prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/2264/1/2507S.pdf · CERTIFICATE This thesis, submitted by MR. RIZWAN HAIDER (Registration No. 2141-NIBGE/Ph.D-2007) is accepted

BIOSOLUBILIZATION AND BIOGASIFICATION

OF INDIGENOUS LOW RANK COAL

FOR ITS APPLICATIONS

A Dissertation

Submitted to

Quaid-i-Azam University, Islamabad In Partial Fulfillment of the Requirements

For the Degree of

Doctor of Philosophy

In

BIOTECHNOLOGY

By

RIZWAN HAIDER

NATIONAL INSTITUTE FOR BIOTECHNOLOGY

AND GENETIC ENGINEERING (NIBGE), FAISALABAD

&

QUAID-I-AZAM UNIVERSITY, ISLAMABAD, PAKISTAN

2014

CERTIFICATE

This thesis, submitted by MR. RIZWAN HAIDER (Registration No. 2141-NIBGE/Ph.D-

2007) is accepted in its present form by the National Institute for Biotechnology and

Genetic Engineering (NIBGE), School of Biotechnology, Quaid-i-Azam University,

Islamabad, Pakistan, as satisfying the thesis requirement for the degree of Doctor of

Philosophy in Biotechnology.

Supervisor ___________________________

(Dr. M. Afzal Ghauri)

External Examiner-I ___________________________

(Prof. Dr. Ikram-ul-Haq, S.I)

External Examiner-II ___________________________

(Prof. Dr. Haq Nawaz Bhatti)

Chairman ___________________________

(Dr. Shahid Mansoor, S.I)

Dated ___________________________

DECLARATION

I hereby declare that the work presented in the following thesis is my own

effort, except where otherwise mentioned/acknowledged, and that the

thesis is my own composition. No part of this thesis has been previously

presented for any other degree.

RIZWAN HAIDER 2014

“(O Mankind) We established you on the earth & placed resources in it for your life”

Al-Quran (Sura-tul-A’raf: 10)

To

Unraveling the Enduring Mysteries of Earth;

Endowed with Huge Natural Resources,

ACKNOWLEDGEMENTS

Through the moments of great pleasure and satisfaction, I realize that I would not have been able to do anything without the blessings of ALLAH. My faith and belief remains incomplete without the respect for Holy Prophet Muhammad (PBUH), the great universal messenger and Saviour of Humanity, who conveyed the message of truth and showed us the right way to live. My Parents, who always considered the parenthood as an adventure, are my source of inspiration. Their endless trust and love was a full package, which kept me moving and moving even in the hard times. The support of “Gulshan-e-Alvi” during the whole course of time is worth-mentioning. Almost six years ago, the phone call of Dr. M. Afzal Ghauri, my PhD supervisor, happened to be a crossroad in my academic and research career. Right from that day, his ever-increasing support and belief in me is my strength and vigor. The way he has been trimming my personality in every aspect, including my research capabilities and sophistication, is one of the most precious assets of my life. I am entitled to pay special thanks to Dr. Shahid Mansoor, S.I. (Director, NIBGE) for his never-ending encouragement, support and trust in me. I am sure he would be successful in upholding the distinction of NIBGE in R & D activities. I would not be able to forget my days at United States Geological Survey, (USGS), USA, and I am thankful to Dr. William H. Orem for making my stay convenient and amazing at USGS laboratories. His help regarding analytical facilities was fabulous. I feel proud of working with John SanFilipo, the true discoverer of Thar coal field, I believe. The work related to anaerobic processing of coal was not possible without the help of the beautiful lady, Elizabeth Jones. The motherly nature of Kathleen Spiegelberg, my landlady, made me feel like being home at 1160 Lisa Street, Herndon, USA. I also feel pleasure to mention the name of ex-director, Dr. Zafar M. Khalid, for his patronage at the times, when I joined this programme. Dr. Kalsoom Akhtar has been great help regarding experimentation with fungi, for which I am highly obliged to her. Her guidance has been paving the way for the achievement of my research objectives. Though, being biological, I was not a man of biological sciences and in this regard, it was really a privilege to learn from Dr. Nasrin Akhtar, who is responsible for my basic skills in microbiology and molecular biology work. The scientific discussions with her have always been fruitful and informative. I am also thankful to Dr. Munir A. Anwar, Ms. Shazia Khaliq, Farooq Aslam, Ijaz Bano, and Niaz Muhammad for a wonderful and pleasing time in the form of their company. The technical support and efforts of all research assistants, particularly, Faqir Muhammad, Syed Habib-ur-Rehman, Amer Zahid, and Muhammad Ejaz, are highly appreciated, as doing the whole lot of work without their help had not been so easy. One of the comforting things, during my stay at NIBGE, was a wide variety of my friends with whom I shared one of the best days of my life. These guys understood and accepted me, for which I am grateful to Rizwan Subhanii, Amir Raza, Tahir Naqqash, Muther Mansoor Qaisrani, Arshad Murtaza, Amir Iqbal, Atif Iqbal, Ahmad Zaheer, Nouman Tahir, Muhammad Ikram Anwar, Asif Habeeb, Abdul Jabbar, Mehboob Islam and many more. The financial support of Higher Education Commission (HEC), Pakistan, was really very appreciable for making smooth trailing of my research objectives possible here in NIBGE and getting me into the laboratories of USGS, USA. Finally, in completing and finalizing this thesis, the toleration of Ayesha for my continuous working/reading again and again on my laptop, particularly in the evenings and nights, is admirable.

Haider

LIST OF ABBREVIATIONS % Percentage

L Microliters

m Micrometer

moles Micromoles

13C-NMR Carbon-13 Nuclear Magnetic Resonance

1H-NMR Hydrogen-1/Proton Nuclear Magnetic Resonance

2D Two Dimensional

3D Three Dimensional

AS Alkali Solubilized

ASTM American Society for Testing & Materials

BP British Petroleum

BTU British thermal unit

CBM Coalbed Methane

CEOM Coal Extractable Organic Matter

d,mmf Dry Mineral Matter Free

DCM Dichloromethane

DNA Deoxyribonucleic Acid

DOC Dissolved Organic Carbon

EDTA Ethylenediaminetetraacetic Acid

EEMS Excitation-Emission Matrix Spectroscopy

EI Electron Ionization

FID Flame Ionization Detector

FPAS Fungal Pretreated Alkali Solubilized

FTIR Fourier-Transform Infrared Spectroscopy

GC Gas Chromatography

GC-MS Gas Chromatography-Mass Spectrometry

GSP Geological Survey of Pakistan

HRP Horseradish Peroxidase

IDGCC Integrated Drying Gasification Combined Cycle

IGCC Integrated gasification Combined Cycle

ITS Internal Transcribed Spacer

LiP Lignin Peroxidase

M Molar

m,mmf Moist, Mineral Matter Free

MEA Malt Extract Agar

mg Milligram

mL Milliliters

mm Millimeter

mM Millimolar

mmf Mineral Matter Free

MnP Manganese Peroxidase

MQW MilliQ Water

MSTFA N-Methyl-N-(Trimethylsilyl)-Trifluoracetamide

Mt Million Tonnes

MW1 Montana White-1

nm Nanometers

NPOC Non-Purgeable Organic Carbon

NSO Nitrogen-Sulphur-Oxygen

NTA Nitrilotriacetic

PAH Polyaromatic Hydrocarbon

PCR Polymerase Chain Reaction

PES Polyethersulfone

ppm Parts Per Million

RNA Ribonucleic Acid

RPM Revolutions per Minute

SBP Soybean Hull Peroxidase

SCF Standard Cubic Foot

SEM Scanning Electron Microscope

USGS United States Geological Survey

UV-Vis Ultraviolet-Visible

WBC-2 West Branch Canal-2

TABLE OF CONTENTS I

TABLE OF CONTENTS

LIST OF FIGURES V

LIST OF TABLES VIII

ABSTRACT IX

1 Introduction & Review of Literature 1

1.1 LOW RANK COAL 3

1.2 UTILIZATION OF LOW RANK COAL 4

1.3 STRUCTURE OF LOW RANK COAL 5

1.4 MACERALS IN COAL 8

1.5 COAL CONVERSION TECHNOLOGIES 9

1.6 BIOLOGICAL TRANSFORMATION OF LOW RANK COAL 10

1.6.1 Aerobic Degradation of Coal 11

1.6.1.1 Microorganisms Involved in Coal Solubilization and Depolymerization 12

1.6.1.2 Mechanisms of Coal Bioconversion 14

1.6.1.2.1 Alkaline Substances 14

1.6.1.2.2 Chelators 15

1.6.1.2.3 Hydrolases 15

1.6.1.2.4 Oxidative Enzymes 16

1.6.2 Anaerobic Degradation of Coal 18

1.7 COALBED METHANE (CBM) 19

1.7.1 Gas Quality 19

1.7.2 Thermogenic CBM 20

1.7.3 Biogenic CBM 21

1.7.4 Intermediates Involved in Pathways for Biogenic CBM Generation 21

1.7.4.1 Aromatics 22

1.7.4.2 Aliphatics 23

1.7.4.3 Heteroatoms 23

1.8 MICROBIAL COMMUNITIES RESPONSIBLE FOR DEEP SURFACE COAL

METHANOGENESIS

24

1.9 STIMULATION OF BIOLOGICAL METHANE GENERATION FROM COAL 27

TABLE OF CONTENTS II

1.10 SCOPE OF BIOLOGICAL BENEFICIATION OF LOW RANK COALS IN PAKISTAN 28

1.11 OBJECTIVES OF THE STUDY 30

2 Materials & Methods 32

2.1 ORIGIN OF COAL SAMPLES 32

2.2 PREPARATION OF REPRESENTATIVE COAL SAMPLES 36

2.3 ULTIMATE ANALYSIS OF COAL SAMPLES 36

2.4 PROXIMATE ANALYSIS OF COAL SAMPLES 36

2.4.1 Moisture Content (ASTM D-3302) 36

2.4.2 Volatile Matter Content (ASTM D-3175) 37

2.4.3 Ash Determination (ASTM D-3174) 37

2.4.4 Fixed Carbon (ASTM D-5142) 37

2.5 MACERAL ANALYSIS OF COAL SAMPLES 37

2.5.1 Sample Preparation and Analysis 37

2.5.2 Random Reflectance Analysis 38

2.6 COAL CLASSIFICATION 38

2.7 FUNGAL ISOLATION 40

2.8 COMPOSITION OF MALT EXTRACT AGAR (MEA) MEDIUM 41

2.8.1 Purification and Storage of Fungal Isolates 41

2.9 SCREENING OF FUNGAL ISOLATES FOR COAL DEGRADATION 41

2.10 TAXONOMIC EVALUATION OF FUNGAL ISOLATES 42

2.10.1 Morphological Identification 42

2.10.2 Molecular Identification 42

2.10.2.1 Fungal ITS Sequence PCR Amplification 43

2.10.2.1.1 Preparation of 1% Agarose Gel Electrophoresis 44

2.10.2.1.2 PCR Product Purification 44

2.10.2.2 DNA Sequencing of the PCR Products 45

2.10.2.2.1 Cycle Sequencing 45

2.10.2.2.2 Ethanol/EDTA/Sodium Acetate Precipitation 46

2.10.2.2.3 DNA Sequence Analysis 46

2.10.2.3 Phylogenetic Analysis of Fungal Strains 46

TABLE OF CONTENTS III

2.11 COAL DEGRADATION EXPERIMENTS 48

2.11.1 Filtration of Supernatants 48

2.12 ANALYTICAL INVESTIGATIONS 48

2.12.1 Spectrophotometric Studies 48

2.12.2 Non-Purgeable Organic Carbon (NPOC) Determination 49

2.12.3 pH Determination 49

2.12.4 Gas Chromatography-Mass Spectrometry (GCMS) 49

2.13 WBC-2 Based Bioassay 50

2.13.1 Growth of Anaerobic Microorganisms 51

2.13.1.1 Amorphous Ferric Oxyhydroxide (FeOOH) 51

2.13.1.2 Ferric Citrate Medium 51

2.13.1.3 Fe (III) Nitrilotriacetic (NTA) Stock 51

2.13.1.4 Fresh Water Medium 52

2.13.1.4.1 Composition of Trace Minerals and Vitamin Solution 52

2.13.2 Bioassay Experimental Design for Methane Generation from Released Organics 53

2.13.3 Bioassay Experimental Design for Native Coal Samples 54

2.13.4 Methane Determination 55

2.13.5 Spectrophotometric Investigations of Residual Supernatants in Serum Bottles 55

2.14 HUMIC ACID EXTRACTION FROM LOW RANK COAL 55

2.14.1 Extraction of Humic Acids from Coal by Alkali Solubilization 56

2.14.2 Extraction of Humic Acids from Fungal-Transformed Lignite 57

2.15 ANALYSIS OF HUMIC MATERIALS 58

2.15.1 Elemental Analysis 58

2.15.2 Spectrophotometric Analysis 58

2.15.3 Fourier-Transform Infrared Spectroscopy (FTIR) 58

3 Results & Discussion 59

3.1 GEOLOGY OF THE COAL SAMPLES 59

3.2 CHEMICAL CHARACTERIZATION OF THE COAL SAMPLES 62

3.3 MACERAL ANALYSIS OF THE COAL SAMPLES 69

3.4 FUNGAL ISOLATES 74

TABLE OF CONTENTS IV

3.4.1 Sample Collection and Origin of Fungal Isolates 74

3.4.2 Primary Screening of Isolates for Coal Degrading Activity 75

3.4.3 Optimization of Coal Degrading Activity 77

3.4.3.1 Effect of Glucose 79

3.4.3.2 Effect of Incubation Time 81

3.4.3.3 Effect of Coal Loading Ratio 82

3.4.4 Characterization of MW1 84

3.4.5 Phylogenetic Analysis of MW1 85

3.5 FUNGAL PRETREATMENT OF COAL SAMPLES 88

3.6 ANALYTICAL INVESTIGATIONS OF EXTRACTS 89

3.6.1 Excitation-Emission Matrix Spectroscopy (EEMS) 89

3.6.2 Non-Purgeable Organic Carbon Determination 98

3.6.3 pH of the Extracts 99

3.6.4 Gas Chromatography-Mass Spectrometry (GC-MS) of Degraded Extracts 101

3.7 METHANE GENERATION FROM RELEASED ORGANICS 130

3.8 METHANE GENERATION FROM NATIVE COAL SAMPLES 136

3.9 ANALYSIS OF RESIDUAL FILTRATES OF BIOASSAY EXPERIMENTS 144

3.10 EXTRACTION OF HUMIC ACID FROM LOW RANK COAL 147

3.10.1 Chemical Characterization 147

3.10.2 Fourier-Transform Infrared Spectroscopy (FTIR) 149

4 General Discussion & Conclusions 153

5 References 162

6 Appendices 188

LIST OF FIGURES | V

LIST OF FIGURES

Figure 1.1 | Primary Energy Consumption of the World and Pakistan; A Comparison (BP Statistical

Review, 2013)

2

Figure 1.2 | World Coal Reserves in Billion Tonnes (Catelin, 2010) 3

Figure 1.3 | Usage of Low Rank Coal around the Globe 4

Figure 1.4 | Molecular Representation of Brown Coal adapted from Wender (1976)-(With **, as

Connectivity Points)

7

Figure 1.5 | Lignite Structure Model by Tromp and Moulijn (1987) 7

Figure 1.6 | Periodic Unit in the Structure of Yallourn Brown Coal by Kumagai et al. (1999) 7

Figure 1.7 | Coal Reserves of Pakistan 29

Figure 1.8 | Schematic Illustration of Research Outline 31

Figure 2.1 | Geographical Setting and Borehole Location for the Samples used in the Study 33

Figure 2.2 | Experimental Design for WBC-2 Based Bioassay of Released Organics 54

Figure 2.3 | Experimental Design for WBC-2 Based Bioassay of Native Coal Samples 54

Figure 2.4 | Approaches for Humic Acid Extraction from Low Rank Coal 56

Figure 3.1 | Photomicrographs of Thar Coal Sample (TP-3-2X) 73

Figure 3.2 | Growth of MW1 in the Presence of Coal 76

Figure 3.3 | UV-Vis Scan Pattern of Released Organics from Coal by MW1 Isolate 76

Figure 3.4 | UV-Vis Scan Pattern for Controls 78

Figure 3.5 | Effect of Glucose Concentration on Release of Organics 80

Figure 3.6 | Effect of Incubation Time on Release of Organics 81

Figure 3.7 | Effect of Coal Loading Ratio on Release of Organics 83

Figure 3.8 | Morphological Features of MW1 84

Figure 3.9 | PCR Amplifications of ITS Regions 85

Figure 3.10 | Inferred Relationship Based on Partial ITS Sequences of MW1 to other Fungal Isolates 87

Figure 3.11 | Excitation-Emission Matrix Spectra for MW1 Culture (Control) 90

Figure 3.12 | Excitation-Emission Matrix Spectra for TP-1-1.1 91

Figure 3.13 | Excitation-Emission Matrix Spectra for TP-1-5.2 91

Figure 3.14 | Excitation-Emission Matrix Spectra for TP-3-2B 92

Figure 3.15 | Excitation-Emission Matrix Spectra for TP-3-2X 92

Figure 3.16 | Excitation-Emission Matrix Spectra for TP-3-2K1 93

Figure 3.17 | Excitation-Emission Matrix Spectra for TP-4-2A 93

Figure 3.18 | Excitation-Emission Matrix Spectra for TP-4-10 94

Figure 3.19 | Excitation-Emission Matrix Spectra for LS-4-1 94

LIST OF FIGURES | VI

Figure 3.20 | Excitation-Emission Matrix Spectra for LS-4-2B 95

Figure 3.21 | Excitation-Emission Matrix Spectra for UAS-4-2E 96

Figure 3.22 | Excitation-Emission Matrix Spectra for UAJ-1-1 96

Figure 3.23 | Excitation-Emission Matrix Spectra for UAK-1-4 97

Figure 3.24 | GC-MS Scan Pattern for Culture Growth of MW1 (Control) 102

Figure 3.25 (A) | GC-MS Scan Pattern of Organics from UAS-4-2E after Fungal Pretreatment 103

Figure 3.25 (B) | GC-MS Scan Pattern of Organics from UAS-4-2E without Fungal Pretreatment 104

Figure 3.26 (A) | GC-MS Scan Pattern of Organics from UAK-1-4 after Fungal Pretreatment 105

Figure 3.26 (B) | GC-MS Scan Pattern of Organics from UAK-1-4 without Fungal Pretreatment 106

Figure 3.27 (A) | GC-MS Scan Pattern of Organics from UAJ-1-1 after Fungal Pretreatment 107

Figure 3.27 (B) | GC-MS Scan Pattern of Organics from UAJ-1-1 without Fungal Pretreatment 108

Figure 3.28 (A) | GC-MS Scan Pattern of Organics from LS-4-1 after Fungal Pretreatment 109

Figure 3.28 (B) | GC-MS Scan Pattern of Organics from LS-4-1 without Fungal Pretreatment 110

Figure 3.29 (A) | GC-MS Scan Pattern of Organics from LS-4-2B after Fungal Pretreatment 111

Figure 3.29 (B) | GC-MS Scan Pattern of Organics from LS-4-2B without Fungal Pretreatment 112

Figure 3.30 (A) | GC-MS Scan Pattern of Organics from TP-1-1.1 after Fungal Pretreatment 114

Figure 3.30 (B) | GC-MS Scan Pattern of Organics from TP-1-1.1 without Fungal Pretreatment 115

Figure 3.31 (A) | GC-MS Scan Pattern of Organics from TP-1-5.2 after Fungal Pretreatment 116

Figure 3.31 (B) | GC-MS Scan Pattern of Organics from TP-1-5.2 without Fungal Pretreatment 117

Figure 3.32 (A) | GC-MS Scan Pattern of Organics from TP-3-2B after Fungal Pretreatment 118

Figure 3.32 (B) | GC-MS Scan Pattern of Organics from TP-3-2B without Fungal Pretreatment 119

Figure 3.33 (A) | GC-MS Scan Pattern of Organics from TP-3-2X after Fungal Pretreatment 120

Figure 3.33 (B) | GC-MS Scan Pattern of Organics from TP-3-2X without Fungal Pretreatment 121

Figure 3.34 (A) | GC-MS Scan Pattern of Organics from TP-4-2A after Fungal Pretreatment 122

Figure 3.34 (B) | GC-MS Scan Pattern of Organics from TP-4-2A without Fungal Pretreatment 123

Figure 3.35 (A) | GC-MS Scan Pattern of Organics from TP-3-2K1 after Fungal Pretreatment 125

Figure 3.35 (B) | GC-MS Scan Pattern of Organics from TP-3-2K1 without Fungal Pretreatment 126

Figure 3.36 (A) | GC-MS Scan Pattern of Organics from TP-4-10 after Fungal Pretreatment 127

Figure 3.36 (B) | GC-MS Scan Pattern of Organics from TP-4-10 without Fungal Pretreatment 128

Figure 3.37 | Methane Generation from Organic Fractions of TP3 Borehole Samples 130



Figure 3.40 | Methane Generation from Organic Fractions of UAS-4-2E, UAK-1-4 and UAJ-1-1

Samples

133

Figure 3.41 | Methane Generation from Organic Fractions of Lakhra Coal Samples 134

LIST OF FIGURES | VII

Figure 3.42 | Methane Generation from Coal Samples of TP1 Borehole 136



Figure 3.45 | Methane Generation from Lakhra Coal Samples 139

Figure 3.46 | Methane Generation from Sonda Coal Samples 140

Figure 3.47 | Methane Generation from Samples of Khost, Salt Range, Makarwal, Indus East and

Metting-Jhimpir Coal Fields

141

Figure 3.48 | UV-Vis Scan Pattern from Residual Filtrates of TP1 Borehole Samples 146

Figure 3.49 | UV-Vis Scan Pattern from Residual Filtrates of TP3 Borehole Samples 146

Figure 3.50 | FTIR Spectra of Coal Sample (TP-31) 150

Figure 3.51 | FTIR Spectra of Alkali Solubilized Humic Acid 151

Figure 3.52 | FTIR Spectra of Fungal-Pretreated Alkali Solubilized Humic Acid 152

Figure 4.1 | Laboratory Scale Model for Biotransformation of Coal into Methane 158

Figure 4.2 | Correlation of Physical Parameters of Coal; With Reference to CBM Origin 160

LIST OF TABLES | VIII

LIST OF TABLES

Table 1.1 | Top 10 Brown Coal Producers 5

Table 1.2 | Microorganisms Involved in Solubilization and Depolymerization of Coal 13

Table 1.3 | Methanogens Reported in Coal Formation Waters and Coal Mines 26

Table 2.1 | Origin of Coal Samples 34

Table 2.2 | ASTM Classification of Coal 39

Table 2.3 | Rank Classification by Vitrinite Reflectance 39

Table 2.4 | Coal Ranks with their Designations 40

Table 2.5 | Recipe for Cycle Sequencing (BigDye® Terminator v3.1 Cycle Sequencing Kit) 45

Table 2.6 | Accession Numbers for the Partial Sequences of ITS Regions of Other Fungal

Strains

47

Table 2.7 | Composition of Trace Minerals 53

Table 2.8 | Composition of Vitamin Solution 53

Table 3.1 | Geological Information of Coal Samples 61

Table 3.2 | Chemical Characterization of the Coal Samples 65

Table 3.3 | Atomic Indices of the Coal Samples 68

Table 3.4 | Petrography of the Coal Samples 71

Table 3.5 | Percentage Sequence Identity in ITS Regions of Selected Isolates with MW1 86

Table 3.6 | NPOC of Released Organics Extracts 98

Table 3.7 | pH of Released Organic Extracts 100

Table 3.8 | Released Organic Fractions from Coal Samples 129

Table 3.9 | Average Methane Generation from Coal Samples 142

Table 3.10 | E4/E6 Ratio of Residual Filtrates from Methane Generating Coal Samples 145

Table 3.11 | Elemental Analysis, Atomic Ratios and Spectroscopic Characteristics of AS-Humic

Acid and FPAS-Humic Acid

148

ABSTRACT | IX

ABSTRACT

Coal has been ensuring energy security and stability for a long period of time in terms

of increasing exploitation and excavation of high rank coals, thus, leaving behind huge

unattended low rank coal reserves. Furthermore, thermal systems were designed on the

basis of physico-chemical properties of high rank coals. However, temporarily, low rank

coals were abandoned as these had been found to be associated with high moisture

contents and low calorific values, which posed their unsuitability for application in

conventional thermal systems. The present study was aimed at investigating and

exploring the prospects of possible intervention of biotechnological approaches into

conventional fuel sciences for the extraction of alternative fuel options like methane and

value added products such as humic acids.

In this regard, fungal degradation of coal can serve as a pretreatment step in order to

make coal a suitable substrate for biological beneficiation. Thirty one (31) coal samples,

originating from eight coal areas of Pakistan and majority from Thar coal field, were

subjected to detailed chemical analyses including maceral analysis and these indicated

that geological stage of Thar coal, which might be susceptible to biological

modification. A fungal strain MW1, identified as Penicillium chrysogenum on the basis

of fungal ITS sequences, was isolated from a core sample of sub-bituminous coal and

twelve (12) low rank coal samples were treated with this isolate for further studies.

Liquid extracts were analyzed through Excitation-Emission Matrix Spectroscopy

(EEMS) to obtain qualitative estimates of solubilized coal; these analyses exhibited the

release of complex organic moieties.

In addition, analysis of these extracts, based on Gas Chromatography-Mass

Spectrometry (GC-MS), confirmed the presence of single ring aromatics, polyaromatic

hydrocarbons (PAHs), aromatic nitrogen compounds and some aliphatics.

Subsequently, the released organics were subjected to a bioassay WBC-2, based on

mixed bacterial consortium, containing methanogenic and non-methanogenic types, for

the generation of methane, which conferred the potential application of fungal

degradation as pretreatment.

ABSTRACT | X

The native coal samples were also subjected to WBC-2 culture based bioassay for

determining the methane generation. Upon incubation with WBC-2 culture under

anaerobic conditions, methane was produced from Thar coal samples in the range of 3.7

to 23.2moles per gram of coal (2.13 to 16.33scf/ton of coal). The susceptibility of

methane generation from Thar coal samples may also be considered as an indicative

index of the possible presence of biogenic coalbed methane (CBM) in Thar coal seams.

Vitrinite reflectance values for Thar coal samples, which were less than 0.4%,

corresponded to biogenic hydrocarbon generation range.

In addition, the residual coal after fungal pretreatment was processed for the extraction

of humic acids from Pakistani brown coal. Extracted humic acid were analyzed on the

basis of UV-Vis Spectroscopy for determining the E4/E6 ratio, which appeared to be in

the range of 6 to 7. Fourier Transform Infrared Spectroscopy (FTIR) showed typical

intensified absorption bands related to aliphatic stretching (2917-3335cm-1) and C=O

stretching of COOH and ketones (1607-1698cm-1).

Conclusively, on the basis of present study and some of the previous reports, a

laboratory model for biotransformation of coal into methane was proposed, which gave

an insight of the underlying mechanisms operative in this conversion. This proposed

model provided a route for comparing two types of initial biological degradation

approaches i.e., bacterial and fungal. Besides, fungal mediated degradation may be

prospected for extracting chemical entities like humic acids from brown coals with high

huminite content as shown by petrographic studies.

1

Introduction & Review of Literature

Energy security, after food and shelter, is considered to be one of the basic and

fundamental mainstays of the modern society. Today, without energy, it is impossible to

access the opportunities, which are coming up in this modern era. Besides, a stable, secure

and indigenous energy system with logical energy mix may contribute to addressing the

problems and challenges caused by the poverty.

According to BP Statistical Review (2013), the global primary energy

consumption is increasing on yearly basis and for the year of 2012, this digit increased by

1.8%, with oil as the leading energy source worldwide. Fossil fuels, the compendium of

oil, natural gas and coal, are meeting energy demands of the world to the maximum, which

happens to be 87% share in world’s energy mix. Among fossil fuels, coal relished the 2.5%

increase in consumption during last year (British Petroleum, 2013). Having depleted oil

and natural gas resources and decreasing reserve-to-production ratios of these, coal has

emerged as the fastest growing form of energy in this context.

Comparing the world’s energy mix with that of Pakistan (Figure 1.1), it is evident

that the share of coal is anomalous, contrary to the huge reserves of indigenous coal, which

have been estimated to be more than 185 billion tonnes (Ghaznavi, 2002). Besides, the

enormous and irrational dependence on natural gas (54% share in energy mix) has resulted

in the drastic shortfalls in gas supplies, thus aiding to the prevailing energy crisis in

Pakistan.

2 Introduction & Review of Literature |

Figure 1.1 | Primary Energy Consumption of the World and Pakistan; A Comparison

(BP Statistical Review, 2013)

However, a number of issues intensified the country’s ever-worsening energy crisis

and these include short term energy policies and some technical constraints in the

applications of coal in conventional thermal applications. Amongst the practical

limitations, one of the major factors is that almost 99% of our coal resources come under

the category of low rank coal, which have been associated with high moisture contents and

low heating values (Fasset and Durrani, 1994) and these have not been exploited for any

use so far. In addition to this, high sulphur content of coal has also limited the application

of indigenous resources. Above all, political and governmental regimes of the country were

unable to develop appropriate policy frameworks for the deployment of comprehensive

energy infrastructure at the time when it was direly required.

Alongside, the increased use of coal, in terms of global scenario, has forced us to

take a couple of things into the consideration, which include the use of coal with

environmental responsibilities and the role of huge reserves of low rank coals as leftover

after massive excavation of high rank coals. In this perspective, there is a need to focus on

developing the mechanisms and approaches for the efficient utilization of low rank coals,

under the present circumstances, which demand meeting environmental concerns, as well.

This would, possibly, enable us in tapping sole resources of the lignite coal in the countries

like Pakistan. In short, coal-dependent energy portfolio of the countries is contributing

significantly in economic and social uplift of the nations.

29

54

62

9

33

24

30

47 2

Oil

Natural Gas

Coal

Nuclear Energy

Hydroelectricity

Renewables

WORLD PAKISTAN


1.1 LOW RANK COAL

Generally, the rank of any coal type is determined by the degree of metamorphosis

through which the plant material undergoes the coalification route, the process, recognized

as the formation of coal. Low rank coals include lignite and sub-bituminous ranks (World

Coal Association, 2012). Brown coal is an alternative expression, which is specifically

used for lignite. According to American Society for Testing and Materials (ASTM) lignite

is defined as a class of brownish-black, low rank coal having less than 8,300 British

Thermal Unit (BTU) on a moist, mineral matter free basis (Wood et al., 1983). Currently,

around the globe, the share of lignite and sub-bituminous in total coal reserves is about

53% (Catelin, 2010). World coal reserves estimates have been indicated in Figure 1.2.

