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ScienceDirect Energy Procedia 65 (2015) 282 – 291

Conference and Exhibition Indonesia - New, Renewable Energy and Energy Conservation (The 3rd Indo-EBTKE ConEx 2014)

Microbial Methane Potential for the South Sumatra Basin Coal: Formation Water Screening and Coal Substrate Bioavailability Rita Susilawatia,b,*, Joan S. Esterlea, Suzanne D. Goldinga, Tennille E. Maresa a

School of Earth Sciences, The University of Queensland., St Lucia QLD 4072, Australia, b Geological Agency of Indonesia, Jl Sukarno Hatta no 444, Bandung 40254, Indonesia

Abstract Formation water samples collected from five South Sumatra Basin (SSB) coal bed methane (CBM) wells were investigated for the presence of active microbial communities capable of converting coal to methane using culture enrichment studies and molecular phylogenetics techniques. All water samples contained communities capable of methanogenesis using both acetoclastic and hydrogenotrophic pathways. In addition, viable coals to methane communities were detected in four of the water samples. Molecular analysis further confirmed the presence of active microbial methanogen consortia in the tested water samples. Taken together these results highlight the potential of SSB coals to be developed as real time methane bioreactors that may contribute to Indonesia’s future energy supply. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 R. Susilawati, J.S. Esterle, S.D. Golding, T.E. Mares. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014. Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 Keywords: Coal bed methane; microbial methanogen consortia; renewable energy

Nomenclature DNA kPa mM OTU Rv rRNA Scf t μM ΔG

deoxyribonucleic acid kilopascal millimolar operational taxonomic unit vitrinite reflectance ribosomal ribonucleic acid standard cubic feet tonne (metric ton) 103 kg micromolar gibbs free energy

* Corresponding author. Tel.: +61 451 991 371; fax: +61 7365 1277. E-mail address: [email protected]

1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 doi:10.1016/j.egypro.2015.01.051

Rita Susilawati et al. / Energy Procedia 65 (2015) 282 – 291

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1. Introduction Currently, Indonesia’s energy resources are predominantly oil and conventional natural gas, which are no longer enough to feed the increasing national energy demand. As a result, finding new alternative energy is considered a priority. Environmental concerns such as climate change have also increased the national interest in the use of clean sources of energy. CBM is considered to be a cleaner energy source as it produces less CO 2 when burned compared to oil and coal. Indonesia has abundant coal resources, at a depth favorable to CBM that can be used as a source of energy if the gas can be produced. Methane in coal seams is produced either as a by-product of the thermal coalification process (thermogenic) or by microbial activity (biogenic). Over the last decade, several economic biogenic methane resources have been discovered (e.g. Powder River Basin) leading to a number of laboratory and field experiments demonstrating the production of real time biogenic methane from coal [1-5]. Biogenic methane formation in coal requires the involvement of a metabolically diverse microbial community. The multistage transformation of coal to methane at the basin scale is thought to involve Bacteria phyla that hydrolyze and ferment complex organic compound into H 2 and organic acids, as well as methanogenic Archaea, which ultimately transform these fermentative products into methane [6,7]. At the phyla-scale, bacteria from Firmicutes, Proteobacteria, Bacteroidetes, and Spirochaetes have been detected in many biogenic CBM reservoirs along with methanogenic Archaea from the orders Methanomicrobiales, Methanobacteriales and Methanosarcinales [4,7-11]. The most common pathways for biogenic methane production from coal are aceticlastic and hydrogenotrophic, and to a lesser extent methylotrophic. Indonesia’s CBM is partly biogenic in origin [12]. Preliminary culture experiments have indicated viable coal to methane indigenous microbiota residing in SSB CBM reservoirs [12], suggesting there is potential to develop new and renewable biogenic gas resources from Indonesian coals. Using culture enrichment and molecular phylogenetics techniques, this study further investigated the presence of active microbial methanogen consortia that are capable of converting coal to methane as well as testing the bioavailability of the SSB coals. SSB was selected as there was access to formation water, and it is considered to have the greatest potential for CBM development in Indonesia [13]. 2. Materials and methods 2.1. Water and coal sampling Sampling of formation water was conducted in November 2011 and May 2012 from two areas within the SSB, about 60 km apart (Figure 1). Three formation water samples; SSB1, SSB2 and SSB3, were collected from the same area, with each well about two km apart. SSB4 and SSB5 were collected from a different area from wells about 20 km apart. The formation water sampling methodology for enrichment purposes is described in [13]. The formation water samples were imported to Australia following Australian Quarantine and Inspection Service (AQIS) guidelines under permit no IP13002699. Water samples were subjected to water quality analysis (analyzed by the Centre for Environmental Geology Bandung, Indonesia) and used as a source of inoculum for biogenic methane culturing experiments (carried out at The University of Queensland Australia). Fresh coal was sampled from nearby operating mines in May 2012. Two SSB coal samples were collected from the Muara Enim Formation to be used as the main carbon substrate for the culture enrichment studies. Coal characterization included petrography, and proximate and ultimate analysis was conducted at The University of Queensland, Australia and ALS Laboratory, Queensland respectively. 2.2. Culture experiment and molecular analysis The culturing experiments were prepared following the method described by [2] and [12]. In experiment 1, H2CO2 gas (300 kPa) and acetate (sodium acetate trihydrate at 36.7 mM) were used as substrates to assess the active methanogenic pathway in the area while tubes without substrate (no-substrate) were made as a negative control to test the presence of viable methanogen in the water samples (Figure 2). Theoretical methane yields of these substrates were calculated using the stoichiometry given in Table 1. To assess the presence of viable coal to methane consortia and to test the coal bioavailability (experiment 2), two SSB coal at similar rank (Burung and Suban coal, Rv 0.39) were used as the main carbon substrates and inoculated with 3 mL of formation water along with tubes without coal (no-coal) as controls. Additional controls included non-inoculated coal and media only tubes, which were autoclaved and prepared as abiotic controls.

