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    Carbon and hydrogen isotope fractionation during methanogenesis: A laboratory study using coal and formation water Rita Susilawati, Suzanne D. Golding, Kim A. Baublys, Joan S. Esterle, Stephanie K. Hamilton PII: DOI: Reference:

S0166-5162(16)30189-6 doi: 10.1016/j.coal.2016.05.003 COGEL 2635

To appear in:

International Journal of Coal Geology

Received date: Revised date: Accepted date:

16 October 2015 9 May 2016 10 May 2016

Please cite this article as: Susilawati, Rita, Golding, Suzanne D., Baublys, Kim A., Esterle, Joan S., Hamilton, Stephanie K., Carbon and hydrogen isotope fractionation during methanogenesis: A laboratory study using coal and formation water, International Journal of Coal Geology (2016), doi: 10.1016/j.coal.2016.05.003

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ACCEPTED MANUSCRIPT CARBON AND HYDROGEN ISOTOPE FRACTIONATION DURING METHANOGENESIS: A LABORATORY STUDY USING COAL AND FORMATION WATER

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Rita Susilawatia,b*, Suzanne D. Goldinga, Kim A. Baublysa, Joan S. Esterlea and Stephanie K.

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.

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Hamiltona

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ABSTRACT

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Carbon and hydrogen isotope compositions of CH4 generated via methanogenesis in cultures of South Sumatra Basin (SSB) coalbed methane (CBM) formation waters grown on coal, acetate and H2+CO2 were investigated. CH4 production and molecular analysis confirmed the presence of active microbial communities that are able to convert coal into CH4 using both acetoclastic and hydrogenotrophic pathways. The representative bacterial sequences were dominated by Bacteroidetes, Firmicutes and Deltaproteobacteria, while Methanosaeta and Methanosarcina were the most prevalent archaeal methanogens present in the cultures. CH4 produced in this study’s culturing experiments has δ13C values in the range of –50 ‰ to –20 ‰, with most values falling outside the current understanding of the carbon isotopic boundaries for biogenic CH4 (–110 ‰ to –30 ‰). However, the corresponding apparent carbon isotopic α factor (αc = 1.02±0.006), and isotopic effect (εc = –20.1 ‰ ±15.3) showed that CH4 in SSB cultures was predominantly produced by acetoclastic methanogenesis, which is consistent with the results of molecular DNA analysis. In addition, the calculated contribution of CO2 reduction from the δ13C values of coaltreated cultures was overall < 50 %, further confirming the high contribution of the acetoclastic pathway to CH4 production in the SSB cultures. The outcome of this experimental study also suggests that δ2HCH4 values may not provide a reliable basis for distinguishing methanogenic pathways, while apparent carbon isotopic fractionation factor (αc) and isotope effect (εc) are considered more useful indicators of the methanogenic pathway. The high δ13C-CH4 values (>–30 ‰) and the dominance of Methanosaeta over Methanosarcina indicate that methanogens within the SSB cultures were operating at low substrate concentrations. An unusually positive δ13C-CH4 suggests a substrate depletion effect, which is thought to be related to a decrease in the relative abundance of key bacterial coal degraders with formation water inoculum storage time. Closer observation of δ13C-CH4 values during the growth of cultures within a single experiment also showed a 13C-enrichment trend over time. At log phase of growth, the CH4 produced was 13C-depleted when compared to the stationary phase that also indicates substrate depletion effects. Finally, the δ13CCH4 values encountered in this study (as high as –20 ‰) highlight the possible positive extension of δ13CCH4 values of acetoclastic methanogenesis from those currently reported in the literature for natural and experimental samples (as high as –30 ‰). Keywords: Biogenic gas, coal, culture experiment, isotopic composition

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INTRODUCTION Archaeal methanogens are the only known microorganisms capable of forming CH4. These

strictly anaerobic microbes are restricted to utilizing simple compounds that contain no more

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than two carbon atoms. As such, complex organic compounds in anoxic environments (e.g., marine sediments, rice paddies, subsurface shales and coalbeds) are degraded by associations of

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microbes consisting of hydrolytic, fermentative and acetogenic bacteria and methanogenic

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archaea (Schink, 2006; Strąpoć et al., 2011; Winter and Knoll, 1989; Zinder, 1993). This association is often termed a methanogenic consortium. Methanogenesis is the final step in the

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decomposition of organic matter by methanogenic consortia. Because of their vital ecological role in the Earth’s carbon cycling, methanogens have gained attention in the literature,

