Obliqatelv Anaerobic Alkaliphiles from Kenya Soda Lake Sediments Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Gerald G. Owenson B.Sc. (Dundee) Department of Microbiology and Immunology University of Leicester February, 1997. UMI Number: U087829 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U087829 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Statement The work in this thesis was carried out by the author during the period October 1992 to March 1996, under the supervision of Prof. W.D. Grant in the Department of Microbiology and Immunology, University of Leicester. This thesis is submitted for the degree of Doctor of Philosophy at the University of Leicester, and has not been submitted in full or part for any other degree. This thesis may be made available for consultation, photocopying and for use through other lending libraries, either directly of through the British Lending Library. Gerald G. Owenson Obligately anaerobic alkaliphiles from Kenya soda lake sediments Gerald Owenson During the month of December 1992, an expedition was undertaken to collect anaerobic sediment samples from the alkaline lakes of the East African Rift Valley in Kenya. During this expedition, eleven samples were collected from the four non-saline, northern lakes (Lake Bogoria, Lake Nakuru, Lake Elmenteita and Lake Sonachi). A further five samples were obtained from the hypersaline Lake Magadi. Utilising an array of media, the isolation of alkaliphilic, obligate anaerobes, representing one of the major undescribed constituents of the trophic network, was attempted. Extensive investigations into the sulphate-reducing bacteria (SRB) community of the sediment samples failed to provide pure culture isolates. However, successful enrichments utilising a range of substrates were obtained. Ethanol as a substrate resulted in the greatest number of positive enrichments, with representatives from each of the northern lakes visited. These are the first indications that ethanol may be used to enrich for alkaliphilic SRB. Lactate also performed well as a growth substrate, whilst acetate, butyrate, formate and fumarate also resulted in one or more positive enrichments. Contrary to previous findings, these data reveal the presence of alkaliphilic SRB capable of utilising a range of substrates. Although positive enrichments under hypersaline conditions were initially obtained using lactate, these cultures could not be maintained. Using the substrates betaine, trehalose, starch, carboxymethyl cellulose, xylan and guar gum (galactomannan), a number of other organisms were isolated. Despite being enriched under anaerobic conditions, all the isolates were found to be facultatively anaerobic, although exo-enzyme production appeared to take place only under anaerobic conditions. Six alkaliphilic, obligate anaerobes were isolated from samples taken from two of the northern lakes (Lake Elmenteita and Lake Bogoria) using a complex medium with glucose. Phenotypic and taxonomic data indicated the presence of five species belonging to the Clostridium spectrum, although they were found to be phylogenetically distinct from previously described isolates. Each organism showed optimal growth at alkaline pH, and tolerated only low concentrations of NaCl (ca. 8% w/v). Enrichment of three Lake Magadi samples resulted in the isolation of three haloalkaliphilic obligate anaerobes. These organisms had an obligate requirement for high NaCl concentrations (> 16% w/v), and showed a limited pH range for growth in the alkaline region (> pH 9.5). Phylogenetic analysis of 16S rDNA revealed these isolates also clustered within the Clostridium region of the low G+C Gram-positive bacteria, although they were also unrelated to any of the previously described species. Taxonomic proposals for a new genus and several new species are presented. Acknowledgements Completion of this thesis would not have been possible without the assistance of the following individuals, to whom I would like to pass on my gratitude: Prof. W.D. Grant for the supervision of this project. Prof. B.E. Jones and the members of the R&D labs at Gist-brocades B.V. for assistance and support during my work in Holland. Paul Baker, Martin Krsek and Elizabeth Wellington and the University of Warwick for assistance with the DGGE. Terry McGenity and Andy Duckworth for help with the DNA work. The members of Lab 107 and 105, especially Anne Abbott who has helped and encouraged my throughout my time at Leicester, and all the other members of the Department who have helped in their own, special ways! I would especially like to thank my ‘partner in crime’ Chris Jones, who has kept me sane (if not altogether sober) throughout, and also Sarah Rogan for her support and patience. Finally to my parents - without their constant encouragement and support this thesis would never have been submitted. Thankyou, and I hope you think it was worth all the grey hairs! This work was funded by a Natural Environment Research Council CASE award in conjunction with Gist-brocades B.V., The Netherlands. Table of Contents 1 Introduction ........................................................................ 1 1.1 Early investigations ............................................................................. 1 1.2 Geology of the Kenya Rift Valley ......................................................4 1.2.1 Evolution of the rifts ..................................................................... 4 1.2.2 The Lakes of the Kenya Rift ......................................................... 8 1.3 Alkaline environments ...................................................................... 21 1.3.1 Man-made alkaline environments ...............................................21 1.3.2 Alkalinity as a result of biological activity ................................ 21 1.3.3 High Ca2+ environments ..............................................................22 1.3.4 The soda lake environment .........................................................23 1.4 Surviving the alkaline environment ................................................28 1.4.1 pH homeostasis in alkaliphilic micro-organisms ...................... 29 1.4.2 pH homeostasis in anaerobic alkaliphiles ............... 32 1.5 Alkaliphile diversity in soda lakes ...................................................38 1.5.1 Phototrophic Bacteria .................................................................38 1.5.2 Aerobic Chemo-organotrophic Bacteria .................................... 41 1.5.3 Anaerobic Bacteria .....................................................................44 1.5.4 Sulphate-reducing bacteria .........................................................48 1.5.5 Archaea ....................................................................................... 55 1.6 Phylogenetic inference from 16S rRNA sequence data ................ 58 1.6.1 Ribosomal RNA as a phylogenetic marker ................................58 1.6.2 Selection of analytical method ....................................................59 1.6.3 Obtaining 16S rRNA sequences .................................................61 1.6.4 Alignment ................................................................................... 62 1.6.5 Methods of phylogenetic inference ............................................63 1.6.6 Construction of phylogenetic trees from distance matrix data.. 77 1.6.7 Evaluating the robustness of inferred phylogeny ...................... 80 1.7 Aims .....................................................................................................83 2 Materials and Methods.................................................... 84 2.1 On-site Measurements ......................................................................84 2.1.1 Temperature ................................................................................ 84 2.1.2 Conductivity ................................................................................ 84 2.1.3 Dissolved Oxygen .......................................................................84 2.1.4 Redox Potential ...........................................................................85 2.2 Sampling Procedure ..........................................................................86 v 2.3 Enrichment and Cultivation ............................................................87 2.3.1 Sulphate-reducing bacteria (SRB) ..............................................91 2.3.2 Chemo-organotrophic bacteria ...................................................93 2.4 S2' production by SRB ..................................................................... 100 2.5 Viable Counts ................................ 100 2.6 Morphology .......................................................................................101 2.6.1 Colony Morphology .................................................................. 101 2.6.2 Cell Morphology........................................................................101