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Figure 1. Water and coal sampling locations

Figure 2. Experiment design. Experiment 1 screens the methanogenic pathways of all five water samples using media with acetate, H2CO2 and a no substrate control. Experiment 2 screens for the presence of viable coal to methane consortia and assesses the bioavailability of the Suban and Burung coals using all five formation water samples, the two coals and several controls. Red lines indicate cultures used for molecular analysis.

Molecular analysis was conducted on selected tubes at the stationary phase of cultures growth and was performed at The Australian Centre for Ecogenomics, The University of Queensland, Australia. Deoxyribonucleic acid (DNA) was extracted using a modified phenol: chloroform extraction method [15]. For phylogenetic analysis, the 16S ribosomal ribonucleic acid (rRNA) gene was amplified for 30 cycles using fusion primers 926F and 1392R under the reaction conditions of [16]. Amplicons were sequenced on the Roche 454 GS-Flex Titanium platform. The resulted

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sequence reads were demultiplexed, and the UCHIME software [17] was used to detect and remove chimeric sequences while ACACIA [18] was used to correct homopolymer errors. The representative sequences were identified into operational taxonomic units (OTUs) at 97 % cut off by comparison to the Green Genes database. The sequences were tabulated and converted to percentages. Diversity indices (Shannon index) were used to estimate the microbial diversity in the samples and calculated using the program EstimateS [19].

Table 1. Methanogenic reactions of hydrogenotrophic and acetoclastic methanogen [14] ΔG°′ (kJ/mol CH4)

Stoichiometry 4H 2+HCO 3 + H+ → CH 4+3 H 2O -

CH 3COO + H2O→ CH 4 +

HCO-3

-136

(1)

-31

(2)