Figure 1.2 | World Coal Reserves in Billion Tonnes (Catelin, 2010)

In terms of proved recoverable reserves at the end of 2012, Pakistan has

considerable resources of low rank coal with estimated 2070 million tonnes of sub-

bituminous and lignite reserves. Majority of these are concentrated in one province of the

country, i.e. Sindh (British Petroleum, 2013).


1.2 UTILIZATION OF LOW RANK COAL

Generally, the use of low rank coal can be placed into three major categories i.e.,

electricity generation, gasification for the production of synthetic natural gas and

processing to yield fertilizer products (Figure 1.3) (Lignite Energy Council, 2012). In

conventional thermal power generation systems, high moisture brown coals are initially

pulverized and then subjected to combustion (Clark, 1984). However, advanced systems

for brown coal power generation are being employed, which take carbon dioxide (CO2)

emissions into consideration and these systems include Integrated Drying Gasification

Combined Cycle (IDGCC). Substantial efficiency improvements and cost effectiveness are

the major advantages of IDGCC over thermal power generation systems (Johnson and

Young, 1999).

Figure 1.3 | Usage of Low Rank Coal around the Globe

The major contribution of low rank coal is to fertilizer products in terms of

extraction of the humic materials, which are used as soil conditioners. Chemically extracted

liquid humates from brown coal and the direct use of brown coal as soil conditioner have

expanded the utilization of low rank coal (Lignite Energy Council, 2012).

The high reactivity of brown coals has also received much attention for its

application in gasification systems, which was demonstrated in the Morwell Lurgi

Gasification Plant from 1956 to 1970 (Bennett, 1961). Generally, brown coal power

79%

13%

8%

Electricity Generation

Gasification

Processing for Fertilizer

Products


generation, briquetting, and carbonization can be considered as broad uses of low rank

coal. In addition, high moisture content of low rank coals makes them highly porous and

this property can be tailored for the use of these coals for the production of activated carbon

and absorbents, as well.

Low rank coal can serve as chemical feedstock for the extraction of alternative fuel

options and value added products (Scott and Carpenter, 1996). Hydrogenation of brown

coal to produce oil has been experimented long time ago in 1930s (Sinnatt and

Baragwanath, 1938). Binderless briquette formation is also one of the major uses of brown

coal. Several agglomeration technologies have been explored in order to exploit the

briquetting properties of these coals.

In 2011, worldwide lignite production increased by 5.9% to 1041 million tonnes (Mt)

and Germany has been the largest brown coal producer with the production of 176Mt

(Table 1.1) (World Coal Association, 2012). However, the use of lignite in Pakistan is

negligible and has not been reported for utilization, so far. In the foreseeable future, the

major uses of low rank coal will be driven by coal conversion technologies due to

increasing rigorous environmental regulations and depleted high rank coal reserves.

Table 1.1 | Top 10 Brown Coal Producers

Country Production

(Million Tonnes) Country

Production

(Million Tonnes)

Germany 176 Mt Australia 69 Mt

China 136 Mt Poland 63 Mt

Russia 78 Mt Greece 59 Mt

Turkey 74 Mt Czech Republic 43 Mt

USA 74 Mt India 41 Mt

1.3 STRUCTURE OF LOW RANK COAL

Lots of efforts have been made for the elucidation of chemical and structural

representation of coals for about last 60 years. However, obscurity has been observed in

the reported structures except few ones. Wender (1976) published first and the simplest


structure of low rank coal (Figure 1.4). For understanding the behavior of coals during

pyrolysis, a model lignite structure (C161H185O48N2S1M4) was presented, which may also

be considered as a building block of transformed lignin. On the contrary to Wender’s

structure, this model structure indicated the aromatic clusters, linked through methoxy and

hydroxyl substitutions and reflected ester linked aliphatic side chains with heteroatoms

bridging the clusters (Tromp and Moulijn, 1987) (Figure 1.5). This model appeared as a

tool for formulating an improved and appropriate model for understanding the origin of

lignite and reaction behavior of coal, together (Tromp and Moulijn, 1987). In the mid-

1990s, three dimensional (3D) molecular modeling was also carried out for determining

the structure with the help of computational chemistry (Carlson, 1992). Kumagai et al.

(1999) described a 3D periodic unit in the structure of Yallourn brown coal, which was

C21H20O7 (Figure 1.6).

The carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy and detailed

elemental analysis have also been applied to the construction of two dimensional (2D)

structural representation of coal (Allardice et al., 2004). However, this model could be a

best fit for coals with high moisture level up to the 60% because this model takes water

molecules into the consideration. In addition, relative energy of the structure can also be

calculated with the help of this model (Allardice et al., 2004). A series of models has also

been discussed describing uniformity in the structures from lignin to sub-bituminous coal,

with the intermediates of brown coal i.e., lignite B and lignite A, on the basis of the

experimental data obtained from solid state hydrogen-1 nuclear magnetic resonance (1H-

NMR), conducted by Adler (1977). Throughout the whole process of coalification, the

chemical changes, which occurred, were also elaborated (Hatcher et al., 1989; Hatcher,

1990; Hatcher et al., 1992; Faulon et al., 1993). The interaction between inorganic cations

and molecular structure of coal has also been discussed by Domazetis et al. (2005). This

coal model (C258H256N2O78S) was based on previously reported data and structures

(Domazetis et al., 2005). Lignin based structural investigations have also been made

(Salmon et al., 2009). In this regard, angiosperm wood part from Australian Morwell

brown coal was extracted and a model was established on the basis of previously reported

structures of lignin in literature (Nimz, 2003).


Figure 1.4 | Molecular Representation of Brown Coal adapted from Wender (1976)

(With **, as Connectivity Points)

Figure 1.5 | Lignite Structure Model by Tromp and Moulijn (1987)

Figure 1.6 | Periodic Unit in the Structure of Yallourn Brown Coal by Kumagai et al. (1999)


1.4 MACERALS IN COAL

Coal macerals correspond to the contribution of plant composition and parts to the

formation of coal. These are the organic part of coal, which can be observed with the help

of microscopes. The factors, which help in distinguishing coal macerals group, are

morphology, shape, color, the extent of preservation of cell structure, reflectance level and

intensity of fluorescence (Strapoc et al., 2011).

Macerals have been classified into three groups, i.e., 1) Vitrinite 2) Liptinite and 3)

Inertinite. The woody plant material for example trunks, branches, stems and roots, which

are mainly composed of cellulose and lignin, give rise to vitrinite/huminite macerals

(Taylor et al., 1998). Vitrinite has also been divided into subdivisions on the basis of degree

of cell preservation structure. Liptinite macerals originate from spores, waxes, resins,

pollens and cuticles. This maceral group is rich in aliphatics, which can also be linked to

the greater hydrogen content as compared to other maceral groups (Taylor et al., 1998).

Besides, liptinite is more resistant to physical and chemical changes as compared to

vitrinite group. The origin of Inertinite macerals is same as that of vitrinite and liptinite in

terms of plant matter, however, inertinite group of macerals has been characterized with

greater content of condensed aromatic rings (Taylor et al., 1998; Scott and Glasspool,

2007).

There exists a range of differences in chemical properties of maceral groups

because of the fact that these groups originate from different plant matter and composition.

This difference in properties of maceral groups is also a function of coal rank. The

significant differences in carbon and oxygen contents have been associated with the rank

of coal. In case of high volatile bituminous coal, lowest carbon content has been observed

(Strapoc et al., 2011).

The abundance of functional groups also determines the elemental changes.

Fusinite, a subdivision of inertinite maceral, exhibits more aromatic character as compared

to sporinite, which is a sub-group of liptinite. On the other side, sporinite has been

associated with major peaks in aliphatic stretching region of 2,800 – 3,000cm-1 (Mastalerz

and Marc Bustin, 1993).


Due to increasing aromaticity, the chemical differences in coal macerals do not

remain distinct as the rank of coal increases. Microprobe studies have showed that vitrinite

and sporinite exhibited same properties on approaching the reflectance of 1.25% and the

carbon content of 88.5%. Similarly, semifusinite corresponded to the properties of vitrinite

and sporinite when carbon content increased to 89.5% with reflectance values ranging from

1.8 – 2.0% (Mastalerz and Marc Bustin, 1993).

1.5 COAL CONVERSION TECHNOLOGIES

The increased moisture content and low carbon content of low rank coals have

forced the technologists to consider coal conversion technologies as an efficient alternative

of conventional applications of coal. Additionally, due to greater contribution of fossil fuels

to CO2 emissions, the conventional use of coal seems to be changed in the conceivable

future. Climate protection from greenhouse gas emissions demand the use of fuel, which

are based on low carbon content.

Chemical gasification is considered to be one of the most common routes for the

extraction of a wide variety of fuels and chemicals from coal. After gasification, the

combustible gas, often known as syngas, can be utilized for power generation directly

through integrated gasification-combined cycle (IGCC) power plants (Shinada et al.,

2002). Gasification of coal offers a practical way of utilizing coal with environmental

responsibilities. The problem of high sulphur, present in coal, can also be addressed

because sulphur gets converted into hydrogen sulphide (H2S) and some minor sulphur

compounds, which can be extracted easily from the gas stream (Rao, 2005).

In principle, steam and oxygen are used as the medium of gasification and solid

fuels like coal and char can be converted into the gaseous products. After the production

of syngas, there is a need to remove excessive CO2, H2S, HCN and particulate matter from

the gas stream. After cleaning the syngas, Fischer-Tropsch synthesis is employed for

conversion of syngas into other fuels, which include methane, gasoline, diesel etc.

(Adesina, 1996). There have been lots of developments in designing of the gasifiers with

special reference to the properties of coal for the efficient transformation and gasification.

These gasifiers include fixed-bed pressure gasifiers, fluidized bed gasifiers i.e., Winkler


Process (Keller, 1990), and entrained flow gasifiers i.e., Koppers-Totzek Process (Rosen

and Scott, 1987). A wider variety of coals can be gasified, however, these conventional

chemical approaches for gasification involve high temperature and pressure applications,

which make these cost intensive. Besides, in these processes high amounts of CO2 are

generated and for that we need CO2 sequestration mechanisms to address greenhouse issues

(Maurstad, 2005).

However, in recent years, some novel approaches based on biotechnology for the

transformation of coal into alternative fuels have paved the way for the application of

biological processes to fuel sciences, especially for the beneficiation of coal (Catcheside

and Ralph, 1999; Igbinigie et al., 2010).

1.6 BIOLOGICAL TRANSFORMATION OF LOW RANK COAL

Biological transformation of coal into alternative fuel options and value-added

products has gained interest because of the cost effectiveness and mild conditions involved

in these processes (Engesser et al., 1994). High temperature and pressure conditions, which

are required in conventional gasification and conversion processes, have necessitated the

shift to mildly-operated and novel coal conversion technologies (Fakoussa and Hofrichter,

1999). Interestingly, low rank coals may be more amenable to biological attack as

compared to higher rank coals. Higher moisture contents, associated with low rank coals,

may also be a supporting factor for microbial growth. The reasons, behind the suitability

of low rank coals for biological modifications, can be summarized as follows (Ralph and

Catcheside, 1997; Laborda et al., 1997; Gokcay et al., 2001);

1. Higher oxygen content; which provides possible ‘biological doorways’ for

degradation

2. Increased solubility; improves the potential of coal as microbiological substrate

and, thus, some of the organic fractions within the coal matrix get exposed to

transformations

3. Resemblance with lignin on the basis of chemical structure; provides an opportunity

to lignin-degrading microbes to play critical role in degradation


However, hard coal or higher rank coal may not be a suitable substrate for microbes

because of condensed aromatic clusters in the structure, higher hydrophobicity and lesser

oxygen content of these coals (Strapoc et al., 2011).

Fakoussa (1981), for the first time, demonstrated the nature of coal as a substrate

for bacteria and he reported that certain bacteria were able to utilize some of the organic

extracts released from coal matrix. Cohen and Gabriele (1982), further, explored the

potential of wood-decaying basidiomycetes, which converted leonardite into black

droplets. These two reports set the pace for isolating and identifying microorganisms

(biological agents), including white-rot and brown-rot fungi, for determining the potential

of biological conversion of coal.

This biological-mediated transformation of coal can be categorized into two routes

on the basis of the microbes involved in degradation and the product obtained after

degradation.

1. Aerobic Degradation – Biosolubilization and Depolymerization of Coal

Though, biosolubilization and depolymerization of coal have been considered to

be two distinctive terms but both of these processes are mediated by micro-fungi, aerobic

bacteria and basidiomycetes. These processes yield humic acid and fulvic acid-like

products (Fritsche et al., 1999).

2. Anaerobic Bioconversion of Coal

Coal has also been subjected to anaerobic bioprocessing for the generation of

methane using anaerobic mixed cultures and the concept of biogenic coalbed methane

(CBM) generation from coal has gained sudden attention nowadays. This conversion

expedites the role of methanogens for consuming coal as carbon source (Gupta et al.,

1988; Ulrich and Bower, 2008).

1.6.1 Aerobic Degradation of Coal

Mainly, biosolubilization, leading to the formation of black droplets from coal, has

been considered as a non-enzymatic process of coal degradation, which generally occurs

at higher pH values, ranging from 7 to 9 (Hofrichter et al., 1997a; Catcheside and Ralph,

1999; Fritsche et al., 1999; Klein, 1999). Microorganisms, involved in this process,


produce alkaline substances, chelators and surfactants, which facilitate the degradation of

coal. However, there have been some reports of hydrolytic enzymes, which initiated coal

degradation (Yuan et al., 2006a). Generally, solubilization does not follow the significant

decrease in molecular mass of resulting products, which are normally humic substances.

On the other side, the depolymerization of coal is considered to be an enzymatic

process, which generally occurs at lower pH values ranging from 3 to 6. This degradation

follows the cleavage of bonds inside the coal matrix, which results in the formation of

yellowish fulvic acid like macromolecules, relatively with lower molecular masses as

compared to the products formed in biosolubilization (Fakoussa and Hofrichter, 1999).

It has been observed that the extent of aerobic degradation of coal, especially in the

case of solubilization, can be increased by prior oxidative pretreatment of coal because

naturally oxidized coals have been found to be solubilized to greater extent (Hofrichter et

al., 1997b). Coal can be treated with oxidizing agents such as nitric acid (HNO3), hydrogen

peroxide (H2O2), ozone (O3) or radiation can also be employed (Achi, 1994; Hofrichter et

al., 1997b). Aerobic degradation of coal has found applications in the extraction of humic-

acid like substances from brown coal, which can serve the purpose of soil conditioning,

thus facilitating the fertilization of the agricultural lands (Aleksandrov et al., 1988).

1.6.1.1 Microorganisms Involved in Coal Solubilization and Depolymerization

Primarily, micro-fungi (molds and yeasts) and bacteria (actinomycetes and

pseudomonads) solubilize low rank coal while wood-decaying and litter-decomposing

fungi depolymerize the coal (Table 1.2).

However, overlapping of these two degrading abilities has been observed in a

number of microorganisms. For example, among white-rot fungi, Phanerochaete

chrysosporium and Coriolus versicolor have been reported to depolymerize and solubilize

coal under certain conditions (Pyne Jr et al., 1987; Fakoussa and Frost, 1999).


Table 1.2 | Microorganisms Involved in Solubilization and Depolymerization of Coal

Known for Solubilization References Known for

Depolymerization References

Bacteria (Actinomycetes) Streptomyces spp.,

Streptomyces setonii

Gupta et al. (1988)

Strandberg and Lewis (1987)

Eubacteria Bacillus spp.

Bacillus lichiniformis,

Pseudomonas cepacia

Quigley et al. (1989)

Polman et al. (1994a)

Crawford and Gupta (1991)

Basidiomycetous fungi

(Wood-decaying White

Rot)

Nematoloma frowardii

Phanerochaete

chrysosporium

Torzilli and Isbister (1994)

Cohen and Gabriele (1982)

Clitocybula dusenni

Nematoloma frowardii

Phanerochaete

chrysosporium

Ziegenhagen and

Hofrichter (2000)

Hofrichter and Fritsche

(1996)

Fakoussa and Frost

(1999)

Litter Decomposing Fungi Agrocybe praecox

Stropharia

rugosoannlata

Hofrichter and Fritsche

(1996)

Willmann and Fakoussa

(1997)

Wood Decaying Brown

Rot

Poria monticola Cohen and Gabriele (1982)

Deuteromycetous and

Ascomycetous Fungi

Alternaria sp.

Fusarium oxysporum

Neurospora crassa

Paecilomyces spp.

Penicillium citrinum

Trichoderma atroviride

Penicillium decumbens

Hofrichter et al. (1997b)

Holker et al. (1995)

Patel et al. (1996)

Ward (1985)

Polman et al. (1994b)

(Holker et al., 1997; Holker et al.,

1999; Silva-Stenico et al., 2007)

Yuan et al. (2006a)

Yeast-like Fungi Candida bombicola Breckenridge and Polman (1994)

Zygomycetous Fungi Cunninghamella sp. Ward (1993)


1.6.1.2 Mechanisms of Coal Bioconversion

Various mechanisms have been described, which may be involved in the aerobic

bioconversion of coal. The role of alkaline substances, chelators, hydrolases and oxidative

enzymes has been investigated in this regard (Willmann and Fakoussa, 1997; Yuan et al.,

2006a).

1.6.1.2.1 Alkaline Substances

Several reports have demonstrated the role of alkaline substances in microbial

conversion of coal (Quigley et al., 1989; Torzilli and Isbister, 1994). Microorganisms can

create alkaline conditions in two ways. The production and secretion of alkaline

metabolites such as amines or ammonium ions can be used to adjust alkaline conditions.

This situation is created in case of high levels of nitrogen in the medium. In most of the

solubilization studies Sabouraud-medium has been used, which contains larger amounts of

nitrogen-based compounds like peptone and nitrate etc. Glutamate and nitrate based media

have also been used (Faison, 1991b; Faison, 1991a; Ward, 1993; Monistrol and Laborda,

1994; Holker et al., 1995).

Nitrate is converted to ammonium within the cells, which is further consumed in

the synthesis of amino acids. The pH of the medium increases because of the release of

excessive ammonium ions into the media. The effect of nitrogen source on the

solubilization may be drastic and these effects have been studied (Hofrichter et al., 1997a).

Furthermore, Hofrichter et al. (1997b) reported that out of 728 strains of filamentous fungi,

44 solubilized the coal in the presence of nitrogen-rich medium while in nitrogen reduced

medium, only 10 strains were able to degrade coal.

Microorganisms may use organic acids as substrate thus leaving behind free bases

and this also results in the increased pH of the medium. This can be associated with the

increased solubilization tendency of Fusarium oxysporum in the presence of sodium

gluconate as a carbon source (Holker et al., 1995).


1.6.1.2.2 Chelators

In chemical structure of low rank coals, different metal ions play the role of bridge

within the coal matrix and the influence of chelators on solubilization has been reported.

Quigley et al. (1988) suggested the effect of different cations for example Ca2+, Fe3+, Al3+

etc. in the solubilization of lignites. Cohen et al. (1990) described that Trametes versicolor

excreted ammonium oxalate, which was the cause of biosolubilization of highly oxidized

lignite.

In comparison studies, it was found that oxalate was the most efficient solubilizing

agent for lignite as compared to nitrate, tartrate and dihydroxybenzoic acids. Oxidative

pretreatment has also significant effect on the enhanced solubilization ability of chelators

(Fakoussa, 1994). However, in certain cases the reverse effect was observed when

untreated German lignite was solubilized through 3,5-dyhydroxy benzoic acid.

Physiological concentration of chelators is also a function of solubilization and it has been

shown that increase of chelators’ concentration to certain extent may be critical for

enhanced solubilization (Fakoussa, 1994).

1.6.1.2.3 Hydrolases

Hydrolytic enzymes, particularly esterases, have been found to be involved in the

solubilization of coal by cleaving ester bonds within the coal matrix. The esterase activity

in Trametes versicolor for solubilization of leonardite was reported by Campbell et al.

(1988). But the solubilization was less efficient in case of chelators (Cohen et al., 1990).

Holker et al. (1999) reported an inducible agent from Trichoderma atroviride, which was

heat-sensitive and found to be responsible for the solubilization of Rhenish lignite. This

mold released a particular esterase, which was able to cleave the esters, present within the

coal matrix. Furthermore, it was suggested that the solubilization ability of this fungus

could be because of the combined effect of ‘non-enzymatic’ actions and enzymatic activity

(Holker et al., 1999). Penicillium decumbens P6 has also been experimented to degrade

lignite involving the combination of different mechanisms including release of alkaline

substance, surfactants and enzymes (Yuan et al., 2006a).


1.6.1.2.4 Oxidative Enzymes

Because of the major part of lignin in wood chemical structure, it has been

postulated that the lignin-like chemicals can be the significant aromatic and structural

moieties in brown coal. It appeared that ligninolytic activity of enzymes in microorganisms

could be a potential and promising aspect for investigations. However, this ability of

degrading lignin is restricted to very few groups of microorganisms and among these

basidiomycetes are the most active ones, which cause white-rot of wood (Kirk and Farrell,

1987; Blanchette, 1991; Hatakka, 1994). Some filamentous bacteria have also been

reported to break lignin network to limited extent (Crawford et al., 1983).

The enzyme systems, which are involved in ligninolytic activity, are as follows;

1. Peroxidases (manganese peroxidase, lignin peroxidase, other peroxidases)

2. Phenol Oxidases (laccases)

3. Supporting Enzymes (H2O2 generating enzymes)

Lignin peroxidase (LiP; EC 1.11.1.14), being a glycoprotein, contains iron

protoporphyrin IX (heme) as prosthetic group and H2O2 is required for the catalytic activity

of LiP (Tien and Kirk, 1984). This enzyme can oxidize phenolic rings and non-phenolic

rings (Odier et al., 1988). The effect of LiP on the constituents from brown coal was first

investigated by Wondrack et al. (1989). The North Dakota lignite and sub-bituminous coal

from Germany were pretreated with nitric acid and subjected to depolymerization by

partially purified LiP from Phanerochaete chrysosporium. The coal was transformed into

smaller water soluble fragments. Ralph and Catcheside (1994) subjected the alkali-

solubilized coal to depolymerization by ligninolytic cultures of Phanerochaete

chrysosporium. The fungus transformed 85% coal upon incubation of 16 days, however,

that solubilized coal was not recoverable by alkali solubilization and acid precipitation.

Manganese peroxidase (MnP; 1.11.1.13) holds resemblance with LiP and for the

first time it was reported in the liquid cultures of Phanerochaete chrysosporium (Kuwahara

et al., 1984). It is glycosylated and an extracellular enzyme, which has heme as prosthetic

group. The catalytic activity of this enzyme is same as that of LiP (Wariishi et al., 1988).

The basidiomycete strain RBS 1k was used by Willmann and Fakoussa (1997) for the


bleaching of water soluble coal macromolecules by stimulating the MnP secretion.

Optimum bleaching was reported at 0.5g/L concentration of coal humic acid. There have

been some investigations regarding the close cooperation of both of these enzymes i.e.,

MnP and LiP. The agaric basidiomycetes Nematoloma frowardii and Clitocybula dusenii

depolymerized the coal humic substances when the cultures were supplemented with Mn2+

and low molecular weight fulvic acids like substances were obtained. The MnP has also

been reported to attack ground low rank coal. Coal particles were incubated with MnP

extracted from Nematoloma frowardii and in the result fulvic acid and small humic acid

molecules were formed (Hofrichter et al., 1999).

In other peroxidases, soybean hull peroxidase (SBP) and horseradish peroxidase

(HRP) have been investigated for their ability to modify the structural matrix of coal. High

molecular weight fraction from a Spanish brown coal was extracted and partial

depolymerization was observed upon incubation with SBP (Blinkovsky et al., 1994).

Likewise, sub-bituminous brown coal was oxidized with an acid and treated with HRP. An

increase in solubility of coal was observed and 44% of the coal was converted into soluble

products (Scott et al., 1990).

In lignin and wood degradation, another important class of enzymes, which belongs

to the blue copper oxidases is laccases (EC 1.10.3.2) (Reinhammar and Malmstrom, 1981).

These are polyphenol-oxidizing enzymes, glycosylated proteins, and mostly produced by

ligninolytic and wood degrading fungi for example Trametes versicolor, (Fakoussa and

Frost, 1999), Phlebia radiate (Kantelinen et al., 1989), Pycnoporus cinnabarinus (Eggert

et al., 1996), and Nematoloma frowardii (Hofrichter and Fritsche, 1997). In biological

conversion of coal, the role of laccases has been determined by Cohen et al. (1987) and it

has been suggested that this enzyme is responsible for solubilization of lignite. Pyne Jr et

al. (1987) purified laccases later and showed their activity towards leonardite degradation.

Besides solubilization, this enzyme is also involved in depolymerization of coal and humic

substances extracted from coal. In addition to fungal laccases, tyrosinase, which is phenol

oxidases, has also been reported to have coal modifying ability. This oxidase is widely

distributed among fungi, plants, animals and actinomycetes (Naidja et al., 1998). The coal


solubilizing activity of tyrosinase-like enzyme has been reported in which the coal was

transformed to black particles on agar plates (Odom et al., 1991; Patel et al., 1996).

Generally, the oxygen content of coal decreases with the increasing rank of coal

and in lignites most of the oxygen is bound in carboxylic functionalities (Huttinger and

Michenfelder, 1987). Removal of COOH groups can be done by thermal or chemical

means but these are very expensive methods (Fristad et al., 1983; Banerjee et al., 1989).

However, the use of decarboxylases is cost effective and can be carried out at moderate

temperatures. The solubilized fraction from lignite was demonstrated to be hydrophobic

and precipitated after decarboxylation. In turn, the calorific value was increased and

oxygen content was decreased. Decarboxylases can find some potential applications in this

regard, however, there are certain concerns over isolation as these are intracellular enzymes

and their extraction is cost-intensive (Fakoussa and Hofrichter, 1999).

1.6.2 Anaerobic Degradation of Coal

Brown coal can be converted to methane with the help of anaerobic bacteria and

this conversion may offer certain advantages over conventional chemical gasification of

coal. Methane cannot be obtained directly in chemical gasification processes. The syngas

need to be converted into methane through Fischer-Tropsch synthesis, which is an energy

intensive approach (Crawford et al., 1990; Leuschner et al., 1990; Isbister et al., 1993).

Microorganisms from different sources, such as cow dung, paddy field mud, gut of

termite and wood eating insects, have been used for the biogasification of coal (Gupta and

Birendra, 2000). Microbial degradation of coal to methane follows a complex pathway

involving initial breakdown of the macromolecular matrix of coal, and fermentation of

intermediates to smaller organic molecules, which can then undergo further transformation

to produce methane. Some models have also been reported, which propose metabolic

pathways involved in biodegradation of coal into methane (Jones et al., 2010).

However in recent years, underground anaerobic carbon degradation to methane in

coals, shales and ocean floor sediments and the concept of biogenic coalbed methane has

enforced the suitability of coal as methanogenic substrates (McIntosh et al., 2002;

Thielemann et al., 2004; Newberry et al., 2004). The biogenic origin of methane in coal


seams indicates that coal, itself, can also serve as a substrate for microorganisms. The

production of significant amounts of methane in abandoned coal mines has been attributed

to the presence of acetogens and methanosarcinales (Kruger et al., 2008). A wide variety

of organics, majorly aliphatics and aromatics, have been observed in coal seam formation

waters, coal-extractable organic matter (CEOM) and methanogenic coal incubations (Orem

et al., 2007; Orem et al., 2010). Before understanding the concept of biogenic methane, it

is important to describe coalbed methane and the origin of this gas.

1.7 COALBED METHANE (CBM)

Being an unconventional gas reserve, coalbed methane has appeared as an

emerging and promising natural resource (Palmer, 2010). United States, Australia, India,

Canada, and China are major CBM producers. In these countries, deep surface coalbeds

and abandoned mines have also been exploited for the production of methane. Coalbed

methane (CBM) is being transported in gas pipelines for electricity generation especially

in United States, Australia, India and China (Moore, 2012). Additionally, this coal mine

gas has also been notorious for causing mine explosions (Flores, 1998; Gentzis and Bolen,

2008).

1.7.1 Gas Quality

The value of the CBM gas can be determined by analyzing the proportion of the

mixed gases. A CBM reservoir can have two types of gasses; 1) Productive 2) Inert.

Productive gases include methane and higher gaseous hydrocarbons (ethane, propane) and

the increased concentrations of these gases give economic value to CBM reservoir

(Whiticar, 1994; Moore, 2012). While inert gases are nitrogen, carbon dioxide and

hydrogen sulphide, which are also present in gas mix and as these gasses are not

combustible so the greater concentration of these gasses can decrease the overall value of

the reserve or coal seam gas (Whiticar, 1994). Hydrogen sulphide is generated due to the

process of devolatilisation during coalification. Because of the presence of H2S in the gas

stream, coal seam gas may be very toxic and corrosive, that is why this can pose some

serious problems in gas pipelines during transportation. Thus, the removal of H2S is


required before putting the seam gas into main stream. The gas with high content of H2S is

considered to be sour while the one with that of lower content is sweet gas (Hunt, 1996).

The energy potential of CBM has proved itself as a potential energy source. There

has been massive discussion and research over the origin and constituents of this gas. The

varying compositions of other gases except methane depend on the nature of the CBM

reserve. Two primary origins of CBM have been described universally; 1) Thermogenic,

2) Biogenic (Strapoc et al., 2011).

1.7.2 Thermogenic CBM

During the process of coalification, when coal acquires the high volatile bituminous

rank and vitrinite reflectance values appear in the range of 0.5-0.6%, there are chances of

adsorbed thermogenic CBM in coalbeds (Clayton, 1998). The concomitant effect of

pressure, heat and time results in the generation of gases including methane, nitrogen,

carbon dioxide, traces of hydrogen sulphide, ethane and propane. The deeper coals have

been associated with the production of thermogenic CBM (Hackley et al., 2009). The

factors, which affect the initiation of thermogenic CBM generation normally, depend upon

particular coal basin, burial history and organic composition of coal basin (Whiticar, 1994).

