- Email: [email protected]
Enhancing biogenic methane generation from a brown coal by combining different microbial communities
Enhancing biogenic methane generation from a brown coal by combining different microbial communities Han Wang, Hai Lin, Carly P. Rosewarne, Dongmei Li, Se Gong, Philip Hendry, Paul Greenfield, Neil Sherwood, David J. Midgley PII: DOI: Reference:
S0166-5162(15)30090-2 doi: 10.1016/j.coal.2015.12.006 COGEL 2565
To appear in:
International Journal of Coal Geology
Received date: Revised date: Accepted date:
6 August 2015 14 December 2015 14 December 2015
Please cite this article as: Wang, Han, Lin, Hai, Rosewarne, Carly P., Li, Dongmei, Gong, Se, Hendry, Philip, Greenfield, Paul, Sherwood, Neil, Midgley, David J., Enhancing biogenic methane generation from a brown coal by combining different microbial communities, International Journal of Coal Geology (2015), doi: 10.1016/j.coal.2015.12.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enhancing biogenic methane generation from a brown coal by combining different microbial communities
1
University of Science and Technology, Beijing, China CSIRO, Australia. 3 Plant Health & Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.
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Corresponding Author: Han Wang Tel: +8615650785032 Email: [email protected] Address: NO.30, Xueyuan Road, Haidian District, Beijing 100083, China
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*
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2
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Han Wang*1,2, Hai Lin1, Carly P. Rosewarne2, Dongmei Li3, Se Gong2, Philip Hendry2, Paul Greenfield2, Neil Sherwood2 & David J Midgley2
Abstract
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To investigate the potential of artificially enhancing methanogenesis in brown coal, microbial communities from coal formation water and a mangrove swamp were used as a treatment. After 30 days of incubation with this ‘mixed’ amendment, both the rates and
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yields of methane generation was enhanced compared to microbial enrichment cultures
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having a single origin. The microbial community derived from a mangrove swamp alone, appeared to lack the ability to degrade coal. Additionally, the pH of the mixed origin
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treatment was favourable for growth of the mangrove derived microbial community at an early stage, which may also affect gas yield.
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Keywords: Coal seam methane; microbial enhancement; 1. Introduction
Methane is commonly generated under anaerobic conditions (Bohutskyi and Bouwer, 2013) in terrestrial environments such as rice fields (Chen et al., 2013; Neue et al., 1996), wetlands, the gastrointestinal tract of animals, and within with coal or oil deposits (Jones et al., 2008; Moore, 2012). Concerns about climate change has seen rising interest in methane as a ‘bridge’ fuel, spanning the gap between emissions intensive energy generation from coal and renewable energy (Flores et al., 1997; Flores, 1998; Hamawand et al., 2013; Park and Liang, 2015; Ritter et al., 2015; Wang et al., 2011). Coal seam methane (CSM) accounted for approximately 40% of total production (by volume) from all gas wells in the United States in 2011 (Strapoc et al., 2011). CSM is produced by both thermogenic and microbial
ACCEPTED MANUSCRIPT processes, the latter often termed biogenic production. Biogenic methane represents approximately 30% of all CSM (Flores, 2008; Strapoc et al., 2011) and its production may occur in coal irrespective of its rank. Given the importance of CSM as an energy source, the
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potential to increase microbial generated CSM production is of great economic and
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environmental interest. Previous published studies have attempted stimulate methane
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production by manipulating native microbial communities using techniques such as adjusting pH, temperature, coal surface area and adding nutrients (Green et al., 2008; Jin et al., 2009; Liu et al., 2013; Midgley et al., 2010; Opara et al., 2012; Pfeiffer, 2011; Shumkov et al., 1999; Unal et al., 2012; Wawrik et al., 2012). All of these studies showed shifts in the
MA
mechanisms remain largely unknown.
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microbial communities that resulted in changes in gas production, though the underlying
Intuitively it seems likely that the microbial communities found in coal seams would be best adapted to that environment, and that introduction of exogenous bacterial consortia would
D
not result in improved gas production. In 2010 however, Jones et al., demonstrated that
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adding exogenous methanogenic communities to a non-CH4-producing coal microbial community resulted in higher rates of methane generation than the addition of nutrients
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(Jones et al., 2010). Similarly, a microbial community derived from an American wetland sediment was demonstrated to be more efficient at the conversion of organic matter in brown coal to methane than the coal’s indigenous community (Opara et al., 2012). The
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present study sought to determine whether a composite microbial community, consisting of a coal seam microbial community mixed with mangrove sediment, was able to improve gas yields compared to the coal seam community alone, in an in vitro system. 2. Materials and methods 2.1 Samples The coal seam microbial community was collected in formation water from a medium volatile bituminous rank Permian coal (∼700 m subsurface) in the Sydney Basin, Australia (34.111478⁰ S, 150.737096⁰E). The sample was collected in pre-sterilized, 1 L bottles, to which a reductant and indicator solution were added as described in Midgley et al., (2010). Within an hour, the sample was transferred to an anaerobic glovebox with an atmosphere of 95% Ar and 5% H2 and then degassed under this atmosphere.
