Fuel 104 (2013) 717–725

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Fuel

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Fungal degradation of coal as a pretreatment for methane production

Rizwan Haider a, Muhammad A. Ghauri a,⇑, John R. SanFilipo b, Elizabeth J. Jones b, William H. Orem b,Calin A. Tatu b, Kalsoom Akhtar a, Nasrin Akhtar a

a Industrial Biotechnology Division, National Institute for Biotechnology & Genetic Engineering (NIBGE), P.O. Box No. 577, Jhang Road, Faisalabad, Pakistanb United States Geological Survey (USGS), 956 National Center, Reston, VA, USA

h i g h l i g h t s

" Fungal isolate MW1 liberated complex organic compounds from coal matrix." MW1 was isolated from a core sample of coal." Variety of aliphatics, aromatics and aromatic nitrogen compounds were identified." Methanogenesis of released organics generated significant methane in some samples.

a r t i c l e i n f o

Article history:Received 1 January 2012Received in revised form 2 May 2012Accepted 3 May 2012Available online 17 May 2012

Keywords:Coal biosolubilizationPenicillium chrysogenumCoal methanogenesis

0016-2361/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fuel.2012.05.015

⇑ Corresponding author. Tel.: +92 41 2550814; fax:E-mail address: maghauri@nibge.org (M.A. Ghauri

a b s t r a c t

Coal conversion technologies can help in taking advantage of huge low rank coal reserves by convertingthose into alternative fuels like methane. In this regard, fungal degradation of coal can serve as a pretreat-ment step in order to make coal a suitable substrate for biological beneficiation. A fungal isolate MW1,identified as Penicillium chrysogenum on the basis of fungal ITS sequences, was isolated from a core sam-ple of coal, taken from a well drilled by the US. Geological Survey in Montana, USA. The low rank coalsamples, from major coal fields of Pakistan, were treated with MW1 for 7 days in the presence of 0.1%ammonium sulfate as nitrogen source and 0.1% glucose as a supplemental carbon source. Liquid extractswere analyzed through Excitation–Emission Matrix Spectroscopy (EEMS) to obtain qualitative estimatesof solubilized coal; these analyses indicated the release of complex organic functionalities. In addition,GC–MS analysis of these extracts confirmed the presence of single ring aromatics, polyaromatic hydrocar-bons (PAHs), aromatic nitrogen compounds and aliphatics. Subsequently, the released organics were sub-jected to a bioassay for the generation of methane which conferred the potential application of fungaldegradation as pretreatment. Additionally, fungal-mediated degradation was also prospected for extract-ing some other chemical entities like humic acids from brown coals with high huminite content espe-cially from Thar, the largest lignite reserve of Pakistan.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Increased excavation and utilization of high rank coals havesparked interest in efficient utilization of massive resources of lowrank and brown coals. With the increasing global energy demand,it is becoming indispensable to tap the colossal reserves of low rankcoals. Transformation of coal into methane could help in providingenergy for countries like Pakistan, which is experiencing an intenseenergy crisis in spite of a reserve of 185 billion tons of low rank coals[1]. Extraction of alternative fuels, particularly methane, is currentlygaining interest due to technical constraints involved in exploita-tion and utilization of low rank coals. Furthermore, increasinglystringent environmental requirements may help make economical

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+92 41 2651472.).

the conversion of coal into alternative fuels like methane, whichburns cleanly.

Naturally occurring coal bed methane (CBM) or coal seam gas,an unconventional gas, is emerging as an important energy sourceworldwide [2]. The biogenic origin of methane within the coalseams makes it plausible in theory to stimulate new methane gen-eration in existing wells or in split process systems. On the otherhand, coal is a recalcitrant geopolymer, and may not be readilydegradable by microorganisms, especially methanogens. Mecha-nisms by which bacteria degrade coal to methanogenic substratesand finally into methane for biogenic generation are not com-pletely understood, though in recent years there has been focuson developing some models for metabolic pathways involved inthe biodegradation of coal to methane [3,4]. However, in all modelsof microbial production of methane from coal, the rate limitingsteps involve the solubilization and degradation of coal to the

718 R. Haider et al. / Fuel 104 (2013) 717–725

substrates that can be utilized by methanogens for methane gener-ation [5].

