AROMATIC HYDROCARBON DEGRADATION BY HALOALKALIPHILIC AND METAL REDUCING BACTERIA SAAD M. FAROOQUI MSc BIOTECHNOLOGY AND MOLECULAR BIOLOGY (Hons 1A) School of Biomolecular and Physical Sciences Faculty of Science, Environment, Engineering and Technology GRIFFITH UNIVERSITY Queensland, Australia A thesis submitted in fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY SEPTEMBER, 2013 STATEMENT OF ORIGINALITY This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. Saad M. Farooqui II ACKNOWLEDGEMENTS First and foremost I would like to thank Dr Tony Greene for giving me the opportunity to work with him and for inspiring and motivating me with his continual enthusiasm, support and calmness. I would also like to thank Prof Bharat Patel for advising and supporting me throughout my research, especially for his help with genome sequencing and analysis. I would also like to thank Mr Mitchell Wright, Dr Chris Ogg, Dr Lokesh Kori, Mr Subash Baral, Mr Joseph Adelskov, Mr Adam Mazins, Mr Tim Owen, Mr Anto Prgomet, Ms Yuswita Pratiwi and Ms Lindi Olivier for their assistance and friendship during the course of my studies. I appreciate the friendship and help of Mr Mark Haase, the visitors from Nanyang Polytechnic, Singapore, Mr Randall Chan, Ms Yi Jie, Mr Mitchell Wong, Mr Leon Oh Yong Ji, Mr Joshua Ong, Ms Ralene Fok, Ms Ginette, Mr Zeus Nair, Ms Dianne Lee and Ms Geraldine Loke. I gratefully acknowledge the financial support from Griffith University for awarding the Griffith University Postgraduate Research Scholarship and Griffith University International Postgraduate Research Scholarship along with the funding to complete my studies. I would also like to thank the administrative staff in the School of BPS. Finally, I would like to thank my family, especially my parents and brother and friends for their endless love, patience, motivation and support in all that I have achieved and all that I will achieve. III PUBLICATIONS ARISING FROM THIS THESIS IN PREPARATION 1. Farooqui, S. M, Greene A. C. and Patel B. K. C. Draft genome sequence of Halomonas sp. strain ML-15, an aromatic hydrocarbon degrading haloalkaliphilic bacterium. Journal of Bacteriology. 2. Farooqui, S. M, Greene A. C. and Patel B. K. C. Draft genome sequence of Trabulsiella sp. strain BP5, an aromatic hydrocarbon degrading bacterium. Journal of Bacteriology. 3. Farooqui, S. M, Greene A. C. and Patel B. K. C. Degradation of anthracene, pyrene and phenanthrene by haloalkaliphilic Halomonas sp. strain ML-15. Applied and Environmental Microbiology. 4. Farooqui, S. M, Greene A. C. and Patel B. K. C. Degradation of polycyclic aromatic (PAHs) by facultative anaerobic Trabulsiella sp. strain BP5 coupled with simultaneous Fe(III) reduction. Applied and Environmental Microbiology. 5. Farooqui, S. M, Greene A. C. and Patel B. K. C. Halomonas aromaticans sp. nov., a polycyclic aromatic hydrocarbon (PAH) degrading haloalkaliphilic bacterium isolated from Mono Lake. CONFERENCE Presented a poster titled “Degradation of Hydrocarbons by Metal Reducing Bacteria” at the Australian Society for Microbiology Annual Scientific Meeting, 2012. IV ABSTRACT A wide variety of contaminants have been introduced in the environment by anthropogenic activities or natural processes. Pollution not only affects humans but also severely hampers the ecosystem by destroying habitats of the affected flora and fauna. In addition, a variety of environments such as subsurface aquifers, oceans, anoxic soils, sediments and soda lakes have been affected. Contaminants such as polycyclic aromatic hydrocarbons (PAHs) are of significance as they have been identified as mutagenic, carcinogenic and teratogenic. Methods such as use of dispersants, solvent