COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). DIVERSITY AND BIOTECHNOLOGY APPLICATIONS OF THERMOPHILIC BACTERIA FROM HOT-SPRING WATER IN LIMPOPO SOUTH AFRICA RELATING TO WASTEWATER BIOREMEDIATION AND WATER SAFETY Jocelyn Leonie Jardine Thesis for the degree of Doctor of Technology Department of Biotechnology Faculty of Science University of Johannesburg, South Africa October 2017 Supervisor: Dr Eunice Ubomba-Jaswa, PhD Co-supervisor: Dr Vuyo Mavumengwana, PhD i ABSTRACT Hot-spring environments are valuable resources for novel and useful bacteria in biotechnology. In South Africa, hot springs in Limpopo Province have been investigated by metagenomics but not by culture-based methods. This study was performed in order to describe the diversity of cultured bacteria from hot springs, and to comment on their relevance to public health and antibiotic resistance. Furthermore, potential bacterial candidates were screened for characteristics associated with wastewater (WW) bioremediation and applications. Cultured bacteria were identified by a combination of tools using the 16S rDNA sequence by comparison with public databases, percent guanine-cytosine content, amplified rDNA restriction analysis (ARDRA) and phylogeny. Resistance against ten antibiotics (carbenicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, ceftriaxone, co-trimoxazole, nalidixic acid and norfloxacin) and eight heavy metal ions (Al, Cr, Cu, Fe, Hg, Mn, Ni and Pb) were tested using disk diffusion assays. Isolates were screened for production of enzymes and biophysical characteristics (bioflocculant and biosurfactant activities, biosorption, bioassimilation, anti-biofilm and antimicrobial properties) and selected cell free culture supernatants (CFCS) were processed by tandem LC-MS for identification of the bioactive molecules. CFCS were exposed to pollutants (pigmented food, textile dyes, polyaromatic hydrocarbons) and food WW samples to establish whether there was a reduction in turbidity or phenol levels. Forty-three (43) Gram-positive isolates were of the phylum Firmicutes with the majority of the genus Bacillus (n = 31). Different species were identified as Anoxybacillus flavithermus, Anoxybacillus rupiensis, Bacillus subtilis, Bacillus licheniformis and Brevibacillus spp. Single isolates of Gram-positive Kocuria sp. and Arthrobacter sp., and Gram-negative Cupriavidus sp., Ralstonia sp., Cronobacter sp., Tepidimonas sp., Hafnia sp. and Sphingomonas sp. were identified, previously reported to be emerging opportunistic pathogens, and an absence of Legionella sp. was reported. Low levels of antibiotic resistance (AR) were reported. Only 2.5% (n = 40) of the isolates were multiple antibiotic resistant against >3 antibiotics, while 37.5% were found for both resistance against one or two antibiotics. Resistance was found against ceftriaxone (52.5%), nalidixic acid (37.5%) and carbenicillin (22.5%). All 29 isolates tested heavy metals were tolerant to ≥2 heavy-metal salts. No association was observed between antibiotic resistance and heavy-metal tolerance. Amylase and protease positive isolates, and isolates that could discolorize bromothymol blue were detected by conventional agar plate assays. The CFCS of A. rupiensis 19S was selected for ii tandem LC-MS analysis and potential bioremediation enzymes were identified (amylase, proteases, catalase peroxidase, superoxide dismutase, azoreductase, quinone oxidoreductase, ribonucleases and inorganic pyrophosphatase). Potential dehydrogenase enzymes for biomonitoring of environmental pollutants were also reported. Bioaccumulation of triphenylmethane dye (bromothymol blue) was a property of B. subtilis (9T), while B. subtilis (20S) was able to bioflocculate kaolin clay reducing turbidity by 30%. Biosorption resulted in reductions of Cr and Cu using CFCS of four isolates (7T, 9T, 30M and 83Li). Four different isolates (16S, 71T, 76S, 85Li), were positive for biosurfactant properties by emulsion activity of paraffin oil, sunflower seed oil and petroleum, and drop collapse assay. Brevibacillus sp. (16S) was able to inhibit biofilm formation of B. subtilis. The CFCS fraction of Bacillus mojavensis (biosurfactant positive) 76S inhibited growth of 76% of an AR environmental bacterial panel as well as human Gram-positive pathogens, but not Gram-negative pathogens and acid-fast Mycobacterium smegmatis. Tandem