By working on the organic composition of coal basin and formation temperature, certain

practical and economical aspects of this type of natural gas reserve can be determined

(Hunt, 1996). Vitrinite reflectance analysis can help out in determining these factors as

Zhang et al. (2008) have worked on specific kinetic and laboratory models, which can

predict the volume of the gas. Vitrinite reflectance was found to be directly related to the

concentration of CO2, methane and higher hydrocarbon gases. Increasing reflectance

values, showing increasing rank of coal, are observed to be indicative of decreasing CO2

and increasing methane (Zhang et al., 2008).

So far, it is assumed that the higher rank coals are capable of producing thermogenic

CBM to the greater extent as compared to that of biogenically originated CBM. This aspect

can be attributed to the fact that the higher rank coals have increased gas holding capacity

because of reduced moisture content, which is sometimes very high in low rank coals.

Secondly, devolatilisation in coalbeds also results in enhanced thermogenic CBM


generation. But this can never be implied that low rank coals generate less CBM because

there are certain factors, which can be operative in reducing the saturation of CBM at a

particular reserve. Some of those factors are elevated pressure and temperature, tectonic

fracturing and migration of gasses. In short, it is generally accepted that biogenic CBM is

associated with low rank coals while thermogenic CBM production is a function of

increasing rank of coal (Moore, 2012).

1.7.3 Biogenic CBM

The deep surface life underneath our earth is driving anaerobic carbon recycling.

On the surface of earth, microbial world is decaying the plant matter; side by side these

microorganisms are involved in the conversion of peat and low rank coal into methane

(Strapoc et al., 2008b). Biogenic methane, caused by the action of microbes, has been

reported to contribute towards 20% of natural gas reserves of the world (Riva, 1995). A

number of research groups have observed and reported microbial ecological profiles in

deep surface coalbeds, ranging from 500 to 1000m, and in coal formation waters (Hendry

et al., 2007; Green et al., 2008; Strapoc et al., 2008a; Li et al., 2008; Midgley et al., 2010;

Penner et al., 2010).

While studying deep surface microbial ecology, a variety of taxa may be observed

but in the methanogenesis of coal, the key role is played by methanogenesis, though this

transformation is based on syntrophic cooperation (Strapoc et al., 2008b). In general, the

precursors for methane production are acetate, hydrogen and methylotrophic substrates. It

is believed that in the generation of biogenic methane, two domains of life are involved; 1)

Bacteria, 2) Archaea. The ones who start degrading coal are bacteria, which by this

degradation provide substrate to methanogens, the Archaea, for finalizing the ultimate

conversion of coal into methane (Green et al., 2008).

1.7.4 Intermediates involved in Pathways for Biogenic CBM Generation

In recent years, lots of efforts have been made in order to identify the intermediates,

which may be formed during the conversion of coal to methane. Metabolic profiling has

been done to determine the classes of compounds in coal formation waters. On the basis of

chemical and structural features, coal matrix is formed by the linked clusters of aromatic


structures and aliphatic hydrocarbons. Some heterocyclic compounds containing, nitrogen,

sulphur and oxygen as heteroatoms are also present (Kabe et al., 2004). Because of

hydrophobicity and heterogeneity of coal matrix, it is a harsh microbial substrate and

degradation under anoxic conditions requires a syntrophic cooperation of a variety of

microorganisms.

Generally, in conversion of complex organic matter into methane, primary or

preliminary degradation is mediated by fermentative bacteria for transformation of

organics polymers to fatty acids, alcohols, organic acids, carbon dioxide and hydrogen.

Further subsequent degradation is carried out by secondary fermentative bacteria, which

include homoacetogenic bacteria and methanogens (acetoclastic, methylotrophic and

hydrogenotrophic) (Schink, 2006). The same degradation pattern has been hypothesized

for the conversion of coal into methane. In recent years, some models have also been

reported, which proposed metabolic pathways involved in biodegradation of coal into

methane (Jones et al., 2010; Strapoc et al., 2011). Besides, in the degradation of coal into

methane, there has been found a cluster of organics, which may be considered as

intermediates in the involved pathways.

1.7.4.1 Aromatics

A wide range of aromatic compounds has been reported in coal formation waters,

methanogenic incubations of coal, and coal-extractable organic matter, which include

polyaromatic hydrocarbons (PAHs) and derivatives of PAHs, phenols, biphenyls, benzene

derivatives and aromatic amines (Orem et al., 2007; Ulrich and Bower, 2008; Orem et al.,

2010). Phenols and benzoates have been the most prevalent constituents of aromatic cluster

of coal. Wawrik et al. (2012) demonstrated the presence of the metabolites derived from

the addition of alkanes and alkenes to fumarate.

Organic extractions have been carried out from Powder River Basin coal (a low

rank coal reservoir) and from San Juan Basin (a mature coal reserve) in order to determine

the preferential specialization of biological degradation of aromatics over aliphatics

(Formolo et al., 2008). However, it has been suggested that the recalcitrant nature of


aromatic clusters reduces the rate of overall degradation process, and with increasing rank

of coal the rate of methanogenesis becomes slower.

1.7.4.2 Aliphatics

A wide range of saturated hydrocarbons including long chain alkanes and long

chain fatty acids like hexadecanoic acid with significant concentrations has also been

observed in coal formation waters. Biomarkers such as hopanes and terpanoids have also

been reported (Formolo et al., 2008; Orem et al., 2010). In San Juan Basin, the

intermediates of biodegradation of n-alkane such as methyl alkyl succinates were detected,

which could be transformed to methanogenic substrates upon further degradation

(Warwick et al., 2008). Vieth et al. (2008) and Glombitza et al. (2009) have reported the

identification of low molecular weight organics, which were water soluble. This release of

organics was found to be the function of rank of coal, which suggested that a certain extent

of extractable organics from low rank coal could serve as substrates for methanogens

(Vieth et al., 2008; Glombitza et al., 2009).

Orem et al. (2007) suggested that the presence of intermediates such as fatty acids

may be due to the biological degradation of cyclic and aliphatic hydrocarbons. However,

the inhibition of methanogenesis because of the potential toxicity of some of the

intermediates such as fatty acids, have also been reported. The accumulation of organic

acids may cause decrease in pH (Jones et al., 2010).

1.7.4.3 Heteroatoms

In the structural matrix of coal, especially in lower rank coals, heteroatoms have

been found to be linkage site for joining the aromatic clusters and these may also provide

a target site for biodegradation. The significance of coal depolymerization, stimulated by

heteroatom, was described by Orem et al. (2007) by detecting the aromatic Nitrogen-

Sulphur-Oxygen (NSO) compounds in produced waters from CBM wells in Powder River

Basin, USA. The specific site with functional groups of methoxy, hydroxyl, methyl and

carboxyl groups can be initially attacked by breaking the linkages between aliphatic and

aromatic clusters in coal. For example, Liu and Suflita (1993) have suggested the

demethoxylation of aromatic rings as possible step in biodegradation. Liptinite macerals


have been reported to be rich in heteroatoms, thus being more prone to biological

degradation as compared to vitrinite and inertinite, which are least rich in heteroatoms (Liu

and Suflita, 1993). Sheremata (2008) proposed the possibility of attractive heteroatom

bonds, which may be thermodynamically favourable to be attacked for degradation just

like the mechanism of thermal decomposition of asphaltenes, structural analogs to lignin.

1.8 MICROBIAL COMMUNITIES RESPONSIBLE FOR DEEP SURFACE

COAL METHANOGENESIS

Coal methanogenesis follows degradation mediated by complex microbial

community for which the complete analysis requires a collection of approaches including

physiological studies, molecular based investigations and analytical probes (Gieg and

Suflita, 2002). Some enrichment studies have been conducted in order to explore the

presence of methanogenic Archaea in coal formation waters (Shimizu et al., 2007). For

studying microbial ecology around the coal environment, microbiologists have relied on

coal formation water (from CBM wells) and fresh coal core samples, which must be intact.

In order to determine the physiological requirements of methanogenic

microorganisms, there is a need to pursue enrichment studies under anaerobic conditions

(Wiegel et al., 2006). A number of research groups have carried out the direct

bioconversion of coal (Shumkov et al., 1999; Thielemann et al., 2004; Green et al., 2008;

Harris et al., 2008; Kruger et al., 2008; Penner et al., 2010). Thielemann et al. (2004)

reported the presence of methanogenic Archaea in coal-mine water collected from Ruhr

River Basin, Germany. Likewise, enrichment studies have also been performed using mine

timber and hard coal, which showed the presence of acetoclastic and hydrogenotrophic

methanogens (Kruger et al., 2008). In enrichment studies, stimulation of methane

generation has also been carried out by adding hydrogen and carbon dioxide under

anaerobic conditions, though there was no significant effect of addition of acetate. The

addition of inorganic nutrients has also been reported to stimulate the biogenic methane

generation at laboratory scale (Harris et al., 2008). Molecular analyses have been done for

having an insight into the composition of microbial communities and metabolism

associated with biogenic CBM generation. Mostly, microbial community profiles have

been determined by analyzing the sequences of 16S ribosomal RNA genes. On the basis of


these analyses, phylogenic trees have been constructed for the microbial communities,

inhabiting coal environment. Shimizu et al. (2007) reported first archaeal sequences,

related to the genera Methanoculleus and Methanolobus, which were hydrogenotrophic

and methylotrophic Archaea, respectively. Among bacteria, the identified genera were

closely related to Acetobacterium and Syntrophus. Other firmicute and proteobacterial

genera were also detected (Shimizu et al., 2007).

Li et al. (2008) reported the presence of Clostridiales and α-protobacterial lineages

in Australian coalbeds. In coalbed water from Illinois Basin, USA, the genus

Methanocorpusculum was detected and the analysis of 16S rRNA gene showed the

presence of the microbial communities, which belonged to Firmicutes, α-Proteobacteria,

Spirochaetes and Clostridia (Strapoc et al., 2008b). Almost same bacterial communities

have also been found in coal samples collected from Waikato coal fields in New Zealand

(Fry et al., 2009). In formation water produced from Alberta Basin, Canada,

Methanosarcina and Methanobacteriales lineages have been detected (Penner et al., 2010).

Methanobacterium and Methanothermococcus have been reported to be detected in

coal core and methanogens were detected in formation waters samples obtained from the

same well in Wyoming, USA (Klein et al., 2008). In short, a variety of methanogenic

microorganisms has been detected in coalbeds and the most common lineages are

Methanobacteria, Methanolobus, Methanosaeta, Methanococci, Methanocorpusculum,

Methanoregula, and Methanoculleus (Table 1.3). Firmicutes, Spirochetes, Bacteroidetes,

and Proteobacteria represent dominant bacterial phyla found to be associated with coal

formation waters produced in CBM wells.


Table 1.3 | Methanogens Reported in Coal Formation Waters and Coal Mines (Strapoc et al., 2011)

Genus Species/Strain Accession Numbers

Methanolobus

Methanolobus sp., str., R15 EF202842

Methanolobus oregonensis, str. WAL1 U20152

Methanolobus vulcani, str. PL-12/M U20155

Methanolobus sp., SD1 EU711413

Methanolobus bombayensis, str. B-1 U20148

Methanosarcina

Methanosarcina acetivorans, str. C2A AE010299

Methanosarcina frisia M59138

Methanosarcina mazei, str. OCM26 AJ012095

Methanosarcina sp., HB-1 AB288262

Methanosarcina barkeri, str. OCM38 AJ012094

Methanosarcina barkeri M59144

Methanosarcina thermophila M59140

Methanosarcina lacustris, str. MM AY260430

Methanosarcina baltica, str. GS1-A AJ238648

Methanosaeta

Methanosaeta sp. Str. 6Ac AY970347

Methanothrix thermophila, str. PT AB071701

Methanothrix thermophila, str. CALS-1 M59141

Methanocorpusculum Methanocorpusculum parvum AY260435

Methanocorpusculum sp., T07 AB288279

Methanoculleus

Methanoculleus thermophilicus M59129

Methanoculleus sp., str. IIE1 AB8089178

Methanoculleus marisnigri, str. CoCam AF028693

Methanoculleus chikugoensis, str. MG62 AB038795

Methanoculleus bourgensis, str. NF-1 DQ150254

Methanoculleus bourgensis, str. CB1 AB236081

Table 1.3 Continued…


1.9 STIMULATION OF BIOLOGICAL METHANE GENERATION FROM

COAL

In a number of studies, microbial methane generation from coal has been stimulated

using coal as sole carbon source without any addition of acetate and hydrogen (Shumkov

et al., 1999; Menger et al., 2000; Green et al., 2008; Harris et al., 2008; Jones et al., 2008;

Kruger et al., 2008; Pfeiffer et al., 2010). Mostly, slow transformation of coal into methane

has been reported requiring even months for significant yields. Thus, there is a need to find

a way for generating methane quickly and to determine the exact type of the coal fraction,

which could be converted into methane preferentially.

The addition of nutrients for stimulating the methane generation has also been

suggested but it seems that methane yield will remain the function of coal rank. Chemical-

based stimulation has been recommended and number of chemicals for example phosphate,

tryptone, ammonia, agar, vitamins, and trace metals etc. have been tested (Jin et al., 2007;


Genus Species/Strain Accession Numbers

Methanoregula Methanoregula boonei, str. 6A8 NC_009712

Methanosphaerula palustris, str. E1-9c EU156000

Methanobacterium

Methanobacterium sp., str. GH EU333914

Methanobacterium curvum AF276958

Methanobacterium congolense AF233586

Methanobacterium formicicum, str. FCam AF028689

Methanobacterium subterraneum DQ649330

Methanobacterium thermoautotrophicum X68716

Methanococcus

Methanococcus jannachii, str. DSM 2661 NC000909

Methanococcus infernus AF025822

Methanococcus igneus M59125

Methanococcus aeolicus, str. Nankai-3 NC_009635


Pfeiffer et al., 2010). Prior fungal treatment has also been reported for the release of

organics, which could possibly serve as methanogenic substrates (Haider et al., 2013).

Another aspect, which can be an important factor for enhanced methane generation

is the microbial consortium. Indigenously isolated consortia from coal environment can be

important consideration for stimulating conversion of coal into methane. Termite gut flora

have also been used for the generation of methane from coal (Srivastava and Walia, 1997;

Menger et al., 2000) while in some studies consortium, isolated from an abandoned coal

mine, has also been employed for the production of methane (Volkwein, 1995).

In the CBM wells, where biogenic methane is being produced naturally, in situ

stimulation has also been proposed (Scott et al., 1994; Volkwein, 1995; Scott, 1999;

Menger et al., 2000; Budwill, 2003; Faiz et al., 2003; Scott and Guyer, 2004; Thielemann

et al., 2004; Jin et al., 2007). Menger et al. (2000) patented the application of underground

chamber with acid formers and methanogens, obtained through termite gut microflora, for

digestion of coal. For subsurface enhancement, nutrient delivery and surface area of coal

can be improved by fracturing the coal reservoir (Jin et al., 2007). In Powder River Basin,

USA, biogenic methane generation has been increased by augmenting the reserve with

phosphate nutrient, which caused the stimulation of CBM production (Pfeiffer et al., 2010).

1.10 SCOPE OF BIOLOGICAL BENEFICIATION OF LOW RANK COALS IN

PAKISTAN

Pakistan has been blessed with huge reserves of coal but unfortunately, majority of

these comes under the category of low rank (Ghaznavi, 2002). Geographical mapping of

coal reserves of Pakistan has been well documented (Figure 1.7). On the contrary, Pakistan

is undergoing a severe energy crisis, which reflects in the form of continuous shortfalls in

electric and gas supplies and the current scenario, regarding crisis, has necessitated the

increased share of indigenous coal in energy portfolio. So far, the use of indigenous coal

in conventional thermal applications has been negligible due to the technical constraints,

related to high sulphur content and low rank nature of coals, though, some efforts are being

carried out at national level including underground coal gasification at Thar, Sindh and

setting up coal power plants in Punjab. But concomitant applications of appropriate


technologies, addressing variety of coals with different physico-chemical properties, are

required. In this regard, biological beneficiation of coal can be advantageous over

conventional technologies, which require intensive energy application in terms of

temperature and pressure. Biological cleaning of local coals has been explored

successfully, achieving significant removal of sulphur up to 75% (Ghauri et al., 2009).

However, the conversion of low rank coals has been unattended so far, particularly for

Thar, one of the world’s largest lignite reserves.

Coalfields Coal Resources

(Million Tonnes) Total Coal Reserves

Salt Range 596

Jherruk 1,323

Lakhra 1,328 ~ 186 Billion Tonnes Sonda-Thatta 3,700

Thar 175,500

Figure 1.7 | Coal Reserves of Pakistan

The associated high moisture content, low aromaticity and less maturity of low rank

coals make these less recalcitrant to biological modification as compared to high rank coals

in terms of chemical structure. Low rank coals from Pakistan, which have not been

subjected to any biological modification effort, may find an application for the extraction


of value added products from lignite. There is a need to determine the potential of lignite

samples from Pakistani coal fields, particularly from Thar, for determining the suitability

of these coals to bio-based applications like biosolubilization and biogasification. In this

regard, preliminary fungal degradation of lignite may be prospected for some potential of

releasing some organic functional moieties from the coal matrix, which could be

investigated for determining their role as methanogenic substrates. Besides, the

methanogenesis of native coal samples, as direct approach without any prior fungal

pretreatment, may also be an alternative route for coal-to-methane transformation. This

direct route will be based on the phenomenon of underground anaerobic carbon recycling,

followed by the conception of biogenic coalbed methane, which is on-going in coal seams

of low rank coals’ reservoirs in real time.

1.11 OBJECTIVES OF THE STUDY

Keeping in mind the need of initial degradation of coal as rate limiting step, two

major biological degradation approaches (bacterial and fungal degradation) were employed

for determining the methane generation potential of indigenous low rank coals. However,

concisely, the main objectives of this study were as follows;

1. Isolation and Identification of Coal Degrading Fungi

2. Optimization of Coal Degradation Process for the Release of Organics

3. Pretreatment of Coal Samples with Screened/Coal Degrading Fungal Isolate

4. Chemical Investigations of the Released Organics/Solubilized Coal after Fungal-

Pretreatment for Determining the Nature of Organics as Methanogenic Substrates

5. Methanogenesis of Released Organics

6. Methanogenesis of Native Coal Samples (Direct Approach)

7. Preliminary Investigations of Residual Coal (after Fungal Pretreatment) for the

Extraction of Value-Added Products like Soil Condition Agent

Representative low rank coal samples will be collected from various coal fields of Pakistan

and analyzed chemically and petrographically. Some of the samples will be pretreated with

isolated fungal strains (coal degrading). The released organics and native coal samples will


be subjected to methanogenesis for comparing these two approaches. Residual coal after

fungal pretreatment would be processed for humic acid extraction.

The schematic illustration of research outline has also been shown in Figure 1.8.

Figure 1.8 | Schematic Illustration of Research Outline

Low Rank Coal

Origin: Pakistan

Proximate & Elemental Analyses, Calorific Value, Geological Info Petrographic Studies

Methanogenesis

Methane Determination

Fungal Pretreatment

Released Organics

Analytical Investigations of Released Organics

(UV Spectra, EEMS, GC-MS, NPOC)

Fungi: From Coal Habitats, Wood Degrading, Lignin Degrading

Molecular Typing

Residual Coal (After Fungal Pretreatment)

Preliminary Chemical Investigations of Derived Humic Acid

2

Materials & Methods

2.1 ORIGIN OF COAL SAMPLES

Because of very limited mining activity at Thar, which was the prime focus of this

study, coal samples had to be obtained from other resource laboratories/organizations for

a detailed study. For this purpose, thirty (30) coal samples were obtained from archives of

drilled cores collected by the United States Geological Survey (USGS), USA and

Geological Survey of Pakistan (GSP) between 1986 and 1992. These samples were ground

splits (<850m) that had been sealed in polyethylene and archived at USGS since shortly

after drilling. Except for some secondary sulphate formation, much of which probably

occurred shortly after core recovery, the samples did not appear to have appreciably

degraded during storage. The origin of the coal samples has been reported in Table 2.1 and

Figure 2.1.

Another core sample of Thar coal was obtained from Dr. Sarfraz Hussain Solangi

(Professor/Director, Center for Pure and Applied Geology, University of Sindh, Pakistan).

Though, complete geological information was not provided for this sample but this sample

was analyzed and used in experiments, which were related to preliminary investigations of

extraction of humic acid from lignite. For all those thirty samples, specific sample IDs were

used, which indicated the borehole and seam information. However, for the sample,

provided by Dr. Solangi, the code ‘TP-31’ was assigned (Table 2.1). As a whole, samples

came from eight coal areas of Pakistan (17 from Thar, 4 from Lakhra, 4 from Sonda, 2

from Indus East, 1 from Khost, 1 from Makarwal, 1 from Salt Range and 1 from Metting-

Jhimpir).

33 Materials & Methods |

Figure 2.1 | Geographical Setting and Borehole Locations for the Samples used in the Study

The sample IDs in the tables generally reflect the borehole number and sample bench.

(e.g. UAS-4-2E = borehole UAS-4, seam 2, bench E). Coal field boundaries are

approximate.


Table 2.1 | Origin of Coal Samples

Sr. No. Sample ID Province Coal Field

1 7 (BLCH) Baluchistan Khost

2 DSA-23-4 Punjab Salt Range

3 MKCT-6 Punjab Makarwal

4 UAL-15-1 Sindh Lakhra

5 UAL-15-2 Sindh Lakhra

6 LS-4-1 Sindh Lakhra

7 LS-4-2B Sindh Lakhra

8 UAS-4-1 Sindh Sonda

9 UAS-4-2E Sindh Sonda

10 UAT-4-1 Sindh Sonda

11 UAT-4-4 Sindh Sonda

12 UAK-1-4 Sindh Indus East

13 UAK-1-8 Sindh Indus East

14 UAJ-1-1 Sindh Meting-Jhimpir

15 TP-1-1.1 Sindh Thar



On provincial basis, 28 samples were from Sindh, 2 samples and 1 sample were

from Punjab and 1 sample was from Baluchistan. Majority of the samples were from Sindh

Province, and among those 17 samples were from Thar.


Sr. No. Sample ID Province Coal Field





20 TP-3-2B Sindh Thar

21 TP-3-2D Sindh Thar

22 TP-3-2K1 Sindh Thar

23 TP-3-2R Sindh Thar

24 TP-3-2X Sindh Thar

25 TP-3-2AG Sindh Thar

26 TP-4-1A Sindh Thar

27 TP-4-2A Sindh Thar

28 TP-4-6 Sindh Thar



31 TP-31 Sindh Thar


2.2 PREPARATION OF REPRESENTATIVE COAL SAMPLES

Each coal sample was poured, as received, into a cone shaped pile on Kraft-paper.

Different portions from random locations in the sample were separated and collected with

a scoop or spatula. Afterwards, air drying of these fractions was carried out at 30C. The

dried collected coal sample was crushed and 500g was split out for grinding in Braun

pulverizer (Ohio, USA), which was equipped with ceramic sieves set to pass through 80

mesh size. The representative ground samples were further used for various analyses

including chemical, proximate and petrographic.

2.3 ULTIMATE ANALYSIS OF COAL SAMPLES

The chemical analyses of these representative coal samples were performed on

Thermo Scientific FLASH 2000 series (Waltham, Massachusetts, USA) and PerkinElmer

PE 2400 CHNS/O (Waltham, Massachusetts, USA) analyzers for determining the % age

composition of carbon, nitrogen, hydrogen, sulphur and oxygen. Gross calorific values of

all coal samples were determined using LECO AC500 Isoperibol Calorimeter (Saint

Joseph, Michigan, USA). All characterizations were made in triplicates and reported as the

mean values.

2.4 PROXIMATE ANALYSIS OF COAL SAMPLES

Proximate analysis represents determination of moisture, volatile matter, ash and

fixed carbon contents of coal. All of these, except fixed carbon, were determined

experimentally in accordance with ASTM standards. Fixed carbon was determined by

difference method after obtaining empirical values of moisture, volatile matter and ash

contents. All characterizations were made in triplicates and reported as the mean values.

2.4.1 Moisture Content (ASTM D-3302)

For determining total moisture content, 1g of ground coal sample was heated at

110°C for approximately one hour. The weight of sample was noted until the weight

difference remained constant. The weight difference was reported as % age of moisture

content on as-received basis.


2.4.2 Volatile Matter Content (ASTM D-3175)

In this test method, 1g of ground coal was weighed and placed in pre-weighed

platinum crucible (10 to 20mL in capacity, 25 to 35mm in diameter, and 30 to 35mm in

height) with a close-fitting cover. The crucible was then suspended at a specified height in

furnace chamber. The temperature of the region in the furnace was maintained at 950 ±

20°C. The weight loss was calculated, which was reported as % volatile matter content.

2.4.3 Ash Determination (ASTM D-3174)

The determination of mineral ash in coal was carried out by heating (burning) an

accurately weighed 1g of sample of the coal in an adequately ventilated muffle furnace at

temperature in the range of 700 to 750°C for four hours or until the weight difference

remained constant. Afterwards, the weight difference was measured, and the residue (Ash)

was reported as % ash after calculations.

2.4.4 Fixed Carbon (ASTM D-5142)

Fixed Carbon, basically, represents that solid combustible material in coal, which

is left behind after the removal of volatile matter and this is a parameter for considering the

suitability of a coal in combustion systems (Speight, 2012). It was determined by difference

method as per following formula.

Fixed Carbon (%) = 100 ̶ (% Moisture + % Volatile Matter + % Ash)

2.5 MACERAL ANALYSIS OF COAL SAMPLES

Vitrinite reflectance and maceral analysis for all coal samples were carried out in

Center for Applied Research, at University of Kentucky, USA and Organic Petrology

Laboratory, U. S. Geological Survey, Reston, Virginia, USA.

2.5.1 Sample Preparation and Analysis

The samples were ground and sieved using sample dividers, sieves, Buehler

SimpliMet® 3000 (Lake Bluff, Illinois, USA) automatic mounting press and believed to

be the representative of a grab sample from coal exploration core, which was subjected to

fungal degradation studies. The sample preparation was carried out according to the


standardized method of American Society for Testing and Materials (ASTM D-2797),

which described preparation of coal samples for microscopic analysis by reflected light.

The sample was mounted in 1-inch mold using a heat-setting thermoplastic resin medium.

The examination surface was ground and polished prior to overnight desiccation. Buehler

Ecomet 4 variable speed grinder-polisher (Lake Bluff, Illinois, USA) was used for this

purpose.

2.5.2 Random Reflectance Analysis

Random reflectance analysis was performed using ASTM standard test method (D-

2798), which defined protocol for microscopic examination of coal for vitrinite reflectance.

Fluorescence microscopy was used to identify organic materials and fluorescence

photographs with accompanying photographs of the same field in white light under oil

immersion were taken.

For reflectance analysis, Leitz Orthoplan microscope (Solms, Germany) with

photometer and peak detector was used. Additionally, a Leica DMRX (Solms, Germany)

with mechanical stage, tungsten halogen, and xenon gas discharge light sources were used

for incident light compositional analysis. Zeiss AxioImager (Oberkochen, Germany)

equipped with tin halide illumination and a photodiode array was used for light detection,

and a Leitz Orthoplan was employed for examination of rock thin sections in transmitted

polarized light.

2.6 COAL CLASSIFICATION

Coal classification was carried out on the basis of vitrinite reflectance values and

ASTM classification method, which takes volatile matter, fixed carbon contents and

calorific value into the consideration for designating the rank of coal (Tables 2.2 and 2.3)

(Stach and Murchison, 1982; ASTM Standards, 2011). The abbreviations for the

designation of coal ranks have been described in Table 2.4.


Table 2.2 | ASTM Classification of Coal

Class Group Fixed Carbon

d,mmf basis*, %

Heating Value

m,mmf basis

Agglomerating

Character

Anthracite

Meta-anthracite

Anthracite

Semi-anthracite

96

92-96

86-92

Non-agglomerating

Bituminous Low volatile bituminous

Medium volatile bituminous

High volatile A bituminous

High volatile B bituminous

High volatile C bituminous

78-86

69-78

69

14,000

13,000-14,000

11,500-13,000

Commonly

agglomerating

Agglomerating

Subbituminous Subbituminous A

Subbituminous B

Subbituminous C

10,500-11,500

9,500-10,500

8,300-9,500 Non-agglomerating

Lignite Lignite A

Lignite B

6,300-8,300

6,300

Non-agglomerating

*Dry Mineral Matter Free (d,mmf) Moist Mineral Matter Free (m,mmf)

Table 2.3 | Rank Classification by Vitrinite Reflectance

Ranks Reflectance, %

Lignite 0.35

Subbituminous 0.300.50

High Volatile Bituminous 0.501.12

Medium Volatile Bituminous 1.121.51

Low Volatile Bituminous 1.511.92

Semi-anthracite 1.922.50

Anthracite 2.50


Table 2.4 | Coal Ranks with their Designations

Coal Rank Designation Coal Rank Designation

Meta-Anthracite ma Anthracite an

Semianthracite sa Low Volatile Bituminous lvb

Medium Volatile Bituminous mvb High Volatile A Bituminous hvAb

High Volatile B Bituminous hvBb High Volatile C Bituminous hvCb

Subbituminous A subA Subbituminous B subB

Subbituminous C subC Lignite A ligA

Lignite B ligB

2.7 FUNGAL ISOLATION

Isolations were carried out from various habitats like degrading trees, coal mine

soils, drilled coal cores etc. in order to isolate fungal strains capable of degrading coal. In

case of samples from degrading trees, coal mine soils, and weathered coal samples, 50%

(w/v) suspensions were made in sterilized water and incubated for 2 days at 25C at 120

rpm shaking. After 2 days, the suspension was diluted 100 times and spread on malt extract

agar plates. In case of core samples of coal, an indigenous sample was removed from the

center of the intact core using sterile technique for the isolation of fungi. The piece of intact

core was first sterilized with ethanol and then holes were drilled in the core using a

sterilized screw. The powder sample from inside core was suspended in 100mL of minimal

salts medium and incubated for 2 days at 25C at 120 rpm shaking. After 2 days, the

suspension was diluted 100 times and spread on malt-extract agar plates. The pH was

maintained at 5.0.