3. Results and discussion 3.1. Assessment of viable methanogens and coal bioavailability The tested SSB waters are primarily sodium bicarbonate-chloride types (Table 2), which have very low concentration of sulfate, low concentrations of sodium and magnesium and higher concentrations of chloride and sodium bicarbonate, such characteristics are typical of CBM formation water [2,20-22]. In general, the SSB CBM wells contain formation water whose temperature, pH and salinity range are suitable for methanogen optimal growth [23]. The results of experiment 1 showed that all waters contained viable methanogens, which produced methane through acetoclastic and hydrogenotrophic pathways (Figure 3). The theoretical yield of methane production from H2-CO2 (300 kPa) and acetate (36.7 mM) in 14 mL of headspace based on Eq. 1 and Eq. 2 (Table 1) is 343 μM tube–1. In this study, methane production from acetate treatment cultures (363 μM tube–1 to 642 μM tube–1 or 105 % to 187 % of theoretical yield) are somewhat higher than methane produced from H2-CO2 cultures (68 μM tube–1 to 318 μM tube–1 or 20 % to 93 % of theoretical yield), which indicated the predominance of acetoclastic over hydrogenotrophic methanogens in all the tested SSB water samples. Less methane is produced from all H 2-CO2 treatment cultures than is predicted as its theoretical yield. This result, along with the dominance of acetoclastic methanogen suggests a low concentration of hydrogenotrophic methanogens in all the tested water which likely was not sufficient to metabolize all of the available substrates. Little methane was produced from abiotic control tubes in experiment 2 (data was not reported) suggesting that the majority of methane produced in the coal treated cultures was biogenic in origin and was not from methane adsorbed in the substrate coal. Maximum methane production from cultures with coal were higher than cultures without coal (no-coal control), suggesting the ability of the consortia to biodegrade the coal (Table 3). Methane detected in the no-coal control cultures was thought to be related to the activity of microbial methanogen consortia that utilized many organic constituents available from continued microbial metabolism in the inoculum. Fine coal particles carried over in the water possibly provided substrate for the methanogen growth. The results of experiment 2 suggest that not all water samples tested contained microbial communities capable of coal to methane conversion. Of the five waters tested, only the SSB2 cultures did not show activity of coal to methane consortia as shown by the lower methane production from the SSB2 coal culture in comparison to the methane produced from the no-coal control culture (Table 3). There are several possible explanations for this phenomenon. First, this is thought to be related to a low concentration of critical coal to methane degrading microbial members present in the inoculum, which may not be sufficient to metabolize the coal. Another possibility is that, during storage, a key nutrient required for the rapid growth of the microbial methanogen community (available in the initial formation water) became depleted or that toxic by-products of microbial metabolism were accumulated, which reduced the growth of microbial communities capable of converting coal to methane. Alternately, the SSB2 water has the highest water salinity (Table 2). As significant methane was produced by the

286


SSB2 cultures when grown on both H2-CO2 and acetate, it is also possible that the high water salinity provided unfavorable conditions for the growth of coal degrader bacteria, whereas the methanogen population seemed to be unaffected. It has been previously proposed that salinity affects the growth of consortia with the ability to convert coal to methane [2,24]. The negative number of net methane yield in SSB2 coal treated culture may also indicate microbial methane oxidation. Anaerobic methane oxidation involving Methanoarchaea has been reported in both field and laboratory study [25-27]. As neither geochemical nor microbial community data are available to confirm this hypothesis, anaerobic oxidation in SSB2 culture remains to be tested.

Table 2. Water quality analysis CBM wells Characteristics SSB1

SSB2

SSB3

SSB4

SSB5

34. 0

33. 0

33. 0

39. 0

32. 0

7. 1

6. 9

6. 8

7. 0

6. 6

1 388. 4

3 715. 8

1 098. 7

936. 9

134. 8

4. 1

10. 9

2. 9

1. 9

3. 3

nd

nd

nd

nd

nd

1 246. 6

416. 0

490. 1

1 132. 2

942. 5

3. 9

1. 3

2. 4

10. 5

3. 0

Chloride (mg · L–1)

273. 1

2878. 0

480. 2

30. 1

439. 5

Calcium (mg · L–1)

