University of Canberra. This thesis is available in print format from the University of Canberra Library.

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1 University of Canberra This thesis is available in print format from the University of Canberra Library. If you are the author of this thesis and wish to have the whole thesis loaded here, please contact the University of Canberra Library at Your thesis will then be available on the www providing greater access.

2 METAL-REDUCING MICROORGANISMS IN PETROLEUM RESERVOIRS SHAHRAKBAH YACOB MASTER OF APPLIED SCIENCE UNIVERSITY OF CANBERRA 2000

3 ACKNOWLEDGEMENTS I am deeply indebted to a number of people with whom I have worked during my project and from whom, by association and discussion, I have learnt so much that has helped me in my task. First of all, I am very grateful to Land & General (Malaysia) Bhd. for sponsoring the research project. In this assignment, much support was received from Dr. Baharuddin Abd Ghani and Mr. Zainol Alang, Chief Operating Officer of Research & Development and Azerbaijan Operations, respectively. Special word of thanks to the Director of MRU, who is also my primary supervisor, Professor Alan Sheehy, for his excellent editorial and guidance and for the time and trouble taken to read through the manuscript. I wish most particularly to express my gratitude to Dr. Tony Greene, who is my co-supervisor, and gave me a basic training, the value of which I have appreciated more and more with the passing years, also for his invaluable criticisms, suggestions and demonstrated great patience while waiting for the thesis to be completed. I am very much indebted to three wonderful people, Dr. Gino Grassia, Dr. Gavin Rees, and Dr. Tony Dimichel, who gave valuable technical and moral support when things did not develop as they were expected to. Special thanks are also due to staff of Faculty of Applied Science and to those who experienced the MRU, Monica Cole, Tony Hancock, Kathryn Russel, Karyn Wilde, Claire Batum, Elaine Wyatt and Kathryn Smith. Many thanks are forwarded to Dr. Bharat Patel of Griffith University, Roger Heady and Lily Shen of Australian National University for their collaboration and significant contribution to the thesis. Most of all I am indebted to my wife for her unfailing patience, encouragement and immeasurable support. S. Yacob, Malaysia. iii

4 ABSTRACT Metal-reducing microorganisms reduce a variety of metals in metabolic processes coupled to the oxidation of organic compounds. These bacteria play an important role in the biogeochemical cycling of metals and organic matter in anaerobic aquatic and sediment ecosystems. It has been proposed recently that metal-reducing microorganisms also are active in deep subsurface environments such as petroleum reservoirs. Only two metal-reducing bacteria have been isolated from petroleum reservoir fluids, Shewanella putrefaciens and Deferribacter thermophilus. This project studied the occurrence and distribution of metal-reducing microorganisms in petroleum reservoirs. The research focused on the isolation, characterisation and identification of anaerobic bacteria from petroleum reservoirs that were capable of reducing metals and the potential roles of these isolates in the microbial ecology and biogeochemical cycling of petroleum reservoirs. Petroleum reservoirs were selected for this study on the basis of physio-chemical conditions such as temperature, salinity, ph and the presence of organic and inorganic compounds, that were likely to provide a suitable environment for anaerobic bacteria capable of reducing metals. Factors such as the stratigraphic features of the sedimentary basin, age of reservoir and past oil field practices also were considered in choosing the reservoir for study. Seven petroleum reservoirs in the USA and Azerbaijan were chosen for extensive investigations. The physico-chemical conditions in these reservoirs varied substantially. A systematic study of the production water from these petroleum reservoirs revealed a consistent presence of iron- and manganese-reducing microorganisms. It was found that salinity and temperature play a significant and defining role in the occurrence and distribution of these metal-reducing microorganisms. Biotic metal reduction was detected from production waters from all but one of the oil wells sampled. It was significant that the water from this well (Neftcala #1074) was the most saline (78 g/l NaCI). Metal-reducing activity was detected at temperatures up to 70 C. Two pure cultures, strains RED1 for Redwash petroleum reservoir (USA) and NEF1 from the Neftcala petroleum reservoir (Azerbaijan) were isolated and characterized.