The solubilization of lignite (low rank coal) and subsequentbreakdown into low-molecular weight aromatic and aliphatic com-pounds can be an indirect option for extracting some material capa-ble of being fermented by anaerobic microorganisms [6]. Thisindirect step could be achieved by various methods including (i)extraction using organic solvents, (ii) enhanced bacterial decompo-sition of coal matrix, or (iii) fungal-mediated degradation. Additionof a solubilization step could serve as a pretreatment to enhanceoverall methane production. Fungal degradation of coal is espe-cially interesting as it is well known that coals, particularly of lowrank, contain some lignin-derived structures within the coal matrixwhich are susceptible to fungal attack [7]. Phanerochaete chrysospo-rium, Penicillium species and Trichoderma atroviride are some exam-ples of fungi that are involved in solubilization of coal [8–10].However, the role of fungi in the process of biogenic methane for-mation is unclear. Though in a recent report bacteria, archaea andfungi have been reported to be involved in methane release in aban-doned coal mines where weathering of coal and timber is initiatedby fungi and bacteria under a suboxic atmosphere [11]. But com-plete understanding of the mechanism for underground anaerobiccarbon recycling and anaerobic degradation of coal is still needed

Indus

River

MancharLake

KalriLake

ARABIAN SEA

UAJ-1

UAS-4UAK-1

LS-4

LakhraCoal Field

Lakhra southcoal area

Indus Eascoal area

SondaCoal Field

Meting-JhimpirCoal Field

KARACHI

Hyderabad

Thatta

Nawabshah

Dadu

Bad

natsihcolaB

Sind

h

0 20 3010 MILES

0 20 3010 KILOMETERS

Intermittentestuary

Swamp

Coal field

Selectedborehole

TP-1

City

67° 68° 69

27°

26°

25°

24°

Fig. 1. Geographic setting and borehole locations for the samples used in this study. Th(e.g. UAS-4-2E = borehole UAS-4, seam 2, bench E). Coal field boundaries are approxima

in order to develop effective methods for stimulating new biogenicmethane production from the coal matrix.

The objective of this study was to investigate the fungal-mediated degradation of some representative lignite samples fromPakistani coal fields and to analyze those extracts for their poten-tial to support subsequent methanogenesis based on previouslyreported models and using a bioassay to measure methanogenicpotential of the fungal coal extract.

2. Materials and methods

2.1. Coal samples

2.1.1. Geological settingTwelve low-rank coal samples from Sindh Province, Pakistan

were subjected to fungal degradation in the laboratory. The sam-ples were obtained from drill-core collected by the United StatesGeological Survey (USGS) and the Geological Survey of Pakistan(GSP) between 1986 and 1992. The samples used in this study wereground splits (<850 lm) that had been sealed in polyethylene andarchived at USGS since shortly after drilling. Except for some sec-ondary sulfate formation, much of which probably occurred shortly

Rann of Kutch

TP-1

TP-4

TP-3t

TharCoal Field

Mithiin

INDIAPAKISTAN

Names and boundary representation arenot necessarily authoritative.

AFGHANISTAN

PAKISTAN

A R A B I A N S E A

INDIA

IRAN

TURKMENISTAN

UZBEKISTAN

TAJIKISTAN KYRGYZSTAN

CHINA

Maparea

° 70° 71°

e sample numbers in Table 1 reflect the borehole number and sample benchte.

Table 1Geological setting of samples taken from various coal fields of Pakistan.

Sample Coal field/area

Depth(m)

BTU(m, mmf)b

ASTMrank

Mean randomreflectancein oil (Rm) %

Rmrank

UAS-4-2E Sonda 183.58 8042 ligA 0.47 subBUAK-1-4 Indus east 183.50 8274 ligA 0.45 subBUAJ-1-1 Jhimpir 119.90 7206 ligA 0.42 subCLS-4-1a Lakhra south 172.96 8220 ligA 0.51 subALS-4-2Ba Lakhra south 191.68 9056 subC 0.48 subBTP-1-1.1 Thar desert 146.38 5978 ligB 0.37 ligATP-1-5.2 Thar desert 178.91 6074 ligB 0.30 ligBTP-3-2Ba Thar desert 147.19 5705 ligB 0.27 ligBTP-3-2K1* Thar desert 153.92 5948 ligB 0.22 ligBTP-3-2Xa Thar desert 165.52 6312 ligA 0.30 ligBTP-4-2A Thar desert 192.05 5874 ligB 0.34 ligBTP-4-10 Thar desert 272.05 5239 ligB 0.34 ligB

ASTM rank from ASTM (2011), D388-05.Deutsches Institut für Normung, (Rm) Rank estimated from Stach (1982) [27].

a Desorbed samples.b Moist, mineral matter free (m, mmf).