28. 2

14. 5

8. 3

13. 8

9. 7

6. 6

3. 3

28. 1

13. 6

6. 2

512. 5

1001. 1

421. 5

360. 0

577. 9

99. 9

1555. 0

20. 2

55. 5

126. 1

Cu (mg · L–1)

0. 003

0. 017

0. 006

0. 029

0. 025

Zn (mg · L–1)

0. 110

0. 245

0. 388

0. 140

0. 318

Ni (mg · L–1)

0. 029

10. 380

5. 670

0. 058

58. 660

Co (mg · L–1)

0. 017

nd

nd

0. 019

2. 970

–1

0. 090

nd

nd

0. 060

0. 000

Temperature pH Salinity (mg · L–1) Conductivity (mS · cm–1) Carbonate alkalinity (mg · L–1) –1

Bicarbonate alkalinity (mg · L ) Sulfate (mg · L–1)

Magnesium (mg · L–1) Sodium (mg · L–1) Potassium (mg · L–1) Trace elements

Pb (mg · L )

CH4 μM/tube

700 600 500 H2CO2 H2-CO2

400 300

acetate Acetate

200 100

noNo substrate control substrate control

0 SSB1

SSB2

SSB3

SSB4

SSB5

Theoritical yield theoretical yield

Formation water

Figure 3. Results of the methanogenic pathway screening of SSB formation water

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Both the Burung and Suban coals were found to be biodegradable (Table 3). A comparable volume of methane was generated from both coals, although the net yields are at best semi quantitative because of issues with high methane production from no coal controls and the possible presence of other sources of carbon. Overall the data suggest that the Suban coal produced a slightly greater amount of methane than Burung coal. Since the two coals are of similar rank, it is likely that differences in coal type can explain the difference in the bioavailability between the two coals. Studies have suggested that vitrinite rich coal with very low inertinite content is more suitable for organic matter biodegradation and microbial methanogenesis than coal with lower vitrinite and higher intertinite content [28], which is consistent with this study as the Suban coal has higher vitrinite and lower inertinite content than the Burung coal (Table 4). The Suban coal hydrogen content is also slightly higher than the Burung coal, which may also have some influence. Clearly more detailed study is required to fully understand factors that influence the bioavailability of the two coals.

Table 3. Maximum methane production yield and rate from cultures of SSB formation water grown on SSB coal

Formation water (Inoculum)

Burung coal substrate

Suban coal substrate No coal Control

Coal treated culture

Coal treated culture

No coal Control

gross yield

net yield

rate

yield

gross yield

net yield

rate

yield

SSB1

133.0

84.7

15.7

48.3

177.0

128.7

3.1

48.3

SSB2

63.3

-16.1

3.2

79.4

48.9

-30.4

4.9

79.3

SSB3

145.5

78.6

4.6

66.9

171.0

104.0

4.1

67.0

SSB4

253.1

138.3

5.2

114.8

153.0

64.0

5.6

89.0

SSB5

178.7

108.9

3.1

69.8

294.0

179.6

26.7

114.4

Methane yield: μM · g–1 of coal, Methane rate: μM · g–1 of coal day–1, Net yield = gross yield - control yield

Table 4. Coal chemical and physical characteristics Analysis Proximate % (a.d.b) Moisture Ash yield Volatile matter Fixed carbon Total sulfur Ultimate % (d.a.f) Carbon Hydrogen Nitrogen Sulfur Oxygen (By difference) Petrography (%) Rank (Rv) Maceral composition Vitrinite Inertinite Liptinite Mineral matter

