School of Biomedical Sciences Acidihalobacter: Novel Halotolerant Iron- and Sulfur-oxidizing Acidophiles with Potential for Saline Water Bioleaching Himel Nahreen Khaleque This thesis is presented for the Degree of Doctor of Philosophy of Curtin University September 2017 Dedication To my beloved parents and my wonderful husband. i Acknowledgements First of all, I would like to thank my supervisor and mentor, Professor Elizabeth Watkin. Throughout this journey, you have supported me, motivated me, guided me and been there to celebrate my victories and help me through the tough times. Working with you has been one of the greatest experiences in my life and I will be forever grateful for all that you have done for me. From the bottom of my heart, I thank you. I would also like to extend my sincere gratitude to my co-supervisors, Anna, Naomi and Josh. Your support and knowledge have been invaluable throughout the course of this project. Professor David Holmes and Carolina, thank you so much for all your help and generosity. My trip to Chile was life-changing and I am truly grateful to you both. To my wonderful, amazing mother - Mom, words can’t express what you mean to me. To say the least, you are my inspiration, my role model and everything I ever wanted to be. Thank you for making me who I am and for loving me unconditionally. You are everything to me. Dad, not one day goes by that I don’t miss you. I am proud to be your daughter and to be able to follow in your footsteps. I hope I have made you proud. I love you both more than anything. Doyel, you have looked after me, protected me, loved me and been my best friend all my life. Without your support and love, I would not have made it this far. Thank you for everything. Raihan, my dearest husband, you are truly my better half and the best thing that has ever happened to me. You have made so many sacrifices so that I could chase my dreams. Thank you for your endless love, support and encouragement. You are the wind beneath my wings. To my dear friend, Abhi - your support and friendship over the past few years has been invaluable. You have truly been the friend who has been there each and every day, through all the good and the bad. I can’t thank you enough for everything. This journey wouldn’t have been the same without you. ii Homayoun, Kofi and Anna - thank you for always being there for me and believing in me. You have been a constant source of strength and comfort and I honestly don’t know what I would do without you guys. Thiru, Felipe, Imran, Jennifer, Troy and Lynda - thank you for your friendship and support throughout the years, it means so much to me. Abhishek, Sam, Sarmed and W. Woodcraft, thanks for the good times and the laughs, without which this journey would have been incomplete. I would like to extend my heartfelt thanks to all the past and present members of Lab 10 and those in Building 305. Rob W., thank you for being an inspiration to me and teaching me to believe in myself and my science. Mel, Josh, Rob S. and Carl, thank you for always being there when I have needed advice, cheering up or just a good laugh. Thank you to my family back home in Dhaka– Abba, Amma, Neela Apu, Upal Bhai, Shompa Bhabi, Kuntol, Zahira. I love you all and your support means the world to me. Last but not least, a big thank you to Toffee, for all the cuddles and comfort you have given me over the last few years. Your Khalamoni loves you. Apollo, Phoenix, Nitua and Soda, my fur babies in heaven, I miss you dearly and will always love you. iii List of Publications This thesis is assembled by publications (either published, accepted, submitted or prepared for submission) which form the individual chapters of the thesis. The publications are listed as follows: Chapter 2 Khaleque HN, Kaksonen AH, Boxall NJ, Watkin ELJ. Chloride ion tolerance and pyrite bioleaching capabilities of pure and mixed halotolerant, acidophilic iron- and sulfur-oxidizing cultures. Minerals Engineering (accepted for publication in Minerals Engineering). Chapter 3 Khaleque HN, Ramsay JP, Murphy RJ, Kaksonen AH, Boxall NJ, Watkin EL. Draft Genome Sequence of the Acidophilic, Halotolerant, and Iron/Sulfur-Oxidizing Acidihalobacter prosperus DSM 14174 (Strain V6). Genome Announcements. 2017 Jan 19;5(3):e01469-16. Khaleque HN, Ramsay JP, Murphy RJ, Kaksonen AH, Boxall NJ, Watkin EL. Draft Genome Sequence of Acidihalobacter ferrooxidans DSM 14175 (Strain V8), a New Iron-and Sulfur- Oxidizing, Halotolerant, Acidophilic Species. Genome Announcements. 2017 May 25;5(21):e00413-17. Khaleque HN, Corbett MK, Ramsay JP, Kaksonen AH, Boxall NJ, Watkin ELJ. Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite. Journal of Biotechnology, Elsevier, 2017, 262, pp 56-59. iv Chapter 4 Khaleque HN, González C, Kaksonen AH, Boxall NJ, Holmes DS, Watkin ELJ. Genome-based reclassification and renaming of two extremely acidophilic, iron- and sulfur-oxidizing halophiles Acidihalobacter prosperus strain V6 and 'Acidihalobacter ferrooxidans' strain V8 as two new species, Acidihalobacter aeolianus sp. nov., and Acidihalobacter vulcanensis sp. nov., respectively. International Journal of Systematic and Evolutionary Microbiology (under revision for publication). Khaleque HN, González C, Kaksonen AH, Boxall NJ, Holmes DS, Watkin ELJ Genome-based reclassification of the extremely acidophilic, iron- and sulfur-oxidizing halophile Acidihalobacter prosperus strain F5 as a new species, Acidihalobacter yilgarnensis sp. nov. International Journal of Systematic and Evolutionary Microbiology (prepared for submission). Chapter 5 Khaleque HN, Kaksonen AH, Boxall NJ, Watkin ELJ. Metabolic capabilities and adaptive mechanisms of the members of the Acidihalobacter genus: A comparison of four Acidihalobacter genomes (prepared for submission). Chapter 6 Khaleque HN, Kaksonen AH, Boxall NJ, Watkin ELJ. Identifying the mechanisms of osmotic stress tolerance in the halotolerant, extreme acidophile, ‘Acidihalobacter aeolianus’ DSM 14174T (prepared for submission to Research in Microbiology). v Statement of Contribution by Others I hereby declare that the work presented in this thesis was primarily designed, experimentally executed, interpreted, and written by the first author of the individual manuscripts (Himel Nahreen Khaleque). Contributions by colleagues are described in the following. The signed statement by co-authors are in Appendix 2. Chapter 2 ELJW, AHK and NJB provided significant contributions to the conception and design of this study. They also provided intellectual input on the interpretation of the results and critical revision of the manuscript. Chapter 3 JPR assisted with genome assembly and annotations. RJM performed the Miseq sequencing of the genomic DNA for ‘Ac. ferrooxidans’ DSM 14175 (strain V8) and Ac. prosperus strain F5. ELJW, JPR, AHK and NJB provided intellectual input on the interpretation of the results and critical revision of the manuscripts. MKC provided data for the bioleaching studies on Ac. prosperus strain F5. Chapter 4 DSH and ELJW provided significant contributions to the conception and design of this study. CG helped in bioinformatics analyses and generation of images. ELJW, DSH, AHK and NJB provided critical revision of the manuscripts. vi Chapter 5 ELJW provided significant contributions to the conception and design of this study. ELJW, AHK and NJB provided intellectual input on the interpretation of the results and critical revision of the manuscript. Chapter 6 ELJW provided significant contributions to the conception and design of this study. ELJW, AHK and NJB provided intellectual input on the interpretation of the results and critical revision of the manuscript. vii Abstract In this study, the tolerance of seven environmental acidophilic cultures to increasing chloride ion concentrations (2-45 g/L chloride ion) was tested. From these, the four mesophilic cultures that showed highest chloride ion tolerances were further screened for their ability to oxidize 1% w/v pyrite at 9, 15 and 30 g/L chloride ion. The results showed that cultures V6 and V8 (pure cultures of Acidihalobacter prosperus DSM 14174 strain V6 and ‘Ac. ferrooxidans’ DSM 14175 strain V8, respectively) showed the maximum ability to tolerate high concentrations of chloride ion (up to 45 g/L) as well as the ability to oxidize pyrite at up to 30 g/L chloride ion. Furthermore, the mixed culture 14C, which was dominated by Acidihalobacter spp. (93% relative abundance) was also able to tolerate higher chloride concentrations than the other mixed cultures. Another iron-and sulfur- oxidizing Ac. prosperus isolate, Ac. prosperus strain F5, was included into the study due to its ability to tolerate and oxidize pyrite, chalcopyrite and pentlandite at up to 45 g/L chloride ion. The genomes of Ac. prosperus DSM 14174, ‘Ac. ferrooxidans’ DSM 14175 and Ac. prosperus strain F5 were sequenced using PacBio long read sequencing technology, which allowed for the public release of the high quality genomes at the National Centre for Biotechnology Information (NCBI). Phylogenomic studies of the newly sequenced genomes was undertaken using both sequence and non-sequence based approaches. The results of the analyses revealed that the isolates belonged to separate species of the Acidihalobacter genus. Accordingly, the isolates were reclassified as ‘Ac. aeolianus’ DSM 14174T (previously Ac. prosperus DSM 14174), ‘Ac. vulcanensis’ DSM 14175T (previously ‘Ac. ferrooxidans’ DSM 14175) and ‘Ac. yilgarnensis’ strain F5T (previously Ac. prosperus strain F5), with each representing type strains of their species. The genomes of all the members of the Acidihalobacter genus (including Ac. prosperus DSM 5130T) were compared to increase the understanding on their mechanisms of nutrient assimilation, energy acquisition and tolerance to acid, osmotic, metal and oxidative stresses.