R. Haider et al. / Fuel 104 (2013) 717–725 719

after core recovery, the samples do not appear to have appreciablydegraded during storage.

The samples came from five coal areas in lower Sindh Province(Fig. 1 and Table 1). Four areas near the Indus River are laterallycontiguous, but one of these, the Meting-Jhimpir coal field, isstratigraphically higher than the other three. The sample from thisfield (UAJ-1-1) is from the Sohnari member of the Laki Formation,which is generally considered of Eocene age [12], but may in factbe a tongue of the Bara Formation [13]. The samples from the Lakh-ra and Sonda coal fields are from the Paleocene Bara Formation.Thar coal field is covered with dune sand and has no bedrockexpression [14]. Bara Formation and possibly Sohnari coals arelikely to be continuous from the Indus Basin to the Thar Desertin the subsurface, but the age and correlations of the Thar coalshave not been precisely determined. The samples used in thisstudy were collected from drilling-depths ranging from 120 m(UAJ-1-1) to 272 m (TP-4-10). Sindh coals are typically less thanthree meters thick [15], but the main seam at Sonda was over6 m thick at borehole UAS-4, and the main seam intercepted inTP-3 was over 29 m thick [16].

2.1.2. Chemical characterizationSindh Province coals generally range in rank from lignite to sub-

bituminous (Table 1). Thar coals are very high moisture brown coalswith a conspicuous woody texture. The sulfur content of Sindhcoals is typically medium to high [17], but is laterally and verticallyvariable, and can be <1% for some benches of the thicker seams (Ta-ble 2). Adjusting to sulfate-free ash would decrease the values of thegross calorific value on the moist, mineral-matter free basis (BTU/

Table 2Characterization of various coal samples used in this study.

Sample %C %H %N %O %S Moisture (%) Ash (%) Volat

UAS-4-2E 44.27 3.13 0.70 8.90 0.77 38.62 3.61 29.18UAK-1-4 37.56 2.96 0.60 5.63 6.13 31.41 15.71 29.25UAJ-1-1 24.67 2.42 0.40 6.20 6.75 26.90 32.66 22.97LS-4-1 43.06 2.96 0.93 7.78 3.71 33.63 7.93 29.65LS-4-2B 45.00 3.18 0.85 7.31 3.44 29.50 10.72 30.26TP-1-1.1 31.42 2.34 0.40 7.73 1.26 47.62 9.23 24.41TP-1-5.2 33.42 2.42 0.44 7.23 1.42 49.45 5.62 25.73TP-3-2B 28.18 2.12 0.31 6.24 4.61 45.66 12.88 23.29TP-3-2K1 28.61 2.26 0.28 7.74 1.55 44.81 14.75 23.91TP-3-2X 34.65 2.76 0.34 8.48 0.40 50.24 3.13 26.99TP-4-2A 30.57 2.37 0.41 7.42 2.08 47.38 9.77 24.21TP-4-10 26.04 1.79 0.40 7.19 2.27 45.85 16.46 20.14

a Mineral matter free (mmf).

lb m, mmf) shown in Table 1, but would not change the ASTM rankfor any of the samples for which the required data are available. Ashyield is quite variable between sample benches (Table 2), but tendstoward the low end of the medium ash range (8–15%) for the mainLakhra–Sonda seams and the high end for other seams [17]. Theaverage as-received ash for Thar coals is about 9% [18], but the mid-dle part of the main seam is typically less than 8%. In situ methanedesorption was conducted for a few of the Lakhra South and Tharcoal samples [16]. Although only a small amount of gas was de-tected in the desorbed samples, additional testing under more rig-orous technical and geologic constraints is needed in terms ofextensive drilling activities in perspective of CBM occurrence po-tential [19]. The samples selected for this study were chosen to in-clude typical coals from the various stratigraphic intervals and coalfields for which well-preserved samples were available, along witha few high-ash samples and desorbed samples (Tables 1 and 2).