ACCEPTED MANUSCRIPT The mangrove swamp microbial community was obtained by collection of ~200g of sediment , from a depth of 10-15cm, near Meadowbank, Sydney (-33.819865⁰ S,151.091569⁰E), using a clean, surface sterilized trowel. The collected sediment was then
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transferred to anaerobic glove box under conditions described above. The physicochemical
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Measurement Institute (Sydney, Australia) (Table S1).
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characteristics of the formation water and swamp sediment were measured by National
2.2 Feedstock coal and characterisation
A homogenised and sieved (<6mm) sample of Latrobe Valley, Cenozoic brown coal was used
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as the carbon source for experiments. The ‘run-of-mine’ sample was taken from the Loy Yang open cut located in the Gippsland Basin, Victoria, Australia. A representative
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subsample of the coal was characterised using conventional organic petrological methods, according to the Australian Standard AS2856.2 (1998) and AS2856.3 (2000). For the maceral analysis ~600 point-counts were carried out, with examinations using both reflected white
D
light and incident ultraviolet light (UV)/blue light excitation for fluorescence mode. About 60
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measurements were made for vitrinite reflectance analysis. The proximate analysis was carried out by Australian Laboratory Services (ALS), Carrington, New South Wales, Australia,
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according to Australian Standard AS1038.3-2000. Before the coal was used as a carbon source, it was transferred to the anaerobic chamber under conditions described previously
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and allowed to degas.
2.3 Generation and characterisation of enrichment cultures Two enrichment cultures were established, one from the Permian coal seam and one from the mangrove sediment. The formation water enrichment culture was established by adding 20mL of the formation water to 1L of minor modified minimal salts (MS) medium (as described in Midgley et al., 2010) with 10g of the Loy Yang coal. One ml of filter sterile 100 μM Na2S solution was used as a reductant. The mangrove swamp enrichment culture was generated by adding 10g of fresh sediment to 1L of MS medium. After two weeks, 50mL of the initial enrichment was transferred to fresh, degassed 1L of MS medium supplemented with 10g of Loy Yang coal and supplemented with 0.5g of yeast extract. Both the formation water and the mangrove sediment enrichment culture were incubated with non-sterile
ACCEPTED MANUSCRIPT feedstock coal, as autoclaved coal did not support the growth of microbes. Both enrichment cultures were incubated at 33°C for a minimum of four weeks prior to the start of the gas production tests. All culturing work was undertaken inside the anaerobic chamber under an
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atmosphere of 95% Ar and 5% H2 atmosphere.
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2.4 Gas production tests
To investigate whether the two enrichment cultures enhanced methane production, 2g of coal and 50ml of MS medium were added to 120 ml sterilized serum vials, and degassed in
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the anaerobic chamber. After degassing, either 1ml of formation water enrichment culture, 1ml mangrove sediment enrichment culture or 0.5mL of both were added. An un-inoculated control was also included. The vials were sealed using butyl rubber septa and aluminium
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crimps inside the anaerobic chamber and removed for incubation at 33°C in the dark, in an inverted position. Triplicates were established in all treatments, including controls. The
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headspace gases of all bottles were assayed at 10, 20 and 30 days using a gas-tight syringe
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as described in Midgley et al., 2010. After gas sampling (which occurred inside the anaerobic chamber), the bottles were unsealed and allowed to equilibrate for 3 mins in the
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atmosphere of the anaerobic chamber, prior to being resealed with a new butyl-rubber septum and aluminium crimp and being returned to the 33°C incubator. Effectively resetting the headspace to that present in the anaerobic chamber.
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Sampled gas was analysed on an Agilent Technologies 490 Micro Gas Chromatograph (Micro-GC). The samples were injected into the front injection port of the Micro-GC using a gas-tight syringe and a motorised syringe pump. The Micro-GC is equipped with four different column modules: a 10 m Molesieve 5Å column for separating O 2/Ar, N2, CH4 and CO, a 10 m Pora Plot Q column for separating CO2, C2H6 and C3H8, a 10 m CP-Sil-5CB column for separating C4-C5 hydrocarbon gases and H2S and a 20m Molesieve 5Å column for separating H2 and He using argon carrier gas. The temperature of these four columns was set to 90°C, 70°C, 60°C and 90°C, respectively. A typical analysis time was 3 min for a single sample injection. 2.5 Statistical analysis
ACCEPTED MANUSCRIPT One-way ANOVA and the Tukey HSD post hoc test (Viotti et al., 2009) were used for testing significant differences of CH4 and CO2 concentrations between each time point and different treatments. All statistical analyses were performed in R (version 3.1.0; R Development Core
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Team, Vienna, Austria). 3. Results and discussion
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3.1 Coal petrology
The feedstock Loy Yang brown coal comprised mainly telovitrinite and detrovitrinite, in near equal abundances, with major amounts of liptinite and minor amounts of inertodetrinite,
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funginite and minerals (Table 1). The liptinites mainly comprised sporinite and liptodetrinite with lesser amounts of cutinite, suberinite and resinite (Table 1, Fig 1). The mean random
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vitrinite reflectance, including measurements on both telovitrinite and detrovitrinite was 0.35%. Consistent with this brown coal rank, the moisture content was ~39%, the volatile
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matter yield was ~33% (dry basis) and the fixed carbon content was ~28%. The high
3.2 Gas production results
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moisture, may facilitate production of films from microbial growth (Faison, 1991).
