Molecular Studies of Bacterial Communities in the Great Artesian Basin Aquifers Author Kanso, Sungwan Published 2004 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Biomedical Sciences DOI https://doi.org/10.25904/1912/2158 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/366613 Griffith Research Online https://research-repository.griffith.edu.au Molecular Studies of Bacterial Communities in the Great Artesian Basin Aquifers A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University. Sungwan Kanso, B.Sc. (Biotech.) Hons School of Biomolecular and Biomedical Science Faculty of Science, Griffith University, Nathan Campus Queensland, Australia March 2003. i This thesis is dedicated to the Divine Essence within us. ii 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. Sungwan Kanso iii ACKNOWLEDGEMENTS I thank my supervisor, Associate Professor Bharat Patel, for the opportunity to work with him. His advice and support have been greatly appreciated. I thank Dr. Robert Huestis for grammatical correction and proof reading of this thesis. I appreciate the advice, assistance and friendship of Dr. Mark Spanevello, Dr. Ben Mijts, Dr David Innes, Dr Hok-Sin (Tony) Woo, Marwan Abu-Halaweh, Dr. Lyle McMillen and Kathy Hampson throughout the course of my Ph.D. These people have been so wonderful to me at all times in every way. I gratefully acknowledge financial support from the Thai Government and the Australian Government. Finally, I thank my immediate and extended families for their love and encouragement. The friendships and generosities of the Huestis, Wrathmall, Hobba and Teakle families have been so incredibly beyond belief that it is impossible to express in words. I especially thank Robert Huestis, Frank Wrathmall, the Didis, the Dadas and all my Margii friends for their belief in what I have chosen to do and their support for what I will do in the future. Their unconditional love, belief and faith in me have been an enormous encouragement that has kept me going during all these years. iv PUBLICATIONS AND CONFERENCES PROCEEDINGS ARISING FROM THIS THESIS Publications Kanso, S., Patel, B. K. C. (2003). Microvirga subterranea gen. nov., sp. nov., a moderate thermophile from a deep subsurface Australian thermal aquifer. International Journal of Systematic and Evolutionary Microbiology. 53, 401-406. Kanso, S., Greene, A.C. and Patel, B.K.C. (2002). Bacillus subterraneus sp. nov., an iron- and manganese-reducing bacterium from a deep subsurface Australian thermal aquifer. International Journal of Systematic and Evolutionary Microbiology. 52, 869-874. Conference Proceedings Kanso, S., and Patel, B. K. C. (2001). Studies of Molecular Microbial Ecology of the Great Artesian Basin Aquifer. Twenty-seventh Science and Technology Symposium of Thailand, 16th-19th October, Hat-Yai, Thailand. v ABSTRACT 16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the α-, β- and γ-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the δ-proteobacteria and ε- proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide vi enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB’s sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores. vii TABLE OF CONTENTS STATEMENT OF ORIGINALITY................................ III ACKNOWLEDGMENTS ..............................................IV PUBLICATIONS AND CONFERENCES PROCEEDINGS ARISING FROM THIS THESIS................................... V ABSTRACT................................................................ VI TABLE OF CONTENTS............................................VIII LIST OF FIGURES.................................................... XV LIST OF TABLES ..................................................XVIII ABBREVIATIONS ...................................................XIX CHAPTER 1: INTRODUCTION ....................................1 Diversity and classification of living organisms ................... 1 Methods of classification and taxonomy of prokaryotes (Domains Archaea and Eubacteria)...................................... 2 Polyphasic taxonomy................................................................. 3 Main phenotypic properties used in polyphasic taxonomy ............. 3 Classical phenotypic analysis...................................................... 4 Cell wall composition................................................................