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 accumulation of peat was transformed into the coals of the SSB

during the regressive phase of the Neogene depositional cycle (de Coster, 1974). A major regressive

sequence of Late Miocene-Pliocene sediments were deposited as the Barisan mountains uplifted

resulting in the deposition of the Muara Enim Formation, the main coal bearing unit in the SSB

(Koesoemadinata, 2000). During the deposition of the Muara Enim Formation, the tectonic setting of

the SSB produced a laterally extensive coal in respect of thickness, mineral matter and split pattern,

caused by exceptionally consistent subsidence coupled with the right type of climate (Stalder, 1976).

Coalification in the Muara Enim Formation on a regional scale was controlled predominantly by

burial depth variations as the geothermal gradient was relatively constant throughout the basin

(Stalder, 1976). The higher gradient encountered in some areas results from the localised effects of

igneous intrusions (Amijaya and Littke, 2006; Moore et al., 2014; Susilawati and Ward, 2006).

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Coal of the Muara Enim Formation has been exploited for decades at the Bukit Assam field,

where the three main coal seams (Mangus, Suban and Petai) have an average aggregate thickness

from 30 to 40 m. Several minor seams (eg. Benuang and Burung), lenticular shales, sandstones,

carbonaceous mudstones, and tuffaceous marker horizons are interbedded within these coals

(Figure 1).

3. Indonesian CBM

Indonesia is only just beginning to develop its CBM resources, and the SSB has been

highlighted as the most promising CBM basin in Indonesia (Figure 1), estimated to hold around 183

Tcf of CBM resources (Stevens and Hadiyanto, 2004). The coal in the SSB ranges from in depth 300-

1000 m and is mostly sub bituminous (Rv 0.4-0.5%) in rank with high vitrinite contents (> 90%) and

extremely low ash yields (< 5%). Most Indonesian low rank coals are composed of hydrogen rich

organic components (Davis et al., 2007) that are thought to make them excellent substrates for

biogenic methane generation.

To date, only two studies have reported the potential for biogenic CBM in Indonesia coal

basins. Gas isotopic analysis and preliminary enrichment work by Susilawati et al. (2013) identified a

biogenic origin for CBM in the SSB, and highlighted the potential of its coal and formation water for

future natural gas regeneration. Additionally Fallgren et al. (2013b), demonstrated methane

production from an Indonesia South Sumatra lignite and formation water for which there is no

previous history of gas production. To date, no study has reported the microbial methanogen

community structure in SSB CBM reservoirs.

3. Materials and methods

3.1. Coal sampling and analysis

Collection of coal directly from producing CBM reservoirs was unfortunately not possible, at

such, coal was sampled in May 2012 at PT Bukit Asam, a nearby coal mine in Tanjung Enim SSB.

Three Bukit Asam coals representing different coal seams; Burung sub-bituminous (Burung SB),

Mangus sub-bituminous (Mangus SB) and Mangus anthracite (Mangus A) were used in this study

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(Figure 1, Table 1). The rank of the two Mangus coals differed due to the effects of an igneous

intrusion into the coal seam. Coal samples were collected from the inner side of a fresh mining face

after digging out about 10 cm of surface coal to minimize the chance 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 4oC for one week before being shipped to Australia for culture

enrichment purposes. In the laboratory, coal samples were stored in an anaerobic chamber, to

avoid further oxidation processes. Time from collection to anaerobic chamber storage was less than

2 weeks.

The outer layer of the coal was pared away before the coal was crushed in the anaerobic

chamber using a mortar and pestle, milled using an electric coffee grinder and sieved to collect the

220 to 350 μm fractions. Subsequent petrographic analysis did not show any identifiable oxidation.

The crushed coal was then split into two parts using a riffle splitter, for chemical-physical analysis,

and for enrichment studies (used as sole carbon and energy substrate). ALS Laboratory, Queensland,

conducted coal chemical analyses (proximate and ultimate) while coal physical analyses (rank and

petrographic composition) were conducted using a Polarised Light microscope Leica DM4500 P LED

at the School of Earth Sciences, the University of Queensland, and Australia.

3.2. Formation water collection

Formation water for microbial culturing was collected anoxically from a CBM pilot

production well SSB5, located approximately 300 km south of Palembang, South Sumatra, Indonesia,

in May 2012. The formation water came from a depth of 609 m where the SSB5 well intersects the

Mangus seam. The formation water was flushed for one minute through a Norprene® rubber tube

connected to the SSB5 well water line before the water was collected into multiple 1L Mason jars by

overflowing the jar twice and sealing to exclude air. The Mason jars had been previously autoclaved

and flushed with nitrogen gas and filled with 1 ml reduction solution which consisted of 3 g sodium

sulphide nonahydrate, 0.675 g sodium hydroxide, 2.69g L-cysteine hydrochloride and 20 µL resazurin

dissolved in 30 ml of anoxic deionized water. The formation water samples were imported to

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Australia following Australian Quarantine and Inspection Service (AQIS) guidelines under permit no

IP13002699. In the laboratory, the formation water was stored in an anaerobic chamber (COY

laboratory Products, MI, USA) with an atmosphere of 95% nitrogen and 5% hydrogen.

A water sample from the SSB5 pilot well was also collected for anion and cation

concentration analysis. The analysis was conducted at the Centre of Groundwater Resource and

Environmental Geology in Bandung, Indonesia.

3.3. Enrichment culture experimental conditions

Experiments were designed to assess community changes during the process of biogenic

methane formation in culture enrichments of SSB5 formation water as inoculant and three SSB coals

as carbon substrates. Basal coal-based medium, as described in Papendick et al. (2011) and

Susilawati et al. (2013), was used for the microbial enrichment culture work and was prepared under

anoxic conditions according to the methods outlined by these authors.

Culture tubes were prepared in 26 mL tubes (Bellco Glass) where 0.25 g of coal substrate

(<300 μm) was combined with 9 mL of basal medium and capped with a butyl rubber stopper and

then removed from the chamber. Outside the chamber, using the gasing manifold and syringe, the

headspace was vacuumed and flushed with nitrogen 3 times to remove oxygen, then pressurized to

5 psig with nitrogen before being autoclaved at 121oC for 15 min. Culture tubes without added coal

(no-coal controls) were prepared as negative controls to test the ability of the microbial community

to form methane from coal. Other tubes with both coal and 20 mM 2-bromoethanesulfonic acid

(BESA) were also prepared as abiotic controls. Inside an anaerobic chamber and using a sterile 21-

gauge syringe needle, 3 mL of formation water 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 37oC until they

reached the stationary phase where cumulative methane concentrations no longer increased.