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Taken across the entire time course, significantly greater total yields of methane (p<0.0001) were generated from the mixed origin enrichment culture (~162µmol CH4 g-1 coal) than from either the formation water (~126µmol CH4 g-1 coal) or mangrove enrichment cultures
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(~13µmol CH4 g-1 coal; Fig 2a). Only trace amounts of methane (<0.1µmol CH4 g-1 coal) were observed in desorption controls, indicating that the methane observed in all of the mixed origin, formation water and mangrove enrichment cultures was produced biogenically. The reasons for the increase in methane yields by the mixed origin enrichment culture are unknown but were presumably related to changes in the microbial community in the enrichment cultures. It is worth noting that a small amount of yeast extract (totalling 0.1 mg carbon) was present at the start of the mixed origin enrichment cultures. Assuming that all the carbon in this yeast extract was converted to methane, without any losses to biomass or other gases, it could account for the presence of 8.3µmol of the additional methane produced in the mixed origin enrichment culture. Removing this amount of methane from the total and retesting for significantly different total yields of methane between the treatments did not substantively alter the p-value. Methane yields from this carryover yeast
ACCEPTED MANUSCRIPT extract, however, could account for about half of the methane produced by the mangrove sediment enrichment culture. It is also worth noting that an average of 34µmol H2 was present in headspace at each time point (due to the atmosphere in anaerobic chamber; see
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Fig 2c). This amount of H2, assuming no losses of H2 to other processes and the availability of
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sufficient carbon, would be sufficient to account for the production of ~17µmol CH4. The
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low production of methane by the mangrove enrichment culture would then imply that the microbes in this community lacked the ability to access carbon in the feedstock coal, and that the microbes in the mixed origin enrichment and formation water enrichment cultures
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are accessing both carbon and hydrogen from the coal.
Along with changes in yields of methane, different patterns of methane generation were
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observed for the formation water, mangrove and mixed origin enrichment cultures (Fig 2a). The mixed origin enrichment culture started generating methane quite early, and methane production climbed steadily thereafter. In contrast, there was little sign of early
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methanogenesis with either the formation water or mangrove enrichment cultures, but
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methane generation markedly increased after day 20. Methanogenesis normally occurs after the exhaustion of other terminal electron acceptors, and the faster onset of methane
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production in the mixed microbial culture may be related to a rapid consumption of the energetically more profitable terminal electron acceptors by the more diverse community. That is, any nitrates, sulphates or iron (III) present in the inoculum may be more rapidly
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consumed with the greater diversity of organisms in the mixed community treatment or it may simply a productive of the composition of the mixed community. Differences in the pH of the samples may also have been an important influence on methane yield, especially the rapid onset of methane production seen in the mixed microbial samples. The initial pH value of mangrove sediment and formation water were 5.9 and 9.2 (Table S1), respectively, and Victorian Loy Yang (LY) brown coal has been shown to have a low pH of ~4.2 (Qi and Chemistry, 2004; Yuliani et al., 2012). This is close to the initial pH of the mangrove sediment and this may also have contributed to the earlier onset of methane generation observed in the mangrove sediment microbial community. All treatments yielded measurable quantities of CO2 (Fig 2b). The formation water enrichment culture produced the greatest yield of CO2 (~119µmol CO2 g-1 coal), significantly
ACCEPTED MANUSCRIPT more CO2 (p<0.0001) than was produced by the mixed origin enrichment culture (~88µmol CO2 g-1 coal). The little CO2 produced by the mangrove enrichment culture (~4µmol CO2 g-1 coal) was not significantly different (p>0.6) to the desorbed CO2 observed in the desorption
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control (~2µmol CO2 g-1 coal), adding further weight to the proposal that the microbes in the
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mangrove sediment enrichment culture were unable to access carbon in the feedstock coal.
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4. Conclusions
Methane is an important energy source as it offers economic and environmental benefits over traditional coal-fired power generation. A significant proportion of coal seam methane
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is biogenic in origin, being produced by the syntrophic and synergistic activities of complex microbial consortia. The results presented here suggest that manipulating microbial
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communities by bioaugmentation may provide methods that can be used to improve the biogenic production of methane from coal. Additionally, physicochemical properties of the carbon source may be a critical factor in the success of ex situ bioaugmentation as it
Acknowledgments
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provides the actual living environment for the microbial community.