2.8 COMPOSITION OF MALT EXTRACT AGAR (MEA) MEDIUM

Composition of malt extract agar medium was as follows (g/L);

Glucose 10.00

Malt Extract 5.00


Peptone 1.00

Agar 10.00

Distilled Water 1L

2.8.1 Purification and Storage of Fungal Isolates

Growth colonies obtained on malt extract agar plates were purified by sub-culturing

on solid media plates serially. Purified strains were grown on malt agar slants and stored

at 4ºC. Stocks were prolonged by sub-culturing on monthly basis. Besides, isolated fungal

strains were also transferred to liquid media for observation of growth in submerged

conditions (with minimal salts medium and pH 5.5) for which the composition was as

follows (g/L) (Silva-Stenico et al., 2007);

Ammonium Sulphate 1.00

Magnesium Sulphate 0.52

Dihydrogen Potassium Phosphate 5.00

Zinc Sulphate 0.0003

Ferrous Sulphate Decahydrate 0.0005

Malt Extract 30.00

Distilled Water 1L

2.9 SCREENING OF FUNGAL ISOLATES FOR COAL DEGRADATION

Fungal isolates were grown on malt extract agar plates for four days until the fungal

mycelia developed completely. Each 1g of coal particles (0.3 to 0.5mm in diameter) was

spread on the mycelia of fungal cultures in the Petri dishes. The Petri dishes were checked

after specific time intervals for any liquid products over the coal particles or any diffused

region around the coal particles over fungal mycelia. Afterwards, those, primarily screened,

fungal isolates were processed further for coal degradation experiments. When it was not

possible to determine any liquid droplets over coal particles or any dispersed region around

the coal particles, UV-Vis spectrophotometric scan in the range of 200 to 700nm was

carried out for the primary screening. Those fungal isolates were selected for coal

degradation studies, which produced significant peaks in the range of 240 to 350nm against


two controls. These two controls were medium with coal but without inoculation and

medium with inoculation but without coal.

2.10 TAXONOMIC EVALUATION OF FUNGAL ISOLATES

Taxonomic evaluation of the fungal strains was carried out on the basis of

microscopic morphological features, colony forms and PCR amplifications of Internal

Transcribed Spacer (ITS) regions.

2.10.1 Morphological Identification

Fungal isolates were grown on malt extract agar plates and morphological features

such as colony mycelia formation, color, colony textures, and spore formation were

observed. Phase contrast microscope (Zeiss Axiovert, MC80, Oberkochen, Germany) was

used to study the cell morphology, conidiophores structures, color and shape of hyphae and

photographs were taken.

2.10.2 Molecular Identification

For the extraction of total genomic DNA, Fast DNA Spin Kit for Soil (MP

Biomedicals, Solon, OH, USA) was used. However, the detailed stepwise protocol for the

extraction of DNA was as follows;

1. Agar plug from freshly grown isolate was added to the multimix 2 tissue matrix

tube (the ones with beads, not more than 7/8 filled).

2. Sodium phosphate buffer (978L) and MT buffer (122L) were added.

3. The mix in matrix tube was processed in bead beater 3 times for 90 seconds. Tubes

were centrifuged at 14,000xg for 30 seconds.

4. Supernatant was transferred to a clean tube. PPS-reagent (250L) was added and

mixed by inverting the tube by hand 10 times. Tubes were centrifuged at 14,000xg

for 5 minutes to pellet the precipitate.

5. Supernatant was transferred to a clean 15mL tube (Binding matrix suspension was

re-suspended before use) and 1mL binding matrix suspension was added.


6. Tubes were placed on a rotor or inverted by hand for 2 minutes to allow binding of

DNA to the matrix. Tubes were placed in a rack for 3 minutes to allow settling of

silica matrix.

7. Supernatant (500L) was removed being careful to avoid settled binding matrix.

Supernatant was discarded.

8. Binding matrix in the remaining amount of supernatant was re-suspended.

9. Approximately 600L of the mixture was transferred to a spin filter and centrifuged

at 14,000xg for 1 minute.

10. Catch tube was emptied. The remaining supernatant was added to the spin filter and

spun again.

11. SEWS-M (500L) was added to the spin-filter and centrifuged at 14,000xg for 1

minute. The flow-through was decanted and the spin filter was put back in catch

tube. Catch tube was centrifuged at 14,000xg for 2 minutes to dry the matrix of

residual SEWS-M wash solution.

12. Spin-filter was removed, placed in a fresh kit-supplied catch tube and then, air dried

for 5 minutes at room temperature.

13. DES (DNAse/Pyrogen free water; 50L) was added and matrix on the filter was

stirred gently with a pipette tip or vortex/finger flips to re-suspend the silica for

efficient elution of the DNA. Catch tube was incubated around 20 minutes at 65C

to elute. Tube was centrifuged at 14,000xg for 1 minute to transfer eluted DNA to

catch tube.

2.10.2.1 Fungal ITS Sequence PCR Amplification

For molecular typing of fungal strains, ITS internal regions were amplified through

PCR using universal primers ITS1, ITS4 and Taq polymerases (Invitrogen, San Diego, CA,

USA) (Silva-Stenico et al., 2007).

ITS1 (Forward Primer): TCC GTA GGT GAA CCT GCG G

ITS4 (Reverse Primer): TCC TCC GCT TAT TGA TAT GC

PCR conditions were as follows (Silva-Stenico et al., 2007);


94ºC for 3 Minutes

94ºC for 30 Seconds

56ºC for 1 Minute 30 Cycles

72ºC for 1 Minute

72ºC for 10 Minutes

2.10.2.1.1 Preparation of 1% Agarose Gel Electrophoresis

The PCR products were confirmed through 1% agarose gel electrophoresis. The

agarose gel was prepared as follows;

1. Agarose (1%), in respective amount, was dissolved in 1.0X TAE Buffer and heated

to the level of boiling, approximately, in microwave oven. Agarose was cooled

down to almost 50ºC and poured into the gel casting tray with comb inserted in the

tray. Gel was allowed to solidify at room temperature.

2. The gel casting tray was placed in the electrophoretic tank having 1.0X TAE buffer.

Ethidium bromide was added in the buffer already present in the tank.

3. PCR Products/DNA samples were mixed thoroughly with loading dye and loaded

into the wells with micropipette.

4. Voltage was regulated at 70V. Movement of dye indicated the migration of DNA

from anode to cathode through gel. When dye covered the distance sufficient for

separation of DNA fragments, the power supply was turned off. DNA fragments

were visualized under UV light in gel documentation system (Bio-Rad®, Hercules,

California, USA) and photographed.

2.10.2.1.2 PCR Product Purification

PCR products were purified using Wizard® PCR Preps DNA Purification System

(Promega®, Fitchburg, Wisconsin, USA). The protocol was as follows;

1. Direct PCR purification buffer (100µL) was dispensed into a tube. Then, 50µL of

the PCR product was added and mixed through vortex.

2. Resin (1mL) was added and vortexed briefly 3 times over a 1-minute interval. A

Wizard® Minicolumn for each sample to be purified was prepared.


3. Syringe barrel was attached to the Minicolumn. Resin/DNA mix was added to the

syringe barrel. Plunger was inserted, and the resin/DNA slurry was pushed into the

Minicolumn. Syringe was detached from Minicolumn, plunger was removed from

syringe barrel and barrel was reattached to Minicolumn.

4. Isopropanol (80%; 2mL) was added. Plunger was inserted and the isopropanol was

pushed through the Minicolumn. Syringe was removed. The Minicolumn was

transferred to a 1.5mL microcentrifuge tube and centrifuged at 10,000xg for 2

minutes. The Minicolumn was transferred to a clean 1.5mL microcentrifuge tube.

Nuclease-Free water or TE Buffer (50µL) was added. Microcentrifuge tube was

centrifuged at 10,000xg for 20 seconds at room temperature.

5. Minicolumn was removed and discarded. PCR products/DNA samples were stored

at ˗20ºC.

2.10.2.2 DNA Sequencing of the PCR Products

Purified DNA products were sequenced through BigDye Terminator v3.1 Cycle

Sequencing Kit (Applied Biosystems®, Foster City, California, USA) using ITS1 and ITS4

primers.

2.10.2.2.1 Cycle Sequencing

Cycle sequencing was carried out using the BigDye Terminator v1.1/3.1

Sequencing Buffer (Applied Biosystems®, Foster City, California, USA), which was

supplied at 5X concentration. Composition of reaction mix has been shown in Table 2.5.

Table 2.5 | Recipe for Cycle Sequencing (BigDye® Terminator v3.1 Cycle Sequencing Kit)

Reagent Volume

Ready Reaction Premix 2µL

BigDye Sequencing Buffer 1µL

Primers 1µL

Template 2µL

Water 4µL

Final Volume 10µL


2.10.2.2.2 Ethanol/EDTA/Sodium Acetate Precipitation

After cycle sequencing reaction, precipitation of extension product was carried out.

The protocol was as follows;

1. PCR tubes, containing the extension products, were removed from thermal cycler and

briefly spun. EDTA (125mM; 1L) and sodium acetate (3M; 1L) was added into each

tube.

2. Ethanol (100%; 25L) was added into each tube. Tubes were sealed, wrapped with

aluminum foil and mixed by inverting 4 times. Tubes were incubated at room

temperature for at least 30 minutes.

3. Tubes were spun for 20 minutes and the supernatant was pulled off. Ethanol (70%;

200L) was added and vortexed quickly for almost 5 minutes. Supernatant was pulled

off and tubes were dried. TSR (20L) was added and vortexed for 1 minute.

4. Extension products, in tubes, were denatured at 93˗95ºC for 3˗4 minutes. Tubes were

cooled down on ice for 2 minutes and sample was loaded into the sequencer.

2.10.2.2.3 DNA Sequence Analysis

Samples obtained after ethanol/EDTA/sodium acetate precipitation were loaded on

ABI Prism 3700 DNA analyzer (Applied Biosystems®, Foster City, California, USA) and

Sequence Analysis Software was used for acquiring the sequences of the samples. Error

rate in DNA sequencing was found to be less than 0.5% and two reads per sample were

made.

2.10.2.3 Phylogenetic Analysis of Fungal Strains

After getting the sequence of partial ITS regions of fungal isolates, phylogenetic

studies were conducted in order to derive evolutionary relationship. The ITS sequences

were compared in GenBank database (www.ncbi.nlm.nih.gov). Besides, ITS sequences for

other reported coal solubilizing and wood degrading fungi were also acquired from

GenBank database and aligned with the sequenced ITS regions of isolated fungal strains

using ClustalX (Table 2.6). A distance matrix was developed using aligned sequences and

afterwards exploited for the construction of phylogenetic tree by neighbor joining method

http://www.ncbi.nlm.nih.gov/


using TreeView software (ver 1.6.5) (Thompson et al., 1997). The partial sequences of ITS

regions of coal degrading fungal strains in this study have been submitted to GenBank

database (Appendix I).

Table 2.6 | Accession Numbers for the Partial Sequences of ITS Regions of Other Fungal Strains

(Used for the Construction of Phylogenetic Tree)

Organism Accession Number Reference

MW1 JN572146 Haider et al. (2013)

Trichoderma atroviride ES11 DQ489298 Silva-Stenico et al. (2007)

Hypocrea lixii TZ1 FJ645728 Tao et al. (2010)

Trichoderma sp. AH - Shi et al. (2009)

Penicillium chrysogenum QML-2 JF907010 Zhang and Sang (2012)

Penicillium chrysogenum GW20-4 JQ670962 Unpublished

Penicillium chrysogenum strain SGE6 JQ776534 Unpublished

Trametes versicolor CTB 863 EF524042 Unpublished

Fungal sp. AB 37 FJ235970 Unpublished

Penicillium chrysogenum WGS11799 JX406503 Unpublished

Penicillium sp. KU-DFR9 JX519346 Unpublished

Penicillium sp. SGE28 JX232275 Unpublished

Penicillium decumbens CBS230.81 AY157490 Weber et al. (2003)

Aspergillus Oryzae DT1 JN572145 Unpublished

Trichoderma reesei KC013272 Unpublished

Hypocrea rufa strain AN242 JX184121 Unpublished

Paraconiothyrium sp. GHJ-4 GQ331986 Unpublished

Ceriporiopsis subvermispora isolate DLL2010-072 JQ673086 Brazee et al. (2012)

Phlebia brevispora AB519182 Unpublished

Phlebia sp. b19 EF491864 Hilden et al. (2008)


2.11 COAL DEGRADATION EXPERIMENTS

Coal degradation experiments were carried out for the release of organics from coal

matrix, which could be subjected to subsequent methanogenesis for the generation of

methane. For this purpose, degradation through fungi was optimized for the significant

release of organics and this extent was determined on the basis of UV-Vis

spectrophotometric scan in the wavelength range of 200 to 700nm. Different parameters

were studied for this purpose, which included the effects of incubation time, coal loading

ratio (coal concentration), and concentration of glucose. Once the conditions were

optimized, major degradation experiments were performed in triplicates, though variation

was statistically non-significant. Fungal isolates were grown in minimal salt medium

supplemented with glucose (1%, w/v) and malt extract (0.3%, w/v) for 4 days at 25°C. For

the inoculation, 2.0mL of freshly grown culture (4 day growth) was used in subsequent

experiments for determining the effect of each parameter. All incubations were conducted

for seven days in sterile aerobic glass flasks, which were pre-combusted at 500ºC.

Following controls were also treated under the same conditions:

1. Medium with coal but without inoculation of fungal culture

2. Medium with inoculation of fungal culture but without coal

2.11.1 Filtration of Supernatants

After 7 days, supernatants from treatments were filtered for analytical

investigations using Whatman Glass Fiber Filters (Pore Size, 2.7µm) and for subsequent

methanogenesis. All the glassware used in the degradation studies and fiber filters were

pre-combusted at 500ºC in order to avoid any interference of the trace organics, which

could possibly be already present or sticking to the glassware.

2.12 ANALYTICAL INVESTIGATIONS

2.12.1 Spectrophotometric Studies

For the qualitative estimation of released organics in supernatants from coal,

filtrates were analyzed on Excitation-Emission Matrix Spectroscopy (EEMS). Filtrates

were scanned over the range of 200 to 700nm in terms of wavelengths. For EEMS studies,


Agilent Cary Eclipse Fluorescence Spectrophotometer (Santa Clara, California, USA) was

used.

2.12.2 Non-Purgeable Organic Carbon (NPOC) Determination

Non-purgeable organic carbon (NPOC) and dissolved organic carbon (DOC) are

identical notions [Shimadzu (Japan) uses the NPOC term]. For dissolved organic carbon

(DOC), samples were filtered using Whatman Polyethersulfone (PES) 0.2µm syringe

filters and were kept refrigerated (4ºC) until analyzed. Samples’ DOC was determined

using Shimadzu TOC-VCPH analyzer (Shimadzu® Scientific Instruments, Columbia,

MD, USA), equipped with a catalytically-aided 680ºC combustion chamber and normal

sensitivity platinum catalyst.

Standardization was based on a 6 point calibration curve (from 1 to 100ppm) using

a potassium phthalate DOC standard (100ppm, or 10ppm depending on the DOC range of

the samples) and the detection limit was 100ppb. MilliQ water (MQW), the ultrapure water,

was used as blank and each sample was injected at least two times, to ensure data

consistency and a maximum coefficient of variation (CVmax) of the integrated areas below

2. Samples with content higher than 100ppm DOC were diluted before injection for

obtaining measurements within the calibration curve range.

2.12.3 pH Determination

The pH of the filtrates, obtained after incubation of 7 days, with released organics

was determined using pH 20 Hanna Instruments (Woonsocket, Rhode Island, USA).

2.12.4 Gas Chromatography-Mass Spectrometry (GCMS)

For a detailed profile of released organics and having an insight into the nature of

liberated fractions through coal matrix, supernatants were also analyzed through Gas

chromatography-mass spectrometry (GC-MS).

Released organics, after the fungal treatment of coal, were sequentially liquid/liquid

extracted from 45mL of filtrate using pesticide-grade dichloromethane (DCM). The extract

was concentrated (to 1˗2mL) through rotoevaporation and further reduced to 200µL under


a gentle stream of N2. Generally, 1µL was used for gas chromatography/mass spectrometry

(GC-MS) analysis of each 200µL.

The GC-MS analysis was carried out using Hewlett-Packard 6890 series gas

chromatograph and 5973 Electron Ionization (EI) Mass Selective Detector (Agilent

Technologies, Santa Clara, California, USA), which was operated in Scan Mode. An HP-

5MS column (95% Dimethyl, 5% Diphenyl Polysiloxane), with 0.25mm x 30m x 0.25µm,

was used. An injection volume of 2µL was used. The NIST 02 mass spectral library was

used for identification of the compounds from the mass spectral data. Complete GC-MS

programme profile has been provided in Appendix I.

2.13 WBC-2 BASED BIOASSAY

A mixed methanogenic culture (WBC-2) was employed as a bioassay for

determining the methane generation potential from released organics of coal and from

native coal samples. The WBC-2 has been previously enriched from peat sediment

collected in tidal, organic-rich wetland supporting grasses, sedges, cattails, arrowhead,

pickerelweed and phragmites in West Branch Canal Creek, near Aberdeen, Maryland,

USA. The WBC-2 has been maintained in culture since 2003 at Voytek microbiology

laboratory (USGS, USA) in anaerobic, bicarbonate-buffered medium with lactate and

tetrachloroethane as carbon sources and <1% remnant sediment (Jones et al., 2006).

The WBC-2 microbial consortium is primarily involved in the fermentation of

complex organic matter and it contains Clostridium sp., Bacteroides sp. Acetobacterium

sp., and bacterial species that can produce acetate, either from H2 and CO2 or from some

aromatic acid and aliphatic compounds. The consortium also includes methane-forming

Achaea (methanogens capable of generating methane from both H2 + CO2 and from

acetate) and sulphate reducing bacteria (e.g., Desulfobulbus sp.).

The stock culture produces approximately 1mmol methane per liter of culture per

day during its normal feeding cycle (Jones et al., 2006). For each experiment, culture was

freshly prepared and for the purpose of bioassay application, after one week of feeding

cycle the stock was shaken and allowed to settle down. Culture, devoid of sediments, was

transferred to an anaerobic bottle and pre-incubated until use.


2.13.1 Growth of Anaerobic Microorganisms

Anaerobic microorganisms were grown with Fe (III) as a terminal electron

acceptor. An anaerobic-gas setup was established to flush oxygen out of the medium and

buffer the pH. Containers with a gas tight seal (thick butyl rubber stoppers) were used.

2.13.1.1 Amorphous Ferric Oxyhydroxide (FeOOH)

In case of availability of acetate as electron donor, FeOOH was used as electron

acceptor (Neilands, 1981). The stock solution for FeOOH was prepared as follows;

1. Sodium hydroxide (NaOH) was added to a 0.2M solution of FeCl3 carefully in order

to adjust the pH to 7 (but not over 7).

2. Resulting suspension was washed by centrifuging, decanting and adding deionized

water.

3. Washing step was repeated until the concentration of chloride (Cl-1) was less than

1mM.

4. It was stored as a suspension in refrigerator.

5. It was added into the medium in the ratio of 1:9.

6. The medium was autoclaved, afterwards.

2.13.1.2 Ferric Citrate Medium

For sulphate-reducing bacteria, hydrogen has appeared to be one of the most

important electron donors (Nedwell and Banat, 1981). For this purpose, ferric citrate

medium was prepared;

1. Ferric citrate (50mM) was dissolved in water by heating.

2. Sodium hydroxide was added to increase the pH to 6.

3. Other media components were added and flushed with N2:CO2 (80:20).

4. Medium was sealed and autoclaved.

5. Final pH was 6.8.

2.13.1.3 Fe (III) Nitrilotriacetic (NTA) Stock

Fe (III)-NTA (100mM) stock solution was prepared as follows during the use of

ethanol as electron donor (Neilands, 1981). The preparation was made as follows;


1. Sodium bicarbonate (1.64g) was added in 100 mL of distilled water.

2. Nitrilotriacetic (2.56g) was added.

3. FeCl3 (2.70g) was added.

4. Mixture was gassed out with N2:CO2 (80:20).

5. Filter sterilization was carried out into a sterile, sealed, anaerobic 100mL bottle

(Final pH was 7).

6. Sterile stock was added into the sterile medium in the ratio of 1:9.

2.13.1.4 Fresh Water Medium

Fresh water medium was used in all WBC-2 based bioassay experiments in which

coal was used as sole carbon source (Jones et al., 2006). The composition of fresh water

medium is as follows (g/L);

NaHCO3 2.5

NH4Cl 0.5

NaH2PO4 0.5

KCl 0.1

Trace Minerals 10mL

Vitamins 10mL

Distilled Water 1L

For buffering, medium was flushed with N2/CO2 (80:20), thus maintaining the final

pH of 6.8.

2.13.1.4.1 Composition of Trace Minerals and Vitamins Solution

The composition of trace minerals (g/L) and vitamins solution has been described

in Table 2.7 and Table 2.8, respectively (mg/L) (Jones et al., 2006). For each solution, all

ingredients were mixed thoroughly in 1L of distilled water.


Table 2.7 | Composition of Trace Minerals

Ingredient Concentration

(g/L) Ingredient

Concentration

(g/L)

Nitrilotriacetic Acid 1.5 MgSO4.7H2O 3.0

MnSO4.H2O 0.5 NaCl 1.0

FeSO4.7H2O 0.1 CaCl2.2H2O 0.1

CoCl2.6H2O 0.1 ZnCl2 0.13

CuSO4.5H2O 0.01 A1K(SO4)2.12H2O 0.01

H3BO3 0.01 Na2MoO4 0.025

NiCl2.6H2O 0.024 Na2WO4.2H2O 0.025

Table 2.8 | Composition of Vitamin Solution

Ingredient Concentration

(mg/L) Ingredient

Concentration

(mg/L)

Biotin 2 Folic Acid 2

Pyridoxine-H2O 10 Riboflavin 5

Thiamine 5 Nicotinic Acid 5

Pantothenic Acid 5 Cyanocobalamin 0.1

p-Aminobenzoic Acid 5 Thioctic Acid 5

2.13.2 Bioassay Experimental Design for Methane Generation from Released Organics

The potential of released organics from coal (through fungal-mediated degradation)

to serve as precursors in the methanogenic pathway was assessed using microbial

consortium WBC-2 as a bioassay (Jones et al., 2008). After fungal pretreatment, the filtered

supernatants containing released organics from respective coal samples were purged with

N2/CO2 (80:20) in serum bottles (125mL) and 10% (v/v) WBC-2 culture, trace minerals

and vitamin solution were added. Bottles were sealed with a Teflon coated stopper (West

Co, Lionville, PA, USA) and aluminum crimp (Figure 2.2). Incubation time was extended

to 35 days and experiments were conducted in triplicates. However, methane generation


Teflon Stopper

Serum Bottle

(125mL)

Released Organics

WBC-2

Methane

Accumulation

was statistically non-significant on the contrary to methane generation from native coal

samples.

Figure 2.2 | Experimental Design for WBC-2 Based Bioassay of Released Organics

2.13.3 Bioassay Experimental Design for Native Coal Samples

In anaerobic chamber, 1 or 3g of coal was added to serum bottle (125mL) and 50mL

of fresh water medium was added (Figure 2.3). A 10% inoculum of the culture WBC-2,

trace minerals and vitamins solution, were added to the medium containing coal. Bottles

were sealed with a Teflon coated stopper (West Pharmaceutical Services, Exton,

Pennsylvania, USA) and aluminum crimp. Incubation time was extended to 35 days and

experiments were conducted in triplicates. Quantification of generated methane was carried

out and significant differences were measured.

Figure 2.3 | Experimental Design for WBC-2 Based Bioassay of Native Coal Samples

Aluminum Crimp

Teflon Stopper

Serum Bottle

(125mL)

Fresh Water Medium

WBC-2

Ground Coal

Methane

Accumulation

Aluminum Crimp


2.13.4 Methane Determination

The monitoring of methane was carried out by removing 0.3mL of the headspace

with the help of a gas tight syringe supported with a pressure lock and analyzing by gas

chromatography (GC).

The samples, which produced methane in concentration greater than 0.2mol % were

analyzed on Shimadzu model GC-17A (Shimadzu®, Columbia, MD, USA) split with

isothermal (100ºC) separation of Rt Q-plot (Restek®, Bellefonte, PA, USA) column (30m

x 0.32mm), through flame ionization detector (FID) with 10 times signal reduction. The

samples, which produced methane in concentration less than 0.2mol%, were analyzed

using Hewlett-Packard 5890A (Agilent Technologies, Santa Clara, California, USA) in

splitless mode with isothermal (100C) separation on VOCOl capillary column (Supelco®,

Bellefonte, PA, USA) and an FID. Both GC signals were analyzed using VP Class 7.3

software (Shimadzu®, Columbia, MD, USA).

Methane standards were used for the standardization of instrument responses (Scott

Specialty Gas®, Plumsteadville, PA, USA). As per calculation using Henry’s Law constant

of 28.5, it was assumed that the bottle represented a two-phase (gas/solution) system. The

values measured in the headspace and reported accounted for 95% of the total methane in

the system (Jones et al., 2008).

2.13.5 Spectrophotometric Investigations of Residual Supernatants in Serum Bottles

The residual brown colored supernatant was analyzed on the basis of UV-Vis

spectrophotometry using GENESYSTM 10 UV/Vis Spectrophotometer (Thermo Fisher

Scientific®, Waltham, Massachusetts, USA) for determining the absorption patterns in the

range of 200 to 700nm.

2.14 HUMIC ACID EXTRACTION FROM LOW RANK COAL

Another important facet of fungal treatment of low rank coals was to investigate

the potential of humic acid extraction from lignite after modification of low rank coal

through its pretreatment with fungi. Humic acids were extracted from lignite using two


approaches. First approach was considered as ‘direct approach’, or ‘chemical approach’

in which humic acids were extracted through alkali solubilization of lignite, without any

pretreatment.

In the second approach, coal samples were first treated with fungi and after this

pretreatment step, fungal-transformed lignite was treated with alkali for extraction of

humic acids. After extraction of these two kinds of humic acids from these two approaches,

some of the chemical properties were compared for these in order to develop an assessment

to determine the quality of humic acid. A sketch has also been drawn in order to

differentiate between these two approaches (Figure 2.4).

Figure 2.4 | Approaches for Humic Acid Extraction from Low Rank Coal

2.14.1 Extraction of Humic Acids from Coal by Alkali Solubilization

Coal sample, TP-31, was used for the humic acid extraction. The detailed procedure

of the extraction of humic acid from this coal sample was as follows;

1. Two grams of lignite powder were suspended in 100mL of 0.1M NaOH and stirred at

20ºC for 20 hours.

Low Rank Coal

Alkali Solubilization Alkali Solubilization

Fungal Pretreatment

Humic Acid

Extraction Humic Acid

Extraction


2. Suspension was transferred into 50mL centrifuge tubes, which were centrifuged at

6000xg for 15 min.

3. Supernatant was filtered through Whatman No. 1 paper and the pH was adjusted to 1.8

with 6.0M HCl.

4. The solution was allowed to settle for at least 12 hours and was centrifuged at 8000xg

for 5 minutes to precipitate humic acid.

5. The precipitated humic acid was washed with distilled water three times and dried at

60ºC.

6. Dried humic acid was stored at 4ºC for further analyses.

2.14.2 Extraction of Humic Acids from Fungal-Transformed Lignite

For the extraction of humic acids from fungal transformed lignite, coal/lignite

samples were treated with fungal isolate, which were able to solubilize coal and for this

purpose, a biosolubilization assay was employed (Prakash et al., 2010). The coal residue

was further processed for the extraction of humic acid after fungal pretreatment.

The same coal sample was used as described in section 2.14.1. Fungal isolates were

screened primarily for biosolubilization activity. The step-wise protocol for bioassay was

as follows;

1. Liquid medium with lignite as carbon source was inoculated with coal degrading

fungal isolate. The composition of media was as described as in section 2.8.1.

However, the concentration of glucose was reduced to 0.1% (w/v) and malt extract

was eliminated. Coal was added at the concentration of 2%.

2. Inoculated flasks were kept at 25ºC in a shaker set at 180 rpm.

3. After the incubation period, broth was filtered through Whatman No. 1 filter paper

and the filtrate was used for biosolubilization assay.

4. Concentrated HCl (1mL) was added to the collected filtrate and stirred with

magnetic stirrer. Formation of dense precipitate indicated solubilization of lignite

to humic acid.

5. The fungal isolates, which showed positive results, were further processed.


The residual coal, after fungal pretreatment of coal, was processed for the extraction

of humic acid. After incubation period, liquid media with degraded lignite was suspended

in 100mL of distilled water, stirred and centrifuged at 8000xg for 15 min. The precipitate

was collected to extract humic acid through alkali solubilization as described earlier.

2.15 ANALYSIS OF HUMIC MATERIALS

2.15.1 Elemental Analysis

Elemental analysis of humic acids was carried out in order to determine the

percentage compositions of Carbon (C), Hydrogen (H), Nitrogen (N), Sulphur (S) and

Oxygen (O). PerkinElmer PE 2400 CHNS/O (USA) analyzer was employed for

determining the elemental composition. Percentages of C, H, N and O were determined

experimentally while oxygen was determined by difference method.

% OOxygen = 100 ‒ (%C + %H + %N + %S)

2.15.2 Spectrophotometric Analysis

For the light absorbance studies, extracted humic acid samples were dissolved in

0.05M NaHCO3 and spectra was obtained in the range of 200 to 700 nm using

GENESYSTM 10 UV/Vis Spectrophotometer (Thermo Fisher Scientific®, Waltham,

Massachusetts, USA). The E4/E6 ratio was also determined, which was obtained by

dividing the absorbance at 465nm by absorbance at 665nm.

2.15.3 Fourier-Transform Infrared Spectroscopy (FTIR)

Extracted humic acid samples were ground finely and analyzed on Alpha FT-IR

spectrometer (Bruker®, Billerica, Massachusetts, USA).

3

Results & Discussion

3.1 GEOLOGY OF THE COAL SAMPLES

The Sindh Province samples used in this study were collected from drilling-depths

ranging from 120m (UAJ-1-1) to 304m (TP-4-10). Sindh coals are typically less than three

meters in thickness but the main seam at Sonda was over 6m thick at UAS-4 borehole, and

the main seam intercepted in Thar at TP-3 was over 29m thick (SanFilipo et al., 1989;

SanFilipo et al., 1994a)

The coal samples originated from five coal areas in lower Sindh Province (Table

3.1). Four of these areas near the Indus River are laterally contiguous, but one of them (the

Meting-Jhimpir coal field) is stratigraphically higher than the other three. The sample

(UAJ-1-1) from this field belongs to the Sohnari Member of the Laki Formation, which is

generally considered of Eocene age, but may in fact be a tongue of the Paleocene Bara

Formation (Shah, 1977; SanFilipo et al., 1994b). Metting-Jhimpir coal basin lies in

between Sonda and Lakhra coal fields. The Laki formation have been divided into four

members, which include Meting limestone, Laki limestone member, Shale member, and

Sohnari member (Fatmi et al., 1995). The Sohnari member has been reported to be

comprised of sandstone and lateritic clay, which has been characterized by reddish brown

to yellowish brown in colour (Schweinfurth and Hussain, 1988). Lacustrine conditions may

be the cause of the deposition of Sohnari coalbeds and this environment sustained during

early Eocene timescale before accumulation of marine regime. Later, swamp vegetation,

which kept growing and buried along with brackish water, resulted in the formation of coal

(Outerbridge et al., 1991).