5 The strains had diverse physiological and metabolic properties including the ability to oxidize a wide range of carbon compounds and reduce a variety of metals. Their temperature, salinity and ph optima varied markedly. Phylogenetic analyses of the 16S rrna of strain RED1 showed that the strain represented a new species of a new genus in the domain Bacteria. The bacterium most closely related to strain RED1 is the fermentative Fe(III)-reducer, Pelobacter acetylenicus (similarity value, 92.8%). Strain NEF1 possesses a unique combination of phenotypic traits and a low mol % G+C. From preliminary analyses and comparative biochemistry, NEF1 appears to be a novel metal-reducing bacterium of the Flexistipes group. The bacteria isolated in this study were able to grow at temperatures and salinities consistent with the reservoir from which they were isolated. This indicated that petroleum reservoirs are a new source of physiologically diverse, novel, metal-reducing microorganisms. The bacteria isolated also demonstrated a number of characteristics that would enable them to survive and persist in extreme subsurface conditions and develop a selective ecological advantage in petroleum reservoir environments. Significantly, the metal-reducing bacteria isolated were able to utilize an array of metabolic products produced by bacteria indigenous to petroleum reservoirs. This has resulted in a new proposed model for the ecological succession of bacteria in petroleum reservoirs.

6 CONTENTS ABSTRACT i ACKNOWLEDGMENTS iii 1. INTRODUCTION General introduction Biogeochemical cycling of organic matter coupled to iron and 2 manganese reduction 1.3 Dissimilatory metal-reducing microorganisms Iron reduction Manganese reduction Selenium reduction Chromium reduction Molybdenum reduction Petroleum reservoirs Petroleum formation and accumulation Petroleum reservoir environment Petroleum reservoir ecology Sulfate-reducing bacteria (SRB) Methanogens Fermentative microorganisms Aerobic microorganisms Chemical composition of petroleum reservoir fluids Inorganic compounds Organic compounds Occurrence of metal-reducing microorganisms in petroleum 31 reservoirs 1.6 Aims MATERIALS AND METHODS Media Media components Media preparation Supplements to media 35 iv

7 2.1.4 Metals/electron acceptors Amorphous Fe(lll) oxyhydroxide Amorphous manganese dioxide Solid media Agar plates Agar tubes Screening of petroleum reservoir fluids Reservoirs tested Sample collection Screening procedures Enrichment and isolation of metal-reducing bacteria Enrichment Isolation and purification Characterization of bacterial strains Morphological characteristics Gram reaction Electron microscopy Colony morphology Growth studies Growth curves Determination of temperature optimum and range Determination of ph optimum and range Determination of salinity optimum and range Nutritional studies Organic acids Alcohols Amino acids Biological extracts H 2 +CO Antibiotics susceptibility Molecular characterization Deoxyribonuclease acid (DNA) base composition S rrna analysis Dissimilatory Fe(lll) and Mn(IV) reduction studies 56

8 2.5.1 Preparation of Fe(lll) forms Ferrihydrite Geothite Preparation of Mn(IV) forms Vemadite Colloidal Mn(IV) Comparison of different Fe(lll) and Mn(IV) species as 57 electron acceptors Morphology Of Metal Oxides Alternative electron acceptors to Fe(lll) and Mn(IV) Carbon utilization Analytical techniques Fe(lll) assay Mn(IV) assay HPLC analyses Cell density RESULTS Screening of petroleum reservoir waters Metal reduction by microorganisms Enumeration of metal-reducing microorganisms in petroleum 66 reservoir fluids Enrichment for metal-reducing microorganisms Redwash petroleum reservoir fluids Neftcala #752 petroleum reservoir fluids Isolation and purification of bacterial strains Redwash petroleum reservoir fluids Neftcala #752 petroleum reservoir fluids Characterisation of metal-reducing bacteria from redwash# B and Neftcala # Cellular features Growth studies Growth curves Temperature optimum and range for metal- 78 reducing microorganisms vi

9 ph optimum and range for metal-reducing 80 microorganisms Salinity optimum and range for metal-reducing 81 microorganisms Nutritional studies Antibiotic susceptibility Molecular characterization Deoxyribonuclease acid (DNA) base composition S rrna sequencing Bacterial metal reduction studies Dissimilatory iron reduction Dissimilatory manganese reduction Comparison study between metal-reducing strains Electron acceptors utilized by RED1 and NEF Utilization of carbon compounds DISCUSSION CONCLUSIONS REFERENCES APPENDICES 1. Characteristics of the Xilli oilfield reservoirs Characteristics of the Neftcala oilfield reservoirs Standard curve for quantification of Fe(lll), developed on a Varian Cary double beam spectrophotometer 4. Standard curve for quantification of Mn(IV), developed on a Varian Cary double beam spectrophotometer 5. Standard curve for acetate measured on a Waters High Performance 156 Liquid Chromatography using a Maxima Chromatography Version 3.3 (Waters) 6. Thermal denaturation curve for strain RED1 DNA performed on Varian 157 Cary 3 double beam spectrophotometer at 260 nm 7. Thermal denaturation curve for strain NEF1 DNA performed on Varian 158 Cary 3 double beam spectrophotometer at 260 nm VII