As previous experiments using the SSB5 formation water revealed that the growth of the

culture is in the range of 20-25 days (data not reported), eight replicate tubes for each coal

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enrichment culture were prepared to capture the different growth phases (lag, log, peak and

stationary). Changes in the relative abundance of the microbial communities during growth on each

of the coal enrichment cultures were assessed using a molecular analysis method. For this purpose,

one tube that produced the second highest methane yield from each set of coal enrichment cultures

was terminated every 2-3 days starting at day six. Terminated cultures were transferred into sterile

15 ml falcon tubes and centrifuged at 3270 × g for 30 min. The supernatant was discarded, and the

pellet frozen at -20°C until required for DNA extraction.

3.4. Measurement of Enrichment Culture Parameters and statistical analyses

Methane measurement was conducted every 2-3 days on all replicates tubes for each

culture to generate methane production profiles. Headspace gas was drawn from culture tubes

using an aseptic, anoxic technique. Headspace gas was drawn from the tubes using a Hamilton 1710

gas tight syringe and manually injected into a Varian 9700 Gas Chromatograph equipped with a 1177

column and a flame ionization detector (FID). Methane was calibrated using a 1%, 4% and 10% CH4

standard (Scientific and Technical Gases Ltd, UK).

Statistically significant differences of means in all culture treatments 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 comparison tests, each row was

analysed individually, without assuming a consistent standard deviation (SD). The means of methane

production and 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).

3.5. Growth curve and microbial community analysis

For the terminated enrichment culture tubes, the four that corresponded to the lag, log,

peak and stationary phases were selected for DNA extraction and community profiling. For

comparison, microbial community analysis was also performed on initial formation water (day 0) and

no-coal control cultures (day 10 and 20). DNA was extracted from the frozen enrichment culture cell

pellets using the phenol:chloroform:isoamyl alcohol method of Robbins et al. (in preparation) that

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was developed specifically for coal extractions. For community profiling, approximately 20 ng of

extracted DNA from enrichment cultures was used as a template for polymerase chain reaction PCR.

The 16S rRNA gene was PCR amplified using the universal primers 926F and 1392R under the

reaction conditions described in (Mondav and Woodcroft et al., 2014). Amplicons were sequenced

on the Roche 454 GS-FLX Titanium platform at the Australian Centre for Ecogenomics, University of

Queensland, Australia.

The resulting sequence reads were demultiplexed and chimeric sequences 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 operational taxonomic units

(OTUs) at a 97% cut-off. A representative sequence was then randomly selected within each cluster

which is then given a taxonomic affiliation by performing a BLASTN search against the Greengenes

16S rRNA gene database (DeSantis et al., 2006). The results in the form of the abundance of

different OTUs and their taxonomic assignments in each sample are tabulated. To avoid misleading

statistical comparisons that could results from different sequencing depths and comparisons of

diversity with the bias of uneven sampling effort (a higher number of low-abundance OTUs will be

recovered by chance with increasing sequencing effort), the number of reads was normalized to

1150, to match the sample with the lowest number of sequences. Measures of microbial diversity

(Shannon index), species community richness (Chao 1) and composition skew (Evenness) were

estimated using the program EstimateS.

4. Results

4.1. Water chemistry

The SSB5 formation water is sodium bicarbonate-chloride type, and has very low

concentration of sulphate and low concentration of calcium and magnesium (Table 1). These

signatures are typical of formation water associated with CBM (Papendick et al., 2011; Taulis and

Milke, 2007; Van Voast, 2003). The dissolution of carbonate minerals such as calcium carbonate and

magnesium carbonate may contribute to the calcium, magnesium and bicarbonate levels in the

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water. However, significantly high bicarbonate alkalinities of SSB5 water may also indicate that the

source of dissolved inorganic carbon is from other than carbonate dissolution. The same phenomena

was also noticed in the Forest City Basin (McIntosh et al., 2008). It has been suggested that a high

concentration of bicarbonate in the water indicates the microbial decomposition of reduced carbon

stored in organic matter. The SSB5 water has higher total dissolved solid as shown by higher

concentration of sodium and chloride. Sulphate in SSB5 water was only detected in very low

concentration and that may not favour the growth of bacterial sulphate reducers.

4.2. Coal characterisation

Petrographic analysis (Table 2) showed that the Burung SB coal was the 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 Mangus SB coals or vitrinite in Mangus A coal were the major maceral groups present in the

coals and accounted for 81-97% of maceral composition. Liptinite group content is 6-7%, and

inertinite group content varies from 2-12% (Table 2). All three coals had very low mineral matter (≤

1%; corroborated by low ash yields <10%), which is typical for Indonesian coal. The anthracite by

comparison had much lower moisture and higher reflectance and as a result, higher vitrinite content

as the liptinite group macerals were devolatilised.

4.2. Methane production from coal enrichment cultures

Negligible amounts of methane produced from BESA treated abiotic control tubes (data

were not presented) suggested that little methane in cultures was from methane adsorbed in the

substrate coal. Overall, methane from each of the enrichment cultures increased slowly until day 6.

After day 6, methane production increased significantly and continued until around day 17 when the

peak of methane yield was achieved (Figure 2). After day 10, methane production rate started to

decline and was confirmed by day 20 when no increases in methane were seen for all coal types and

no-coal controls (Figure 2).

Using two-factor Anova, the difference between the methane production yields from the

four culture treatments (three cultures with coal substrate and 1 control) over time was analysed.

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The Anova test produced P values less than 5% for the three parameters being measured

(interaction, row and column factor) (Table 3) suggesting that methane production in this

experiment was affected by both time and substrate. Although the mean and maximum methane

production in all coal cultures were higher than no coal control culture, post hoc multiple

comparison test showed that only the Burung cultures consistently showed statistically significant

differences in their methane concentration compared to the no-coal control cultures whereas no

significant differences were observed in the mean between both Mangus cultures and no-coal

control cultures (Table 4). At peak production (day 17), the methane produced by the Burung

cultures was also significantly higher than other coal treated cultures (Figure 2, Table 4). This result

suggests there is some ability of the SSB5 microbial community to metabolise the coal and indicates

the influence of coal substrate to methanogenesis in SSB5 cultures. Even though differences in

methane concentration among the 3 coal cultures were not all statistically significant according to

the ANOVA test, the mean of methane production data indicated declining methane production

yields with increased rank (Figure 2). Burung SB coal culture has the highest methane production

yield, followed by the Mangus SB and Mangus A coal as the least (Figure 2). However, the rank trend

observed in the maximum total production rate is not as evident in the net rate (treatment ─

control) (Figure 3).