60 Results & Discussion |

Lakhra and Sonda coal field samples are from the Bara Formation, which is mainly

composed of lignite, shale and sandstone (Siddiqui et al., 2011a). Basically, sandstone is

granular containing sub rounded pyrite and glauconite. However, lignite seams were found

in upper 30m (Siddiqui et al., 2011a). Thar coal field is, largely, covered with dune sand

and has no bedrock expression (SanFilipo and Khan, 1994). The overburden is typically

composed of dune sand, Paleocene to Eocene sedimentary rock and alluvium (Fasset and

Durrani, 1994). The age of the thickest coal in Thar has been reported to be of Jurassic age

by Ahmad and Zaigham in 1993, however, these coal-bearing strata may be of late

Paleocene to early Eocene age on the basis of palynological studies of some coal bearing

samples in the close vicinity of borehole TP1 (Ahmad et al., 1993). Fasset and Durrani

(1994) also concluded the age of Thar coal rocks of late Paleocene to early Eocene.

Preliminary evidences indicated the deposition of Thar coal in a raised-bog environment

landward (east) of a north-trending coastline (Fasset and Durrani, 1994).

Precise thicknesses of the samples from Northern Pakistan are not available and

their correlation with Sindh coals is not firmly established. Sample MKCT-6 is from the

Paleocene Hangu Formation of the Makarwal coal field, which is probably correlative with

the Bara Formation of Sindh (Haider et al., 2013). Sample DSA-23-4 is from the Paleocene

Patala Formation of the Salt Range, which could be related to the upper Bara Formation or

the Sohnari interval.

Coalbeds in Patala Formation in the eastern part of Salt Range coal field were

appeared to be deposited in mires of back-barrier environments that were landward of

foreshore and near-shore marine sediments deposited unconformably on Cambrian and

Permian sedimentary rocks (Warwick and Wardlaw, 2007). Warwick and Wardlaw (2007)

also mentioned the average coalbed thickness 0.5m and it was generally greater where the

beds superimposed sandstone bodies, which suggested that sandstone bodies might have

served as platforms for the development of thick peat bodies. The Sample 7 (BLCH) is

from the Eocene Ghazij Formation of the Khost coal field, which may also be related to

the Sohnari Member, or possibly a facies in an overlaying part of the Laki Formation

(Fasset and Durrani, 1994).


Table 3.1 | Geological Information of Coal Samples

Samples Coal Field Depth (m) ASTM Rank*

7 (BLCH) Khost ND hvBb

DSA-23-4 Salt Range 75.66 hvCb

MKCT-6 Makarwal ND hvCb

UAL-15-1 Lakhra 175.58 subC

UAL-15-2 Lakhra 183.10 subC

LS-4-1 Lakhra 172.96 ligA

LS-4-2B Lakhra 191.68 subC

UAS-4-1 Sonda 166.40 ligA

UAS-4-2E Sonda 183.58 ligA

UAT-4-1 Sonda 204.26 subB

UAT-4-4 Sonda 304.46 subC

UAK-1-4 Indus East 183.50 ligA

UAK-1-8 Indus East 249.96 ligA

UAJ-1-1 Meting-Jhimpir 119.90 ligA

TP-1-1.1 Thar 146.38 ligB

TP-1-3.3 Thar 164.97 ligA







Samples Coal Field Depth (m) ASTM Rank*

TP-3-2B Thar 147.90 ligB

TP-3-2D Thar 148.64 ligB

TP-3-2K1 Thar 153.92 ligB

TP-3-2R Thar 160.02 ligB

TP-3-2X Thar 165.52 ligA

TP-3-2AG Thar 173.83 ligB

TP-4-1A Thar 180.91 ligB

TP-4-2A Thar 192.05 ligB

TP-4-6 Thar 223.78 ligB



TP-31 Thar ND ligB

*ASTM Rank from ASTM (2011), D388-05

Samples, with not designated depth (ND), are from underground mines; remaining samples are drilled cores.

3.2 CHEMICAL CHARACTERIZATION OF THE COAL SAMPLES

Chemical characterization, including proximate and ultimate analyses, has been

listed in Table 3.2. The Sindh Province coals, which are the main focus of this study, are

low rank coals, generally ranging in rank from lignite to subbituminous (Table 3.1). The

Thar coals in particular are very young in age; they are high-moisture brown coals with a

conspicuous woody texture somewhat resembling dried peat. The moisture content for the

samples from Thar was observed in the range of 40 to 52% approximately. The sulphur

content of Sindh coals is typically medium (1-3%) to high (>3%) but is laterally and


vertically variable, and can be <1% for some benches of the thicker seams, especially the

Thar “sweet spot” (e.g. the TP-1-3 and TP-3-2 series; Table 3.2) (Wood et al., 1983).

Adjusting to sulphate-free ash would decrease the values of the gross calorific value on the

moist, mineral-matter free basis (BTU/lb m,mmf; Table 3.2) , but with one exception

(UAT-4-1; subB) would not change the ASTM rank for the twelve samples for which the

required data are available.

Ash yield was quite variable between the sample benches (Table 3.2), but tended

towards the lower end of the medium ash range (8-15%) for the main Lakhra-Sonda seams

and the high end for other seams. The average as-received ash for Thar coals is about 9%

but the middle part of the main seam (e.g. samples TP-3-2R to 2AG; Table 3.2) has

typically much lower value (Fasset and Durrani, 1994). The samples from Punjab and

Baluchistan appeared from the areas, which have been more tectonized than the Sindh coal

fields and are of somewhat higher rank (high volatile bituminous, Table 3.2), but are

otherwise similar to Lakhra-Sonda coals. In situ methane desorption was conducted for a

few of the Lakhra South and Thar coal samples (SanFilipo et al., 1994a). Although only a

small amount of coalbed gas was detected in the desorbed samples, SanFilipo (2000) has

suggested additional testing under more rigorous technical and geological constraints.

For elemental analysis of coal, water was excluded from the oxygen and hydrogen

content of the samples. So to make proximate and elemental addition up to 100, moisture

and ash contents were also included to get the final summation of 100 (Table 3.2). The

carbon contents in samples from Sindh were in the range of 24 to 44%, approximately,

however the samples from Khost and Makarwal contained higher carbon contents,

indicative of higher rank nature. All these values for chemical analyses comply with a

number of other analytical investigations over local coal fields of Pakistan, conducted by

various government organizations like Geological Survey of Pakistan (Warwick and Javed,

1990; Fasset and Durrani, 1994).

In terms of heating properties of all these coal samples, the three samples had

calorific value greater than 10,000BTU, i.e., 7 (BLCH), MKCT-6 and DSA-23-4. Other

samples from Sindh coal fields, except from Thar, were found to have heating value in the

range of 8000 to 9000BTU (Table 3.2). However, the Thar coal field samples contained


the lowest calorific values, which may be due to the highest moisture content of these

samples and this is also one of the major technical constraints posed by these low rank

coals. Based upon the quality and chemical characteristics of these coal samples and

comparing these with some initial studies, as well, it can be concluded that the coals of

northern and western Pakistan appeared to be of high rank with high calorific values.

However, the coals from Sindh Province come under the category of low rank, ranging

from lignite to subbituminous and having heating values in the range of 6500 to 7500BTU

(Warwick and Javed, 1990). Particularly, the characteristics of Thar coal field resemble to

the coal fields of west-central India. One of those coal fields is the Panandhro lignite field,

which is 160km southwest of the southwest corner of the Thar field, is considered to be the

third largest lignite reserves in India (Misra, 1992). The moisture content of these coals is

35% with heating values of 6800BTU and ash is 8%, on as received basis (Gowrisankaran

et al., 1987). Likewise, Eocene-age lignite beds have been found to be present in the

Barmer Basin of India, which is 85km northeast of the northeastern corner of the Thar coal

field (Mukherjee et al., 1992). This reserve has been found to be associated with 41 to 50%

moisture content, 12% ash content and average calorific values of 4500BTU

(Gowrisankaran et al., 1987). The similarities of Thar coal to the coals in west central India

are more prominent as compared to other coal fields of Pakistan (Fasset and Durrani, 1994).

However, contrary to the coals of Sindh Province, the coals originating from Northern

Pakistan are high in sulphur and ash contents, though these are medium rank coals.

Atomic oxygen and hydrogen indices with respect to carbon (O/C and H/C) have been

found to be in the range of 0.09 to 0.22 and 0.76 to 1.22, respectively and these values have

appeared to be variable, thus, not following any particular trend (Table 3.3). Generally,

with increasing rank of coal, these indices decrease because of the increase in carbon and

decrease in oxygen and hydrogen contents. With the process of coalification, hydrogen and

oxygen rich components are driven off, thus leaving behind majority of the organic matter

concentrated in fixed carbon. In some of the samples, the values of these indices for Sindh

coals were found to be a bit higher, though not following any strict pattern, than those for

the three samples, i.e., MKCT-6, DSA-23-4 and 7 (BLCH), which appeared to be high rank

coals as compared to other samples for Sindh coal fields (Table 3.3).


Table 3.2 | Chemical Characterization of the Coal Samples

Samples % C % H % N % O % S Moisture

%

Ash

%

Volatile

Matter

%

Fixed

Carbon

%

Calorific Value

(BTU)

m,mmf

7 (BLCH) 66.89 4.28 1.52 8.22 5.67 6.46 6.96 38.07 48.51 13019

DSA-23-4 40.80 3.37 0.47 7.12 3.84 7.05 37.35 26.51 29.09 12563

MKCT-6 59.91 4.67 0.74 13.24 4.09 6.17 11.18 38.56 44.09 12349

UAL-15-1 29.55 2.79 0.59 7.54 2.81 21.84 34.88 25.63 17.65 8679

UAL-15-2 41.43 3.39 0.74 6.50 6.81 25.17 15.96 31.18 27.69 9299

LS-4-1 43.06 2.96 0.93 7.78 3.71 33.63 7.93 29.65 28.79 8220

LS-4-2B 45.00 3.18 0.85 7.31 3.44 29.50 10.72 30.26 29.52 9056

UAS-4-1 43.48 2.95 0.89 9.10 0.93 37.60 5.05 28.75 28.60 8107

UAS-4-2E 44.27 3.13 0.70 8.90 0.77 38.62 3.61 29.18 28.59 8042

UAT-4-1 41.99 3.65 0.67 7.44 4.51 22.22 19.52 37.33 20.93 9835


66 Results and Discussion |



%

Ash

%

Volatile

Matter

%

Fixed

Carbon

%

Calorific Value

(BTU)

m,mmf

UAT-4-4 29.37 2.99 0.53 7.04 4.70 20.72 34.65 25.89 18.74 8758

UAK-1-4 37.56 2.96 0.60 5.63 6.13 31.41 15.71 29.25 23.63 8274

UAK-1-8 29.68 2.69 0.53 5.92 5.20 26.74 29.24 24.91 19.11 7900

UAJ-1-1 24.67 2.42 0.40 6.20 6.75 26.90 32.66 22.97 17.47 7206

TP-1-1.1 31.42 2.34 0.40 7.73 1.26 47.62 9.23 24.41 18.74 5978

TP-1-3.3 37.96 2.85 0.46 9.14 0.31 44.92 4.36 29.58 21.14 6888

TP-1-4.5 31.83 2.16 0.40 7.32 3.10 46.97 8.22 24.71 20.10 6029

TP-1-5.2 33.42 2.42 0.44 7.23 1.42 49.45 5.62 25.73 19.20 6074

TP-1-6.1 32.01 2.16 0.47 8.26 0.72 49.87 6.51 23.40 20.22 5790

TP-3-2B 28.18 2.12 0.31 6.24 4.61 45.66 12.88 23.29 18.17 5705


67 Results and Discussion |



%

Ash

%

Volatile

Matter

%

Fixed

Carbon

%

Calorific Value

(BTU)

m,mmf

TP-3-2D 31.31 2.31 0.32 7.63 1.79 50.57 6.07 24.07 19.29 5781

TP-3-2K1 28.61 2.26 0.28 7.74 1.55 44.81 14.75 23.91 16.53 5948

TP-3-2R 33.81 2.67 0.38 8.87 1.05 48.71 4.51 26.26 20.52 6193

TP-3-2X 34.65 2.76 0.34 8.48 0.40 50.24 3.13 26.99 19.64 6312

TP-3-2AG 30.89 2.41 0.43 9.21 0.38 51.33 5.35 23.07 20.25 5632

TP-4-1A 26.50 2.25 0.38 6.73 2.38 47.62 14.14 22.14 16.10 5484

TP-4-2A 30.57 2.37 0.41 7.42 2.08 47.38 9.77 24.21 18.64 5874

TP-4-6 32.59 2.54 0.46 8.48 0.16 51.25 4.52 25.07 19.16 5895

TP-4-8 28.41 2.28 0.38 8.06 0.39 41.81 18.67 23.72 15.80 6073

TP-4-10 26.04 1.79 0.40 7.19 2.27 45.45 16.46 20.14 17.95 5239

TP-31 27.37 2.79 0.43 7.03 1.75 46.94 13.69 22.70 16.4 5786

* Mineral Matter Free (mmf)


Table 3.3 | Atomic Indices of the Coal Samples

Sample ID Atomic

H/C

Atomic

O/C Sample ID

Atomic

H/C

Atomic

O/C

7 (BLCH) 0.76 0.09 TP-1-4.5 0.81 0.17

DSA-23-4 0.98 0.13 TP-1-5.2 0.86 0.16

MKCT-6 0.93 0.16 TP-1-6.1 0.80 0.19

UAL-15-1 1.12 0.19 TP-3-2B 0.90 0.17

UAL-15-2 0.98 0.12 TP-3-2D 0.88 0.18

LS-4-1 0.82 0.14 TP-3-2K1 0.94 0.20

LS-4-2B 0.84 0.12 TP-3-2R 0.94 0.20

UAS-4-1 0.81 0.16 TP-3-2X 0.95 0.18

UAS-4-2E 0.84 0.15 TP-3-2AG 0.93 0.22

UAT-4-1 1.03 0.13 TP-4-1A 1.01 0.19

UAT-4-4 1.21 0.18 TP-4-2A 0.92 0.18

UAK-1-4 0.94 0.11 TP-4-6 0.93 0.19

UAK-1-8 1.08 0.15 TP-4-8 0.96 0.21

UAJ-1-1 1.17 0.19 TP-4-10 0.82 0.21

TP-1-1.1 0.89 0.18 TP-31 1.22 0.19

TP-1-3.3 0.89 0.18 ‒ ‒ ‒


3.3 MACERAL ANALYSIS OF THE COAL SAMPLES

Maceral groups and mineral contents have been represented in volume percentages

(volume %), along with maximum vitrinite reflectance (Rmax, %) and mean random

vitrinite reflectance (Rm, %; Table 3.4). However, on the basis of mineral matter free

(mmf), these values have been tabulated in Appendix II.

Mean random huminite reflectance (Rm) in oil ranges from 0.22 to 0.51% and by

convention, the maceral group is referred to as huminite if reflectance is less than 0.5;

otherwise it is categorized as vitrinite (Table 3.4). Keeping this convention in mind, only

three samples 7(BLCH), MKCT-6 and LS-4-1 were found to have vitrinite content with

the random vitrinite reflectance values of 0.74, 0.63 and 0.51% respectively, while all other

samples reflected huminite content. However, in the sample, TP-31, the presence of highly

structured vitrinite was observed with approximately equal proportions of telo- vs.

detrovitrinite. Generally, it is believed that huminite/vitrinite group of maceral originate

from woody plant material such as stems, roots, branches etc., usually derived from

cellulose and lignin part of the plant (Taylor et al., 1998). Vitrinite may be subdivided on

the basis of the degree of the preservation of cell structure. For all samples from Thar coal

field, huminite content ranged from 34.0 to 79.2% (on volume percent basis).

Inertinite group of maceral is derived from plant material that is highly altered

during peat diagenesis and this is associated with high degree of aromatization and

condensation (Taylor et al., 1998; Scott and Glasspool, 2007). In addition, inertinite is

believed to have greater carbon content as compared to the respective liptinite and huminite

contents because of the high degree carbonization and biological modification before

undergoing the coalification process (Scott and Glasspool, 2007). In one sample from

Lakhra, LS-4-2B, a greater extent of inertinite content was observed (34.2%) as compared

to the general trend from all of the samples used. Likewise, for liptinite content, TP-1-5.2

and TP-3-2X were observed to have 21.2 and 21.0%, slightly deviating from the general

trend (Table 3.4).

Liptinite is thought to be originated from pollen, spores, cuticles, waxes and resins

etc. Furthermore, liptinite contains more hydrogen and aliphatic moieties within the


structure as compared to other coal maceral groups (Taylor et al., 1998). However, in

comparison with vitrinite/huminite, the contents of liptinite are more resistant to the

chemical or physical change (Scott, 2002). The sample, TP-31, had liptinite and Inertinite

content less than 10% by volume. Mineral matter content was 11.7% by volume. However,

Rmax value, which was 0.53, tended to be higher as compared to all other Thar coal

samples from USGS (USA) archives. This may be due to the possible increased weathering

of the sample, TP-31, as compared to the other samples from the same coal field. For

mineral content in petrographic analysis, a mixed trend was observed with maximum

volume percentage of 68.6 in case of UAK-1-8 (Table 3.4).

Though, previously, detailed petrographic studies of coal basins in Pakistan were

not conducted at the time when these coals were discovered and even later, however,

recently a number of other research groups have also tried to cover this aspect. Nawaz et

al. (2010) reported the mineral matter, in the form of shale, coarse and fine grained pyrite

and specks of kaolinite, in a sample, which originated from Makerwal coal field. The

vegetal matter found to be related to good grade macerals and on the basis of X-ray

diffraction (XRD) and microscopic studies, it was concluded that Makerwal sample

belonged to subbituminous rank (Nawaz et al., 2010). Rizvi (2011) described some

preliminary petrological investigations of basement rocks at Thar Basin. The basement

rocks were identified as alkali-feldspar granite, granodiorite, rhyolite, rhyodacite, and

aplite (Rizvi, 2011).

Cleat fractures and matrix porosity were determined in Lakhra and Thar coals using

scanning electron microscopy (SEM) in order to investigate the potential and presence of

possible coalbed methane (CBM) (Siddiqui et al., 2011b). The porosity in coal is developed

by micropores (matrix) and cleats facilitate the permeability for the flow and recovery of

coalbed methane. Siddiqui et al. (2011b) suggested the prospects of methane gas from

macro cleats in Lakhra.

Representative photomicrographs of Thar coal sample (TP-3-2X) have been shown

in Figure 3.1, representing the fluorescent liptinite maceral. Under UV fluorescence,

liptinite appeared as a strong yellow or green, vitrinite showing poor fluorescence while

inertinite gave nothing.


Table 3.4 | Petrography of the Coal Samples

Sample Maceral Groups and Mineral Content (Volume %)

RMax

(%)

RRan

(%)

Rm Rank

of Coal Vitrinite/Huminite Inertinite Liptinite Minerals

7 (BLCH) 91.2 2.0 1.6 5.2 0.77 0.74 hvBb

DSA-23-4 50.4 14.6 2.4 32.6 0.45 0.42 subC

MKCT-6 77.0 7.8 3.8 11.4 0.65 0.63 hvCb

UAL-15-1 30.0 6.2 3.4 60.4 0.49 0.45 subB

UAL-15-2 52.2 11.4 7.8 28.6 0.49 0.45 subB

LS-4-1 79.2 10.8 3.6 6.4 0.54 0.51 subA

LS-4-2B 44.2 34.2 8.8 12.8 0.51 0.48 subB

UAS-4-1 76.0 14.0 5.4 4.6 0.53 0.49 subB

UAS-4-2E 72.8 17.0 6.6 3.6 0.51 0.47 subB

UAT-4-1 52.6 12.6 15.0 19.8 0.50 0.46 subB

UAT-4-4 33.0 0.4 5.2 61.4 0.46 0.41 subC

UAK-1-4 69.2 6.8 4.6 19.4 0.49 0.45 subB

UAK-1-8 20.4 9.2 1.8 68.6 0.43 0.39 subC

UAJ-1-1 34.4 0.0 5.6 60.0 0.47 0.42 subC

TP-1-1.1 48.8 0.8 3.6 46.8 0.40 0.37 ligA

TP-1-3.3 54.0 2.4 5.6 38.0 0.30 0.27 ligB




Sample

Maceral Groups and Mineral Content (Volume %) Rmax

(%)

Rm

(%)

Rm Rank

of Coal Vitrinite/Huminite Inertinite Liptinite Minerals

TP-1-4.5 74.8 3.6 12.0 9.6 0.33 0.28 ligB

TP-1-5.2 71.6 0.0 21.2 7.2 0.35 0.30 ligB

TP-1-6.1 79.2 1.6 12.0 7.2 0.35 0.31 ligB

TP-3-2B 69.5 1.3 10.6 18.6 0.31 0.27 ligB

TP-3-2D 68.4 2.4 14.4 14.8 0.32 0.27 ligB

TP-3-2K1 41.0 0.0 12.0 47.0 0.26 0.22 ligB

TP-3-2R 76.0 1.2 16.8 6.0 0.34 0.30 ligB

TP-3-2X 34.0 7.0 21.0 38.0 0.33 0.30 ligB

TP-3-2AG 68.4 1.2 4.8 25.6 0.33 0.28 ligB

TP-4-1A 52.0 1.6 6.8 39.6 0.35 0.30 ligB

TP-4-2A 60.4 1.6 6.0 32.0 0.39 0.34 ligB

TP-4-6 71.2 2.0 7.2 19.6 0.37 0.33 ligB

TP-4-8 34.8 2.8 10.0 52.4 0.38 0.34 ligB

TP-4-10 73.6 0.4 9.6 16.4 0.38 0.34 ligB

TP-31 77.3 2.6 8.4 11.7 0.53 0.48 SubB


Figure 3.1 | Photomicrographs of Thar Coal Sample (TP-3-2X)

(Strong yellow or green fluorescence showing liptinite maceral)


3.4 FUNGAL ISOLATES

3.4.1 Sample Collection and Origin of Fungal Isolates

Total fifty three (53) fungal strains were isolated from various environmental

habitats, which included samples from degrading trees, coal mine soil, weathered coal

lumps, and drilled coal core. Twenty nine (29) isolates originated from three (3) degrading

tree samples, collected from trees alongside Canal Road, Lahore. The degrading trees offer

an opportunity for the isolation of fungal species, possibly, involved in lignin degradation.

These fungal isolates may also be capable of modifying the structure of coal because of the

resemblance of low rank coal to lignin on the basis of chemical structure. Likewise, twenty

one (21) fungal distinctive colonies appeared from five (5) coal mine soil samples, which

were obtained from Lakhra coal field. The soils in coal mines are enriched with coal as

carbon source and the isolated fungi from these soils may have better ability to degrade

coal, particularly low rank coals.

One (1) fungal isolate, initially designated as MW1, was isolated from drilled core

of sub-bituminous coal sample, which originated from a well drilled by United States

Geological Survey (USGS), in the Tongue River portion of the Powder River Basin,

Montana, USA. It was stored in an anaerobic chamber for about a year. Two (2) other

significant fungal strains, HC1 and KB1, were also isolated from two Romanian brown

coals (samples originated from coal fields of Husnicioara and Kolubara, Romania) on

which the presence of fungal spores was quite evident and these coals were observed to

contain apparent conspicuous woody structure. On the basis of the attachment of microbes

to coal surfaces, drilled cores have also been found to be one of the most appropriate

habitats for isolating coal solubilizing fungi (Yossifova et al., 2011). Pokorny et al. (2005)

isolated various fungal isolates from freshly excavated lignite from Zahori coal mine,

Slovakia.

The adaptation of these fungal isolates to the coal environment may be an important

selection criterion for conducting fungal degradation studies. Although, in majority of

reports, coal solubilization was carried out by the fungal isolates, which were cultured from

mine soil and rotten wood samples (Yuan et al., 2006b; Yin et al., 2009a; Tao et al., 2009).


However, fungal strains, isolated from coal environment may have enhanced capability for

degradation of coal (Tao et al., 2010).

3.4.2 Primary Screening of Isolates for Coal Degrading Activity

The primary screening for coal degrading activity was determined on the basis of

the formation of black droplets from coal particles, which were sprinkled over fungal

mycelia, grown on malt extract agar (MEA) medium. The extent of release of organics was

estimated on UV-Vis spectrophotometer (Section 2.9). The minimal slats medium,

supplemented with ammonium sulphate, has been reported to be one of the most effective

medium used for coal degradation (Silva-Stenico et al., 2007; Oboirien et al., 2008).

This primary screening on the basis of spectrophotometric properties has been the

most widely used methodology for determining the coal degradation activity (Gokcay et

al., 2001; Yuan et al., 2006a; Elbeyli et al., 2006; Yin et al., 2009a; Tao et al., 2009). In

the presence of coal, the growth of fungal isolates in submerged conditions was

characterized by the presence of fungal mycelia trapping coal particles inside, which were

also observed under phase contrast microscope (Figure 3.2).

Upon screening all fungal isolates, the formation of black or brown droplets on coal

particles was not significant, rather water droplets surrounded coal particles over fungal

mycelia. However, the release of organics in the range of 250 to 350nm was comparable

among all these isolates and HC1, KB1 and MW1 appeared to liberate significant organics

in these ranges with intensified peaks in the range of 200 to 350nm on the basis of UV-Vis

scan patterns. The absorbance intensity at 450nm for MW1 in the presence of coal was

higher as that for HC1 and KB1. This release of organics may be attributed to the origin of

these isolates from coal environment. Among these three fungal isolates, MW1 appeared

to be the most potent in terms of absorbance intensities for the organics, released during

the incubation time of 7 days, in the above mentioned spectrophotometric range, which

was also comparable with some previous studies (Tao et al., 2009; Yin et al., 2009a)

(Figure 3.3).


. Figure 3.2 | Growth of MW1 in the Presence of Coal (A) In Submerged Conditions

(B) Fungal Mycelia Trapping Coal Particles (Under Phase Contrast Microscope (100X))

Figure 3.3 | UV-Vis Scan Pattern of Released Organics from Coal by MW1 Isolate

Maximum absorption intensity was at 290nm with 0.211 by the treatment with

MW1, however, in case of HC1 and KB1, though the peaks were observed in this range

but those were not that much intensified and their absorption intensity did not exceed 0.1.

Yin et al. (2009a) reported the significant release of organics in the range of 200 to 300nm,

Wavelength (nm)

Ab

sorp

tion

Inte

nsi

ty

Coal Particles Fungal Mycelia

(A) (B)


which was indicative of the existence of unsatisfied chemical bonds in the liberated organic

moieties. Particularly, the presence of aromatics was responsible for the peaks in the range

of 260 to 300nm (Yin et al., 2009a). Tao et al. (2009) also reported the presence of alkyl

substituted unsaturated aldehyde and ketones, which showed peaks at 240nm after 15 days

of incubation time. In case of MW1 fungal isolate, the presence of peak at 240nm was also

observed, which could be related to the presence of organic functionalities as described

above.

By comparing the intensities of these peaks by MW1 with other fungal isolates HC1

and KB1, the isolate MW1 seemed to be most efficient in terms of providing microbial

substrates from coal matrix for subsequent transformation. Though, HC1 and KB1, were

also isolated from coal lumps, however, those coal samples belonged to very low rank and

somewhat resembling to dried peat, which retained conspicuous woody structure, as well.

So they might not be regarded as pure coal environment, however, MW1 was isolated from

inside core of subbituminous coal, which may be true representative of low rank coal

family. In short, primary screening indicated MW1 as a viable fungal isolate for the

extraction of microbial substrates for subsequent methanogenesis.

3.4.3 Optimization of Coal Degrading Activity

The pretreatment of coal with fungi needed to be optimized for enhanced release of

organics for subsequent methanogenesis. The considerable factors, which could affect the

growth and coal degrading ability of fungal isolate, MW1, were studied and optimized.

These included incubation time of pretreatment, coal loading ratio and the concentration

of glucose, which have been reported to be significant and key-factors, affecting the whole

process of coal solubilization (Gokcay et al., 2001; Silva-Stenico et al., 2007; Yin et al.,

2009a). For determining the growth and activity of fungi for releasing organics from coal,

absorbance intensity was determined at 240nm as also adopted by Tao et al. (2009). In all

pretreatment experiments, ground coal particles (≤850) were used instead of smaller coal

chunks as carbon source, as reduced particle size have already been reported to be more

prone to enhanced degradation (Oboirien et al., 2008). Suspensions of coal were treated

with fungal isolate using methods described in section 2.9. The two controls were medium

with coal but without inoculation and medium with inoculation but without coal. The


intensity of the release of organics in controls was not comparable with that of fungal

pretreatments of coal in the required UV-Vis spectrophotometric region (Figure 3.4A). The

major peak at 300nm in the control, containing coal in the medium, may be the possible

release of hydrophilic humic contents in coal, which could be regarded as the function of

medium. However, in this control, the absorbance at 240nm was negligible, thus, indicating

the absence of smaller organic fractions (Figure 3.4B).

Figure 3.4 | UV-Scan Pattern for Controls

(A) Medium with Inoculation but without Coal

(B) Medium with Coal but without Inoculation


3.4.3.1 Effect of Glucose

The presence of glucose, as supplemental carbon source, at varying concentrations

of 0.1%, 0.5% and 1.0% (w/v), was investigated for determining the extent of release of

organics with coal loading ratio of 1.0% (Figure 3.5). Coal pretreatments were carried out

in the absence of glucose as well. In terms of absorption intensity at 240nm, 0.1% glucose

concentration appeared to be optimum for which the release of organics was significant.

On the other side, in the absence of glucose, absorption intensity decreased, which showed

the possible effect of glucose, to particular extent, on coal degradation activity. The

presence of glucose in media with coal as primary carbon source has been reported to

enhance coal degradation (Gokcay et al., 2001; Silva-Stenico et al., 2007). Silva-Stenico

et al. (2007) reported the addition of glucose at 5g/L (0.5%) for enhanced degree of coal

solubilization with coal loading ratio of 10%. However, the increase of glucose addition

up to the level of 1% and 2% has also been reported to decrease the efficiency of coal

degradation (Silva-Stenico et al., 2007).