LC-MS identified the biosurfactant, subtilisin, and possible responsible biomolecules with antimicrobial activity of isolate 76S as proteases, subtilisin BM1 and bacillolysin. A reduction in turbidity of dairy and brewery WW was observed when exposed to CFCS fractions of these isolates. Anoxybacillus sp. 19S and Brevibacillus sp. 84Li were found to be the most effective in reducing toxic phenol by at least half in phenol red broth media, food WW samples, in river water contaminated with industrial effluents, and poly aromatic hydrocarbons. In the simulated pollutant experiment, phenols were reduced by more than 54% in coffee, soya sauce, and a commercial textile dye. This is the first report describing cultured aerobic bacteria from hot springs in South Africa. Identification using a combination of molecular tools was useful in discerning differences between the Bacillus and Bacillus-related bacteria. Emerging opportunistic pathogens were isolated and identified, having implications for water safety and public health. Low levels of AR could be useful as a baseline measure of intrinsic environmental AR. Isolates were identified that produced relevant enzymes, and with biophysical properties useful for WW bioremediation. Exposure of CFCS to pollutants and WW samples reduced the toxic phenol concentrations, substantiating the belief that South African hot springs are a valuable resource of potentially important bacteria that can be used for several applications in WW bioremediation. iii DECLARATION I, Jocelyn Leonie Jardine, do hereby declare that this thesis is my own, unaided work. The thesis is presented in fulfilment of the requirement for the degree of D Tech in Biotechnology, Faculty of Science, University of Johannesburg, and it has not been submitted before any degree or examination in any other Technikon or University. Signature: Date: 23/11/2017 iv DEDICATION To my parents with all my Love, Leo Paul Jardine and Sue Phyllis Jardine (1936-2017) v ACKNOWLEDGEMENTS I wish to acknowledge and thank the following people for their contribution to this research. Family (especially my mother who did not live long enough to see this completed) and friends who gave me constant encouragement, support and help. Supervisor Dr Vuyo Mavumengwana who started the ball rolling, Dr Eunice Ubomba-Jaswa who kept it going, and for submission of manuscripts to journals. From the department of Biotechnology UJ, Ms Malie King (retired departmental secretary) and Mr Erick Van Zyl (retired Head of Department). For assistance with statistics : Dr Gill Hendry; RT-PCR : Dr King Abia; LC-MS/MS : Dr Stoyan Stoychev at CSIR Biosciences; DNA sequencing : Prof Michelle van der Bank and Ronny Kabongo at the African Centre for DNA Barcoding, UJ, SA. University of Johannesburg for student funding in the form of Global Excellence Scholarship over a period of 3 years. Conference travel bursaries awarded from South African society for Microbiology (SASM) 2016 and Centre for Microbial Ecology and Genomics (CMEG), University of Pretoria and Thermophiles 2017 conference committees. Sponsorship from Dr Patrik Njobeh of Food Technology, UJ, for attendance to Food Security and Safety (SFAS) 2016 conference. Owners and managers of the hot spring resorts : Tshipise, Siloam, Mphephu, Lekkerrus and Libertas. Assistance from Dr Sudarshan Sekar and Dr Mavumengwana in field sample collection. Dr Irwin Juckes for industrial wastewater collection from a stormdrain in Edenvale, South Africa, Mr Andrew Edwards at Douglasdale dairy for wastewater samples and Mr Vincent Le Roux of the Brew Keg, Kyalami for supply of brewing wastewater and sample of amylase. Colleagues and staff in the Dept of Biotechnology and Food Technology, UJ; in the Dept Physics, UJ; and staff at Doornfontein and Auckland Park Libraries, Post Grad Centre UJ, Faculty of Science Offices, UJ and staff at Natural Resource Environments, CSIR Pretoria. vi PUBLICATIONS Jocelyn L. Jardine, Akebe Luther King Abia, Vuyo Mavumengwana, Eunice Ubomba-Jaswa. (2017). Phylogenetic analysis and antimicrobial profiles of cultured emerging opportunistic pathogens (phyla Actinobacteria and Proteobacteria) identified in hot springs. International Journal of Environmental Research and Public Health 14, 1070; doi10.3390/ijerph14091070. PUBLICATIONS SUBMITTED 1. Identification of Bacillus and closely related Bacillus species