It has been reported that headspace gas composition and media bicarbonate concentrations

can have a significant effect on gas and methane production in culturing experiments (Lin et al.,

2013; Patra and Yu, 2014). The concentration of bicarbonate in our media was shown to have no

deleterious effect on coal to methane rate and yields (Papendick et al., 2011). Bicarbonate in the

inoculum may provide excess CO2, which in the presence of H2 rich substrates may stimulate

methanogenesis. The headspace gas containing H2 can provide hydrogen needed by

hydrogenotrophic methanogens to reduce CO2 to produce methane (Patra and Yu, 2014). Although

we use H2N2 as a gas mix in our anaerobic vessel, the presence of this gas in our culture tubes was

replaced by N2, as such the influence of additional H2 to the methane production in our experiment

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can be eliminated. Nitrogen can be a limiting factor (in the hydrolysis phase) but also as a source for

fermentation for subsequent methane production by methanogenic archaea (Wagner et al., 2012).

We did not test the effect of N2 gas on our methane production. However, since all cultures,

including the no-coal control, were treated similarly with 125 kPa N2, the presence of this gas in our

culture headspace will not affect treatment comparisons.

4.3. Microbial community

4.3.1. Microbial diversity

At the 97% cut-off score for an operational taxonomic unit (OTU), a total of 46 bacterial and

10 archaeal genera were observed in the SSB5 microbial communities grown on the Burung SB,

Mangus SB and Mangus A coals. Those OTUs over 1% are shown in Tables 5 and 6. The microbial

community of the Burung SB enrichment cultures had both the highest bacterial and archaeal

diversities among the coal enrichment cultures (Table 7 and 8), and produced the greatest quantity

of methane (Figure 2). For all three coals rank enrichment cultures, the bacterial diversity and

community skew, measured by evenness, decreased. Archaeal diversity and skew remained static

across all time points (Table 7 and 8).

The SSB water bacterial sequences were dominated by members of the Pelobacter and

Acetobacterium genera across all cultures and time points, while other species belonging to the

orders Bacteriodetes, Spirochaetes, Synergistetes and Tenericutes were consistently present at low

levels (Table 5). For the archaeal component of the communities, all enrichment cultures were

dominated by acetoclastic methanogens from the genera of Methanosaeta (Figure 4 and Table 6).

Lower levels of Methanosarcina, Methanobacterium, Methanospirillum and Candidatus

Methanoregula were also detected across all enrichment cultures whereas members belonging to

Methanofollis, Methanomethylovorans and Thermoplasmata were only found at very low

abundance in most cultures (<1%) and are grouped in Table 6 as “other archaea”.

4.3.2. Temporal changes in community structure

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The initial formation water contained a community composed of 14% bacteria and 86%

archaea. Culturing successfully enriched the bacterial communities, and by day six (the beginning of

log phase), the relative abundance of bacteria had almost doubled. At day six, the bacterial

population was at its highest proportion in all three cultures (bacteria 43-48% and archaea 52-57%)

and the community structure was similar between all three cultures, owing to the same inoculum

source. Over time the relative abundance of the bacterial population tended to decline, dropping

from 43-48% at day 6 to 15-29% by day 20. In contrast, in the no-coal controls, during the log phase

(day 10) bacteria occupied 17%, but increased to 28% at stationary phase (day 20), whereas archaea

decreased from 83% at day 10 to 71% at day 20.

In the Burung SB culture, the proportion of Acetobacterium and Pelobacter were similar with

both tending to decrease with time, whereas in the Mangus SB, Mangus A and no-coal control

cultures, Pelobacter is more dominant than Acetobacterium. The Burung SB culture had the highest

Bacteriodales population. Molecular analysis showed significant variations in methanogen

communities, with shifts in the dominance of different groups (Figure 4). It appeared that in the

Burung SB culture, most of the methane production was associated with growth of the obligate

acetoclastic methanogen, Methanosaeta. At day six, in both Mangus SB and Mangus A, the

acetoclastic (Methanosaeta) and hydrogenotrophic methanogens (Methanoregula and

Methanospirllium) were present in almost the same proportion but then decreased toward the end

of growth. In contrast, despite its low proportion at the early stage of growth, Methanosarcina was

present in significant proportions at the stationary phase. In the no-coal control culture,

Methanosaeta was the dominant methanogen until day 10. However, at the end of experiment

Methanosarcina and Methanobacteria were found to be equivalent in abundance. The

Methanobacteriales population was only observed in the early growth stage of Burung SB culture

and at the late stage of no-coal control culture. Its closest match was to the Methanobacterium

formicicum type strain.

5. Discussion

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5.1. Methane production

Although the differences are not considered significant by the ANOVA tests, the total and

net methane production from the experiments increased consistently over time, with some variation

attributable to the rank of the coal substrates. The small differences between methane yield from

coal tubes and no-coal controls in our experiment was likely to be a result of low concentration of

critical coal degrading bacterial consortium in the initial formation water inoculum, as such only

small amount of coal organic material can be biodegraded. Moreover, during storage, it is likely that

a key nutrient needed for the rapid growth of the microbial community available in the initial

formation water becomes depleted, or toxic by-products of microbial metabolism were

accumulated, which further influenced the growth of microbial communities capable of converting

coal to methane. Additionally, it seems that many organic constituents are also available as

substrates in the initial formation water, likely from continued microbial metabolism. This product

along with fine coal particles carried over in the water can provide substrates for metabolism in coal

and no-coal enrichment cultures.

The variation in methane production amongst experimental coal and no-coal control

replicate tubes may relate to the concentration of microbial methanogen consortium member

present in each 3ml of the inoculum added to these experimental systems. Each tube likely

contained slightly varying concentrations of the critical microbial methanogen consortium member,

which then led to differences in the methane yield. It can be assumed that the tube with the highest

methane production contains the highest concentration of critical consortium members. The fact

that differences showed a consistent change with rank could be coincidental, or reflect the influence

of coal on the process.

Our data suggests that the source of carbon for methane production in all SSB5 cultures was

from both the additional coal substrate and the inoculum (Figure 3). The greatest influence of coal

substrate occurred from day 6 to 10. In general, total production rate was higher than net

production rate, indicating that the organic precursor in the inoculum was easier to biodegrade than

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the additional coal substrate. This is not surprising since the consortia may well have adapted to the

native source of carbon present in the water.