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The authors would like to thank CSIRO-Chinese Academy of Science Collaboration Fund and China Scholarship Council for the support of this project. We thank Ms. Jan Shaw for her assistance with the statistical analysis used in this study, Dr. Stephen Sestak for helpful
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discussions on the experimental design and Dr. Nai Tran-Dinh for his helpful thoughts on the manuscript. We also thank Emma Qi for providing the brown coal.
Figure and Table Legends Figure 1. Photomicrograph of Loy Yang brown coal, Gippsland Basin, under reflected white light (a) and fluorescence mode (b).
Figure 2. Gas production and consumption by the coal formation water enrichment culture, the mixed origin enrichment culture and the mangrove sediment enrichment culture at 10, 20 and 30 days.
ACCEPTED MANUSCRIPT Table 1. Maceral Composition for Loy Yang brown coal.
Table S1. Physicochemical characteristics of the mangrove sediment and Sydney Basin
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Table S2. Proximate analysis for the Loy Yang brown coal.
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formation water sample.
ACCEPTED MANUSCRIPT References
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Bohutskyi, P., Bouwer, E., 2013. Biogas Production from Algae and Cyanobacteria Through Anaerobic Digestion: A Review, Analysis, and Research Needs, in: Lee, J.W. (Ed.), Advanced Biofuels and Bioproducts. Springer New York, pp. 873-975.
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Chen, H., Zhu, Q.A., Peng, C.H., Wu, N., Wang, Y.F., Fang, X.Q., Jiang, H., Xiang, W.H., Chang, J., Deng, X.W., Yu, G.R., 2013. Methane emissions from rice paddies natural wetlands, lakes in China: synthesis new estimate. Global Change Biol 19, 19-32. Faison, B.D., 1991. Microbial Conversions of Low Rank Coals. Nature biotechnology 9, 951-956.
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Flores, A., Chet, I., Herrera-Estrella, A., 1997. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Current genetics 31, 30-37. Flores, R.M., 1998. Coalbed methane: From hazard to resource. Int J Coal Geol 35, 3-26.
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Flores, R.M., 2008. Microbes, methanogenesis, and microbial gas in coal. Int J Coal Geol 76, 1-2. 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. Int J Coal Geol 76, 34-45.
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Hamawand, I., Yusaf, T., Hamawand, S.G., 2013. Coal seam gas and associated water: A review paper. Renew Sust Energ Rev 22, 550-560.
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Jin, S., Bland, A.E., Price, H.S., 2009. Biogenic Methane Production Enhancement Systems. Google Patents. Jones, D.M., Head, I.M., Gray, N.D., Adams, J.J., Rowan, A.K., Aitken, C.M., Bennett, B., Huang, H., Brown, A., Bowler, B.F.J., Oldenburg, T., Erdmann, M., Larter, S.R., 2008. Crude-oil biodegradation via methanogenesis in subsurface petroleum reservoirs. Nature 451, 176-U176.
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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. Appl Environ Microb 76, 7013-7022. Liu, Y.P., Urynowicz, M.A., Bagley, D.M., 2013. Ethanol conversion to methane by a coal microbial community. Int J Coal Geol 115, 85-91. 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. Int J Coal Geol 82, 232-239. Moore, T.A., 2012. Coalbed methane: A review. Int J Coal Geol 101, 36-81. Neue, H.U., Wassmann, R., Lantin, R.S., Alberto, M.A.C.R., Aduna, J.B., Javellana, A.M., 1996. Factors affecting methane emission from rice fields. Atmos Environ 30, 1751-1754. Opara, A., Adams, D.J., Free, M.L., McLennan, J., Hamilton, J., 2012. Microbial production of methane and carbon dioxide from lignite, bituminous coal, and coal waste materials. Int J Coal Geol 96-97, 1-8.
ACCEPTED MANUSCRIPT Park, S.Y., Liang, Y., 2015. Biogenic methane production from coal: A review on recent research and development on microbially enhanced coalbed methane (MECBM). Fuel.
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Pfeiffer, R.S.P., CO, US), Ulrich, Glenn A. (Golden, CO, US), Debruyn, Roland P. (Highlands Ranch, CO, US), Vanzin, Gary (Arvada, CO, US), 2011. Chemical amendments for the stimulation of biogenic gas generation in deposits of carbonaceous material. Luca Technologies, Inc. (Golden, CO, US), United States.
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Qi, Y., , 2004. Characterisation of Organic and Inorganic Components in Process Water from a Novel Lignite Dewatering Process. Monash University.
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Ritter, D., Vinson, D., Barnhart, E., Akob, D.M., Fields, M.W., Cunningham, A.B., Orem, W., McIntosh, J.C., 2015. Enhanced microbial coalbed methane generation: A review of research, commercial activity, and remaining challenges. International Journal of Coal Geology 146, 28-41.