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particularly in the environmental and bioenergy areas (Conrad et al., 2011; Demirel and Scherer, 2008; Milkov, 2011; Miller, 2004; Wawrik et al., 2012). Methanogen substrates, in general, can be grouped into competitive and non-competitive

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substrates. The first category relates to substrates that are also utilized by other groups of

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microbes. These substrates include acetate, formate and H2+CO2. Depending on the environment, bacteria can successfully out-compete methanogens for competitive substrates

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(e.g., sulfur reducing bacteria in a sulfate-rich zone) (Whiticar, 1999). Non-competitive substrates are substrates that are available for methanogens but less attractive to other microbes (Whiticar, 1999). Included in this group are methanol and methylated amines. When utilizing substrates to produce CH4, methanogens mainly use two primary pathways, acetate fermentation (Equation (1)) and CO2 reduction (Equation (2)) (Strąpoć et al., 2011; Whiticar, 1999; Zinder, 1993). Studies have documented that CO2 reduction is the dominant pathway in the marine environment, while acetate fermentation is the major pathway in freshwater environments (Schoell, 1980; Whiticar et al., 1986). However, in coalbed environments, it has been recognized that the CO2 reduction pathway predominates, even when the waters are quite fresh (Golding et al., 2013). CH3COOH → CO2 + CH4

(1)

CO2 + 4H2 → 2H2O + CH4

(2)

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ACCEPTED MANUSCRIPT Stable isotopes are a valuable tool for the identification of CH4 gas origin and generation pathways (Flores et al., 2008; Hamilton et al., 2014; Kinnon et al., 2010; Strąpoć et al., 2007). The method has been used to evaluate CH4 dynamics both in ecosystems and laboratory

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engineered systems. Organic matter degradation by microbial activity in an anoxic environment produces CH4 depleted in 13C relative to CH4 produced thermogenically (Faiz and Hendry, 2006;

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Golding et al., 2013; Whiticar, 1999). This is because microbes preferentially utilize the lighter

generally

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isotope of C (12C) that has lower bond energies, and therefore biogenically derived CH4 is C-depleted. Isotope fractionation is maximized under conditions where the

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methanogenic substrate is in abundant supply, and depends on several factors including temperature, microbial growth phase, the hydrogen supply, the methanogen species, and the

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methanogenic pathways involved (Botz et al., 1996; Conrad, 2005; Penning et al., 2005). Moreover, isotopic fractionation of biogenic CH4 may increase throughout the culture’s exponential growth phase before stabilizing during the stationary phase (Valentine et al., 2004).

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The current understanding of the isotopic composition of biogenic CH4 is in the range of –

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110 ‰ to ‒50 ‰ and ‒400 ‰ to ‒150 ‰, for δ13C-CH4 and δ2H-CH4, respectively (Conrad, 2005; Golding et al., 2013; Whiticar, 1999; Whiticar et al., 1986). In addition, CH4 produced 13

C-depleted relative to that produced from acetate (Chanton et al.,

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from H2+CO2 is typically

2004; Conrad et al., 2011; Whiticar, 1999). Hydrogenotrophic methanogenesis derived CH4 typically has δ13C-CH4 between –110 ‰ and ‒60 ‰, while acetoclastic methanogenesis produced CH4 has δ13C between ‒60 ‰ and ‒45 ‰ (Conrad et al., 2011; Whiticar, 1999). Small isotopic fractionation factor α values ( 0 ‰ (Table 9). Similarly, for the H2+CO2-

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treated culture, the use of αmc = 1.072 also gave unreasonably low ƒmc values, while calculation using αmc = 1.041 gave ƒmc values ranging from 0.99 to 1, suggesting a high contribution from the CO2 reduction pathway to CH4 generation in this culture as expected. The somewhat different fractionation factors for hydrogenotrophic methanogenesis in the coal-treated cultures and the H2+CO2-treated culture were likely caused by differences in H2 concentration. As previously mentioned in the methods section, a high concentration of H2 (80 %) was injected into the H2+CO2-treated culture. A previous study showed that under high H2 concentration (unlimited substrate), the hydrogenotrophic methanogens fractionated carbon isotopes less than when H2 concentration was low (Valentine et al., 2004). That may explain why the H2+CO2treated culture had a lower αc value (1.041) compared to the coal-treated culture (1.072). In addition, studies have noted that such differences in fractionation factors for the same pathway may be related to differences in microbial community structures and culture conditions. In the case of our SSB cultures, when different carbon sources were present, their carbon isotopic compositions and differing bioavailability also possibly influenced the isotopic fractionation factor of the samples.