5.2. Microbial methanogen community structure

In the future, CBM resources may become an important energy supplement to meet some

of the global demand for energy resources. Replenishment of current and future CBM resources by

in situ methanogenesis is one way to increase the productive lifetime of these fields but is

dependent on the ability of coal to be degraded by microbial communities. To this end, we tested

the ability of Indonesian coals to provide substrates for a native microbial community from an

Indonesian CBM well. The results of our experiments suggest that these coals can provide carbon

and energy source for a microbial consortium. Lower rank coals produced slightly more methane

than the higher rank coal in the experiments. Whereas cultures grown on coals of similar rank and

type produced different community structures, coal of the same seam with different rank revealed

similarity in their community structures, suggesting that community structure could be dependent

on coal source.

After an initial lag phase, typical of lag phases observed by other researchers for microbial

consortia grown on coal (Green et al., 2008; Papendick et al., 2011), the microbial population was

consistent among all enrichments grown on coal and consisted predominantly of the obligate

acetoclastic Methanosaeta methanogen species. However, due to the time taken to start

enrichment experiments, it is likely that a key nutrient needed to drive the metabolism of the

microbial consortia might have forced the community to this structure. As little methane was

formed during the lag phase, the Methanosaeta members are potentially significant constituents of

the original formation water inoculum, which is consistent with other studies that show

Methanosaeta species as a large proportion of indigenous communities (Jones et al., 2010; Wawrik

et al., 2012). The predominant bacterial members of the community during this lag phase were

Acidaminobacter, Acetobacterium and Pelobacter. The members of these bacteria are known to be

able to hydrolize and ferment aromatic compounds and hydrocarbons (Kreikenbohm and Pfennig,

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1985; Schink, 2006a; Sträuber et al., 2012) and have previously been identified in studies of coal

associated enrichment cultures across several CBM reservoirs (Barnhart et al., 2013; Dawson et al.,

2012; Green et al., 2008; Jones et al., 2010; Penner et al., 2010; Strąpod et al., 2011; Wawrik et al.,

2012). It is likely these bacteria were the primary degraders of coal constituents in our cultures.

Moreover, the involvement of Acetobacterium in the utilization of substrates produced by coal

degradation has been suggested by Barnhart et al. (2013). Consistent with this result, our

experiment showed that Acetobacterium species were always lesser proportions of the no-coal

community over the time series compared to coal containing cultures, suggesting that

Acetobacterium species likely metabolise substrates directly from the coal, even though the exact

mechanisms remain unknown.

After the lag phase, methane production increased rapidly and reached maximum

production rate by day 11 in all enrichment cultures. During this time the relative proportions of the

microbial community measured at days 10 and 17 had changed from day 6, with an increased

archaeal and corresponding decreased bacterial fraction. This result suggested that methanogenesis

was occurring from hydrolysis and fermentation end products produced by the bacterial component.

As the proportion of the obligate acetoclastic Methanosaeta had increased, such as in the Burung

microcosm, it is likely that it syntrophically consumes the acetate being produced by the Pelobacter,

Acetobacterium and Acidaminobacter members, which are known acetogens (Beckmann et al., 2011;

Green et al., 2008; Schink, 1997; Schink, 2006a, b; Stams and Hansen, 1984; Stams, 1994). It has

been reported that acetate production can eventually inhibit the growth of Acetobacterium, but in

the presence of Methanosaeta, acetate is utilised to produce methane and carbon dioxide (Bainotti

et al., 1997; Winter and Knoll, 1989). Acetobacterium would likely compete with hydrogenotrophic

methanogens for hydrogen when grown on coal which may explain why the proportion of

hydrogenotrophic methanogens only increased in the no-coal controls culture in the near absence of

Acetobacterium. Based on the ability of Methanosaeta to outcompete Methanosarcina species for

acetate (Liu and Whitman, 2008), the dominance of the Methanosaeta members over

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Methanosarcina species suggests that in our experiment acetate was only present at low

concentrations. Furthermore, Methanosarcina members observed in our study were closely related

to Methanosarcina mazeii, a versatile strain that is able to metabolise H2-CO2, acetate,

methylamines and methanol (Hovey et al., 2005; Jäger et al., 2009), therefore an increase in the

proportion of Methanosarcina species likely accounted for increases in methane concentration and

could be due to these members being able to metabolise methylated compounds such as

methylamines, methyl sulphide and methanol when produced towards the end of fermentation

(Allison and Macfarlane, 1989).

During the times between 10 and 17 days it became apparent that the communities on the

different coals diverged. The microbial community grown with the Mangus SB coal was more similar

to the Mangus A coal, than to the Burung SB coal. Pelobacter species were more dominant in both

Mangus cultures than in Burung SB culture. In the Mangus coal communities, Methanosarcina

became a significant proportion while in the Burung enrichment it was present in low proportion.

The increase of Methanosarcina in both Mangus cultures is concomitant with the decrease of both

Methanosaeta and Methanoregula. We postulated that both Mangus coals substrates reduced the

particular contribution of the obligate acetoclastic methanogen Methanosaeta and the obligate

hydrogenotrophic methanogen Methanoregula to methane production. On the contrary both coal

substrates increased the relative contribution of Methanosarcina to methane production in the

cultures. Methanosarcina has versatile methanogenic pathways, thus depending on the

environment condition these microbes may have advantage to produce methane via an effective

pathway and outcompete the other methanogen genera. In the end, it appeared that Burung coals

led to the growth of Methanosaeta while both of the Mangus coals stimulated the growth of

Methanosarcina. Except that the coals come from the same seam, it is not clear what factors are

responsible for the similarities in the microbial communities of cultures on Mangus SB and A coals.

The two coals have different chemical and petrographic properties due to the significant differences

in rank, but formerly shared the same origin and may have some characteristics that force the

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communities to reach a similar population structure. Although the communities on the Mangus SB

was similar to Mangus A coals, maximum methane produced from the Mangus SB was higher than

Mangus A, suggesting that the coal rank has an effect in the ability of the community to generate

methane. Overall, there also appeared to be an inverse correlation between maximum methane

yield and coal rank which is in agreement with previous studies (Scott, 1999; Strąpod et al., 2011).

The lower rank coal Burung SB culture is likely to be less recalcitrant and is easier to be biodegraded

than higher rank Mangus anthracite coal. It has been reported that an increasing recalcitrant

material has an inhibitory effect on methanogenesis (Minderlein and Blodau, 2010). It is likely that

the coal maturity and the organofacies of the source material govern the dominant extractable

compounds and their concentrations in the coal samples (Glombitza et al., 2009).