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Shumkov, S., Terekhova, S., Laurinavichius, K., 1999. Effect of enclosing rocks and aeration on methanogenesis from coals. Applied microbiology and biotechnology 52, 99-103. Strapoc, D., Mastalerz, M., Dawson, K., Macalady, J., Callaghan, A.V., Wawrik, B., Turich, C., Ashby, M., 2011. Biogeochemistry of Microbial Coal-Bed Methane. Annu Rev Earth Pl Sc 39, 617-656.
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Unal, B., Perry, V.R., Sheth, M., Gomez-Alvarez, V., Chin, K.J., Nusslein, K., 2012. Trace elements affect methanogenic activity and diversity in enrichments from subsurface coal bed produced water. Frontiers in microbiology 3, 175.
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Viotti, R.G., Kasaz, A., Pena, C.E., Alexandre, R.S., Arrais, C.A., Reis, A.F., 2009. Microtensile bond strength of new self-adhesive luting agents and conventional multistep systems. The Journal of prosthetic dentistry 102, 306-312.
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Wang, J., Ryan, D., Anthony, E.J., 2011. Reducing the greenhouse gas footprint of shale gas. Energy Policy 39, 8196-8199. Wawrik, B., Mendivelso, M., Parisi, V.A., Suflita, J.M., Davidova, I.A., Marks, C.R., Van Nostrand, J.D., Liang, Y.T., Zhou, J.Z., Huizinga, B.J., Strapoc, 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, 2642. Yuliani, G., Qi, Y., Hoadley, A.F.A., Chaffee, A.L., Garnier, G., 2012. Lignite clean up of magnesium bisulphite pulp mill effluent as a proxy for aqueous discharge from a ligno-cellulosic biorefinery. Biomass Bioenerg 36, 411-418.
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ACCEPTED MANUSCRIPT
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Fig. 1
Fig. 2
ACCEPTED MANUSCRIPT
Table 1. Maceral Composition for Loy Yang brown coal
3.8
Sporinite
3.6
Suberinite
0.9
Resinite
0.5
Cutinite
<0.2
Alginite
0
Liptodetrinite
4.6
Semifusinite
0
Fusinite
0
Macrinite
0
Micrinite
0
Funginite
1.5 1.7
Minerals
29.1
Total
100
81.9
Liptinite
9.6
Inertinite
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13.5
3.2
4.6
Minerals
29.1
-
100
100
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Inertodetrinite
58.1
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Gelovitrinite
Vitrinite
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33.8
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Detrovitrinite
Volume % (mineral free)
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20.5
Volume %
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Telovitrinite
Maceral Group
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Volume %
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Maceral
S0166-5162(15)30090-2 doi: 10.1016/j.coal.2015.12.006 COGEL 2565
To appear in:
International Journal of Coal Geology
Received date: Revised date: Accepted date:
6 August 2015 14 December 2015 14 December 2015
Please cite this article as: Wang, Han, Lin, Hai, Rosewarne, Carly P., Li, Dongmei, Gong, Se, Hendry, Philip, Greenfield, Paul, Sherwood, Neil, Midgley, David J., Enhancing biogenic methane generation from a brown coal by combining different microbial communities, International Journal of Coal Geology (2015), doi: 10.1016/j.coal.2015.12.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enhancing biogenic methane generation from a brown coal by combining different microbial communities
1
University of Science and Technology, Beijing, China CSIRO, Australia. 3 Plant Health & Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.
NU
Corresponding Author: Han Wang Tel: +8615650785032 Email: [email protected] Address: NO.30, Xueyuan Road, Haidian District, Beijing 100083, China
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*
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2
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Han Wang*1,2, Hai Lin1, Carly P. Rosewarne2, Dongmei Li3, Se Gong2, Philip Hendry2, Paul Greenfield2, Neil Sherwood2 & David J Midgley2
Abstract
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To investigate the potential of artificially enhancing methanogenesis in brown coal, microbial communities from coal formation water and a mangrove swamp were used as a treatment. After 30 days of incubation with this ‘mixed’ amendment, both the rates and
D
yields of methane generation was enhanced compared to microbial enrichment cultures
TE
having a single origin. The microbial community derived from a mangrove swamp alone, appeared to lack the ability to degrade coal. Additionally, the pH of the mixed origin
CE P
treatment was favourable for growth of the mangrove derived microbial community at an early stage, which may also affect gas yield.