mixtures and detergents have been employed for the clean-up of the affected sites. These chemicals may be useful in the short-term but they significantly affect the surviving organisms and alter the food- chain of the ecosystem. In addition, they don’t necessarily destroy and may mobilize contaminants. In recent years, focus has shifted towards natural processes for degradation of contaminants. Bacteria can be employed for successful, sustainable and harmless clean up of such contaminated environments. Attenuation of pollutants including hydrocarbons such as PAHs can be achieved under aerobic conditions by intrinsic bacteria present in the impacted site or by introduction of organisms capable of biodegradation. Few studies have investigated the degradation potential and mechanisms of bacteria in anoxic, saline and alkaline conditions. The aim of the present study was to isolate PAH degraders from anaerobic and saline and alkaline environments to study the degradation potential. Extensive V ABSTRACT enrichments on samples from Great Artesian Basin (Oil Company of Australia bore), Mono Lake (California) and anoxic soil samples from a bioremediation unit in Caltex Oil Refinery (Lytton, Queensland, Australia) were performed. Enrichments were performed on a variety of hydrocarbons including anthracene, pyrene and phenanthrene (PAHs), aerobically and anaerobically using metals as terminal electron acceptors. Two pure PAH degrading cultures were isolated and studied. Strain BP5, a facultative anaerobe and closely related to Trabulsiella odontotermitis was isolated from the Great Artesian Basin samples and was able to achieve almost complete oxidation of anthracene (93.05%), pyrene (87.25%) and phenanthrene (94.53%) as sole carbon source coupled with simultaneous reduction of Fe(III). Aerobic haloalkaliphilic strain ML-15 belonging to the genus Halomonas was isolated from Mono Lake and was also able to achieve substantial degradation of anthracene (93.45%), pyrene (94.76%) and phenanthrene (95.76%). Characterization and optimization of degradation of these two strains was performed. In addition, genomes of strains ML-15 and BP5 were sequenced, analysed and annotated. In all, 4,462 and 3,930 putative genes were predicted in strains ML-15 and BP5, respectively. Analysis of the genomes revealed the presence of a variety of genes involved in the lower degradation pathways of PAH metabolism. Strains ML-15 had 30 coding sequences involved in metabolism of aromatic compounds and strain BP5 had 41 sequences involved in metabolism of aromatic compounds. The predicted PAHs degradation pathways have been described. VI ABSTRACT The results obtained from this study are of utmost importance in the field of bioremediation as strain BP5 is the first reported soluble-iron reducing isolate capable of degrading PAHs and strain ML-15 is one of very few aerobic haloalkaliphilic PAH degraders. This work will serve as an initial study to further in-depth studies of degradation and mechanisms. VII TABLE OF CONTENTS STATEMENT OF ORIGINALITY……………………………………………………II ACKNOWLEDGEMENTS……………………………………………………………III PUBLICATIONS ARISING FROM THIS THESIS……………………...