By day 20, methane production had ceased, and differences between the methane

concentrations of the microbial consortia when grown on the three coal substrates were still

observed when compared to the no coal control, although these differences were not always

significant. Interestingly, in the no-coal control enrichment cultures, the proportion of the

acetoclastic Methanosaeta species decreased significantly whereas the proportion of the obligate

hydrogenotrophic methanogens Methanobacterium increased which suggested that

hydrogen/carbon dioxide and not acetate are more important intermediates in microbial

metabolism in cultures without a coal substrate. Overtime we noted that in all cultures there was a

tendency that the bacterial members decreased in proportion whereas the archaeal component

increased and the increase in methanogen population corresponded with an increase in methane

production yield (Figure 5)

6. Conclusion

In this study, we show that an Indonesian CBM well produced an active methane-forming

microbial community, which adds to the known basins where microbial communities can form

methane from coal. The diversity of the community derived from the Indonesian coal formation

water was similar to previous studies that have enriched for microbial communities from coal basins

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worldwide. Further, to enrichment culture study and microbial community profiling, we show

community structure changes over time, which exhibited more similarity when grown on coal from

the same seam but of different ranks (Mangus SB and Mangus A), than to coal from similar rank

(Mangus SB and Burung SB). It is noteworthy that the enrichment culture grown on the lowest rank

coal produced the greatest quantity of methane, whereas the highest rank coal produced the least

methane per gram of coal. Overall, our results indicate a potential relationship between coal type,

microbial community composition and methane production. Further work should focus on factors

that may enhance or limit coal methanogenesis. We also highlight the need for more detailed

studies on coals of different type and ranks and their potential for microbes to form methane.

7. Acknowledgment

Australian Awards Scholarship provided funding for this research. The TSOP Spackman

award was used to fund part of laboratory consumables and gas isotope analyses for this study. PT

Medco CBM Sekayu and PT Bukit Asam kindly allowed site and sample access. The authors thank

Gordon Southam for constructive review of this manuscript, also Soleh Basuki Rahmat, Eko Budi

Tjahyono and Erlina Lena Ria from Geological Agency of Indonesia for field assistance.

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References

Aklujkar, M., Haveman, S.A., DiDonato, R., Jr., Chertkov, O., Han, C.S., Land, M.L., Brown, P., Lovley,

D.R., 2012. The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and

physiological features. BMC genomics 13, 690.

Allison, C., Macfarlane, G.T., 1989. Influence of pH, nutrient availability, and growth rate on amine

production by Bacteroides fragilis and Clostridium perfringens. Applied and environmental

microbiology 55, 2894-2898.

Amijaya, H., Littke, R., 2006. Properties of thermally metamorphosed coal from Tanjung Enim Area,

South Sumatra Basin, Indonesia with special reference to the coalification path of macerals.

International Journal of Coal Geology 66, 271-295.

Bainotti, A.E., Futagami, K., Nakashimada, Y., Chang, Y.J., Nagai, S., Nishio, N., 1997. pH Auxostat

continuous culture of Acetobacteriumsp. coupled with removal of acetate by Methanosaeta concilii.

Biotechnology Letters 19, 989-993.

Barnhart, E.P., De León, K.B., Ramsay, B.D., Cunningham, A.B., Fields, M.W., 2013. Investigation of

coal-associated bacterial and archaeal populations from a diffusive microbial sampler (DMS).

International Journal of Coal Geology 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 Abandoned Coal Mines. Applied and

environmental microbiology 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.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Bragg, L., Stone, G., Imelfort, M., Hugenholtz, P., Tyson, G.W., 2012. Fast, accurate error-correction

of amplicon pyrosequences using Acacia. Nature methods 9, 425.

Butland, C.I., Moore, T.A., 2008. Secondary biogenic coal seam gas reservoirs in New Zealand: A

preliminary assessment of gas contents. International Journal of Coal Geology 76, 151-165.

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 from Western

Indonesia: Relationship to mire type and sequence stratigraphic setting. International Journal of Coal

Geology 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 Consortia from a Biogenic Gas Field in

Alaska's Cook Inlet Basin. Applied and environmental microbiology 78, 3599-3605.

de Coster, G.L., 1974. The geology of the Central and South Sumatra Basins, 3th Annual Convention

Indonesian Petroleum Association Jakarta, pp. 77-110.

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. International Journal of

Systematic and Evolutionary Microbiology 59, 1064-1069.

Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and

speed of chimera detection. Bioinformatics 27, 2194-2200.

Faison, B.D., 1991. Biological Coal Conversions. Critical Reviews in Biotechnology 11, 347-366.

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.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

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. International Journal of Coal Geology 115, 92-96.

Fallgren, P.H., Zeng, C., Ren, Z., Lu, A., Ren, S., Jin, S., 2013b. Feasibility of microbial production of

new natural gas from non-gas-producing lignite. International Journal of Coal Geology 115, 79-84.

Flores, R.M., Rice, C.A., Stricker, G.D., Warden, A., Ellis, M.S., 2008. Methanogenic pathways of coal-

bed gas in the Powder River Basin, United States: The geologic factor. International Journal of Coal

Geology 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, Including a Previously Deeply Buried

Section (1.6–2.3 km) Above ∼ 150 Ma Basement Rock. Geomicrobiology Journal 26, 163-178.

Ghosh, S., Jha, P., Vidyarthi, A.S., 2014. Unraveling the microbial interactions in coal organic

fermentation for generation of methane — A classical to metagenomic approach. International

Journal of Coal Geology 125, 36-44.

Glombitza, C., Mangelsdorf, K., Horsfield, B., 2009. Maturation related changes in the distribution of

ester bound fatty acids and alcohols in a coal series from the New Zealand Coal Band covering

diagenetic to catagenetic coalification levels. Organic Geochemistry 40, 1063-1073.

Golding, S.D., Boreham, C.J., Esterle, J.S., 2013. Stable isotope geochemistry of coal bed and shale

gas and related production waters: A review. International Journal of Coal Geology 120, 24-40.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Green, M.S., Flanegan, K.C., Gilcrease, P.C., 2008. Characterization of a methanogenic consortium

enriched from a coalbed methane well in the Powder River Basin, U.S.A. International Journal of Coal

Geology 76, 34-45.

Guo, H., Liu, R., Yu, Z., Zhang, H., Yun, J., Li, Y., Liu, X., Pan, J., 2012. Pyrosequencing reveals the

dominance of methylotrophic methanogenesis in a coal bed methane reservoir associated with

Eastern Ordos Basin in China. International Journal of Coal Geology 93, 56-61.