AC
Keywords: Coal seam methane; microbial enhancement; 1. Introduction
Methane is commonly generated under anaerobic conditions (Bohutskyi and Bouwer, 2013) in terrestrial environments such as rice fields (Chen et al., 2013; Neue et al., 1996), wetlands, the gastrointestinal tract of animals, and within with coal or oil deposits (Jones et al., 2008; Moore, 2012). Concerns about climate change has seen rising interest in methane as a ‘bridge’ fuel, spanning the gap between emissions intensive energy generation from coal and renewable energy (Flores et al., 1997; Flores, 1998; Hamawand et al., 2013; Park and Liang, 2015; Ritter et al., 2015; Wang et al., 2011). Coal seam methane (CSM) accounted for approximately 40% of total production (by volume) from all gas wells in the United States in 2011 (Strapoc et al., 2011). CSM is produced by both thermogenic and microbial
ACCEPTED MANUSCRIPT processes, the latter often termed biogenic production. Biogenic methane represents approximately 30% of all CSM (Flores, 2008; Strapoc et al., 2011) and its production may occur in coal irrespective of its rank. Given the importance of CSM as an energy source, the
T
potential to increase microbial generated CSM production is of great economic and
IP
environmental interest. Previous published studies have attempted stimulate methane
SC R
production by manipulating native microbial communities using techniques such as adjusting pH, temperature, coal surface area and adding nutrients (Green et al., 2008; Jin et al., 2009; Liu et al., 2013; Midgley et al., 2010; Opara et al., 2012; Pfeiffer, 2011; Shumkov et al., 1999; Unal et al., 2012; Wawrik et al., 2012). All of these studies showed shifts in the
MA
mechanisms remain largely unknown.
NU
microbial communities that resulted in changes in gas production, though the underlying
Intuitively it seems likely that the microbial communities found in coal seams would be best adapted to that environment, and that introduction of exogenous bacterial consortia would
D
not result in improved gas production. In 2010 however, Jones et al., demonstrated that
TE
adding exogenous methanogenic communities to a non-CH4-producing coal microbial community resulted in higher rates of methane generation than the addition of nutrients
CE P
(Jones et al., 2010). Similarly, a microbial community derived from an American wetland sediment was demonstrated to be more efficient at the conversion of organic matter in brown coal to methane than the coal’s indigenous community (Opara et al., 2012). The
AC
present study sought to determine whether a composite microbial community, consisting of a coal seam microbial community mixed with mangrove sediment, was able to improve gas yields compared to the coal seam community alone, in an in vitro system. 2. Materials and methods 2.1 Samples The coal seam microbial community was collected in formation water from a medium volatile bituminous rank Permian coal (∼700 m subsurface) in the Sydney Basin, Australia (34.111478⁰ S, 150.737096⁰E). The sample was collected in pre-sterilized, 1 L bottles, to which a reductant and indicator solution were added as described in Midgley et al., (2010). Within an hour, the sample was transferred to an anaerobic glovebox with an atmosphere of 95% Ar and 5% H2 and then degassed under this atmosphere.
ACCEPTED MANUSCRIPT The mangrove swamp microbial community was obtained by collection of ~200g of sediment , from a depth of 10-15cm, near Meadowbank, Sydney (-33.819865⁰ S,151.091569⁰E), using a clean, surface sterilized trowel. The collected sediment was then
T
transferred to anaerobic glove box under conditions described above. The physicochemical
SC R
Measurement Institute (Sydney, Australia) (Table S1).
IP
characteristics of the formation water and swamp sediment were measured by National
2.2 Feedstock coal and characterisation
A homogenised and sieved (<6mm) sample of Latrobe Valley, Cenozoic brown coal was used
NU
as the carbon source for experiments. The ‘run-of-mine’ sample was taken from the Loy Yang open cut located in the Gippsland Basin, Victoria, Australia. A representative
MA
subsample of the coal was characterised using conventional organic petrological methods, according to the Australian Standard AS2856.2 (1998) and AS2856.3 (2000). For the maceral analysis ~600 point-counts were carried out, with examinations using both reflected white
D
light and incident ultraviolet light (UV)/blue light excitation for fluorescence mode. About 60
TE
measurements were made for vitrinite reflectance analysis. The proximate analysis was carried out by Australian Laboratory Services (ALS), Carrington, New South Wales, Australia,
CE P
according to Australian Standard AS1038.3-2000. Before the coal was used as a carbon source, it was transferred to the anaerobic chamber under conditions described previously
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and allowed to degas.
2.3 Generation and characterisation of enrichment cultures Two enrichment cultures were established, one from the Permian coal seam and one from the mangrove sediment. The formation water enrichment culture was established by adding 20mL of the formation water to 1L of minor modified minimal salts (MS) medium (as described in Midgley et al., 2010) with 10g of the Loy Yang coal. One ml of filter sterile 100 μM Na2S solution was used as a reductant. The mangrove swamp enrichment culture was generated by adding 10g of fresh sediment to 1L of MS medium. After two weeks, 50mL of the initial enrichment was transferred to fresh, degassed 1L of MS medium supplemented with 10g of Loy Yang coal and supplemented with 0.5g of yeast extract. Both the formation water and the mangrove sediment enrichment culture were incubated with non-sterile
ACCEPTED MANUSCRIPT feedstock coal, as autoclaved coal did not support the growth of microbes. Both enrichment cultures were incubated at 33°C for a minimum of four weeks prior to the start of the gas production tests. All culturing work was undertaken inside the anaerobic chamber under an
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atmosphere of 95% Ar and 5% H2 atmosphere.