On the contrary, Gokcay et al. (2001) suggested the increase of concentration of

glucose up to the level of 1% and 2% for enhanced solubilization of coal. The findings of

Silva-Stenico et al. (2007) were found to be consistent with this study, as well, in which

enhanced glucose concentration resulted in the reduction of absorption intensity at 240nm.

Selvi et al. (2009) also reported the higher support of growth by glucose for depolymerizing

activity of low rank Indian coals. Likewise, Silva-Stenico et al. (2007) reported decreased

solubilization efficiency in the absence of glucose. The presence of glucose may be

involved in supporting the growth of fungi initially, as supplementing the advancement of

coal degradation activity. Afterwards, the reduced glucose concentration and coal-rich

environment could, possibly, induce extracellular enzymes for making coal into utilization

(Selvi et al., 2009; Yin et al., 2009b).

It can be concluded that the 0.1% glucose concentration may be involved in

optimum growth of fungi, leading to increase in coal degrading activity. Higher glucose

concentrations i.e., 5% and 10%, may have led to the formation of flock-like structures of

fungal mycelia due to dense growth, thus posing oxygen as limiting factor for enzymatic

activities of MW1 and decreasing coal degrading activity, ultimately.


Ab

sorp

tion

Inte

nsi

ty

Wavelength (nm) A

bso

rpti

on

Inte

nsi

ty

Wavelength (nm)

Ab

sorp

tion

Inte

nsi

ty

Wavelength (nm)

Ab

sorp

tion

Inte

nsi

ty

Wavelength (nm)

Figure 3.5 | Effect of Glucose Concentration on Release of Organics

Absence of Glucose

Glucose 0.1%

Glucose 0.5%

Glucose 1.0%


3.4.3.2 Effect of Incubation Time

Coal samples were treated with fungal isolate MW1 for 7 days and 14 days. Longer

incubation could have possible effect on release of organics, however, interestingly, the

absorbance at 240nm reduced in case of 14 days (Figure 3.6). Coal was added at the

concentration of 1% and glucose was added as supplemental carbon source with the

concentration of 0.1% into minimal salts medium.

Figure 3.6 | Effect of Incubation Time on Release of Organics

(A) After 7 Days (B) After 14 Days

A (After 7 Days)

B (After 14 Days)

Wavelength (nm)

Wavelength (nm)

Ab

sorp

tion

Inte

nsi

ty

Ab

sorp

tion

Inte

nsi

ty


In case of fungal pretreatment after 14 days, there was significant reduction in the

peaks, indicative of decrease in organics with time, between wavelengths of 200 to 300nm.

Besides, the peak at 240nm was also absent in case of longer pretreatment of coal with

isolate MW1. There may be possibility of the utilization of organics by fungi itself, thus,

leaving less enriched supernatants behind. Such kind of effect of longer incubation and

reduced absorption intensity at 240nm was also observed by Tao et al. (2009). With

extending incubation to 15 and 19 days from 9 days, reduction in absorption intensity at

240nm was observed (Tao et al., 2009).

Initially, in the presence of coal as carbon source, fungal activity may be enhanced

in terms of making coal more bio-available by release of organics from coal, which could

subsequently be used by fungi for the growth. For obtaining, maximum organics for

subsequent biological transformation of coal by methanogens, the cessation of fungal

pretreatment at certain level may be required.

3.4.3.3 Effect of Coal Loading Ratio

The coal loading ratio may have important effect on the extent of coal

solubilization. The coal pretreatments were conducted with MW1 using varying coal

loading ratio including 1%, 2%, and 5% (w/v) (Figure 3.7). The coal loading ratio of 1%

was found to be optimum for the extent of release of organics; however, increasing coal

loading ratio may have inhibitory effect on the growth of fungi, thus resulting in

suppression of the peaks in the range of 200 to 300nm wavelength, indicative of the release

of organics.

In another study, conducted by Yuan et al. (2006b), the coal loading ratio of 1.0%

was found to be optimum for the degradation and extraction of humic acid from low rank

coal. Tao et al. (2009) also used 1.5% (w/v) suspension of low rank coal and inoculated

the suspension with Trichoderma sp. AH, which solubilized coal to greater extent. The

higher coal loading ratio may cause cell damage (Nemati and Harrison, 2000; Harrison et

al., 2003). However, on the contrary to this study, Oboirien et al. (2008) reported optimum

coal loading between 5 to 10% (w/v) for coal solubilization in a slurry bioreactor.


Figure 3.7 | Effect of Coal Loading Ratio on Release of Organics

Wavelength (nm)

Wavelength (nm)

Wavelength (nm)

Ab

sorp

tion

Inte

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Ab

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tion

Inte

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ty

Ab

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tion

Inte

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Coal Loading Ratio 5%




3.4.4 Characterization of MW1

Initially, the fungal colonies of MW1 appeared to be white in color, turning to green

later after 3 to 4 days of growth. The colonies of MW1 on MEA plates appeared to be

filamentous and velvety. It formed septate and colorless hyphae. Under microscopic

examination, the structure of conidiophores was found to be branched (Figure 3.8). The

isolation of MW1 from a coal-bearing environment also suggested that fungal spores were

able to survive in this coal seam over a long period of time, somehow (Haider et al., 2013).

Figure 3.8 | Morphological Features of MW1 (On right, Phase Contrast Image of Mycelia: 100X)

The molecular typing of fungal strain was carried out on the basis of PCR

amplifications of ITS regions after the extraction of genomic DNA from MW1 (Figure

3.9). The length of the PCR product was 510bp. The PCR products were commercially

sequenced and sequences of amplicons were searched for similarities through NCBI

BLASTn. The MW1 appeared to have homology (Maximum Identity of 97%) with

Penicillium chrysogenum QML-2. For MW1 isolate, the sequence of partial ITS region 1,

5.8S ribosomal RNA gene and partial ITS region 2 was submitted to NCBI (Accession

Number JN572146.1).


Figure 3.9 | PCR Amplification of ITS Regions

3.4.5 Phylogenetic Analysis of MW1

For the construction of phylogenetic tree, it was tried to infer a relationship of MW1

with some other already reported coal degrading and wood/lignin degrading fungi. Table

3.5 shows percentage sequence identity in ITS regions of MW1 and other selected isolates

that were selected for the construction of phylogenetic tree using softwares, ClustalX and

TreeView (ver 1.6.5). According to nucleotide BLASTn (NCBI, www.ncbi.nlm.nih.gov)

results for the ITS gene sequence of the isolated fungal strain MW1, the top hit and highest

score was obtained with Penicillium chrysogenum strain QML-2 (Accession Number

JF907010), with 97% sequence identity to the sequence of strain MW1. The percentage

identity values for the ITS gene sequence of MW1 to other coal solubilizing/degrading

species with validly published names were in the range of 90-97% (Table 3.5).

100bp

300bp

500bp 650bp

Lane 1,12,13 = Kb Plus DNA Ladder

Lane 2,3,4 = MW1 Isolate

Lane 5,6,7 = HC1 Isolate

Lane 8,9,10 = KB1 Isolate

Lane 11,14 = FO Isolate

Lane 15 = Positive Control

Lane 16,17,18 = HC2 Isolate

Lane 19 = Negative Control

13 14 15 16 17 18 19

1 2 3 4 5 6 7 8 9 10 11 12

http://www.ncbi.nlm.nih.gov/


Table 3.5 | Percentage Sequence Identity in ITS Regions of Selected Isolates with MW1

Accession Number Isolate % Identity % Gap

JF907010 Penicillium chrysogenum QML-2 97 1

JQ670962 Penicillium chrysogenum GW20-4 97 1

JQ776534 Penicillium chrysogenum SGE6 97 1

DQ489298 Trichoderma atroviride ES11 90 2

FJ645728 Hypocrea lixii TZ1 92 2

EF524042 Trametes versicolor CTB 863 90 1

FJ235970 Fungal sp. AB37 97 1

- Trichoderma sp. AH 92 2

JX406503 Penicillium chrysogenum WGS11799 97 1

JX519346 Penicillium sp. KU-DFR9 97 1

JX232275 Penicillium sp. SGE28 97 1

AY157490 Penicillium decumbens CBS230.81 89 2

JN572145 Aspergillus Oryzae DT1 86 6

KC013272 Trichoderma reesei 92 1

JX184121 Hypocrea rufa strain AN242 90 2

GQ331986 Paraconiothyrium sp. GHJ-4 89 2

JQ673086 Ceriporiopsis subvermispora isolate DLL2010-072 92 1

AB519182 Phlebia brevispora 89 1

EF491864 Phlebia sp. b19 88 1

In order to determine the general phylogenetic position of MW1, the neighbor-

joining tree of the 19 aligned ITS gene sequences (19 reported isolates; Figure 3.10) was

constructed with 100 bootstrap replications and the tree was rooted with Trametes

versicolor strain CTB 863 clone (Accession Number EF524042), one of the strains

commonly used in wood degradation studies. It is evident from the phylogenetic tree that

MW1 did not form the clad with other reported coal degrading fungal strains i.e.,

Trichoderma atroviride ES11, Hypocrea lixii TZ1 and Trichoderma sp. AH; however, it


formed the major clad with some of the Penicillium spp. On the basis of above mentioned

inferred information, it can be concluded that the isolate MW1 may be novel strain in terms

of coal degrading activity.

Figure 3.10 | Inferred Relationship Based on Partial ITS Sequences of MW1 isolate to other Fungal

Isolates (Scale bar represents the number of inferred nucleotide substitutions per site. Bootstrap

values have been shown at the nodes)

0.1

Penicillium chrysogenum WGS11799

Penicillium chrysogenum SGE6

100

Penicillium chrysogenum GW20-4

Penicillium chrysogenum QML-2

92

Fungal sp. AB 37

63

Penicillium sp. KU-DFR9

Penicillium sp. SGE28

88

MW1

96

Penicillium decumbens CBS230.81

98

Aspergillus Oryzae DT1

100

Trichoderma sp. AH

Hypocrea lixii TZ1

100

Trichoderma reesei

67

Trichoderma atroviride ES11

Hypocrea rufa strain AN242

82

100

100

Paraconiothyrium sp. GHJ-4

100

Ceriporiopsis subvermispora isolate DLL2010-072

65

Phlebia brevispora

Phlebia sp. b19

100

Trametes versicolor CTB 863


3.5 FUNGAL PRETREATMENT OF COAL SAMPLES

Twelve low rank coal samples (UAS-4-2E, UAK-1-4, UAJ-1-1, 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 and TP-4-10) were treated

with fungal isolate MW1. The preference for selecting the coal samples for fungal

pretreatment was based on upright position of the coal samples in drilled cores. Besides,

the choice of low rank coal samples was made on the basis of supposed susceptibility of

low rank coals to biological attack or biodegradation. Among these, 4 samples (LS-4-1,

LS-4-2B, TP-3-2B and TP-3-2X) were desorbed samples, which were also tested for any

coal seam gas at the time of drilling. Though only small amount of gas was detected at that

time but the effect of fungal pretreatment of these samples was also worth-determining

(SanFilipo, 2000).

The presence of higher oxygen contents in low rank coals, in the form of oxygen

linkages bridging aromatic clusters, may facilitate fungal attack on structural matrix of

coal, which releases a variety of organics. Low rank coals have always been a target of

interest for microbial modification of coal (Gokcay et al., 2001; Yuan et al., 2006b; Yuan

et al., 2006a; Silva-Stenico et al., 2007; Yin et al., 2009a; Tao et al., 2009; Yin et al.,

2009b). Though, there have been reports of fungal degradation of hard coal, as well, but,

the structural resemblance of low rank coal to lignin makes degradation of low rank coal

more favourable (Igbinigie et al., 2008; Mutambanengwe, 2009; Igbinigie et al., 2010).

The aromatic condensation in higher rank coals may have inhibitory effect on fungal

growth, which could probably result in decreased coal solubilization activity. In this regard,

coal degradation does not seem to be reliant only on finding an appropriate biological

agent; however, it is also a function of the chemical structure and rank of the coal.

The adaptation of MW1 to coal-bearing environment may have eased the enhanced

release of organics from low rank coals, which was shown in preliminary qualitative

investigations of released organics. The probable relatedness of MW1 to Penicillium

species, being ubiquitous soil fungi, also suggested that the fungal spores survived for a

long period of time along with coal debris. Yuan et al. (2006b) also isolated Penicillium

sp. P6 from coal mine soil, which was capable of degrading and solubilizing lignite.

Penicillium species have been reported to degrade coal, however, majority of these were


isolated from coal mine soil and rotten wood etc. (Polman et al., 1994b; Achi, 1994; Yuan

et al., 2006b). On the contrary, MW1, originated from a coal environment (Haider et al.,

2013).

On the basis of optimization studies, in the degradation experiments coal served as

primary source of carbon, however, in the presence of carbon as supplementing agent,

because of proposed effect of glucose on enhanced degradation of coal. During the course

of degradation, coal particles were found to be trapped within fungal mycelia, which can

be attributed to the fact that fungal mycelia might have surrounded the coal particles. In

nature, this mycelia extension may function to break up the coal matrix by releasing

extracellular enzymes. After the incubation of seven days, the color of filtrates appeared to

be light brown, which may be indicative of the release of humic moieties into the media.

Brown coals contain larger fractions of humic acids, in general.

3.6 ANALYTICAL INVESTIGATIONS OF EXTRACTS

3.6.1 Excitation-Emission Matrix Spectroscopy (EEMS)

For the qualitative analysis of coal degradation through fungi, extracts from fungal

pretreatment were analyzed on excitation emission matrix spectroscopy (EEMS).

Fluorescence spectroscopy has appeared to be one of the well-established techniques for

fingerprinting of dissolved organic matters in waters and complex mixtures, containing

variety of chromophores, including humic-like fluorophores (Bieroza et al., 2012). So far

the released organics from fungal degradation or solubilized coal have not been subjected

to such kind of fluorescence analysis.

In number of other reports, UV-Vis spectroscopy has been used widely as

qualitative analytical technique for this purpose (Laborda et al., 1999; Yin et al., 2009a;

Tao et al., 2009). However, as compared to conventional UV-Vis spectroscopy, three

dimensional EEMS offers some advantages as it is considered to be highly sensitive and

selective technique for compounds containing aromatic rings, exhibiting fluorescence

properties (Parlanti et al., 2000). Keeping in view the heterogeneous nature of coal and the

possible release of aromatic moieties after fungal degradation, fluorescence spectroscopy


was employed for determining the presence of organics in filtrates. The Figure 3.11 shows

typical EEM spectra for growth of MW1 as control.

Figure 3.11 | Excitation-Emission Matrix Spectra for MW1 Culture (Control)

Spectra from control (medium with coal but without inoculation, on right side) and

experimental flasks (on left side) have been shown together in order to elucidate the

comparison. In all treatments including controls containing coal, except the control of

MW1 culture (without coal), a major peak was observed in the range of 325nm to 350nm,

which may be characteristic peak of the release of fulvic-acid like structural units into the

media (Ghabbour and Davies, 2009). However, distinctive peaks (encircled black in all

illustrations showing EEM spectra) in the range of 250nm to 350nm, which were absent in

controls, provided a qualitative indication of the release of organics pertaining to aromatic

rings, alkyl substituted unsaturated aldehydes and ketones, after fungal pretreatment (Yin

et al., 2009a; Tao et al., 2009).

Substantial release of organics was observed in representative samples of Thar coal

from borehole TP1 i.e., TP-1-1.1 and TP-1-5.2, after fungal pretreatment. This may,

possibly, be because of lignitic nature of these samples. Besides, this may also be a function

of some particular maceral group. Interestingly, the release of organics in sample, TP-1-

5.2, was comparably higher than that of TP-1-1.1 and this may be because of higher


liptinite content (21.2%) in sample TP-1-5.2 as compared to the sample TP-1-1.1 (Figures

3.12 & 3.13). Higher aliphatic and hydrogen contents in liptinite macerals may facilitate

biological degradation (Scott and Glasspool, 2007).

Figure 3.12 | Excitation-Emission Spectra for TP-1-1.1 (Left) Fungal Treatment (Right) Control

Figure 3.13 | Excitation-Emission Spectra for TP-1-5.2 (Left) Fungal Treatment (Right) Control


From samples of TP3 borehole, three representative samples, TP-3-2B, TP-3-2X

and TP-3-2K1, were treated with MW1 and almost same pattern of peaks was observed in

EEM spectra (Figures 3.14, 3.15 & 3.16).

Figure 3.14 | Excitation-Emission Spectra for TP-3-2B (Left) Fungal Treatment (Right) Control

Figure 3.15 | Excitation-Emission Spectra for TP-3-2X (Left) Fungal Treatment (Right) Control


Figure 3.16 | Excitation-Emission Spectra for TP-3-2K1 (Left) Fungal Treatment (Right) Control

The liptinite contents of the samples TP-3-2B, TP-3-2X and TP-3-2K1were 10.6%,

21.0% and 12.0%, respectively. For the samples from TP4 borehole, i.e., TP-4-2A and TP-

4-10, some organics were observed to be released, though the liptinite contents were not

up to the level as for other samples from TP1 and TP3 boreholes (Figures 3.17 & 3.18).

Figure 3.17 | Excitation-Emission Spectra for TP-4-2A (Left) Fungal Treatment (Right) Control


Figure 3.18 | Excitation-Emission Spectra for TP-4-10 (Left) Fungal Treatment (Right) Control

From Lakhra coal field, two samples i.e., LS-4-1 and LS-4-2B, were experimented

for fungal degradation and released organics were subjected to fluorescence spectroscopy

(Figures 3.19 & 3.20).

Figure 3.19 | Excitation-Emission Spectra for LS-4-1 (Left) Fungal Treatment (Right) Control


Figure 3.20 | Excitation-Emission Spectra for LS-4-2B (Left) Fungal Treatment (Right) Control

Significant organics were also released from these coal samples of Lakhra coal

field, which also appeared to be higher in rank as compared to the samples from Thar coal

field. In this way, it can be suggested that the fungal isolate MW1 was also found to be

capable of degrading coal of subbituminous ranks. Secondly, the pattern of liptinite

content, affecting the degree of organics release, was not followed strictly in one sample

i.e., LS-4-1 with very low liptinite content (3.6%), however the increased vitrinite group

(79.2%) may also affect and aid the process of fungal degradation. By comparing these two

samples with each other, the liptinite content of the sample, LS-4-2B, was 8.8%, which

was greater than that of LS-4-1 and this may be regarded in terms of the more intensified

and resolved peaks, pertaining to the organics, of the sample LS-4-2B as compared to the

sample LS-4-1.

The samples from Metting-Jhimpir (UAJ-1-1) and Indus-East (UAK-1-4)

coalfields did not release any significant organics, however, UAS-4-2E, sample from

Sonda, showed some degradation in terms of the presence of some organics in the filtrate

(Figures. 3.21, 3.22 & 3.23). The liptinite contents for these three samples were in the range

of 4 to 6% approximately.


Figure 3.21 | Excitation-Emission Spectra for UAS-4-2E (Left) Fungal Treatment (Right) Control

Figure 3.22 | Excitation-Emission Spectra for UAJ-1-1 (Left) Fungal Treatment (Right) Control


Figure 3.23 | Excitation-Emission Spectra for UAK-1-4 (Left) Fungal Treatment (Right) Control

In all treatments showing release of organics, the distinctive peaks appeared in the

range of 250 to 350nm, which may be characteristic of complex organics functionalities,

particularly related to the aromatic clusters. Previously, in other reports of coal

solubilization/degradation, release of condensed aromatics has been indicated on the basis

of UV-Vis spectroscopy (Shevla, 1976; Yin et al., 2009a; Yin et al., 2009b; Tao et al.,

2009). The structural integrity of low rank coal is maintained by interconnected aromatic

clusters and through these linkages fungi manage to break up the coal matrix, thereby,

releasing a variety of aromatic moieties into the media, thus reflecting the heterogeneous

nature of coal (Yin et al., 2009a).

Besides, in relation to the petrographic composition of coals, it may be concluded

that the liptinite and huminite contents may determine the susceptibility of coal to fungal

degradation and the major reason behind this could possibly be the increased aliphatic

nature of liptinite and origin of huminite macerals, which made this degradation process

easier, comparatively. Though, this relationship has not been established so far, however,

this study can provide a startup point for exploring this relationship further for its warranty.


3.6.2 Non-Purgeable Organic Carbon Determination

Non-purgeable organic carbon (NPOC), which is also considered to be dissolved

organic carbon (DOC), was also determined in the supernatants of released organics

obtained after fungal pretreatment of coal samples (Table 3.6). The NPOC values of tap

water and MilliQ, ultrapure water, (MQ Water) in ppm were 1.604 and 0.5881,

respectively. The values for all controls were in the range of 350ppm to 485ppm, showing

not any significant difference and for samples these values did not exceed the value of

100ppm.

Table 3.6 | NPOC of Released Organic Extracts

Sample ID

NPOC (ppm)

Organic Extracts Controls

UAS-4-2E 49.63 411.10

UAJ-1-1 69.46 361.10

UAK-1-4 90.46 392.70

LS-4-1 50.78 419.50

LS-4-2B 50.89 416.60

TP-1-1.1 86.15 401.80

TP-1-5.2 55.88 465.85

TP-3-2B 77.19 431.70

TP-3-2X 63.47 423.30

TP-3-2K1 65.32 411.60

TP-4-2A 67.70 471.59

TP-4-10 94.09 485.63


The concentration of dissolved organic carbon in controls was greater as compared

to that of the same sample. This could possibly be linked in such a way that the MW1

utilized the released organics, thus, leaving behind limited DOC contents as compared to

controls. The release of organics into the aqueous media indicated some of the hydrophilic

fractions present within the coal, which were subsequently, utilized by fungi. As low rank

coals are more soluble in water as compared to high rank coals, which suggested the

presence of those fractions in coal and these may be served as microbial substrate (Ralph

and Catcheside, 1996). Now, the utilization of these fractions by fungi would yield further,

less complicated, organic fractions, in terms of possible substrate for next biological

transformation (Wondrack et al., 1989). However, this whole transformation will follow a

very complex mode of pathways, which still need to be explained.

The particle size of coal can also be affecting parameter for the release of organics

from structural matrix of coal (Oboirien et al., 2008). The samples, which were used in this

study, were ground splits and having particle size ≤850m. The reduction in particle size

may have significant effect on degradation of coal, as studied by Oboirien et al. (2008).

This could probably be increasing the surface area of coal making the hydrophilic fractions

of coal more accessible to water and releasing more organics into the aqueous media.

3.6.3 pH of the Extracts

The pH of all the organic extracts, after the incubation time of seven days, including

their controls has been shown in Table 3.7 and pH of the pure MW1 culture was 3.5. In

controls, the pH of samples from Thar was 6 or greater than 6, while for all other samples

pH was less than 5 except UAS-4-2E with 5.0 pH. The variation in controls for pH can be

the result of the release of variety of organics into the aqueous media.

The pH in released organics was reduced thus suggesting that mode of degradation

might be depolymerization, which was generally followed at lower pH relatively and

leading to the formation of organics with less molecular mass (Fakoussa and Hofrichter,

2001).

This has been found to be in accordance with some other reports in which

Penicillium spp. have been reported to degrade coal following various mechanisms


including production of alkaline materials and enzymes (Yuan et al., 2006a; Dong et al.,

2006). In most of the studies Penicillium spp. have been found to be involved in coal

solubilization processes, however, overlapping of these two type of degrading abilities,

depolymerization and solubilization, may be a function of Penicillium spp. (Achi, 1994).

Table 3.7 | pH of Released Organic Extracts

Sample ID pH

Organic Extracts Control

UAS-4-2E 4.5 5.0

UAJ-1-1 3.5 4.5

UAK-1-4 3.6 4.0

LS-4-1 3.4 4.3

LS-4-2B 3.8 4.7

TP-1-1.1 4.1 6.1

TP-1-5.2 4.2 6.4

TP-3-2B 4.2 5.6

TP-3-2X 4.3 6.0

TP-3-2K1 4.1 6.5

TP-4-2A 4.5 6.3

TP-4-10 4.3 6.4

3.6.4 Gas Chromatography-Mass Spectrometry (GC-MS) of Degraded Extracts

The degraded extracts from fungal pretreatment were subjected to GC-MS analysis

for determining the nature of the released organics. The NIST 02 mass spectral library was

used for identification of compounds from the mass spectral data. Released organics, from


fungal degradation of coal, were liquid/liquid extracted from 45ml of filtrate using

pesticide-grade dichloromethane (DCM). Controls were also analyzed using DCM

extractions in order to have a clear picture of the actual released organics from coal matrix

in experimental flasks. The selection of DCM, as solvent for extractions of organics from

coal, was made on its volatility and ability to dissolve a wide range of organic compounds,

which were being expected to be released from heterogeneous structural matrix of coal

(Rossberg et al., 2000).

These DCM extracts from fungal degradation consisted of a variety of organic

compounds including aliphatics, polyaromatic hydrocarbons (PAHs) and primarily single

ring aromatics and aromatic nitrogen compounds (These particular fractions were found to

be absent in controls). Though, the liberated organics were also observed in controls,

however, fungal-mediated released organics varied somewhat in terms of presence or

absence of a particular organic compound, which have been showed in GC-MS

chromatograms. Secondly, the release of organics in coal controls may be a function of the

media, because of the presence of hydrophilic content in the structural matrix of coal. The

GC-MS chromatogram for pure MW1 culture has been shown in Figure 3.24.

In samples, UAS-4-2E (Sonda coal field), UAK-1-4 (Indus East), and UAJ-1-1

(Metting-Jhimpir), no significant organic fractions were observed after fungal

pretreatment, though the presence of ethosuximide and complex aromatic alcohol was

indicated in these samples (Figures 3.25, 3.26 & 3.27). The samples from Lakhra coal

fields, LS-4-1 and LS-4-2B, released primarily single ring aromatics and aromatic nitrogen

compounds including benzaldehyde, benzamide and benzothiazole (Figures 3.28 & 3.29).


Figure 3.24 | GC-MS Scan Pattern for Culture Growth of MW1 (Control)


Figure 3.25 (A) | GC-MS Scan Pattern of Organics from UAS-4-2E after Fungal Pretreatment


Figure 3.25 (B) | GC-MS Scan Pattern of Organics from UAS-4-2E without Fungal Pretreatment (Control)


Figure 3.26 (A) | GC-MS Scan Pattern of Organics from UAK-1-4 after Fungal Pretreatment


Figure 3.26 (B) | GC-MS Scan Pattern of Organics from UAK-1-4 without Fungal Pretreatment (Control)


Figure 3.27 (A) | GC-MS Scan Pattern of Organics from UAJ-1-1 after Fungal Pretreatment


Figure 3.27 (B) | GC-MS Scan Pattern of Organics from UAJ-1-1 without Fungal Pretreatment (Control)


Figure 3.28 (A) | GC-MS Scan Pattern of Organics from LS-4-1 after Fungal Pretreatment


Figure 3.28 (B) | GC-MS Scan Pattern of Organics from LS-4-1 without Fungal Pretreatment (Control)


Figure 3.29 (A) | GC-MS Scan Pattern of Organics from LS-4-2B after Fungal Pretreatment


Figure 3.29 (B) | GC-MS Scan Pattern of Organics from LS-4-2B without Fungal Pretreatment (Control)


In case of samples from Thar, cluster of organics were observed in the extracts

obtained after pretreatment with MW1. The sample, TP-1-1.1, released some mid chain

fatty acids in addition to benzaldehyde and phenolic aldehyde (Figure 3.30). However, in

comparison, no significant organics seemed to be released in the sample TP-1-5.2, though

peaks were observed in preliminary qualitative investigations by fluorescence

spectroscopy (Figure 3.31). Some aromatic heterocyclic organic compounds like indole

and quinoline were also observed in samples, TP-3-2B (Figure 3.32), TP-3-2X (Figure

3.33) and TP-4-2A (Figure 3.34). These organics represented the major class of aromatics

and the derivatives of benzene, as expected.

In a number of other studies, the release of aromatics after fungal degradation of

coal has been reported and a variety of organic solvents have been used for determining

the nature of complex organics in solubilized fraction. For example, ethyl esters of benzoic

acid, phthalic acid and benzenetricarboxylic acid were observed, through GC-MS, in

biosolubilized fraction of coal using tetraethyl ammonium hydroxide (Gotz and Fakoussa,

1999). Silva-Stenico et al. (2007) also investigated the nature of released organics,

however, these extracts were derivatized using N-methyl-N-(trimethylsilyl)-

trifluoracetamide (MSTFA), which indicated the presence of ethers, esters, 4-

hydroxyphenylethanol, 1,2-benzendiol, and 2-octenoic acid, in the extracts from coal

degradation by Trichoderma atroviride ES11.

Regardless of the nature of solvent and the primary mode of degradation, whether

it is aerobic or anaerobic, the major class, related to the nature of organics, would be

aromatics. In some other reports, following the investigations of anaerobic degradation of

coal, benzene derivatives (alkyl benzenes and toluene) and PAHs including naphthalenes

and phenanthrenes have been observed (Orem et al., 2007; Ulrich and Bower, 2008; Orem

et al., 2010). After biological degradation of coal, the majority of the organic fractions

would relate to the class of aromatics on the basis of structural composition of coal.

However, the rank of coal may determine the extent of this release, as the aromaticity in

the structure of coal, generally, increases with the coal rank. On the contrary, low rank

coals may yield some aliphatics because of the less condensed aromatic knots.