Hamilton, S.K., Golding, S.D., Baublys, K.A., Esterle, J.S., 2014. Stable isotopic and molecular

composition of desorbed coal seam gases from the Walloon Subgroup, eastern Surat Basin,

Australia. International Journal of Coal Geology 122, 21-36.

Hovey, R., Lentes, S., Ehrenreich, A., Salmon, K., Saba, K., Gottschalk, G., Gunsalus, R.P.,

Deppenmeier, U., 2005. DNA microarray analysis of Methanosarcina mazei Go1 reveals adaptation

to different methanogenic substrates. Molecular genetics and genomics : MGG 273, 225-239.

Jäger, D., Sharma, C.M., Thomsen, J., Ehlers, C., Vogel, J., Schmitz, R.A., 2009. Deep sequencing

analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability.

Proceedings of the National Academy of Sciences 106, 21878-21882.

Jones, E.J.P., Voytek, M.A., Corum, M.D., Orem, W.H., 2010. Stimulation of Methane Generation

from Nonproductive Coal by Addition of Nutrients or a Microbial Consortium. Applied and

environmental microbiology 76, 7013-7022.

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 potential of coal samples.

International Journal of Coal Geology 76, 138-150.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Kinnon, E.C.P., Golding, S.D., Boreham, C.J., Baublys, K.A., Esterle, J.S., 2010. Stable isotope and

water quality analysis of coal bed methane production waters and gases from the Bowen Basin,

Australia. International Journal of Coal Geology 82, 219-231.

Koesoemadinata, R.P., 2000. Tectono-stratigraphic framework of Tertiary coal deposits of Indonesia,

Southeast Asia Coal Geology Conference,. Directorate General of Geology and Mineral Resources of

Indonesia, Bandung Indonesia, pp. 8-16.

Kreikenbohm, R., Pfennig, N., 1985. Anaerobic degradation of 3,4,5-trimethoxybenzoate by a

defined mixed culture of Acetobacterium woodii, Pelobacter acidigallici, and Desulfobacter

postgatei. FEMS Microbiology Letters 31, 29-38.

Kudo, H., Cheng, K.-J., Costerton, J.W., 1987. Interactions between Treponema bryantii and

cellulolytic bacteria in the in vitro degradation of straw cellulose. Canadian Journal of Microbiology

33, 244-248.

Lin, Y., Lü, F., Shao, L., He, P., 2013. Influence of bicarbonate buffer on the methanogenetic pathway

during thermophilic anaerobic digestion. Bioresource Technology 137, 245-253.

Liu, Y., Whitman, W.B., 2008. Metabolic, Phylogenetic, and Ecological Diversity of the Methanogenic

Archaea. Annals of the New York Academy of Sciences 1125, 171-189.

Mares, T.E., Moore, T.A., 2008. The influence of macroscopic texture on biogenically-derived coalbed

methane, Huntly coalfield, New Zealand. International Journal of Coal Geology 76, 175-185.

McIntosh, J., Martini, A., Petsch, S., Huang, R., Nüsslein, K., 2008. Biogeochemistry of the Forest City

Basin coalbed methane play. International Journal of Coal Geology 76, 111-118.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

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 methane reservoir in

the Gippsland Basin, Australia. International Journal of Coal Geology 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 a temperate bog. Soil Biology and

Biochemistry 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. Nature communications 5, 3212.

Moore, T.A., 2012. Coalbed methane: A review. International Journal of Coal Geology 101, 36-81.

Moore, T.A., Bowe, M., Nas, C., 2014. High heat flow effects on a coalbed methane reservoir, East

Kalimantan (Borneo), Indonesia. International Journal of Coal Geology 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 coal seams. International Journal of

Coal Geology 88, 123-134.

Patra, A.K., Yu, Z., 2014. Effects of gas composition in headspace and bicarbonate concentrations in

media on gas and methane production, degradability, and rumen fermentation using in vitro gas

production techniques. Journal of Dairy Science 96, 4592-4600.

Penner, T.J., Foght, J.M., Budwill, K., 2010. Microbial diversity of western Canadian subsurface coal

beds and methanogenic coal enrichment cultures. International Journal of Coal Geology 82, 81-93.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Schink, B., 1997. Energetics of syntrophic cooperation in methanogenic degradation. Microbiology

and Molecular Biology Reviews 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, pp. 1-19.

Scott, A.C., 1999. Improving coal gas recovery with microbially enhanced coalbed methane, in:

Mastalerz, M., Glikson, M., Golding, S.D. (Eds.), Coalbed methane: Scientific, Environmental and

Economic Evaluation. Kluwer, Dordrecht, pp. 89-110.

Singh, D.N., Kumar, A., Sarbhai, M.P., Tripathi, A.K., 2012. Cultivation-independent analysis of

archaeal and bacterial communities of the formation water in an Indian coal bed to enhance

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.J.M., Hansen, T.A., 1984. Fermentation of glutamate and other compounds by

Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black

mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic

bacteria. Archives of Microbiology 137, 329-337.

Stams, A.M., 1994. Metabolic interactions between anaerobic bacteria in methanogenic

environments. Antonie van Leeuwenhoek 66, 271-294.

Stevens, S.H., Hadiyanto, 2004. Indonesia: Coalbed Methane Indicators and Basin Evaluation, SPE

Asia Pacific Oil and Gas Conference and Exhibition. Society of Petroleum Engineers, Perth Australia.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Strąpod, D., Mastalerz, M., Dawson, K., Macalady, J., Callaghan, A.V., Wawrik, B., Turich, C., Ashby,

M., 2011. Biogeochemistry of Microbial Coal-Bed Methane. Annual Review of Earth and Planetary

Sciences 39, 617-656.

Strąpod, D., Mastalerz, M., Eble, C., Schimmelmann, A., 2007. Characterization of the origin of

coalbed gases in southeastern Illinois Basin by compound-specific carbon and hydrogen stable

isotope ratios. Organic Geochemistry 38, 267-287.

Strąpod, 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. Applied and environmental microbiology 74, 2424-

2432.

Sträuber, H., Schröder, M., Kleinsteuber, S., 2012. Metabolic and microbial community dynamics

during the hydrolytic and acidogenic fermentation in a leach-bed process. Energ Sustain Soc 2, 1-10.

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 coals and implications

for a renewable energy source. Journal of Asian Earth Sciences 77, 234-242.