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2.4 Gas production tests
To investigate whether the two enrichment cultures enhanced methane production, 2g of coal and 50ml of MS medium were added to 120 ml sterilized serum vials, and degassed in
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the anaerobic chamber. After degassing, either 1ml of formation water enrichment culture, 1ml mangrove sediment enrichment culture or 0.5mL of both were added. An un-inoculated control was also included. The vials were sealed using butyl rubber septa and aluminium
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crimps inside the anaerobic chamber and removed for incubation at 33°C in the dark, in an inverted position. Triplicates were established in all treatments, including controls. The
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headspace gases of all bottles were assayed at 10, 20 and 30 days using a gas-tight syringe
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as described in Midgley et al., 2010. After gas sampling (which occurred inside the anaerobic chamber), the bottles were unsealed and allowed to equilibrate for 3 mins in the
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atmosphere of the anaerobic chamber, prior to being resealed with a new butyl-rubber septum and aluminium crimp and being returned to the 33°C incubator. Effectively resetting the headspace to that present in the anaerobic chamber.
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Sampled gas was analysed on an Agilent Technologies 490 Micro Gas Chromatograph (Micro-GC). The samples were injected into the front injection port of the Micro-GC using a gas-tight syringe and a motorised syringe pump. The Micro-GC is equipped with four different column modules: a 10 m Molesieve 5Å column for separating O 2/Ar, N2, CH4 and CO, a 10 m Pora Plot Q column for separating CO2, C2H6 and C3H8, a 10 m CP-Sil-5CB column for separating C4-C5 hydrocarbon gases and H2S and a 20m Molesieve 5Å column for separating H2 and He using argon carrier gas. The temperature of these four columns was set to 90°C, 70°C, 60°C and 90°C, respectively. A typical analysis time was 3 min for a single sample injection. 2.5 Statistical analysis
ACCEPTED MANUSCRIPT One-way ANOVA and the Tukey HSD post hoc test (Viotti et al., 2009) were used for testing significant differences of CH4 and CO2 concentrations between each time point and different treatments. All statistical analyses were performed in R (version 3.1.0; R Development Core
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Team, Vienna, Austria). 3. Results and discussion
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3.1 Coal petrology
The feedstock Loy Yang brown coal comprised mainly telovitrinite and detrovitrinite, in near equal abundances, with major amounts of liptinite and minor amounts of inertodetrinite,
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funginite and minerals (Table 1). The liptinites mainly comprised sporinite and liptodetrinite with lesser amounts of cutinite, suberinite and resinite (Table 1, Fig 1). The mean random
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vitrinite reflectance, including measurements on both telovitrinite and detrovitrinite was 0.35%. Consistent with this brown coal rank, the moisture content was ~39%, the volatile
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matter yield was ~33% (dry basis) and the fixed carbon content was ~28%. The high
3.2 Gas production results
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moisture, may facilitate production of films from microbial growth (Faison, 1991).
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Taken across the entire time course, significantly greater total yields of methane (p<0.0001) were generated from the mixed origin enrichment culture (~162µmol CH4 g-1 coal) than from either the formation water (~126µmol CH4 g-1 coal) or mangrove enrichment cultures
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(~13µmol CH4 g-1 coal; Fig 2a). Only trace amounts of methane (<0.1µmol CH4 g-1 coal) were observed in desorption controls, indicating that the methane observed in all of the mixed origin, formation water and mangrove enrichment cultures was produced biogenically. The reasons for the increase in methane yields by the mixed origin enrichment culture are unknown but were presumably related to changes in the microbial community in the enrichment cultures. It is worth noting that a small amount of yeast extract (totalling 0.1 mg carbon) was present at the start of the mixed origin enrichment cultures. Assuming that all the carbon in this yeast extract was converted to methane, without any losses to biomass or other gases, it could account for the presence of 8.3µmol of the additional methane produced in the mixed origin enrichment culture. Removing this amount of methane from the total and retesting for significantly different total yields of methane between the treatments did not substantively alter the p-value. Methane yields from this carryover yeast
ACCEPTED MANUSCRIPT extract, however, could account for about half of the methane produced by the mangrove sediment enrichment culture. It is also worth noting that an average of 34µmol H2 was present in headspace at each time point (due to the atmosphere in anaerobic chamber; see
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Fig 2c). This amount of H2, assuming no losses of H2 to other processes and the availability of
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sufficient carbon, would be sufficient to account for the production of ~17µmol CH4. The
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low production of methane by the mangrove enrichment culture would then imply that the microbes in this community lacked the ability to access carbon in the feedstock coal, and that the microbes in the mixed origin enrichment and formation water enrichment cultures
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are accessing both carbon and hydrogen from the coal.
