Oct 30, 2014 - Please cite this article as: Susilawati, Rita, Evans, Paul N., Esterle, .... Mares and Moore, 2008; StrÄ
pod et al., 2007; Susilawati et al., 2013).
Temporal changes in microbial community composition during culture enrichment 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: DOI: Reference:
S0166-5162(14)00225-0 doi: 10.1016/j.coal.2014.10.015 COGEL 2394
To appear in:
International Journal of Coal Geology
Received date: Revised date: Accepted date:
24 July 2014 30 October 2014 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 changes in microbial community composition during culture enrichment experiments with Indonesian Coals, International Journal of Coal Geology (2014), doi: 10.1016/j.coal.2014.10.015
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ACCEPTED MANUSCRIPT TITLE: Temporal changes in microbial community composition during culture enrichment experiments
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with Indonesian Coals
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CONTRIBUTORS:
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Rita Susilawatia,b,*, Paul N. Evansc, Joan S. Esterlea, Steven J. Robbinsc, Gene W. Tysonc, Suzanne D. Goldinga, Tennille E. Maresa AFFILIATIONS:
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,
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a
The University of Queensland, St Lucia, Queensland 4072, Australia.
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*Corresponding author
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ABSTRACT
Temporal changes in microbial community structures during methanogenesis were investigated in cultures of South Sumatra Basin (SSB) coalbed methane (CBM) formation water
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(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.
ACCEPTED MANUSCRIPT 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
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type (Burung sub bituminous and Mangus sub bituminous). Methanosaeta was the dominant
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methanogen species in the sub bituminous Burung coal culture, but was a lesser proportion in
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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,
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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
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obligate acetoclastic Methanosaeta members while both Mangus coals also favour metabolically versatile Methanosarcina members, and the absence of coal favours hydrogenotrophic
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methanogens. Despite the similarity of communities grown on coals from the same seam, greater
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quantities of methane were generated from the lower rank coals when compared to higher rank
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coals.
Keywords: coal; microbial community; methanogenesis; coal bed methane
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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)
ACCEPTED MANUSCRIPT 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
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from some CBM reservoirs has revealed biogenic origins, and have speculated that real time
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methane could be formed from the in situ coal in those reservoirs (Butland and Moore, 2008; Faiz
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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
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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
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al., 2008). These results suggest the potential exists to develop new and renewable biogenic gas resources from deep coal seams.
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Despite microorganisms being present in coal and its associated formation waters, the
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ability of the microbial community to deconstruct the coal is still poorly understood due to the
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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
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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
ACCEPTED MANUSCRIPT associated with hydrogenotrophic (e.g. Methanobacterium and Methanocorpusculum spp.), acetoclastic (e.g. Methanosaeta and Methanosarcina spp.) and methylotrophic methanogens (e.g.
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Methanolobus spp.) (Doerfert et al., 2009; Ghosh et al., 2014; Green et al., 2008; Midgley et al.,
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2010; Penner et al., 2010; Singh et al., 2012; Strąpod et al., 2008; Tang et al., 2012).
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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
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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
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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
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during growth on three native SSB coals of different rank.
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2. Geology of the South Sumatra Basin
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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
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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).
ACCEPTED MANUSCRIPT 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
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from 30 to 40 m. Several minor seams (eg. Benuang and Burung), lenticular shales, sandstones,
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carbonaceous mudstones, and tuffaceous marker horizons are interbedded within these coals
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(Figure 1). 3. Indonesian CBM
Indonesia is only just beginning to develop its CBM resources, and the SSB has been
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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-
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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
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biogenic methane generation.
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organic components (Davis et al., 2007) that are thought to make them excellent substrates for
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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
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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
ACCEPTED MANUSCRIPT (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
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after digging out about 10 cm of surface coal to minimize the chance of samples being oxidised.
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After coal lithotype description, fresh samples were wrapped with aluminium foil and plastic film,
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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
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2 weeks.
The outer layer of the coal was pared away before the coal was crushed in the anaerobic
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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.
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The crushed coal was then split into two parts using a riffle splitter, for chemical-physical analysis,
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and for enrichment studies (used as sole carbon and energy substrate). ALS Laboratory, Queensland,
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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.
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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
ACCEPTED MANUSCRIPT 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
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laboratory Products, MI, USA) with an atmosphere of 95% nitrogen and 5% hydrogen.
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A water sample from the SSB5 pilot well was also collected for anion and cation
Environmental Geology in Bandung, Indonesia. 3.3. Enrichment culture experimental conditions
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concentration analysis. The analysis was conducted at the Centre of Groundwater Resource and
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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
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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
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anoxic conditions according to the methods outlined by these authors.
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Culture tubes were prepared in 26 mL tubes (Bellco Glass) where 0.25 g of coal substrate
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(