Susilawati, R., Ward, C.R., 2006. Metamorphism of mineral matter in coal from the Bukit Asam

deposit, south Sumatra, Indonesia. International Journal of Coal Geology 68, 171-195.

Tang, Y.-Q., Ji, P., Lai, G.-L., Chi, C.-Q., Liu, Z.-S., Wu, X.-L., 2012. Diverse microbial community from

the coalbeds of the Ordos Basin, China. International Journal of Coal Geology 90–91, 21-33.

Taulis, M., Milke, M., 2007. Coal Seam Gas water from Maramarua, NewZealand: Characterisation

and Comparison to US Analogues Journal of Hydrology New Zealand 46, 1-17.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

Van Voast, W.A., 2003. Geochemical signature of formation waters associated with coalbed

methane. AAPG Bulletin 87, 667-676.

Wagner, A.O., Hohlbrugger, P., Lins, P., Illmer, P., 2012. Effects of different nitrogen sources on the

biogas production – a lab-scale investigation. Microbiological Research 167, 630-636.

Wawrik, B., Mendivelso, M., Parisi, V.A., Suflita, J.M., Davidova, I.A., Marks, C.R., Van Nostrand, J.D.,

Liang, Y., Zhou, J., Huizinga, B.J., Strąpod, D., Callaghan, A.V., 2012. Field and laboratory studies on

the bioconversion of coal to methane in the San Juan Basin. FEMS Microbiology Ecology 81, 26-42.

Wawrik, C.B., Callaghan, A.V., Stamps, B.W., Wawrik, B., 2014. Genome Sequence of Youngiibacter

fragilis, the Type Strain of the Genus Youngiibacter. Genome announcements 2.

Winter, J., Knoll, G., 1989. Methanogens—Synthrophic dependence on fermentative and acetogenic

bacteria in different ecosystems. Advances in Space Research 9, 107-116.

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Figure Captions

Fig.1. Location of the study area: a) Water sampling (SSB5 well: blue dot) and coal sampling (black

dot); b) Coal stratigraphy of Bukit Asam area. Coal samples used in this study are from Burung and

Mangus seams. Stratigraphy of the coal seam is modified from Amijaya and Littke (2006).

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 represent standard deviation

for replicates tubes.

Fig.3. Maximum methane production rate in SSB5 cultures, plotted as total rate (rate from

treatment tube where source of carbon came from both coal substrate and native fine coal particle

or dissolved constituent present in inoculum) and net rate (rate from treatment tubes subtracted by

no-coal control where coal is a major carbon substrate).

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 bituminous, 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 that occupy 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 area with pattern.

Fig.5. Cross plot of methanogen abundance against methane yield in SSB5 cultures. Level of

confidence (P = 0.05) plot as dot line.

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Table Captions

Table 1. Formation water properties.

Table 2. Petrographic and chemical composition of coal samples used in this study. Bases are as

determined (a.d.b) and dry ash free (d.a.f).

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

Table 4. Post Hoc multiple comparison test of methane production in SSB5 culture experiments

Table 5. The dominant bacterial taxa detected in SSB5 cultures and the closest matches at 99%

similarity to known bacteria in GeneBank database.

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

similarity to known bacteria in GeneBank database.

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

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

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Fig 1a

Fig 1.b

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Fig 2

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Fig 3

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Burung (Rv 0.39)

Proteobacteria

Spirochaetes

Firmicutes

Bacteriodetes

Methanosarcinae

Methanosaetaceae

Methanospirillaceae

Methanoregulaceae

Methanobacteriaceae

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Proteobacteria

Spirochaetes

Firmicutes

Bacteriodetes

Methanosarcinae

Methanosaetaceae

Methanospirillaceae

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Fig.4.

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Spirochaetes

Firmicutes

Bacteriodetes

Methanosarcinae

Methanosaetaceae

Methanospirillaceae

Methanoregulaceae

Methanobacteriaceae

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Formation water and no-coal control Proteobacteria

Spirochaetes

Firmicutes

Bacteriodetes

Synergistales

Methanosarcinae

Methanosaetaceae

Methanospirillaceae

Methanoregulaceae

Methanobacteriaceae

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Fig. 5

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Table 1.

SSB5 water parameters Concentration

Temperature 32

pH 6.63

Salinity(ppm) 1350

Conductivity(mS) 3.3

CO32- (mg/L) nd

HCO3- (mg/L) 942.5

SO42-(mg/L) 3

Cl- (mg/L) 439.5

Ca2+ (mg/L) 9.7

Mg2+ (mg/L) 6.2

Na+ (mg/L) 577.9

K+ (mg/L) 126.1

Trace elements

Cu (mg/L) 0.025

Zn (mg/L) 0.318

Ni (mg/L) nd

Co (mg/L) 2.97

Pb (mg/L) 0

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Tabel 2

Analysis Burung Sb Mangus Sb Mangus A

Proximate % (a.d.b)

Moisture 16.8 8 1.2 Ash 2.2 0.7 1.2 Volatile matter 38.1 42.5 14.7 Fixed carbon 42.9 48.8 82.9 Total sulphur 0.83 0.34 1.06

Ultimate % (d.a.f)

Carbon 76.2 78.2 91 Hydrogen 5.14 5.26 3.83 Nitrogen 1.67 1.15 1.56 Sulphur 1.02 0.37 1.09 Oxygen (By difference) 15.9 15.1 2.6

Petrography (%)

Rv (Rank) 0.39 0.5 2.2 Maceral composition

Huminite/Vitrinite

Textinite 6 3

Ulminite 23 22

Attrinite 24 20

Densinite 17 27

Corpohuminite 8 4

Gelinite 4 5

Total 82 81 97 Inertinite

Fusinite 5 3

Semifusinite 1 1

Funginite 2 5

Inertodetrinite 2 3 2 Macrinite 0 0

Total 10 12 2 Liptinite

Sporinite 1 1

Cutinite 1 1

Resinite 1 0

Liptodetrinite 2 2

Suberinite 1 2

Exsudatinite 1 0 0 Total 7 6 0 Mineral matter 1 1 1

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Table 3

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

Interaction 1315 9 146.1 F (9, 62) = 2.414 P = 0.0203

Time 46199 3 15400 F (3, 62) = 254.4 P < 0.0001

Treatments 5659 3 1886 F (3, 62) = 31.16 P < 0.0001

Residual 3753 62 60.53 SS=sum of square, DF= degree of freedom, MS= mean of square, DFn= degrees of freedom numerator, Dfd= degrees of freedom denominator. DFn= a-1, DFd= N-a, a=number of treatments, N= the total number of measurement.