Along with changes in yields of methane, different patterns of methane generation were
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observed for the formation water, mangrove and mixed origin enrichment cultures (Fig 2a). The mixed origin enrichment culture started generating methane quite early, and methane production climbed steadily thereafter. In contrast, there was little sign of early
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methanogenesis with either the formation water or mangrove enrichment cultures, but
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methane generation markedly increased after day 20. Methanogenesis normally occurs after the exhaustion of other terminal electron acceptors, and the faster onset of methane
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production in the mixed microbial culture may be related to a rapid consumption of the energetically more profitable terminal electron acceptors by the more diverse community. That is, any nitrates, sulphates or iron (III) present in the inoculum may be more rapidly
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consumed with the greater diversity of organisms in the mixed community treatment or it may simply a productive of the composition of the mixed community. Differences in the pH of the samples may also have been an important influence on methane yield, especially the rapid onset of methane production seen in the mixed microbial samples. The initial pH value of mangrove sediment and formation water were 5.9 and 9.2 (Table S1), respectively, and Victorian Loy Yang (LY) brown coal has been shown to have a low pH of ~4.2 (Qi and Chemistry, 2004; Yuliani et al., 2012). This is close to the initial pH of the mangrove sediment and this may also have contributed to the earlier onset of methane generation observed in the mangrove sediment microbial community. All treatments yielded measurable quantities of CO2 (Fig 2b). The formation water enrichment culture produced the greatest yield of CO2 (~119µmol CO2 g-1 coal), significantly
ACCEPTED MANUSCRIPT more CO2 (p<0.0001) than was produced by the mixed origin enrichment culture (~88µmol CO2 g-1 coal). The little CO2 produced by the mangrove enrichment culture (~4µmol CO2 g-1 coal) was not significantly different (p>0.6) to the desorbed CO2 observed in the desorption
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control (~2µmol CO2 g-1 coal), adding further weight to the proposal that the microbes in the
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mangrove sediment enrichment culture were unable to access carbon in the feedstock coal.
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4. Conclusions
Methane is an important energy source as it offers economic and environmental benefits over traditional coal-fired power generation. A significant proportion of coal seam methane
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is biogenic in origin, being produced by the syntrophic and synergistic activities of complex microbial consortia. The results presented here suggest that manipulating microbial
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communities by bioaugmentation may provide methods that can be used to improve the biogenic production of methane from coal. Additionally, physicochemical properties of the carbon source may be a critical factor in the success of ex situ bioaugmentation as it
Acknowledgments
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provides the actual living environment for the microbial community.
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The authors would like to thank CSIRO-Chinese Academy of Science Collaboration Fund and China Scholarship Council for the support of this project. We thank Ms. Jan Shaw for her assistance with the statistical analysis used in this study, Dr. Stephen Sestak for helpful
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discussions on the experimental design and Dr. Nai Tran-Dinh for his helpful thoughts on the manuscript. We also thank Emma Qi for providing the brown coal.
Figure and Table Legends Figure 1. Photomicrograph of Loy Yang brown coal, Gippsland Basin, under reflected white light (a) and fluorescence mode (b).
Figure 2. Gas production and consumption by the coal formation water enrichment culture, the mixed origin enrichment culture and the mangrove sediment enrichment culture at 10, 20 and 30 days.
ACCEPTED MANUSCRIPT Table 1. Maceral Composition for Loy Yang brown coal.
Table S1. Physicochemical characteristics of the mangrove sediment and Sydney Basin
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Table S2. Proximate analysis for the Loy Yang brown coal.
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formation water sample.
ACCEPTED MANUSCRIPT References
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Wang, J., Ryan, D., Anthony, E.J., 2011. Reducing the greenhouse gas footprint of shale gas. Energy Policy 39, 8196-8199. Wawrik, B., Mendivelso, M., Parisi, V.A., Suflita, J.M., Davidova, I.A., Marks, C.R., Van Nostrand, J.D., Liang, Y.T., Zhou, J.Z., Huizinga, B.J., Strapoc, 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, 2642. Yuliani, G., Qi, Y., Hoadley, A.F.A., Chaffee, A.L., Garnier, G., 2012. Lignite clean up of magnesium bisulphite pulp mill effluent as a proxy for aqueous discharge from a ligno-cellulosic biorefinery. Biomass Bioenerg 36, 411-418.
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Fig. 1
Fig. 2
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Table 1. Maceral Composition for Loy Yang brown coal
3.8
Sporinite
3.6
Suberinite
0.9
Resinite
0.5
Cutinite
<0.2
Alginite
0
Liptodetrinite
4.6
Semifusinite
0
Fusinite
0
Macrinite
0
Micrinite
0
Funginite
1.5 1.7
Minerals
29.1
Total
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81.9
Liptinite
9.6
Inertinite
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13.5
3.2
4.6
Minerals
29.1
-
100
100
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Inertodetrinite
58.1
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Gelovitrinite
Vitrinite
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33.8
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Detrovitrinite
Volume % (mineral free)
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20.5
Volume %
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Telovitrinite
Maceral Group
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Volume %
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Maceral