Burung

Suban

16.80 2.20 38.10 42.90 0.83

15.50 3.20 29.70 41.60 0.29

76.20 5.14 1.67 1.02 15.90

76.30 5.26 1.28 0.36 16.80

0.39

0.39

82 10 7 1

89 5 5 1

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Muara Enim SSB coals are reported to have gas contents of 0. 4 m3 t–1 to 5. 8 m3 t–1 (14 Scf t–1 to 205 Scf t–1; as received basis) [29]. In this experiment, the SSB5 enrichment culture was the best producer with a maximum coal to methane net yield of 179. 6 μM g–1 of coal (130 Scf t–1) reached in 11 days (Table 3). That yield corresponds to ~63.5 % of the gas content currently present in the Muara Enim SSB coals. It must be acknowledged that the current experiments were conducted under ideal laboratory conditions in which additional nutrient enhanced the growth of consortia and the crushing of the coal provided maximum surface area exposure for the consortia to access. It should also be noted that the laboratory has different pressure and temperature regimes than can be expected in situ. However, although the replication of this laboratory condition to real field remains a challenge, considering the large amount of deep subsurface unutilized SSB coal [13], the results are still promising as the conversion of only a small fraction of that coal to methane may significantly increase the SSB CBM field reserves and reservoir lifetime. 3.2. Microbial diversity Molecular analyses were only undertaken for three of the formation water (SSB1, SSB4 and SSB5). A summary of the results obtained is shown in Figure 4a. The microbial communities identified in SSB formation water were similar to those found previously in other CBM reservoirs [3,5,8,30-33]. However, it is noteworthy that although broad similarities in the structure of the microbial communities exist, each tested water has a unique community structure that differs with respect to its primary community members and their dominance (Figure 4a). Overall it is confirmed that SSB formation waters contain diverse bacterial and archaeal communities that interact in a unique way to generate methane using different substrates. In the SSB cultures, bacterial community profiling generated 15 OTUs (operational taxonomic unit) from seven taxonomic bacterial phyla groups. The bacterial core taxa mainly consist of Proteobacteria (52 %), Firmicutes (26 %), Spirochaetes (7.5 %) and Bacteroidetes (7 %), while the Archaeal communities generated 7 OTU, which confirmed the presence of three methanogen orders Methanosarcinales (80 %), Methanobacteriales (19 %) and Methanomicrobiales (1 %). Molecular analysis on SSB5 acetate and H2-CO2 treatment cultures further confirmed the presence of both acetoclastic and hydrogenotrophic methanogen in the tested waters (Figure 4b). Methanosarcina which can utilize acetate, dominated the archaeal phyla in acetate culture while all archaea present in H 2-CO2 culture belongs to the obligate hydrogenotrophic methanogen from the family of Methanobacteraceae. Methanosaeta, an obligate acetoclastic methanogen, was observed in culture grown on coal but was absent from culture grown on acetate. Study has noted that Methanosarcina are more dominant in high acetate environments while Methanosaeta dominates environments with low acetate concentrations [34]. In the present experiment, cultures grown on acetate had high acetate concentration in comparison to culture grown on coal, which may explain why Methanosarcina dominated the acetate-treated culture while Methanosaeta dominated the coal-treated culture. Shannon diversity index was used to assess the diversity of microbial consortia in the tested SSB water. This index takes into accounts both abundance and evenness of taxa present in the community. Higher diversity index is usually represented by a diverse and equally distributed community, while low diversity is related to a less diverse community or domination of one species over the other. In all enrichment cultures, bacterial communities are more diverse than archaeal communities. The low archaeal diversity in the SSB4 culture seems to correlate with the dominance of Methanosaeta, whereas the even distribution of methanogen taxa in SSB1 is concurrent with the high index of its archaeal diversity. Furthermore, the low bacterial diversity in the SSB1 culture likely correlates to the dominance of Deltaproteobacteria, and the high index of bacterial diversity in SSB4 culture correlates with a more even distribution of its bacterial taxa. Taken together, these result suggest that community evenness or species dominance was greatly influenced the microbial diversity in our enrichment cultures (Figure 4). Cultures that showed the ability to generate methane from coal comprised microorganisms that are known to be capable of hydrolysis, fermentation, acetogenesis, and methanogenesis. Members of Clostridia and Bacteroidia that were observed in all water are well-known fermenters and are major players in the hydrolysis of plant biomass [35,36]. Acetobacterium, the member of Clostridia for instance, is a homoacetogen that can grow chemolithoautotrophically on H2 plus CO2 or heterotrophically on lactate by forming acetate as a sole product (homoacetogenesis) [37]. These groups of bacteria have been previously reported in other CBM methanogenic communities [7,9,38,39]. Members of Deltaproteobacteria, such as Geobacter and Pelobacter, are also known fermenters and were detected in coal associated cultures. They have been suggested as an important secondary fermenter for coal biodegradation [7,30]. Study has suggested that Spirochaetes, a fermenter, plays a role in the solubilization of intermediates from coal [40]. Finally, the SSB water also comprised methanogens both from the obligately acetoclastic Methanosaetaceae, the obligate hydrogenotrophic families Methanobacteraceae,