2.1.3. Maceral analysisVitrinite reflectance (Table 1) and maceral analyses (Table 2)

for these samples were carried out and provided by James C. Howerand Cody D. Patrick of the University of Kentucky, Center forApplied Research.

2.2. Isolation of fungal strain MW1 from a core sample of coal

Fungal strain was isolated from a core sample of sub-bituminouscoal, taken from a well drilled by the US Geological Survey inMontana, USA. The core sample of coal, from which MW1 was iso-lated, was stored in an anaerobic chamber for about a year. Anuncontaminated sample was removed from the center of the intactcore using sterile technique for isolation of fungi and was suspendedin 100 ml of minimal salts medium and incubated for 2 days at 25 �Cwith 120 RPM shaking. After 2 days, the suspension was diluted 100times and spread on malt extract-agar plates. Isolated strains werepurified and cultures were maintained on solid medium (10.0 g maltextract, 5.0 g glucose, 15.0 g agar, and 1L distilled water). The com-position of minimal salts medium was (per L): 1 g (NH4)2SO4,0.52 g MgSO4�7H2O, 5 g KH2PO4, 0.0005 g FeSO4�7H2O, 0.0003 gZnSO4 [20].

2.3. Molecular typing of fungi

For the extraction of total genomic DNA, Fast DNA Spin Kit for Soil(MP Biomedicals, Solon, OH) was used. For molecular typing ofstrains, ITS internal regions were amplified through PCR using uni-versal primers ITS1 and ITS4 [20].

� ITS1: TCC GTA GGT GAA CCT GCG G (Forward Primer)� ITS4: TCC TCC GCT TAT TGA TAT GC (Reverse Primer)

ile matter (%) Fixed carbon (%) Maceral groupsa (vol.%, mmf)

Vitrinite/Huminite Inertinite Liptinite

28.59 75.5 17.6 6.823.63 85.9 8.4 5.717.47 86.0 0.0 14.028.79 84.6 11.5 3.829.52 50.7 39.2 10.118.74 91.7 1.5 6.819.20 77.2 0.0 22.818.17 85.4 1.6 13.016.53 77.4 0.0 22.619.64 54.8 11.3 33.918.64 88.8 2.4 8.817.95 88.0 0.5 11.5

720 R. Haider et al. / Fuel 104 (2013) 717–725

The PCR conditions were as follows: 94 �C for 3 min, 30 cycles of94 �C for 30 s, 56 �C for 1 min, 72 �C for 1 min, followed by 72 �C for10 min [20]. The PCR products were confirmed through agarose gelelectrophoresis and purified using Wizard�PCR Preps DNA Purifi-cation System (Promega, USA). Purified DNA products were se-quenced through BigDye� Terminator v3.1 Cycle Sequencing Kitusing ITS1/ITS4 primers.

2.4. Treatment of coal samples with MW1

Fungal isolate was first grown in minimal salt medium, sup-plemented with glucose (1%, w/v) and malt extract (0.3%, w/v),for 4 days. All incubations were conducted in sterile aerobicflasks. For degradation studies, the concentration of glucose wasreduced to 0.1% and malt extract was eliminated. Coal was addedas the primary source of carbon at the concentration of 1% (w/v)of the solution and final volume of reaction mixture was 130 ml.Flasks were inoculated with 2 ml of freshly grown culture andautoclaved coal was added into the flasks at the time of inocula-tion. Treatments were incubated at 25 �C for 7 days on a rotaryshaker at 150 RPM. Two controls were treated under the sameconditions: (1) medium with coal but without inoculation, (2)medium with inoculation but without coal. After incubation of7 days, supernatants were filtered for analytical investigationsusing Whatman Glass Fiber Filters (Pore Size, 2.7 lm) precombu-sted at 500 �C.

Table 3Released organic fractions from various coal samples.

Sample Organic fractionsa Methane generated

2.5. Analytical investigations of released organics

2.5.1. Excitation-Emission Matrix Spectroscopy (EEMS)Filtrates were analyzed using Excitation-Emission Matrix Spec-

troscopy (EEMS) (Agilent Cary Eclipse Fluorescence Spectropho-tometer) for qualitative estimation of the released organics fromcoal. Filtrates were scanned over the range of 200–700 nm (excita-tion and emission) for a qualitative determination of the nature oforganic fractions being released from coal.