Figure 3.30 (A) | GC-MS Scan Pattern of Organics from TP-1-1.1 after Fungal Pretreatment


Figure 3.30 (B) | GC-MS Scan Pattern of Organics from TP-1-1.1 without Fungal Pretreatment (Control)


Figure 3.31 (A) | GC-MS Scan Pattern of Organics from TP-1-5.2 after Fungal Pretreatment


Figure 3.31 (B) | GC-MS Scan Pattern of Organics from TP-1-5.2 without Fungal Pretreatment (Control)


Figure 3.32 (A) | GC-MS Scan Pattern of Organics from TP-3-2B after Fungal Pretreatment


Figure 3.32 (B) | GC-MS Scan Pattern of Organics from TP-3-2B without Fungal Pretreatment (Control)


Figure 3.33 (A) | GC-MS Scan Pattern of Organics from TP-3-2X after Fungal Pretreatment


Figure 3.33 (B) | GC-MS Scan Pattern of Organics from TP-3-2X without Fungal Pretreatment (Control)


Figure 3.34 (A) | GC-MS Scan Pattern of Organics from TP-4-2A after Fungal Pretreatment


Figure 3.34 (B) | GC-MS Scan Pattern of Organics from TP-4-2A without Fungal Pretreatment (Control)


Two samples, TP-3-2K1 and TP-4-10, released unsaturated hydrocarbons i.e.,

dodecene, typically related to the class of aliphatics (Figure 3.35 & 3.36). In various

reports, the release of aliphatics, belonging to the classes of longer chain alkanes and fatty

acids, was reported due to the biodegradation of cyclic hydrocarbons, which may be the

part of integral structure of coal (Orem et al., 2007). Jones et al. (2008) proposed a model

for the initial degradation of coal, followed by the generation of long chain alkanes and

long chain fatty acids. Though, this degradation was mediated by a mixed consortium of

bacterial and methanogens, but, on the basis of structural insight of coal, the release of

aliphatics was observed. However, as it has been mentioned earlier, the concentration of

aliphatics may depend, ultimately, on the rank of coal as brown coals may be a better source

for aliphatics due to reduced aromatic condensation as compared to high rank coals.

The presence of aliphatics, from some of the samples of Thar coal field, was the

indication of this fact that the cluster of linked and fused rings may sometimes connected

with each other through aliphatic and straight chain linkages that may be saturated and

unsaturated (Fakoussa and Hofrichter, 1999). However, with increasing rank of coal,

aromatic condensation increases, thus, contributing towards the recalcitrant nature of

higher rank coals to biological modification, as well (Strapoc et al., 2011). But, primarily,

the major release of organics was related to aromatics in all of the samples, which were

treated with fungal isolate MW1.

More precisely, it can be concluded that the extent of release of organics was greater

in samples from Thar coal field as compared to the other samples from Lakhra, Sonda,

Metting-Jhimpir and Indus East coal fields. This can be a function of, relatively, more

lignitic nature and high moisture content of Thar coal samples, which tended to be affecting

parameters in the release of organics. Secondly, the distribution of release of organics

among all extracts also indicated the presence of aliphatics, being possibly involved in

interconnected linkages through oxygen to the fused and single ring aromatics in the

structure of coal. The detailed picture of the released organics from each sample has been

shown in Table 3.8.


Figure 3.35 (A) | GC-MS Scan Pattern of Organics from TP-3-2K1 after Fungal Pretreatment


Figure 3.35 (B) | GC-MS Scan Pattern of Organics from TP-3-2K1 without Fungal Pretreatment (Control)


Figure 3.36 (A) | GC-MS Scan Pattern of Organics from TP-4-10 after Fungal Pretreatment


Figure 3.36 (B) | GC-MS Scan Pattern of Organics from TP-4-10 without Fungal Pretreatment (Control)


Table 3.8 | Released Organic Fractions from Coal Samples

Sample ID Organic Fractions

UAS-4-2E Ethosuximide,

(22E)-ergosta-5,7,2,2-trien-3-ol

UAK-1-4 Ethosuximide

UAJ-1-1 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4-H-Inden-4-one

LS-4-1 Benzothiazole,

2-amino benzamide,

LS-4-2B 3-methylbenzaldehyde,

2-amino benzamide

TP-1-1.1 Heptanoic Acid,

2-ethyl hexanoic acid,

Nonanoic acid

3-hydroxy benzaldehyde,

Vanillin,

TP-1-5.2 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4-H-Inden-4-one

TP-3-2B Indole

2-tert-butyl quinoline

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

3-methyl phenol

TP-3-2K1 2,6-bis(1,1-dimethylethyl)-2,5-cyclohexadiene-1,4-dione

Dodecene

TP-3-2X Indole,

Benzothiazole

TP-4-2A 2-tert-butyl quinoline,

2-acetyl-4(1H) quinazolinone

TP-4-10 4-tert-butyl quinoline

Dodecene,

2,4-bis(1,1-dimethylethyl)-phenol


0

2

4

6

8

10

12

5 10 15 20 25 30 35 40

ControlTP-3-2B

TP-3-2XTP-3-2K1

Me

than

e (

mo

les/g

of

Co

al)

Days

3.7 METHANE GENERATION FROM RELEASED ORGANICS

The released organics, from prior fungal treatment of twelve coal samples (UAS-

4-2E, UAJ-1-1, UAK-1-4, LS-4-1, LS-4-2B, TP-1-1.1, TP-1-5.2, TP-3-2B, TP-3-2X, TP-

3-2K1, TP-4-2A and TP-4-10), were subjected to WBC-2 based bioassay for generation of

methane. The organic fractions served as methanogenic substrates while fresh water

medium inoculated with WBC-2 was treated as a control. As for the extraction of organics,

1g of coal was used in fungal pretreatments, so for better understanding and quantification

of methane, the values for methane generation has been reported per gram of coal. The

incubation time was extended up to 35 days. The organics from samples TP-3-2B, TP-3-

2K1 and TP-3-2X have generated maximum methane generation from respective released

organics, which was quantified as 9.67, 7.34 and 10.78 in µmoles/g of coal, respectively

(Figure 3.37).

Figure 3.37 | Methane Generation from Organic Fractions of TP3 Borehole Samples

(Control represents fresh water medium inoculated with WBC-2)


0

0.5

1

1.5

2

2.5

3

3.5

5 10 15 20 25 30 35 40

Control TP-1-1.1 TP-1-5.2

Me

tha

ne (

mo

les

/g o

f C

oa

l)

Days

However, in samples from TP1 borehole i.e., TP-1-1.1 and TP-1-5.2, methane was

generated, insignificantly, as compared to the samples from TP3 borehole, leading to the

maximum methane production of 3.30 and 3.27µmoles/g of coal (Figure 3.38).



Among TP4 borehole samples, from the liberated organics of TP-4-10 sample,

methane generated was 7.51µmoles/g of coal, while in case of TP-4-2A, methane yield was

2.25µmoles per g of coal (Figure 3.39). Keeping in view the nature of organics released

from coal samples (TP-3-2B, TP-3-2K1, TP-3-2X and TP-4-10), which produced

significant methane; it seemed very difficult to find a particular pattern and parameter


0

1

2

3

4

5

6

7

8

5 10 15 20 25 30 35 40

Control TP-4-2A TP-4-10

Me

than

e (

mo

les/g

of

Co

al)

Days

affecting the generation of methane. However, it was evident that released organics could

serve as substrate for methanogenesis, to certain extent.



On the contrary, the organics from samples UAS-4-2E, UAK-1-4 and UAJ-1-1 over

incubation period of 35 days generated less methane 3.69, 1.83 and 1.75µmoles/g of coal,

respectively (Figure 3.40).

On the other side, methanogenesis of organic fractions, released from Lakhra

samples, LS-4-1 and LS-4-2B, methane was produced in the quantity of 2.39 and

2.87µmoles/g of coal of methane, correspondingly (Figure 3.41). Upon approaching the

end of incubation time of 35 days, methane generated in most of the samples either started

to decline or remain almost unchanged.


0

0.5

1

1.5

2

2.5

3

3.5

4

5 10 15 20 25 30 35 40

Control

UAS-4-2E

UAK-1-4

UAJ-1-1

Me

than

e (

mo

les/g

of

Co

al)

Days

Figure 3.40 | Methane Generation from Organic Fractions of UAS-4-2E, UAK-1-4

and UAJ-1-1 Samples (Control represents fresh water medium inoculated with WBC-2)


0

0.5

1

1.5

2

2.5

3

5 10 15 20 25 30 35 40

Control LS-4-1 LS-4-2B

Me

than

e (

mo

les/g

of

Co

al)

Days

Figure 3.41 | Methane Generation from Organic Fractions of Lakhra Coal Samples


Most of the fungal extracts, subjected to methanogenesis, generated methane in the

range of 1 to 4moles/g of coal, but the organics from four samples of Thar (TP-3-2B, TP-

3-2K1, TP-3-2X and TP-4-10) produced methane between 7 and 11moles/g of coal. Now,

these quantities of methane generation have been found to be in the same range as for low


and medium methane producing subbituminous coals in previous WBC-2 based bioassays

and, in a way, this bioassay helped in evaluating the potential of fungal extracts from low

rank coal to support methanogenesis (Jones et al., 2008).

Comparing the amount of methane generated with the types of organics identified

in the fungal pretreatments, it appeared that methane generation may not be limited to the

organic compounds identified previously in methanogenic incubations (Jones et al., 2010;

Orem et al., 2010). The four fungal extracts from Thar coal samples that produced the

highest methane by bioassay included many aromatic nitrogenous compounds, in addition

to unsaturated alkenes, phenols and benzene derivatives. However, the methane could have

been generated from compounds (such as volatile fatty acids) not detectable by methods,

which were used in this study. The heterogeneous nature of coal structure makes it very

complex starting material and therefore it can yield wide-ranging degraded products.

The fungi MW1 released primarily aromatic compounds, which could be

recalcitrant to biodegradation as compared to aliphatics like fatty acids. However, the role

of fungi has also been investigated in real-time release of methane from abandoned coal

mines in terms of releasing reduced substrates, which could be utilized as methanogenic

substrates, thus, suggesting the possible intervention of fungal-mediated coal to methane

transformations (Beckmann et al., 2011). Generally, biogenic methane generation from

coals dominated by condensed aromatic clusters is slower as compared to the coals with

less aromatic and open clusters (Strapoc et al., 2011).

For determining the complete potential of fungal pretreatment of lignite for

generation of methane, the process needs to be optimized by enhancing release of those

organics from structural matrix of coal and the rate limiting steps in this whole

biotransformation pattern needs to be pointed out. Enhancing the initial defragmentation

of coal leading to specific methanogenic substrates may enhance methane generation.

Additional organic analysis may also be helpful to determine the full suite of organics

released by fungal pretreatment. Additionally, testing of individual compounds from the

extract using a bioassay will help to identify novel intermediates between coal and

methane.


0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

Control

TP-1-1.1

TP-1-3.3

TP-1-4.5

TP-1-5.2

TP-1-6.1

Me

than

e (

mo

les/g

of

Co

al)

Days

3.8 METHANE GENERATION FROM NATIVE COAL SAMPLES

Thirty coal samples were subjected to WBC-2 based bioassay for determining the

methane generation potential from native coal samples (without any prior fungal

treatment). Methane generation was quantified in µmoles/g of coal, over the incubation

period of 35 days. From two samples of TP1 borehole, TP-1-1.1 and TP-1-3.3, methane

was generated up to 22.72µmoles/g of coal and 20.94µmoles/g of coal, respectively (Figure

3.42). The sample TP-1-6.1 generated methane relatively in lesser amount, i.e.,

3.65µmoles/g of coal.

Figure 3.42 | Methane Generation from Coal Samples of TP1 Borehole



0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

ControlTP-3-2BTP-3-2D

TP-3-2K1TP-3-2RTP-3-2X

TP-3-2AG

Meth

an

e (

mo

les/g

of

Co

al)

Days

Among TP3 borehole samples, maximum methane generation was observed in

samples, TP-3-2B, TP-3-2D and TP-3-2K1 for which the values were 19.62µmoles/g of

coal, 22.96µmoles/g of coal and 16.77µmoles/g of coal, respectively (Figure 3.43).



From the TP4 borehole samples, there was no significant methane generation as

compared to the samples from TP1 and TP3 boreholes (Figure 3.44). Though, the sample

TP-4-1A, relatively, generated higher methane, approaching the average value of

0.425µmoles/g of coal.


0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35 40

Control

TP-4-1A

TP-4-2A

TP-4-6

TP-4-8

TP-4-10

Me

than

e (

mo

les/g

of

Co

al)

Days



The samples from Lakhra and Sonda coal fields showed the least methane

generation, which was in the range of 0.05 to 0.1µmoles/g of coal, approximately (Figures

3.45 & 3.46). The least methane generation may be considered as a function of coal rank,

which ranged from lignite A to subbituminous A, among all these samples from Lakhra

and Sonda coal fields.


0

0.05

0.1

0.15

0.2

0 5 10 15 20 25 30 35 40

ControlUAL-15-1

UAL-15-2LS-4-1

LS-4-2B

Me

tha

ne (

mo

les

/g o

f C

oa

l)

Days

Figure 3.45 | Methane Generation from Lakhra Coal Samples



0

0.05

0.1

0.15

0 5 10 15 20 25 30 35 40

ControlUAS-4-1

UAS-4-2EUAT-4-1

UAT-4-4

Me

than

e (

mo

les/g

of

Co

al)

Days

Figure 3.46 | Methane Generation from Sonda Coal Samples



0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20 25 30 35 40

Control7 BLCHDSA-23-4

MKCT 6UAK-1-4UAK-1-8

UAJ-1-1

Me

than

e (

mo

les/g

of

Co

al)

Days

For the samples from Khost, Salt Range, Makarwal, Indus East and Metting-

Jhimpir, the yield of methane with the duration of time has been shown in Figure 3.47.

Though, the methane generation was not comparable with the values of TP1 and TP3

boreholes samples, but among these, samples MKCT 6 and 7 BLCH, yielded methane with

average values of 0.27 µmoles/g of coal and 0.24µmoles/g of coal, respectively. The

average methane values for all thirty coal samples have been represented in Table 3.9.

Figure 3.47 | Methane Generation from Samples of Khost, Salt Range, Makarwal,

Indus East and Metting-Jhimpir Coal Fields



Table 3.9 | Average Methane Generation from Coal Samples

Sample Methane

(µmoles/g of Coal)

Sample Methane

(µmoles/g of Coal)

7 BLCH 0.24 TP-1-3.3 20.94

DSA-23-4 0.145 TP-1-4.5 1.00

MKCT-6 0.275 TP-1-5.2 1.58

UAL-15-1 0.03 TP-1-6.1 3.65

UAL-15-2 0.02 TP-3-2B 19.62

LS-4-1 0.065 TP-3-2D 22.96

LS-4-2B 0.065 TP-3-2K1 16.77

UAS-4-1 0.07 TP-3-2R 0.085

UAS-4-2E 0.12 TP-3-2X 0.315

UAT-4-1 0.035 TP-3-2AG 0.065

UAT-4-4 0.025 TP-4-1A 0.425

UAK-1-4 0.055 TP-4-2A 0.065

UAK-1-8 0.025 TP-4-6 0.095

UAJ-1-1 0.04 TP-4-8 0.095

TP-1-1.1 22.725 TP-4-10 0.065

The extent of methane produced by Thar coal samples is comparable with some

samples from Powder River Basin, USA (0 to 23moles/g of coal) for which the same

WBC-2 based bioassay was used (Jones et al., 2008). This similar behavior of the samples

from these two coal fields may be based on resemblance on geochemical basis and age of

these reserves (SanFilipo, 2000). Lignitic nature of Thar coal samples may also be


determining factor in methane generation, as rest of the samples (belonging to other coal

fields) being relatively higher in rank, were not found to be prone to anaerobic degradation

to methane, in these experiments. On the basis of structural formation of brown coals,

methanogenesis of lignite seems to be achievable and number of practices has been carried

out for stimulating methane generation from coal, particularly low rank coals (Shumkov et

al., 1999; Gupta and Birendra, 2000; Kruger et al., 2008; Baysal et al., 2010). As a matter

of fact, underground and natural anaerobic carbon recycling, leading to the formation of

biogenic methane in low rank coalbeds, resulted in rapid research interests for stimulating

methane from coal at laboratory scales (Strapoc et al., 2011). Side by side, several studies

were also conducted for identification of bacterial and archaeal species associated with

degradation of coal and, subsequently, transformation into methane (Shimizu et al., 2007;

Strapoc et al., 2008b; Green et al., 2008; Dawson et al., 2012).

So the notion of biological-mediated extraction of methane has received significant

attention for determining the potential of methane generation from coals, however, this

may be restricted to low rank coals because of their increased susceptibility as compared

to higher rank coals. The susceptibility of Thar coal samples may be driven by the fact that

it still retained that geological stage, which might be susceptible to biological gasification,

as also indicated by Rm values, which were found to be less than 0.4% for the samples

from Thar (Haider et al., 2013).

In this study, the methane potential was generally higher in low rank coals and this

biological conversion may also be a function of liptinite and huminite content. Though,

this relationship has not been elaborated so far, specifically for biological transformations

of coal, however, there were some noteworthy indications. Out of six, three samples from

Thar, TP-1-6.1, TP-3-2B and TP-3-2D, with relatively higher liptinite contents generated

significant amount of methane. The relatively greater portion of aliphatics and increased

hydrogen content in liptinite macerals as compared to other maceral groups may be the key

factors behind susceptibility of lower rank coals for methane generation biogenically

(Taylor et al., 1998). In context of huminite content among six methane generating

samples, TP-1-1.1, TP-1-3.3, TP-1-6.1, TP-3-2B and TP-3-2D had huminite content

greater than 80 volume percent (Table 3.4). The proneness of these samples may be because


of the fact that huminite macerals are derived from cellulose and lignin of the plant tissues,

which can be supportive in providing the best combination of organics source that may

yield some methanogenic substrates upon initial and secondary fragmentation of coal

matrix (Taylor et al., 1998).

Finally, fungal pretreatment of coal, could not enhance methane generation

appreciably and bacterial pretreatment, in terms of direct methanogenesis, appeared to be

more effective in comparison. There may be couple of reasons behind reduced methane

generation from fungal extracts. Firstly, there may be generation of any agent with

inhibitory effect, which could have ceased the methanogenesis process. Secondly, fungi

could have utilized majority of the organics on preferential basis, thus, leaving limited

substrate options behind for subsequent methanogenesis. However, these two reasons need

to be explored on experimental basis in order to elucidate and optimize prior fungal

treatment for enhanced methane generation.

3.9 ANALYSIS OF RESIDUAL FILTRATES OF BIOASSAY EXPERIMENTS

In bioassay experiments, the headspace in serum bottles was analyzed for the

quantification of methane while the residual media with coal started turning into brown

colour over the time with methane generation. That residual suspension was analyzed on

spectrophotometer for determining the qualitative nature of the residues after the release of

methane generation. The suspensions of the six samples, TP-1-1.1, TP-1-3.3, TP-1-6.1,

TP-3-2B, TP-3-2D and TP-3-2K1, which produced significant methane, were analyzed on

spectrophotometer and E4/E6 ratio was also determined, which may be considered as an

index of aromaticity (Table 3.10; Figures 3.48 & 3.49) (Chen et al., 1977). The E4/E6 was

determined by dividing the absorbance of a sample at 400nm by that at 600nm.

In order to develop a comparison between methane producing and non-methane

producing coal samples, UAS-4-2E (a non-methane producing coal sample) was selected

and drawn against all of those methane producing samples. The E4/E6 ratio for sample

UAS-4-2E was 1.15. In samples from TP1 and TP3 boreholes, relatively higher E4/E6 ratios

were observed, which may be due to the release of humic materials after the break-up of

coal matrix for methane generation. This high E4/E6 ratio may correspond to low


aromaticity in the filtrates. In addition, higher the E4/E6 ratio the smaller and more active

are the biomolecules (Malcolm, 1989).

Walia and Srivastava (1994) developed coal biogasification process, MicGAS®

Technology, which described the production of soil conditioning agents and clean fuels

like methane from anaerobic processing of coal. Later in 1997, Srivastava and Walia

(1997), patented this technology, which entailed the extraction of humic acid from residual

coal and filtrates, left behind after coal to methane transformation. The prospects and use

of residual coal, as soil conditioning agent like humic acid, may offer an economical

advantage for application of biological processing of coal, particularly for low rank coals.

The wide-spread use of humic acid (carbon rich macromolecule) in agriculture, as

organic fertilizer, has received significant attention. In this regard, the generation of

methane and production of humic acid from filtrates and the residual coal, from Thar coal

may be a prospective use. However, high-tech analytical facilities may be required for

investigating the exact nature of the filtrates on chemical basis.

Table 3.10 | E4/E6 Ratio of Residual Filtrates from Methane Generating Coal Samples

Sample E4/E6

TP-1-1.1 3.21

TP-1-3.3 2.30

TP-1-6.1 2.83

TP-3-2B 2.99

TP-3-2D 4.24

TP-3-2K1 2.33


Figure 3.48 | UV-Vis Scan Pattern from Residual Filtrates of TP1 Borehole Samples

Figure 3.49 | UV-Vis Scan Pattern from Residual Filtrates of TP3 Borehole Samples

Wavelength

(nm)

Ab

sorp

tion

In

ten

sity

Wavelength

(nm)

Ab

sorp

tion

In

ten

sity


3.10 EXTRACTION OF HUMIC ACID FROM LOW RANK COAL

Humic acid was extracted from coal sample, TP-31, using two approaches. One of

these employed the treatment of coal sample with alkali and, afterwards, humic acid was

extracted, which resulted in 57% yield of humic acid. In the second approach, coal sample

was pretreated with fungal isolate MW1 and the filtered coal particles, after pretreatment,

were processed for the extraction of humic acid. Alkali solubilized humic acid was

designated as AS-humic acid while the one extracted after prior fungal treatment was

designated as FPAS-humic acid (Fungal-pretreated alkali solubilized humic acid). The

yield of FPAS-humic acid was 57.81%, which was almost the same as that for alkali

solubilized coal i.e., 57.19% (Table 3.11).

3.10.1 Chemical Characterization

Chemical characterization, on the basis of elemental analysis, was compared, which

indicated a slight reduction in carbon content in case of FPAS-humic acid (Table 3.11).

However, nitrogen content was increased, which might be due to the incorporated nitrogen

content from the nitrogen source present in the growth media for fungal isolate MW1.

Likewise, increase in oxygen content was also observed in case of FPAS-humic acid and

this increase in nitrogen and oxygen contents may be a function of the incorporation of

nitrogen and oxygen involving functional groups in the humic acid molecule. This increase

has been found consistent with some previous reports (Dong et al., 2006). Slight increase

in H/C ratio was also observed for FPAS-humic acid. Lower H/C ratio indicates higher

molecular mass fraction and aromatic condensation (Dong et al., 2006).

In spectroscopic estimations, E4/E6 was also increased in case of FPAS humic acid

as compared to AS humic acid. Generally, with increasing E4/E6, molecular mass (MM)

content and aromaticity decreases. This also results in increased bioactivity of the molecule

(Malcolm, 1989). In this regard, the FPAS-humic acid can have increased bioactivity as

compared to AS-humic acid, which can be due to the effect of prior fungal treatment of

coal for aiding the reduction of aromatic content and introducing more functional groups

in the acid molecule.


Table 3.11 | Elemental Analysis, Atomic Ratios and Spectroscopic Characteristics of

AS-Humic Acid and FPAS-Humic Acid

AS-Humic Acid FPAS-Humic Acid

Ele

men

tal

An

aly

sis

% C 59.26 57.12

% H 4.07 4.01

% N 0.63 1.16

% S 1.69 1.60

% O 34.35 36.11

Ato

mic

Ra

tio

s

H/C 0.82 0.84

O/C 0.43 0.47

UV-Vis

E4/E6 5.89 6.65

% Recovery 57.19 57.81


3.10.2 Fourier-Transform Infrared Spectroscopy (FTIR)

The FTIR spectra were obtained for lignite sample, TP-31, (Figure 3.50), AS-humic

acid (Figure 3.51) and FPAS-humic acid (Figure 3.52). Both humic acid samples exhibited

typical organic functionalities, generally, found in humic materials (Inbar et al., 1990).

However, it was found that the intensities of the peaks are much clearer and intensified in

the case of FPAS-humic acid, which can be related to the effect of prior fungal treatment.

The typical bands represented the aliphatic stretching (2917-3335cm-1), CO stretching of

COOH and ketones (1607-1698cm-1) and CO bond involving components like ether

linkages (1197cm-1).

Elbeyli et al. (2006) observed bands in lignitic humic acids, from microbial

pretreatment, due to the presence of hydroxyl groups (3400cm-1), aliphatic hydrocarbons

(2926cm-1), ethers (1158cm-1, 1078cm-1) and some aromatic hydrocarbons (1634cm-1), as

well. Dong et al. (2006) have also reported the presence of aliphatic CH stretching at

2932cm-1 and CO stretching at 1720cm-1, in humic acids, which were solubilized through

alkali from lignite samples that were pretreated with Penicillium sp. P6. In a number of

other reports, the effect of pretreatment with fungi has been explained in terms of the

modification of functional groups around the humic acid molecule and these changes may

contribute to increasing the bioactivity of the molecule (Shi et al., 2009; Yin et al., 2009a).

The incorporation of OH and COOH functional groups into humic acid molecule

will be significant and critical in enhancing the chelating abilities of the humic acid

molecule. The pretreatment of lignite samples with fungal isolate MW1 remained effective

in intensifying the absorption of bands, typically related to humic materials, which included

CO stretching and COOH organic functionalities (Dong et al., 2006). However, the

optimization for enhancing the yield of humic acid from lignite may be required along with

the investigations of specific structural functionalities, involved in the activity of the humic

acid molecule.


C=O Functionality Stretching

Minimal Aliphatic Stretching and

Possibly Hydroxyl Group

Figure 3.50 | FTIR Spectra of Coal Sample (TP-31)


Figure 3.51 | FTIR Spectra of Alkali Solubilized Humic Acid

H:\Program Files\OPUS_65\MEAS\HAIDER2.0 HAIDER2 Instrument type and / or accessory 14/10/2011

2974.9

82918.2

4

1700.9

4

1607.5

2

1395.7

1

1185.8

7

1078.8

9

825.2

5760.8

8739.9

9610.8

5569.3

6536.4

9522.1

4

500100015002000250030003500

Wavenumber cm-1

02

04

06

08

01

00

Tra

nsm

itta

nce

[%

]

Page 1/1

Minimal Aliphatic Stretching and


C=O Functionality Stretching


Figure 3.52 | FTIR Spectra of Fungal-Pretreated Alkali Solubilized Humic Acid

Intensified Aliphatic Stretching and


C=O Functionality

Stretching

Enhanced C−O Stretch (Possibly indication of ether

linkages)

H:\Program Files\OPUS_65\MEAS\HAIDERBT.0 HAIDERBT Instrument type and / or accessory 14/10/2011

3335.4

0

2917.9

2

1698.1

5

1607.9

9

1418.0

8

1197.9

2

1076.7

8

523.2

3

500100015002000250030003500

Wavenumber cm-1

02

04

06

08

01

00

Tra

nsm

itta

nce

[%

]

Page 1/1

4

General Discussion & Conclusions

Historically, the role of bacteria as catalytic agents for the oxidation of brown coal

was determined by Potter (1908) for the first time. However, later, fungal degradation of

coal was reported by Fischer and Fuchs (1927) in terms of the presence of growing white

and greenish mycelia on moist coal samples during storage in the laboratory. This initiated

more interests in the observation and study of this phenomenon. Afterwards, several

filamentous micro-fungi, belonging to the deuteromycetes (Aspergillus spp., and

Penicillium spp.), and yeast-like fungi (Torula spp.) were observed to consume a wide

variety of coal-based organic functionalities including coke and coal briquettes (Achi,

1994; Gokcay et al., 2001; Yuan et al., 2006a).

Then, Lieske and Hofmann (1929) examined the microflora from natural coal

deposits and after a couple of years biotechnological applications of coal were prospected.

Lieske and Hofmann (1929) suggested the use of coal as soil conditioning agent after

biological modification. However, in 1981 the investigations of Rene Fakoussa increased

the interests in biological modification of coal as an alternative coal conversion technology.

Afterwards, the bacterial and fungal degradation of coal was investigated in a number of

regions and several microorganisms were reported, which were able to colonize brown and

hard coal (Igbinigie et al., 2008; Yin et al., 2009a). The applications of solubilized coal

can be seen in terms of treating it as chemical feedstock for subsequent chemical or

154 General Discussion & Conclusions |

biological transformations and extracting soil conditioning agents like humic acids from

oxidized residual coal.

The biological modification of low rank coal seems to be favourable because of the

resemblance of chemical structure between lignite and lignin and in this regard lignin

degrading fungi can find an application of coal conversion. Pakistan has huge reserves of

low rank coal, particularly in Thar (SanFilipo and Khan, 1994). However, these coals were

not subjected to any biological modification prior to these studies. The theme of this study

was to investigate the potential of biotechnology for the beneficiation of indigenous low

rank coals as an alternative coal conversion technology. More precisely, these studies were

aimed at isolation of coal degrading fungal isolate, analysis of released/degraded organics

upon fungal pretreatment, and methanogenesis of released organics and native coal

samples. A wide variety of low rank coal samples from Pakistani coal fields were subjected

to degradation by fungal isolate MW1, which resulted in the release of polyaromatic

hydrocarbons, some single ring aromatics, aromatic nitrogen compounds and some

aliphatics (Haider et al., 2013). These results were in accordance with some other studies

in which the release of aromatics and aliphatics from coal matrix was reported (Ward,

1993; Toth-Allen et al., 1994; Silva-Stenico et al., 2007; Orem et al., 2007).

Broadly, the fungal degradation of coal can be a function of the rank of coal and

the fungal isolate. Considerable organics were released from Thar lignite samples through

fungal pretreatment. The MW1 fungal isolate, which was isolated from a bituminous coal

core sample, was found to be related to Penicillium chrysogenum on the basis of

phylogenetic studies (Haider et al., 2013). In a number of other reports, Penicillium species

have been reported to degrade coal (Polman et al., 1994b; Achi, 1994; Yuan et al., 2006a;

Dong et al., 2006).

Methane generation was quantified, which was approximately 11µmoles per gram

of coal, at maximum, from organics released through one of the sample from Thar lignite

(Haider et al., 2013). The methanogenesis was done by a mixed bacterial and archaeal

culture WBC-2, which has already been demonstrated to determine the potential of

biogenic methane generation from bituminous and sub-bituminous coals (Jones et al.,

2008). However, this report happened to be the first for the application of this WBC-2


mixed consortium on lignites. Comparing the amount of methane generated with the types

of organics identified in the fungal treatments, it appeared that methane generation might

not be limited to the organic compounds identified previously. The four fungal extracts that

produced the highest methane by bioassay included many nitrogenous compounds, in

addition to unsaturated alkanes and phenols. However, methane could have been generated

from the compounds (such as volatile fatty acids) not detectable by the methods used here.

The heterogeneous nature of coal structure resulted in the yield of a broad range of

degraded products.