289


Methanoregulaceae and Methanospirillaceae, and the metabolically versatile Methanosarcinaceae which can grow using the three methanogenic pathways [41]. 4. Conclusions This study further confirms the presence of viable methanogen and viable coal to methane consortia in SSB CBM reservoirs as well as providing the first characterization of SSB CBM microbial methanogen communities. The results suggest that there is potential for Indonesia to develop coal resources as methane bioreactors that may be able to provide renewable energy resources for future energy requirements. Further work is still required to fully understand the potential of Indonesia's coal resources.

a)

Methanobacterium

SSB5 (0.93;2.05)

Methanosarcina Methanospirillum Methanosaeta Bacteroidia

SSB4 (0.35;2.79)

Actinobacteria Clostridia Deltaproteobacteria

SSB1 (1.2;0.9)

Spirochaetes Synergistia 0

20

40

60

80

100

Other

Microbial relative abundance (% OTU read)

Unclassified Methanobacteriaceae

b)

Methanobacterium Methanoculleus

SSB5 H2-CO2 SSB5-H2CO2 (1.29;1.95) (1.29 ; 1.95)

Methanofollis Methanosarcina Thermoplasmata Deferribacteres

SSB5-Acetate SSB5-Acetate (0.65 ; 1.51) (0.65;1.51)

Clostridia Deltaproteobacteria Synergistales 0

20

40

60

80

100

Other

Microbial relative abundance (% OTU read)

Figure 4. Microbial diversity in SSB cultures enrichment, a) In SSB1, SSB4 and SSB5 using Suban coal as a substrate, b) In SSB5 using acetate and H2-CO2 as substrates. Number in parenthesis shows the Shannon diversity index for archaea (left) and bacteria (right). Only taxa that have sequence read ≥ 1 % of the total microbial population are included in these profiles. Taxa that have sequence read < 1% are grouped together as other. Bacterial communities are grouped at class level and displayed as an area with solid color. Archaeal communities are grouped at genus level (or at higher level taxa if genus level is undefined) and displayed as an area with the pattern.

Acknowledgements Funding for this research came from Australian Awards Scholarship 2010. PT Medco CBM Sekayu, Lemigas and PT Bukit Asam are gratefully acknowledged for site and samples access. The Australian Centre for Ecogenomics at

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The University of Queensland provided access to its PC2 and quarantine laboratory. The authors also thank Soleh Basuki Rahmat, Eko Budi Tjahyono and Erlina Lena Ria from Geological Agency of Indonesia for field assistance during sampling as well as Steven Robbins and Paul Evans from the Australian Centre for Ecogenomics for assistance in molecular phylogenetic analysis. References [1] McIntosh J, Martini A, Petsch S, Huang R., Nüsslein K. Biogeochemistry of the Forest City Basin coalbed methane play. Int.J. Coal Geol 2008; 76: 111-8. [2] Papendick SL, Downs KR., Vo KD, et al. PC. Biogenic methane potential for Surat Basin, Queensland coal seams. Int.J. Coal Geol 2011; 88: 12334. [3] Fry JC, Horsfiel DB, Sykes R, et al. Prokaryotic populations and activities in an interbedded coal deposit, including a previously deeply buried section (1.6–2.3 km) above 漢 150 Ma basement rock. Geomicrobiol. J. 2009; 26: 163–78. [4] Penner TJ, Foght JM, Budwill K. 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