10 8a. Scanning electron microscope photomicrograph of amorphous Fe(lll) 159 oxyhydroxide crystals 8b. Scanning electron microscope photomicrograph of ferrihydrite crystals 160 8c. Scanning electron microscope photomicrograph of geothite crystals 161 9a. Scanning electron microscope photomicrograph of amorphous 162 manganese dioxide crystals 9b. Scanning electron microscope photomicrograph of vemadite crystals 163 9c. Scanning electron microscope photomicrograph of pyrolusite crystals Jukes-Cantor evolutionary distance matrix table of strain RED Reduction of Fe(lll) by strain RED1 over a range of temperature Reduction of Fe(lll) by strain RED1 over a range of ph Reduction of Fe(lll) by strain NEF1 over a range of temperature Reduction of Fe(lll) by strain NEF1 over a range of ph Growth of strain RED1 on various electron acceptors Growth of strain NEF1 on various electron acceptors The inhibition effect of antibiotics on Fe(lll) reduction of strain RED The inhibition effect of antibiotics on Fe(lll) reduction of strain NEF1 174 LIST OF TABLES Table Page 2.1 Physio-chemical characteristics of the petroleum reservoirs sampled 38 for the presence of metal-reducing microorganisms 3.1 Screening for metal-reducing microorganisms from 7 petroleum 65 reservoir wells 3.2 Reduction of synthetic amorphous manganese dioxide by Fe(lll)- 65 reducing enrichment cultures 3.3 Enumeration of Fe(lll)-reducing microorganisms (cells per litre) in 66 Redwash petroleum reservoir fluids 3.4 Organic acid electron donors utilised by strains RED1 and NEF1 with 84 amorphous Fe(lll) oxyhydroxide as electron acceptor 3.5 Alcohol electron donors coupled with amorphous Fe(lll) oxyhydroxide 85 as electron acceptor 3.6 Growth of strains RED1 and NEF1 in the presence of antibiotics Alternative electron acceptors for strains RED1 and NEF1 with 98 viii

11 acetate as a sole source of carbon 4.1 Freshwater and petroleum reservoir medium typically have the 108 following salt composition 4.2 Comparison of some characteristics of strains NEF1, D. thermophilus, 124 F. sinusarabici and G. ferrireducens LIST OF FIGURES Figure Page 1.1 Oxidation of complex organic matter with Fe(lll) serving as the sole 7 electron acceptor, with the examples of organisms that many catalyse the various reactions (Lovley, 1991) 1.2 Cross-section of petroleum reservoir showing the migration and 17 accumulation of petroleum 1.3 Cross-section showing several types of traps which result in 22 petroleum accumulation 3.1a/b Electron micrograph of strain RED1, which demonstrates cell size, 71/72 structure, arrangement, and flagellation. 3.2a/b Electron micrograph of strain NEF1, which demonstrates cell size, 73/74 structure, arrangement, and flagellation. 3.3 Thin section, electron micrograph of strain RED1, which 75 demonstrates the monolayered cell envelope structure. 3.4 Thin section, electron micrograph of strain NEF1, which 76 demonstrates the monolayered cell envelope structure. 3.5 Growth curve of strain RED Growth curve of strain NEF Temperature optimum and range of strain RED Temperature optimum and range of strain NEF ph optimum and range of strain RED ph optimum and range of strain NEF Salinity optimum and range of strain RED Salinity optimum and range of strain NEF Dendogram showing the phylogenetic position of strain RED Fe(lll) reduction by strain NEF Fe(lll) reduction by strain RED1 91 ix

12 3.16 Reduction of various forms of Fe(lll) by strain RED Reduction of various forms of Fe(lll) by strain NEF Growth of RED1 and reduction of Mn(IV) over time Reduction of various species of Mn(IV) by strain RED Reduction of Fe(lll) by strains BMA, RED1 and NEF Reduction of Mn(IV) by strains BMA and RED Growth of strain RED1 with acetate and amorphous Fe(lll) 100 oxyhydroxide 3.23 Growth of strain NEF1 with acetate and amorphous Fe(lll) 101 oxyhydroxide 4.1 Microbial transformation of crude oil in petroleum reservoirs and the 129 involvement of metal-reducing microorganisms in the ecological succession (Modified from Ivanov et al., 1989)