Table 4

Time (days)

Post hoc multiple comparison test (Holm Sidak method)

Brg vs MSb

Brg vs MA

Brg vs Ncc

MSB vs MA

MSB vs Ncc

MA vs Ncc

MSB vs Ncc

MA vs Ncc

6 < 0.001 < 0.001 < 0.001 0.150 0.370 0.630 0.370 0.630

10 0.020 0.240 0.001 0.600 0.039 0.080 0.039 0.080

17 0.006 0.005 0.002 0.400 0.037 0.181 0.037 0.181

20 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

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Table 5.

No Closest BLAST Sequence match Origin of closest match Affinity

1

Bacteroidetes bacterium AY548787

Metal-Containing Wastewater

Bacteria; Bacteroidetes; Bacteroidia; Bacteroidales; unclassified_Bacteroidales

2

Geovibrio ferrireducens X95744

Surface sediment of hydrocarbon-contaminated ditch

Bacteria; Deferribacteres; Deferribacterales; Deferribacteraceae; Geovibrio.

3

Acetobacterium carbinolicum AB546236 Oil storage tank

Bacteria; Firmicutes; Clostridia; Clostridiales; Eubacteriaceae;Acetobacterium.

4

Acidaminobacter hydrogenoformans AF016691 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.

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Table 6.

No Closest BLAST Sequence match Origin of closest match Taxonomic String

1

Methanobacterium formicicum AF169245

Culture collection, Institute of Microbiology CAS China

Archaea; Euryarchaeota; Methanobacteria; Methanobacteriales; Methanobacteriaceae; Methanobacterium

2

Uncultured Methanospirillaceae archaeon S000591267 river sediment

Archaea; Euryarchaeota; Methanomicrobia; Methanomicrobiales; Methanospirillaceae; Methanospirillum.

3

Methanoregula formicica AB479390 granular sludge

Archaea; Euryarchaeota; Methanomicrobia; Methanomicrobiales; Methanomicrobiales_incertae_sedis; Methanoregula.

4

Uncultured archaeon JN397698 River bank sediment

Archaea; Euryarchaeota; Methanomicrobia; Methanosarcinales; Methanosaetaceae; Methanosaeta.

5

Methanosarcina mazeii DQ987528

Isolated strain from Lake sediment

Archaea; Euryarchaeota; Methanomicrobia; Methanosarcinales; Methanosarcinaceae; Methanosarcina

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Table 7.

Inoculum (Raw Formation

water and No-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

Bacteriodetes

o_Bacteriodales 3.2 1.6 0.4 0.9 0.6 0.3 0.8 0.7 0.7 0.9 1 0.7 0.5 0.4 0.4

Firmicutes

Acidaminobacter 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 2.4

Acetobacterium 2.7 6 0.1 19.6 6.4 4.1 5.0 11.8 4.2 4.1 6.7 14.1 8.2 6.3 7.7

Proteobacteria

f_Comamonadaceae 0.3 0.1 0 0 0.1 0.1 0.2 0.1 0.0 2.3 0 0.1 0.1 0.1 0

Desulfomicrobium 0.0 0.1 0 0.1 0.0 0.1 0.0 0.2 0.0 1.2 0.2 0.0 0.4 0.6 0.1

Desulfovibrio 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 0.5

f_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 0.0

f_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 12.6

Spirochaetes

Treponema 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 0.3

All 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 0.1

All 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 0.1

Other 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 0.7

Total read abundance of Bacteria Taxa (% of normalized 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 0.5

Shannon 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.

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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 has read abundance ≥1% from total are plotted in this table.

Taxa that has read abundance <1% was grouped together. Dominant taxa are highlighted

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Table 8.

Inoculum

(Raw Formation water and No-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

Methanobacteriales

Methanobacterium 0.1 0.0 36.6 9.3 0.0 0.1 0.1 0.2 0.0 0.0 0.1 0.2 0.0 0.0 0.2

Methanomicrobiales

Candidatus Methanoregula 23.5 9.0 0.0 12.3 11.7 7.8 7.2 17.9 14.6 8.4 11.3 21.1 6.8 7.8 9.8

Methanospirillum 8.9 2.4 0.0 2.0 3.7 3.1 1.5 4.3 4.2 8.4 3.2 4.5 2.6 1.3 3.2

Methanosarcinales

Methanosaeta 50.7 65 16.4 26.4 61.2 70.8 71.1 29.2 48.1 43.6 22.9 21.1 38.7 33.8 26

Methanosarcina 1.3 6.0 18.2 6.7 7.0 5.9 4.3 1.8 13.6 0.5 33.6 4.2 14.9 24.4 35.3

Other archaea 1.4 0.3 0 0.5 0.6 0.8 0.6 0.8 0.1 0.2 0.6 1.1 0.7 0.3 0.5

Total read abundance of Archaea Taxa (% of normalised OTU read)

85.9 82.7 71.2 57.2 84.2 88.5 84.8 54.2 80.6 61.1 71.7 52.2 63.7 67.6 75.0

Species richness (Chao 1) 8.0 7.0 7.0 8.0 10.5 7.0 11.0 8.0 8.0 11.0 7.0 8.0 6.0 6.0 7.0

(8.0, (7.0, (6.1, (8.2, (9.2, (7.0, (8.4, (8.0, (8.4, (8.4, (7.0, (8.1, (6.0, (6.1, (7.1,

8.9) 8.0) 19.7) 9.1) 24.1) 8.1) 32.9) 9.4) 9.1) 32.9) 8.4) 9.1) 7.4) 7.2) 8.1)

Evenness

0.5

0.4

0.6

0.7

0.4

0.4

0.3

0.6

0.5

0.5

0.6

0.5

0.6

0.5

0.6

Shannon Diversity Index 1.1 (0.00)

0.8 (0.00)

1.1 (0.00)

1.4 (0.01)

0.9 (0.00)

0.7 (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)

OTU= Operational Taxonomy Unit; RFW =Raw Formation Water; C10 = control at day 10, C20 = control at day 20. p= phylum, c= class, o= order, 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 has read abundance ≥1% from total are plotted in this table.

Taxa that has read abundance <1% was grouped together. Dominant taxa are highlighted.

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Highlights

An active methane-forming microbial community presence in Indonesia CBM reservoir

Microbial community structure changed over time during growth and exhibited more

similarity when grown on coal from the same seam but of different ranks, than to coal from

similar rank and type.

The increase in methanogen population corresponded with an increase in methane

production yield.

The results of this study indicate a potential relationship between coal type and rank,

microbial community composition and methane production.