…………IV ABSTRACT……………………………………………………………………………..V TABLE OF CONTENTS…………………………………………………………….VIII LIST OF FIGURES………………………………………………………………….XIII LIST OF TABLES……………………………………………………………………XVI LIST OF ABBREVIATIONS……………………………………………………...XVIII CHAPTER ONE: INTRODUCTION AND PROJECT OUTLINE 1.1 GENERAL INTRODUCTION……………………………………………………...1 1.2 BACTERIAL GROWTH ON ORGANIC COMPOUNDS……………………….5 1.2.1 AEROBES………………………………………………………………………5 1.2.2 ANAEROBES…………………………………………………………………..7 1.3 HYDROCARBON CONTAMINATION…………………………………………..9 1.4 HYDROCARBON DEGRADATION……………………………………………..11 1.4.1 AEROBIC HYDROCARBON DEGRADATION…………………………….12 1.4.2 ANAEROBIC HYDROCARBON DEGRADATION………………………...16 1.4.3 HYDROCARBON DEGRADATION BY ALKALIPHILES AND HALOPHILES…………………………………………………………………22 1.4.4 HYDROCARBON DEGRADATION PATHWAYS AND GENES INVOLVED……………………………………………………………………25 1.5 MONO LAKE AND ITS ENVIRONMENTAL CONDITIONS………………...29 1.6 THE GREAT ARTESIAN BASIN (GAB)………………………………………...34 1.7 PROJECT OUTLINE……………………………………………………………...37 VIII TABLE OF CONTENTS CHAPTER TWO: GENERAL MATERIALS AND METHODS 2.1 SAMPLE SITE AND SAMPLE COLLECTION TECHNIQUES………………39 2.1.1 MONO LAKE ………………………………………………………………...39 2.1.2 THE GREAT ARTESIAN BASIN……………………………………………41 2.1.3 CALTEX OIL REFINERY SOIL SAMPLE………………………………….42 2.1.4 SAMPLE COLLECTION……………………………………………………..43 2.2 BUFFERS AND MICROBIAL MEDIA…………………………………………..44 2.2.1 BUFFERS……………………………………………………………………..44 2.2.2 MEDIA PREPARATION……………………………………………………..44 2.2.3 MONO LAKE MEDIUM……………………………………………………..46 2.2.4 BASAL SALTS MEDIUM…………………………………………………...47 2.2.5 INOCULATING FLUID…………………………………………………...…48 2.2.6 MacCONKEY AGAR………………………………………………………...48 2.3 STOCK SOLUTIONS, REDUCTION OF ELECTRON ACCEPTOR UTILIZATION AND Fe(III) UTILIZATION ASSAY………………………...49 2.3.1 STOCK SOLUTIONS OF AROMATIC COMPOUNDS……………………49 2.3.1.1 ANTHRACENE, PYRENE AND PHENANTHRENE (PAHs)………...49 2.3.1.2 ELECTRON ACCEPTORS……………………………………………..49 2.3.2 REDUCTION OF ELECTRON ACCEPTORS………………………………50 2.3.3 IRON(III)-REDUCTION ASSAY……………………………………………51 2.4 PURE CULTURE ISOLATION AND LONG TERM STORAGE………….….53 2.4.1 PURIFICATION TECHNIQUES IN LIQUID MEDIA……………………...54 2.4.2 PURIFICATION TECHNIQUES IN SOLID MEDIA……………………….54 2.4.3 DETERMINATION OF CULTURE PURITY……………………………….55 2.4.4 STORAGE OF PURE CULTURES…………………………………………..55 2.5 SUBSTRATE UTILIZATION TEST……………………………………………..56 2.5.1 BD BBL CRYSTALTM IDENTIFICATION SYSTEMS……………………..56 2.5.2 BIOLOG GN2 MicrPlateTM...............................................................................58 2.6 16S rRNA GENE and WHOLE GENOME SEQUENCING……………………61 2.6.1 DNA EXTRACTION FROM PURE CULTURES…………………………..61 2.6.2 AGAROSE GEL ELECTROPHORESIS…………………………………….62 IX TABLE OF CONTENTS 2.6.3 PCR AMPLIFICATION OF 16S rRNA GENES…………………….………63 2.6.4 CLEAN UP OF PCR PRODUCTS…………………………………….……..65 2.6.5 AUTOMATED DYE TERMINATOR CYCLE SEQUENCING……………65 2.6.6 PRECIPITATION OF SEQUENCING REACTION PRODUCTS………….66 2.6.7 16S rRNA GENE SEQUENCING AT AUSTRALIAN GENOME RESEARCH FACILITY (AGRF)…………………………………………...66 2.6.8 SEQUENCE ANALYSIS…………………………………………………….67 2.6.9 ION TORRENT PGM WHOLE GENOME SEQUENCING AT AUSTRALIAN GENOME RESEARCH FACILITY (AGRF)……………..67 2.6.10 WHOLE GENOME SEQUENCE ANALYSIS…………………………….67 2.7 GAS CHROMATOGRAPHY-FLAME IONIZATION DETECTION (GC-FID)…………………………………………………………………………….68 2.7.1 EXTRACTION OF HYDROCARBONS……………………………………...68 2.8 SURFACE TENSION MEASUREMENTS………………………………………69 CHAPTER THREE: RESULTS AND DISCUSSION- SCREENING, ISOLATION AND CHARACTERIZATION OF AROMATIC COMPOUND DEGRADING BACTERIA 3.1 ENRICHMENT CULTURES………………………………………………….…..70 3.1.1 OILS AND NON-AROMATIC HYDROCARBONS…………...…………....71 3.1.2 AROMATIC COMPOUNDS………………………………………………….74 3.1.3 POLYCYCLIC AROMATIC HYDROCARBONS (PAHs)…………………..76 3.2 CHARACTERIZATION OF STRAIN BP5………………………………………79 3.2.1 PHYLOGENETIC ANALYSIS BASED ON 16 rRNA GENE SEQUENCE...79 3.2.2 PHENOTYPIC STUDY: BD BBLCRYSTALTM IDENTIFICATION TEST...81 3.2.3 PHENOTYPIC STUDY: BIOLOG GN2 MICROPLATETM SUBSTRATE UTILIZATION TEST………………………………………………………….83 3.3 CHARACTERIZATION OF STRAIN ML-15…………………………………...86 3.3.1 PHYLOGENETIC ANALYSIS BASED ON 16 rRNA GENE SEQUENCE...86 3.3.2 PHENOTYPIC STUDY: BIOLOG GN2 MICROPLATETM SUBSTRATE UTILIZATION TEST………………………………………………………….88 X