(lmoles/g of coal)

UAS-4-2E Ethosuximide, 3.69(22E)-ergosta-5,7,2,2-trien-3-ol

UAK-1-4 Ethosuximide, 1.83UAJ-1-1 1,2,3,5,6,7-hexahydro-1,1,2,3,3-

pentamethyl-4-H-Inden-4-one1.75

LS-4-1 Benzothiazole, 2.392-amino benzamide,

LS-4-2B 3-methylbenzaldehyde, 2.872-amino benzamide

TP-1-1.1 Heptanoic Acid, 3.302-ethyl hexanoic acid,Nonanoic acid3-hydroxy benzaldehyde,Vanillin,

TP-1-5.2 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-

3.27

4-H-Inden-4-oneTP-3-2B Indole 9.67

2-tert-butyl quinoline

2.5.2. Gas chromatography–mass spectrometry (GCMS)Released organics, after the fungal treatment of coal, were

sequentially, liquid/liquid extracted from 45 ml of filtrate usingpesticide-grade Dichloromethane (DCM). The extract was concen-trated (to 1–2 ml) through rotoevaporation and further reducedto 200 ll under a gentle stream of N2. Generally, 1 ll was usedfor gas chromatography/mass spectrometry (GC/MS) analysis ofeach 200 ll. GC/MS analysis was carried out using Hewlett–Packard(Agilent Technologies, Palo Alto, CA, USA) 6890 series gas chro-matograph and 5973 Electron Ionization (EI) Mass Selective Detec-tor (MSD) which was operated in scan mode. An HP-5MS column(95% Dimethyl, 5% Diphenyl Polysiloxane), with 0.25 mm � 30 m �0.25 lm, was used. An injection volume of 2 ll was used. The NIST02 mass spectral library was used for identification of the com-pounds from the mass spectral data.

N-[2-(1H-indol-3yl)ethyl]-acetamide,3-methyl phenol

TP-3-2K1 2,6-bis(1,1-dimethylethyl)-2,5-cyclohexadiene-

7.34

1,4-dioneDodecene

TP-3-2X Indole, 10.78Benzothiazole

TP-4-2A 2-tert-butyl quinoline, 2.252-acetyl-4(1H) quinazolinone

TP-4-10 4-tert-butyl quinoline 7.51Dodecene,2,4-bis(1,1-dimethylethyl)-phenol

a The fractions which were absent in controls.

2.6. Bioassay for methane generation from released organics

The potential of fungal-released organics to serve as precursors inthe methanogenic pathway was assessed using a microbial consor-tium WBC-2 as a bioassay [21]. WBC-2 is composed of a mixture ofbacteria and methanogens. After fungal treatment, the filteredsupernatants containing released organics from respective coalsamples were purged with N2/CO2 (80:20) in serum bottles and10% (v/v) WBC-2 culture was added. Bottles were sealed with aTeflon coated stopper (West Co., Lionville, PA) and aluminum crimp.Incubation time was extended for 35 days.

2.7. Methane analysis

Methane was analyzed as described by Jones et al. [21]. Serumbottles were monitored for methane by removing 0.3 ml of theheadspace using a gas-tight syringe with a pressure lock and ana-lyzed using a Hewlett–Packard 5890A (split-less) Gas Chromato-graph with a VOCOL capillary column at 100 �C isothermal.

3. Results and discussion

3.1. Petrographic studies of coal samples

Petrographic studies indicated that most of the samples hadhigh huminite content. Mean random huminite reflectance in oil(Rm) ranges from 0.22 to 0.51% (by convention, the maceral groupis referred to as huminite if reflectance is less than 0.5; otherwise itis categorized as vitrinite). Our samples have greater than 80 vol.%content of huminite/vitrinite (Table 2).