Fungal isolate, MW1, released primarily aromatic compounds which were more

recalcitrant than aliphatics like fatty acids. In general, biogenic methane generation from

coals, dominated by condensed aromatic clusters is slower as compared to the coals with

less aromatic and frequent open clusters (Strapoc et al., 2011). Additional organic analyses

may be required for determining the complete profile of the organics. Further work on the

applicability of fungal pretreatment of coal for subsequent methanogenesis is warranted to

examine different coals, the effect of fungi under anaerobic conditions, and treatments with

different fungal species. It is also interesting to note that the fungal isolate MW1used in

this study was recovered from the center of a coal core, stored under anaerobic conditions.

Gokcay et al. (2001) experimented subsequent gasification after solubilizing the

coal through fungi and reported the potential in terms of 21% energy recovery from

solubilized material obtained after fungal degradation. In this study, the released fractions

from fungal degradation of coal were subjected to subsequent methanogenesis for

determining the potential of methane generation by making coal more bioavailable.

Another important factor, gravimetric analyses for the determination of

solubilization extent, can be pivotal for subsequent transformation of solubilized coal into

methane. As a matter of fact, it was not possible to determine the accurate weight loss after

solubilization because the fungal mycelia covered the whole lignite particles and it was,

virtually impossible to sequester coal from the biomass. Though, in a number of previous

studies, extent of solubilization has been reported to approximately 35% using virgin coal

and 100% using chemically pretreated coal (Gokcay et al., 2001; Yin et al., 2009a).


However, the heterogeneous nature of coal presents an obstacle for predicting any

generalized estimates of solubilized coal for any subsequent application.

Native low rank coal samples were also subjected to methanogenesis in order to

compare the results of methane generation and the effect of prior fungal pretreatment. In

this case, maximum methane generation was observed in a sample from Thar coal field

which was approximately 23µmoles per gram of coal. Interestingly, methane generation

was much higher from native coal samples as compared to their respective released

organics. The extent of methane produced by Thar coal samples (0.08 to 23.2µmoles/g of

coal) was also found to be comparable with some samples from Powder River Basin (PRB)

coals, USA (0 to 23µmoles/g of coal) for which the same WBC-2 based bioassay was used

(Jones et al., 2008). Additionally, significant methane was generated from Thar coal

samples and none of the samples from other coal fields indicated methane generation.

Apparently, it seemed prior fungal treatment was not much efficient in terms of the release

of those organics, which could potentially serve as methanogenic substrates.

In case of direct methanogenesis of native coal samples, the pretreatment of coal

may be considered as initiated through bacteria. This study provided a comparison, which

was established between fungal and bacterial pretreatments, for methane generation per

gram of coal. Generally, microbial generation of methane from coal follows three major

steps. In initial degradation, water soluble organics are released from the coal matrix,

belonging to single ring aromatics, long chain fatty acids and long chain alkanes by the

action of fermentative bacteria (Orem et al., 2010). Associated bacterial communities

(Acinetobacter spp. and Hydrogenophaga spp.) with the native coal can also have possible

role in releasing these organics into the media for further degradation (Jones et al., 2010).

In the second degradation phase, these intermediates undergo further degradation

thus releasing less complex intermediates such as mid-chain fatty acids, which are

subsequently degraded to the substrates that can be utilized by methanogens (Evans and

Fuchs, 1988; Zengler et al., 1999; Anderson and Lovley, 2000; Hatamoto et al., 2007a;

Hatamoto et al., 2007b). This degradation may involve the syntrophic cooperation of

microorganisms because of the fact that methanogens, for their growth substrates, depend

on the degrading activity of the bacterial communities, which take part in the first and


second stage degradation of organics released from coal (Zinder, 1993). The syntrophic

bacterium Pelotomaculum spp. was identified in WBC-2 mixed culture, which was capable

of oxidizing phthalates and benzoates, working with hydrogen-consuming methanogen

syntrophically (de Bok et al., 2005; Jones et al., 2006).

The final stage in this biotransformation is methanogenesis i.e., methane formation,

which is generated from the substrates such as hydrogen, carbon dioxide, acetate,

methanol, formate, methylamines or methylated sulphur (Jones et al., 2010). On the other

hand, prior fungal treatment of coal releases, majorly, polyaromatic hydrocarbons and

single ring aromatics (Haider et al., 2013). These organics give light brown coloration to

the filtrate, while dark brown color has been observed in liquid upon treatment of any native

coal sample with WBC-2 consortium. Aromatic clusters seemed to be tough substrate for

bacteria and subsequently methane yields might be restricted (Haider et al., 2013). In

contrast to the WBC-based methanogenesis including bacterial preliminary degradation,

fungal pretreatment of coal and later methanogenesis of released organics follow two-stage

process, which could be energy intensive, as well, because of the involvement of extraction

of organics.

The prior and initial release of organics from coal matrix for subsequent

methanogenesis is generally considered to be the rate limiting step in this over all biological

transformation of coal into methane (Strapoc et al., 2011). Hence, the extent of release of

organics and the nature of organics, whether these can serve as methanogenic substrate or

not, will determine the over-all rate of the process and viability of enhanced methane

generation. Based on previous literature and the experimental design used in this study, a

modified model for biological transformation of coal into methane was proposed for

understanding the mechanisms of the series of the reactions involved in the ultimate

conversion of coal into methane (Figure 4.1) (Jones et al., 2010; Haider et al., 2013). This

model may give an insight into the reaction phases, which may be worked out for enhanced

methane generation, subsequently.


Figure 4.1 | Laboratory Scale Model for Biotransformation of Coal into Methane

Organics

Extraction

Fungal

Degradation

Bacterial

Degradation 1. Fresh Water Medium

2. WBC-2

3. Ground Coal

1. WBC-2

2. Released Organics

Coal

Methanogenesis

Acetate H2 + CO2

CH4

Polyaromatic

Hydrocarbons

Preliminary Degradation

Single Ring

Aromatics Long Chain

Alkanes

Long Chain

Fatty Acids

Single Ring

Aromatics

Mid Chain

Fatty Acids

Propionate, Butyrate

Phenols,

Benzoate

Non-aromatic

Rings Phthalates,

Benzene Derivatives

Non-aromatic

Rings


The petrographic composition and properties of coal may also have some effect on

biogenic methane generation from coal. For all of the samples from Thar coal field, mean

random reflectance (Rm) values were found to be less than 0.4 percent, which may indicate

an early stage of thermal alteration. From this study, it can be concluded that methane

potential was generally higher in low rank coals and this biological conversion may be a

function of liptinite and huminite groups of macerals. Out of six, three samples from Thar

(TP-1-6.1, TP-3-2B, and TP-3-2D) with relatively higher liptinite contents generated

significant methane. Liptinite macerals are made up of waxy and resinous parts of the

plants such as spores, cuticles, and resins, which are resistant to weathering and diagenetic

processes (Taylor et al., 1998). In context of huminite content, among six methane

generating samples, five samples (TP-1-1.1, TP-1-3.3, TP-1-6.1, TP-3-2B and TP-3-2D)

had high huminite content (greater than 80 volume percent). In low rank coals, there may

also be a particular relationship of huminite and liptinite contents, concomitantly, to the

biological transformation and this may be because mainly huminite macerals are derived

from cellulose and lignin of plant tissues while relatively greater portions of aliphatics are

present in liptinite macerals (Taylor et al., 1998).

On the basis of methane generation extent and vitrinite reflectance analysis of

samples from Thar coal field, it can be suggested that Thar coal seams may be at that stage

of geological age where it may be susceptible to biogasification. Another related aspect is

possible presence of coalbed methane (CBM) in Thar, which has never been explored

vigilantly at government level. Recent developments in investigations of mechanisms

involved in the formation of biogenic methane and similarity of some biogenic CBM

producing coal deposits (Powder River Basin, USA) to Thar warrant at least some

investigations for CBM prospects in Pakistan. Additionally, Badin and Thar coals have

been considered to be potential candidates for CBM (Ahmad et al., 2010). Now, these

forecasts along with investigations of these studies including detailed petrographic studies

may confer the possibility of biogenic CBM at Thar (Figure 4.2). The potential of biogenic

CBM needs to be explored at Thar coal reserves as this methane generation through WBC-

2 based bioassay may be an indicative index of the probable presence of adsorbed methane

in Thar coal seams.


Figure 4.2 | Correlation of Physical Parameters of Coal; With Reference to CBM Origin

(Moore, 2012)

The residual coal after methane extraction may find applications in the extraction

of humic acid-based soil conditioning agents. In this study, the residual coal, after fungal

pretreatment, was processed for the extraction of the humic acid through alkali

solubilization, thus indicating the improvement in chemical properties of the humic acids.

In a previous report, the prior fungal treatment modified the structure of coal, which,

eventually, resulted in better distribution of the oxygen containing functional groups

around humic acid molecule (Dong et al., 2006). Generally, the biological treatment of

fungi results in oxidation of low rank coal and in this regard this oxidized lignite can serve

as a suitable raw material for the extraction of humic acids, as leonardite (highly oxidized

lignite) is considered to be one of the best raw materials for the extraction of soil


conditioning agents. On the basis of petrographic studies of lignite samples, the high

huminite content in Thar coals may also make them a potential source of materials for other

useful commodities, such as soil-conditioning agents, though an extensive study for

determining specific structural functionalities would be required for such bioactive agents.

It can be concluded that fungal degradation of coal may find an alternative

application in terms of extracting some other chemical entities like humic acids and this

extraction of humic acids or soil conditioning agents would be an alternative non-fuel

application of low rank coals. Particularly, Thar lignite can have a variety of applications

in this regard and agriculture sector of Pakistan can be boosted, as well. Being an

agricultural country, the backbone of country’s economy is agriculture and this sector can

be improved with the help of indigenously manufactured humic acids, which is not the case

right now.

5

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6

Appendices

189 GC-MS Programme Profile |

APPENDIX-I

GC-MS Programme Profile for the Analysis of Released Organics

GC Method

Oven

Initial Temperature: 50°C Initial Time: 4.00 min

Final Temperature: 350°C Equilibration Time: 0.50 min

Ramps:

No. Rate Final Temp Final Time

1 10.00 150 0.00

2 6.00 230 0.00

3 3.00 300 5.00

4 0.0 (Off)

Post Temperature: 0°C

Post Time: 0.00 min

Run Time: 55.67 min

Front Inlet

Mode: Splitless

Initial Temperature: 280°C

Pressure: 6.49 psi (On)

Purge Flow: 35.0mL/min

Purge Time: 2.00 min

Total Flow: 39.1mL/min

Gas Saver: On

Saver Flow: 25.0mL/min


Saver Time: 3.00 min

Gas Type: Helium

Column

Capillary Column

Model Number: Agilent 19091S-433

HP-5MS, 0.25mm x 30m x 0.25µm

Maximum Temperature: 350°C

Nominal Length: 30.0m

Nominal Diameter: 250.00µm

Nominal Film Thickness: 0.25µm

Mode: Constant Flow

Initial Flow: 0.9mL/min

Nominal Initial Pressure: 6.49 psi

Average Velocity: 35cm/sec

Inlet: Front Inlet

Outlet: MSD

Outlet Pressure: Vacuum

Injector

Sample Washes: 0

Sample Pumps: 3

Injection Volume: 2.0mL

Syringe Size: 10.0mL

Post Injection Solvent A Washes: 2

Post Injection Solvent B Washes: 2

Viscosity Delay: 0


Plunger Speed: Fast

Pre Injection Dwell: 0.00 minutes

Post Injection Dwell: 0.00 minutes

MS Acquisition Parameters

Tune File: atune.u

Acquisition Mode: Scan

Solvent Delay: 5.00 min

EM Absolute: False

EM Offset: 0

Resulting EM Voltage: 2470.6

Scan Parameters

Low Mass: 50.0

High Mass: 550.0

Threshold: 150

MS Zones

MS Quad: 150°C Maximum 200°C

MS Source: 230°C Maximum 250°C

192 Maceral Composition of Coal Samples (Mineral Content Free) |

APPENDIX-II

Maceral Composition of Coal Samples (Mineral Content Free)

Serial No. Sample Maceral Groups and Mineral Content (Volume %)

Vitrinite/Huminite Inertinite Liptinite

1 7 (BLCH) 96.21 02.10 01.69

2 DSA-23-4 74.78 21.66 03.56

3 MKCT-6 86.91 08.80 04.29

4 UAL-15-1 75.75 15.66 08.59

5 UAL-15-2 73.11 15.97 10.92

6 LS-4-1 84.62 11.54 03.84

7 LS-4-2B 50.69 39.22 10.09

8 UAS-4-1 79.66 14.67 05.67

9 UAS-4-2E 75.52 17.63 06.85

10 UAT-4-1 65.59 15.71 18.70

11 UAT-4-4 85.49 01.04 13.47

12 UAK-1-4 85.86 08.44 05.70

13 UAK-1-8 64.97 29.30 05.73

14 UAJ-1-1 86.00 00.00 14.00

15 TP-1-1.1 91.73 01.50 06.77

16 TP-1-3.3 87.10 03.87 09.03

193 Maceral Composition of Coal Samples (Mineral Content Free) |

Serial No. Sample

Maceral Groups and Mineral Content (Volume %)


17 TP-1-4.5 82.74 3.98 13.28

18 TP-1-5.2 77.16 0.0 22.84

19 TP-1-6.1 85.23 1.82 12.95

20 TP-3-2B 85.38 1.60 13.02

21 TP-3-2D 80.28 2.82 16.90

22 TP-3-2K1 77.38 0.0 22.62

23 TP-3-2R 80.85 1.28 17.87

24 TP-3-2X 54.84 11.29 33.87

25 TP-3-2AG 91.62 1.93 6.45

26 TP-4-1A 86.09 2.65 11.26

27 TP-4-2A 88.83 2.35 8.82

28 TP-4-6 88.56 2.49 8.95

29 TP-4-8 73.11 5.89 21.00

30 TP-4-10 88.04 0.48 11.48

31 TP-31 87.54 2.94 9.52

RESEARCH PUBLICATIONS

Haider, R., Ghauri, M.A., Jones, E.J., SanFilipo, J.R., 2014. Methane Generation Potential of Thar Lignite Samples. Fuel Processing Technology (Accepted Manuscript; IF=2.816)

Haider, R., Ghauri, M.A., SanFilipo, J.R., Jones, E.J., Orem, W.H., Tatu, C.A., Akhtar, K., Akhtar, N., 2013. Fungal degradation of coal as a pretreatment for methane production. Fuel 104, 717-725 (IF=3.357)

Ghauri, M.A., Anwar, M.A., Akhtar, N., Haider, R., Tawab, A., 2009. Status of Coal Biotechnology in Pakistan. Advanced Materials Research 71-73, 513-516.

PAPERS/CONFERENCES/ABSTRACTS/SEMINARS

Coalbed Methane (CBM) – Future Prospects in Pakistan

One Day Conference on Coalbed Methane Potential in Pakistan,

Quantum Energy, Islamabad 2014

Exploring and Exploiting Unconventional Natural Gas Resources in Pakistan (vis-à-vis Applications of

Biotechnology in Clean Energy Industry)

1st National Student Conference on Biological Sciences, March 27-28,

2014, NIBGE, Faisalabad, PAKISTAN 2014

Biogenic Methane Generation from Low Rank Coal of Pakistan

UK Pakistan Coal Conference-2012, University of Leeds, UK, (Abstract

Booklet) 2012

Conversion of Coal into Methane (Understanding the Mechanisms behind Biological Degradation of

Coal)

Two Day National Workshop on Bioclean Coal Technologies (A Step

towards the Use of Indigenous Coal Resources), NIBGE, Faisalabad,

PAKISTAN 2012

Methane Generation Potential of Low Rank Coals from Pakistan

Eastern Energy Resources Centre, Unites States Geological Survey,

Virginia, USA 2011

Unravelling the Mysteries of Biogenic Coalbed Methane (With Elizabeth Jones)

USGS Brown Bag Seminar Series, Unites States Geological Survey, Virginia, USA 2011

NATIONAL PRESS

Embarking on an Energy Age

Sci-tech World, Dawn Newspaper January 10, 2009

Coal Biotechnology

Dawn Economic and Business Review, Dawn March 10, 2008

DME-The New Fuel Option

Sci-tech World, Dawn Newspaper August 12, 2006

Let there be Light

Sci-tech World, Dawn Newspaper June 17, 2006

Tapping our Prime Resources

Sci-tech World, Dawn Newspaper April 15, 2006

Biotechnology of Coal and Bioprocessing

Sci-tech World, Dawn Newspaper February 18, 2006

Pollution Free Fuels

Sci-tech World, Dawn Newspaper January 12, 2006

Fuel 104 (2013) 717–725

Contents lists available at SciVerse ScienceDirect

Fuel

journal homepage: www.elsevier .com/locate / fuel

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: [email protected] (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

ll rights reserved.

+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

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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)


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


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


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).


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).


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).


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


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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[3] Jones EJP, Voytek MA, Corum MD, Orem WH. Stimulation of methanegeneration from nonproductive coal by addition of nutrients or a microbialconsortium. Appl Environ Microbiol 2010;76(21):7013–22.

[4] Strapoc D, Picardal FW, Turich C, Schaperdoth I, Macalady JL, Lipp JS, et al.Methane-producing microbial community in a coal bed of the illinois basin.Appl Environ Microbiol 2008;74(12):2424–32.

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[6] Gokcay CF, Kolankaya N, Dilek FB. Microbial solubilization of lignites. Fuel2001;80:1421–33.

[7] Hofrichter M, Bublitz F, Fritsche W. Fungal attack on coal II. Solubilization oflow-rank coal by filamentous fungi. Fuel Process Technol 1997;52(1–3):55–64.

[8] Ralph JP, Catcheside DEA. Transformations of low rank coal by Phanerochaetechrysosporium and other wood-rot fungi. Fuel Process Technol 1997;52(1–3):79–93.

[9] Achi OK. Growth and coal-solubilizing activity of Penicillium simplicissimumon coal-related aromatic compounds. Bioresource Technol 1994;48(1):53–7.

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[11] Beckmann SKM, Engelen B, Gorbushina AA, Cypionka H. Role of bacteria,archaea and fungi involved in methane release in abandoned coal mines.Geomicrobiol J 2011;28:347–58.

[12] Shah SMI. Stratigraphy of Pakistan. Geol surv pak 1977;12:138.[13] SanFilipo JR, Khan SA, Chandio AH. Geological survey of pakistan, United States

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[16] SanFilipo JR, Chandio AH, Khan SA, Khan RA, Shah AA. Results of coalexploratory drilling from February 1992 to July 1992, coal resourceexploration and assessment program (COALREAP). Thar desert, Lakhra south,Indus plain and adjacent areas. US Geological Survey Open-File Reports 94-595; 1994.

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Figure 1. Primary Energy Consumption-Pakistan vs World

Status of coal biotechnology in Pakistan

M. A. Ghauri1, a, M. A. Anwar1,b, N. Akhtar1,c, R. Haider1,d and A. Tawab1,e 1 Industrial Biotechnology Division,

National Institute for Biotechnology and Genetic Engineering (NIBGE),

P. O. Box No. 577, Jhang Road, Faisalabad, Pakistan

Tel: +92-41-2550814, Fax: +92-41-2651472 [email protected], [email protected], [email protected], [email protected],

[email protected]

Keywords: Coal, Biodesulphurization, At. ferrooxidans, At. thiooxidans, S. thermosulfidooxidans. Abstract. Pakistan is endued with 185 billion tons colossal reserves of coal, but only 7.89 % of the country total energy requirements are met by coal. Most of the Pakistani coal reserves are sub-bituminous or lignitic in nature and contain 3-12 % sulphur. Existence of sulphur compounds in coal limits its industrial application due to environmental as well as technical problems. However, coal biotechnology can emerge as panacea for upgrading the huge reserves of coal in Pakistan. In general, coal biotechnology refers to biodesulphurization, biosolubilization and biogasification of coal. NIBGE has long term interests in the field of coal bioprocessing for tapping prime resources of indigenous coal. In NIBGE, lab scale experiments for coal biodesulphurization led to 90% efficiency in sulphur removal. Heap leaching was also carried out at the level of 10 and 20 tons coal heaps with 60% sulphur removal efficiency. Furthermore, a prototype of 300 tons coal heap was set up with a local cement industry and 75% microbial desulphurization was achieved. The league of indigenously isolated chemolithotrophic bacteria was involved in coal desulphurization. On the other side, for making the best use of 175 billion tons of low rank coal reserves, coal biosolubilization and subsequent biogasification is being projected. Consequently, beneficiated coal through biotechnology is supposed to contribute in energy mix of Pakistan for providing electricity requirements of the country and saving huge oil import bills.

Introduction

Almost 30% share of coal and 3.2% annual increase in world energy consumption depicts its importance in energy systems. Coal is mainly being used as a fuel in thermal power generation, cement industries, in brick kilns, and as a chemical feedstock for various industries such as fertilizers and metallurgical operations. Pakistan, with 185 billion tons of coal reserves, has just 7.89% share in terms of coal in total energy consumption (Fig. 1) [1]. However, the high sulphur content in indigenous coal (3-12%) limits its industrial application due to environmental as well as technical problems. Sulphur as a constituent of coal can occur as pyrite, sulphate or organic sulphur containing compounds [2]. Various approaches have been proposed for removing sulphur from coal, which can be mainly categorized as 1) post-combustion 2) during combustion and 3) pre-combustion-relatively more efficient. However, biotechnology can contribute significantly in coal beneficiation processes as pre-combustion approach. It has an edge over post-combustion desulphurization technologies being environment friendly approach, minimal damage of machinery due to emission of corrosive gases and dissolution of minerals present in coal, thus reducing ash

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Table 1. Sulphur Content of Coal Samples

content [3]. Furthermore, it has low capital and operating cost due to mild operating conditions, inexpensive reagents, simplicity of design and construction and less energy consuming than that of chemical processes. Realizing the strategic importance of coal, studies were undertaken to optimize the process parameters for coal biodesulphurization as well as to upscale the process for commercial exploitation, while, studies, regarding coal biosolubilization and subsequent biogasification, have been initiated. Coal. Coal samples were collected from Padhrar, Khushab (Punjab), Harnai (Balochistan) and Lakhra (Sindh) coal mines of Pakistan. The sulphur contents of these samples have been charted in Table 1. Bacterial Cultures. Mesophilic and moderately thermophilic bacteria were isolated from coalmine sites and other extreme habitats of Pakistan. On the basis of cultural and 16S rDNA analyses, these isolates were identified as strains of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans. The origin and some of the characteristics of these isolates are given in Table. 2. Lab Scale Studies. Shake flask studies were conducted in basal salt solution (pH 2) [4] using mesophiles and moderate thermophiles to desulphurize low-grade high sulphur coals (coal mesh size 150 µm). Amongst mesophiles, HC-AF2 was found relatively more active in biodesulphurization through achieving 93% removal of pyritic sulphur from Padhrar coal in 28 days with removal rate of 0.299g L-1 day-1 (Fig. 2).

Origin of Coal

Sulphur Content [%] Total Pyritic Sulphate Organic

Harnai 10 6.5 1.0 2.5

Lakhra 6.5 4.0 0.3 2.2

Padhrar 8.5 5.5 0.5 2.5

Figure 3. Biodepyritization of Coal by Moderately Thermophilic, Iron and Sulphur Oxidizing Bacteria

Bacteria Source Characteristics Mesophiles Iron & Sulphur Oxidizing HC-AF1 Harnai Coal motile rods, Gram–ve, At. ferrooxidans-like HC-AF2 Harnai Coal motile rods, Gram –ve At. ferrooxidans-like MC-AF1 Mach Coal motile rods, Gram–ve At. ferrooxidans-like MC-AF2 Mach Coal motile rods, Gram–ve At. ferrooxidans-like KC-AF1 Khushab Coal motile rods, Gram–ve At. ferrooxidans-like Sulphur Oxidizing HC-AT1 Harnai Coal motile rods, Gram–ve At. thiooxidans-like KC-AT1 Khushab Coal motile rods, Gram–ve At. thiooxidans-like KC-AT2 Khushab Soil motile rods, Gram–ve At. thiooxidans-like Moderate Thermophiles Iron & Sulphur Oxidizing LC-MTH Lakhra Coal motile rods, Gram+ve S. thermosulfido-like MT13 Uranium Mine motile rods, Gram+ve S. thermosulfido-like TH 1 Gift Sulfobacillus thermosulfidooxidans (Dr. Barrie Johnson, Bangor University, UK)

Table 2. Description of Different Bacterial Strains Used in the Study

Figure 2. Biodepyritization of Coal by Mesophilic, Iron and Sulphur Oxidizing Bacteria

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Figure 7. Operation of 300 tons Coal Heap for Biodesulphurization

Similarly, amongst moderate thermophiles, the strain MT-13 removed 54% of pyritic sulphur from coal in three days at a rate of 0.95g L-1day-1 (Fig. 3). Upscaling of Coal Biodesulphurization i) 12L Fermentor Studies. Fermentor (B-Braun) having 12L working volume was run at 20% (w/v) coal pulp density (coal mesh size 150 µm) with air flow rate 600 mL min-1, temperature 45ºC for moderate thermophile (Sulfobacillus thermosulfidooxidans), agitation speed 200 rev min-1 and pH 2. About 77% of pyritic sulphur removal was achieved in 5 days (Fig. 4). The biodepyritization rate in the fermentor was 1.1 gL-1day-1. ii) 50L Airlift Bioreactor Studies (Drum Bioreactor). Bioreactor, with 50L working volume, was fabricated using plastic drum of 70L capacity. Glass tubes were inserted vertically through the lid for the injection of air. The bioreactor was run at 20% (w/v) coal pulp density (coal mesh size 150 µm) in basal salt solution (pH 1.8). About 53% reduction in pyritic sulphur content of coal was observed in 24 days, employing pure culture of Acidithiobacillus ferrooxidans HC-AF2 strain (Fig. 5). However, a mixed consortium of mesophiles (Acidithiobacillus ferrooxidans HC-AF2 and Acidithiobacillus thiooxidans KC-AT2) and a moderately thermophile (S. thermosulfidooxidans MT13) removed about 75% of pyritic sulphur from coal under similar environmental conditions. The rate of sulphur removal by the mixed consortium was also higher (0.177 gL-1day-1) as compared to pure culture (0.128 gL-1day-1). iii) Heap Leaching Studies. Keeping in view the above given results of coal desulphurization studies, the process was scaled up, further, for large-scale desulphurization. Heaps of 10 [dimensions 4.0m x 2.5m x 1m (L x W x H)] and 20 tons [dimensions 5m x 4m x 1.5m (L x W x H)] were established at pilot scale (Fig. 6), while a prototype of 300 tons [dimensions 25m x 10m x 6ft (L x w x H)] coal heap was set up with a local cement industry for coal biodesulphurization at larger scale (Fig. 7).

Figure 4. Biodepyritization of Coal by S. thermosulfidooxidans in 12 L Fermentor

Figure 5. Biodesulphurization of Coal by Pure Culture of At. ferrooxidans (HC-AF2) and its Mixed Consortium with At. thiooxidans (KC-AT2) and S. thermosulfidooxidans (MT13) in 50L Drum Bioreactor)

Figure 6. 20 tons Coal Heap for Biodesulphurization at NIBGE

Advanced Materials Research Vols. 71-73 515



The heaps (coal lump size ~5 cm) were inoculated with a mixed consortium of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans. Provisions were made to remove the exhaust gases from the heaps. Heaps were irrigated vigorously with the culture using overhead showers. In case of 10 and 20 tons coal heap about 60% of total sulphur removal was achieved in 39 days, while in case of 300 tons coal nearly 75% of total sulphur was removed after the same time period at significantly higher rates than the 10 and 20-tons coal heaps (Fig. 8).

Future Prospects of Coal Biotechnology

The transmutation of coal and, especially, lignite (Low Rank Coal) into liquid and gaseous fuels can provide an environmentally safe use of huge reserves of coal, as high rank coals are being exploited at greater rate and soon these reserves will be depleted. Transformation of low rank coals followed by the application of biotechnology would provide us an economic and effective way towards the better utilization of indigenous resources with significant contribution in the production of value-added products [5]. Pakistan has great deal of potential in the form of 175 billion tons of Thar lignite. Keeping in view the significance of this huge lignite reserve, coal biosolubilization and subsequent biogasification is being projected at laboratories of NIBGE for efficient utilization of this huge low rank coal reserve.

Conclusion

Pakistan can be able to resolve issues regarding energy disruptions through productive utilization of high sulphurous coals and huge low rank resources through biotechnological beneficiation. The microbial desulphurization can save the potential in terms of US $ 7 per tone with respect to hydro and flue gas desulphurization. Currently, in Pakistan imported coal is available at the landed cost of US $ 180 per tone as compared to the local with US $ 103 per tone. Keeping in view the total landed cost of imported and local biotreated coal almost US $ 70 per tone can be saved, which makes a handsome amount, regarding high sulphur-coal reserves of Pakistan. The increased share of coal in energy mix is required which would stabilize energy sector by providing continuous energy supply and reducing huge oil import bills.

References

[1] BP Statistical Review of World Energy. June (2008), p. 41 [2] I. P. Ivanov: Solid Fuel Chemistry Vol. 41 (2007), p. 3 [3] P. Prayuenyong: J. Sci. Technol. Vol. 24 (2002), p. 493 [4] W. W. Leathen; N. A. Kinsel and I. A. Braley: J. Bacteriol. Vol. 72 (1956), p. 700 [5] A. Gupta and K. Birendra: Fuel Vol. 79 (2000), p. 103

0.00

0.50

1.00

1.50

2.00

Des

ulfu

rizat

ion

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e [g

/Kg

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oal/d

ay]

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Figure 8. Comparison of Coal Biodesulphurization Rates in Heaps of Various Sizes

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Biohydrometallurgy 2009 doi:10.4028/www.scientific.net/AMR.71-73 Status of Coal Biotechnology in Pakistan doi:10.4028/www.scientific.net/AMR.71-73.513 References[1] BP Statistical Review of World Energy. June (2008), p. 41 [2] I. P. Ivanov: Solid Fuel Chemistry Vol. 41 (2007), p. 3doi:10.3103/S0361521907010028 [3] P. Prayuenyong: J. Sci. Technol. Vol. 24 (2002), p. 493 [4] W. W. Leathen; N. A. Kinsel and I. A. Braley: J. Bacteriol. Vol. 72 (1956), p. 700 [5] A. Gupta and K. Birendra: Fuel Vol. 79 (2000), p. 103doi:10.1016/S0016-2361(99)00097-6

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prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/2264/1/2507S.pdf · CERTIFICATE This thesis, submitted by MR. RIZWAN HAIDER (Registration No. 2141-NIBGE/Ph.D-2007) is accepted