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Table 9. Estimation of CO2 reduction contribution (ƒ mc) for CH4 from SSB H2+CO2 and acetate treated cultures Major source of carbon

ɛma = 10

ɛma = 6.1

ɛma = 0

ɛma = 10

ɛma = 6.1

ɛma = 0

SSB4

H2+CO2

-0.42

-0.18

-1.80

0.99

0.99

1.00

SSB3

Acetate

0.00

-0.15

0.17

-0.01

0.15

0.31

SSB4

Acetate

-0.01

-0.14

0.15

-0.02

0.13

0.28

SSB5

Acetate

-0.01

-0.14

0.16

-0.01

0.14

0.29

1.041

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1.072

CONCLUSIONS

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ƒmc calculated using Eqns 7,8 and 9, with 2 variables of αmc (1.072 and 1.041) and 2 variables of ɛma. Isotopic values are expressed in per mil (‰) notation. Highlighted areas show the most likely contribution of the CO2 reduction pathway. For detailed explanation see text.

Three different experiments were conducted that aimed to determine the isotopic

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composition of microbial CH4 and CO2 produced from cultures of SSB formation waters grown on coal, H2+CO2 and acetate. Overall, the results showed the following: (1) The δ13C-CH4 values in our experiment (–50 ‰ to –20 ‰) mostly fell outside the field currently considered as representative of biogenic CH4. This study hypothesizes two possible causes for the positive δ13C-CH4 values found in SSB cultures; the increasing importance of acetate as a methanogenic substrate and a substrate depletion effect. Substrate depletion possibly occurred due to a decrease in the relative abundance of bacterial coal degraders with formation water inoculum storage time. (2) In these experiments, an apparent carbon isotopic fractionation factor (αc) and isotope effect (εc) were found to be more useful indicators of methanogenic pathways than the absolute δ13C-CH4 values. Overall, the apparent α and ε values calculated in this study suggest the dominance of acetoclastic methanogenesis over hydrogenotrophic methanogenesis, which is consistent with pathway contribution calculation and microbial community data. The values are

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ACCEPTED MANUSCRIPT also in agreement with carbon isotopic fractionation values for acetoclastic methanogenesis reported in the literature. (3) Three cases are thought to have affected the carbon isotopic systematics of SSB coal-

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treated cultures: a) acetate fermentation in cultures that experienced a substrate limitation that

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was not severe enough to exclude isotope fractionation; b) acetate fermentation in cultures that experienced substrate depletion strong enough to exclude isotope fractionation; and c) acetate

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fermentation in cultures that have very severe substrate depletion, which may result in a slightly positive value of ɛma.

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(4) These experimental outcomes support the concept that CO2 reduction can result in CH4 with more negative δ2H values comparable to that found in acetate. Overall, the measurement of

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δ2H suggests that δ2H-CH4 is independent of the methanogenic pathway and may not provide a reliable basis for distinguishing the methanogenic pathway in SSB cultures. In addition, relatively positive δ13C-CH4 values encountered in this study suggest a positive extension of

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δ13C-CH4 values of acetoclastic methanogenesis from those currently reported in the literature. Altogether this study produced results that contribute to knowledge of the isotopic

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composition of CH4 produced from methanogenesis in cultures utilizing coal. The results are also potentially applicable to natural systems and may assist in interpreting the isotopic compositions of field gases.

ACKNOWLEDGEMENTS

Funding for this research came from an Australian Awards Scholarship 2010. PT Medco CBM Sekayu, Lemigas and PT Bukit Asam are gratefully acknowledged for access to sites and samples. TSOP Spackman award was used to partly fund laboratory consumables. The Australian Centre for Ecogenomics at The University of Queensland provided access to its PC2 and quarantine laboratory. The authors thank Sam Papendick, Soleh Basuki Rahmat, Eko Budi Tjahyono and Erlina Lena Ria 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. The authors also thank Tennille Mares, Maria Mastalerz and Anna Martini as well as three other anonymous reviewers for their constructive review of this manuscript. 33

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Methanogenesis investigated in cultures of South Sumatra Basin CBM waters. Cultures grown on South Sumatra Basin coal, acetate and H2+CO2. Stable isotopic and molecular DNA evidence for substrate depletion effects. αc and εc were better indicators of methanogenic pathway than δ13C-CH4 and δ2H-CH4. Possible positive extension of δ13C-CH4 values of acetoclastic methanogenesis.

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