Vitrinite macerals originate from cell wall materials (woody tis-sues) of plants, which are chemically composed of polymeric cellu-lose and lignin (lignocelluloses). Sample LS-4-2B has a highinertinite content (39.2 vol.%), which is normally derived fromplant material that is highly altered during peat diagenesis. Sam-ples TP-1-5.2, TP-3-2K1 and TP-3-2X had relatively high liptinitecontent 22.8, 22.6 and 33.9 vol.%, respectively, which appears tohave greatly impeded the release of organics by our fungal treat-ment (Table 3), possibly because liptinite macerals are made upof waxy and resinous parts of the plants such as spores, cuticlesand resins and which are resistant to weathering and diageneticprocesses. For all of our samples from Thar, Rm is less than 0.4%,which indicates an early stage of thermal alteration. This stage ofcoal is susceptible to biological modification and the conspicuous

R. Haider et al. / Fuel 104 (2013) 717–725 721

woody texture and structures of the Thar coal further indicate thepresence of organic fractions that may be suitable for fungaldegradation.

3.2. Isolation of MW1

MW1 was isolated from a core sample of sub-bituminous coalwhich originated from a well drilled by USGS in the Tongue Riverportion of the Powder River Basin, Montana, USA. The moleculartyping of the fungal strain was carried out on the basis of PCRamplification of ITS regions. Sequences of amplicons were searchedfor similarities through NCBI BLASTn and MW1 appeared to havehomology (Maximum Identity 97%) with Penicillium chrysogenumQML-2 strain.

The Penicillium species have already been reported to degradecoal [9,22,23]. A number of fungal strains from different habitatshave been reported to consume coal as a substrate, but it seemslikely that fungal strains that have been isolated from and areadapted to a coal environment would have greater capacity todegrade coal. Coal degradation is not reliant only on finding anappropriate biological agent. It is also a function of the chemicalstructure and rank of the coal. It is hypothesized that low rank coalsare more biodegradable due to a low aromaticity (less condensedaromatic structure), the presence of lignin-derived molecules andhigh oxygen content (relative to high rank coals).

After 7 days of incubation, coal particles were trapped in fungalmycelia (Fig. 2) which can be attributed to the fact that fungalmycelia surrounded coal particles. In nature, this mycelial exten-sion may function to break up the coal matrix and this also sug-gests that fungal spores were able to survive in this coal seamover a long period of time.

3.3. Coal degradation by MW1 and analyses of organic extracts

After 7 days of incubation, the fungal treatment supernatantswere filtered using glass fiber filters and analyzed for releasedorganics. The results of EEMS analysis are shown in Figs. 3a and3b). The EEMS analyses provided a qualitative estimate of thenature of released organics through fungal treatment. The majorpeak at 350 nm, which was observed in all controls and degradedextracts, might be related to the release of humic materials. How-ever, distinct peaks, in the range of 250 nm and 300 nm, were sig-nificant for samples LS-4-1, LS-4-2B, TP-1-1.1, TP-1-5.2, TP-3-2B,TP-3-2K1, TP-3-2X, TP-4-2A, TP-4-10. For samples UAS-4-2E,UAK-1-4 and UAJ-1-1, no significant peaks were observed in thisrange. The peaks between 250 nm and 300 nm suggest the releaseof some organic compounds which were identified through GC–MS. The DCM extracts, analyzed by GC–MS, consisted of a variety

Coal Particles

Fungal Mycelia

Fig. 2. Coal particles trapped within fungal mycelia (phase contrast microscope100�).

of organic compounds including aliphatics, PAHs and primarily sin-gle ring aromatics and aromatic nitrogen compounds (Table 3).

Examining the particular organic compounds released, it is evi-dent that the fungal isolate MW1 was able to release a mix ofaliphatics, single ring aromatics and PAHs from the coal matrices(Table 3). Several studies have reported the presence of PAHs andtheir functional derivatives, benzene derivatives, phenols and aro-matic amines in coal formation waters from various coal basins[24–26]. In previous studies, Jones [3] proposed a model for thegeneration of biogenic methane from coal which follows exoenzy-matic hydrolysis of coal to yield long chain alkanes, long chainfatty acids and single ring aromatics. In the current study, fungireleased primarily single ring aromatics and amides. Two samplesfrom Thar (TP-3-2K1 and TP-4-10) released the unsaturated alkanedodecene. One sample (TP-1-1.1), released mid chain fatty acids inthe fungal treatment which could lead to the generation of meth-ane under the proposed model. However, no long chain fatty acidswere observed. Based on these analyses, most of the organicsreleased during coal degradation by fungi varied somewhat fromthe model of biogenic methane generation [3], which was basedon experiments with bacteria. It is not clear whether the com-pounds released by fungi could be degraded into methanogenicsubstrates.

Another important factor, gravimetric analyses for the determi-nation of solubilization extent, can be pivotal factor for subsequenttransformation of solubilized coal into methane. As a matter offact, we were not able to determine the accurate weight loss aftersolubilization because the fungal mycelia covered the whole ligniteparticles and it was, virtually impossible to sequester coal from thebiomass. In number of previous reports, extent of solubilizationhas been reported to approximately 35% using virgin coal [28]and 100% using chemically pretreated coal [6]. However, the heter-ogeneous nature of coal presents an obstacle for predicting anyestimates of solubilized coal for any subsequent application.

3.4. Methanogenesis of released organics

The potential of the organics in the fungal extract to supportmethanogenesis was evaluated using a WBC-2 bioassay. Methanewas generated from the organics released in the fungal treatmentof coal (Figs. 4a and 4b and Table 3). Most of the fungal extractstested generated between 1 and 4 lmoles methane per g of coal,but four samples of Thar lignites generated between 7 and11 lmoles methane per g of coal (Fig. 4b). These quantities ofmethane are similar to low and medium producing sub-bitumi-nous coals tested in previous WBC-2 bioassays [21]. Uponapproaching the end of incubation time of 35 days, methane gener-ated in most of the samples either started to decline or remainedalmost unchanged.

Comparing the amount of methane generated with the types oforganics identified in the fungal treatments, it appears that meth-ane generation may not be limited to the organic compounds iden-tified previously. The four fungal extracts that produced thehighest methane by bioassay included many nitrogenous com-pounds, in addition to unsaturated alkanes and phenols. Howeverthe methane could have been generated from compounds (suchas volatile fatty acids) not detectable by the methods used here.The coal structure is very complex and therefore can yield a broadrange of degraded products. In this study, fungi released primarilyaromatic compounds which are more recalcitrant than aliphaticslike fatty acids. In general, biogenic methane generation from coalsdominated by condensed aromatic clusters is slower as comparedto the coals with less aromatic and open clusters. Possibly a longerincubation of coal with fungi could lead to further degradation ofcoal into some other chemical entities which could have potentialto be transformed into methane.

UAS-4-2E UAK-1-4

UAJ-1-1 LS-4-1

LS-4-2B TP-1-1.1

Fig. 3a. Excitation-Emission Matrix Spectra (EEMS) for released organics (samples UAS-4-2E, UAK-1-4, UAJ-1-1, LS-4-1, LS-4-2B, TP-1-1.1).

722 R. Haider et al. / Fuel 104 (2013) 717–725

In order to determine the full potential of fungal pretreatmentof lignite for generation of methane, the process needs to be opti-

mized. The optimum length of time for fungal extraction has yet tobe determined. Additional organic analyses will be needed to

TP-1-5.2 TP-3-2B

TP-3-2K1 TP-3-2X

TP-4-2ATP-4-10

Fig. 3b. Excitation-Emission Matrix Spectra (EEMS) for released organics (samples TP-1-5.2, TP-3-2B, TP-3-2K1, TP-3-2X, TP-4-2A, TP-4-10).

R. Haider et al. / Fuel 104 (2013) 717–725 723

determine the full suite of organics released by fungal treatment.Additionally, testing of individual compounds from the extract

using a bioassay will help to identify novel intermediates betweencoal and methane. Further work on the applicability of fungal

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25 30 35 40

Control (FW+WBC2)

UAS-4-2E

UAK-1-4

UAJ-1-1

LS-4-1

LS-4-2B

Days

Met

hane

( m

oles

/g o

f Coa

l)µ

µ

Fig. 4a. Methane generation from released organics (samples UAS-4-2E, UAJ-1-1,UAK-1-4, LS-4-1, LS-4-2B).

724 R. Haider et al. / Fuel 104 (2013) 717–725

treatment to coal methanogenesis is warranted to examine differ-ent coals, the effect of fungi under anaerobic conditions, and differ-ent fungal species as well. It is interesting to note that the fungusreported on here was recovered from the center of an anaerobiccoal core, and that there was some evidence for anaerobic growth(data not shown).

Employing bioprocesses for the conversion of coal into methanein terms of technological advancements may demand the develop-ment of two stage process units including bioreactors for fungalpretreatment and then subsequent methanogenesis. In slurry bio-reactor, 28% biosolubilization has been reported using coal particlesize of 150–300 lm [29]. Similarly, perfusion fixed-bed bioreactorhas also been employed for coal degradation through fungi [30].However, detailed study pertaining to engineering principles for

0

2

4

6

8

10

12

0 5 10 15 20 25 30 35 40

TP-1-1.1

TP-1-5.2

TP-3-2B

TP-3-2X

TP-3-2K1

TP-4-2A

TP-4-10

Control (FW+WBC2)

Days

Met

hane

( m

oles

/g o

f Coa

l)µ

µ

Fig. 4b. Methane generation from released organics (samples TP-1-1.1, TP-1-5.2,TP-3-2B, TP-3-2X, TP-3-2K1, TP-4-2A, TP-4-10).

the optimization of fungal mediated solubilization of coal is stillrequired while considering the parameters of coal particle sizeand the ultimate objective of this treatment, whether the solubi-lized fraction must be used for further processing like methano-genesis or for the extraction of humic materials from treatedcoal. These coal derived end products may find some applicationsin diversified areas like agents for soil conditioning, alternative fueloptions, chemical feed stocks etc.

4. Conclusions

This study shows that fungal pretreatment has a potential appli-cation for coal solubilization in the process of coal methanogenesis.Bioassay studies indicated that some organics released by fungaltreatment could be converted to methane by a mixture of bacteriaand methanogens under anaerobic conditions. The greatest meth-ane generation was from fungal extracts of Thar lignites, whichreleased a number of nitrogenous, cyclic and aromatic compounds.Although the release of PAHs has been proposed in initial defrag-mentation of coal during biogenic methane generation [4], micro-bial degradation of PAHs in the absence of a terminal electronacceptor has not been demonstrated to date. Fungal treatmentcould be useful if followed by treatment with PAHs degrading aer-obic bacteria and anaerobic steps leading to methanogenesis. Fun-gal pretreatment might be impractical in situ and would most likelyrequire application of a split process system. It is generally assumedthat fungi require oxygen to degrade coal, whereas methanogenesisis strictly anaerobic. Furthermore, because Thar is largely coveredwith dune sand, methane produced in place could not be effectivelytrapped prior to methane recovery.

Fungal degradation of coal can find an alternative application interms of extracting some other chemical entities like humic acids.The high huminite content in Thar coals may also make them apotential source of materials for other useful commodities, suchas soil-conditioning agents, though an extensive study for specificstructural functionalities would be required for such bioactiveagents. Being an agricultural country, the backbone of Pakistan’seconomy is agriculture and this sector can be boosted with indig-enously manufactured humic acid, which is not the case right now.

The fungal isolate MW1 was isolated from a sub-bituminouscoal sample (from a well drilled by USGS in the Tongue River por-tion of the Powder River Basin, Montana, USA) and it was notadapted to the lignite samples which were used in this study. Sothere is a need to study the microbial ecology of the coal seamsand the environment, especially around the Thar seams, whichmay help in finding an indigenous organism suitable for structuralmodification and degradation of coal both as a source for fuel andfor other applications such as the production of humic acids.

Disclaimer

The use of trade names in this report is for the sole purpose ofidentification of methods employed; no endorsement of any prod-uct by the US Geological Survey is implied.

Acknowledgments

The authors wish to thank the GSP staff who drilled and sampledmost of the boreholes used for this study and the United StatesAgency for International Development who funded the drillingand the proximate-ultimate analyses. Coal from the Tongue Riverportion of the Powder River Basin (Montana), and the lab facilitiesand instrumentation used in this study, were provided by the USGeological Survey Energy Resources Program and National ResearchProgram. Vitrinite reflectance and maceral analyses for these

R. Haider et al. / Fuel 104 (2013) 717–725 725

samples were provided by James C. Hower and Cody D. Patrick of theUniversity of Kentucky, Center for Applied Research. Stephen E.Suitt of the USGS provided assistance with Fig. 1. This work wasmade possible by the financial support provided by the Higher Edu-cation Commission (HEC), Pakistan.

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