Diversity and characteristics of yeasts in water sources of the North West Province By Deidré Alima Bregené Van Wyk (B.Sc Honours) Submitted in partial fulfilment of the requirements for the degree Master of Science in Environmental Science, in the School of Biological Science, Faculty of Natural Science, North-West University: Potchefstroom Campus Supervisor: Prof. C.C. Bezuidenhout Co-supervisor: Mr. O.H.J. Rhode Date Submitted: November 2012 ABSTRACT Yeasts form an important part of many ecosystems and significantly contribute to biodiversity. However, yeast biodiversity in the North West Province remains largely unexplored. The aim of this study was to determine the diversity and characteristics of yeasts from water sources in the North West Province, South Africa. Samples were collected over a two year period and included three rivers, a spruit and an inland lake. Temperature, pH, and electrical conductivity (EC) were measured on site using a multi- 2- probe. Nitrate (NO3-N), nitrite (NO2-N) and phosphate (PO4 ) levels were determined in the laboratory using Hatch kits and equipment. The pH ranged from 7.2 to 9.2. Elevated EC levels (36-70 mS) were detected especially at the Harts River and Barberspan (38-165 mS) sites. Physico-chemical parameter levels were higher during the cold dry sampling period compared to the warm rainy sampling period. Levels and diversity of yeasts were determined using the membrane filtration method. The highest level of yeasts was detected in the Mooi River and Schoonspruit during 2010 and 2011 sampling periods. Pigmented and non-pigmented yeasts were enumerated from all samples. Over the two year period the highest number of pigmented yeasts was detected in the Schoonspruit samples. In some cases there were significant (P<0.05) differences between pigmented and non-pigmented yeast levels among the sites. The diazonium blue B (DBB) test was carried out to distinguish between ascomycetous and basidiomycetous yeasts. These isolates were then identified using the API ID 32C system. Yeasts isolates were identified as belonging to the following genera: Candida, Cryptococcus, Pichia, Rhodotorula and Zygosaccharomyces. In addition using 26S rRNA gene sequencing Aureobasidium spp., Clavispora spp., Cystofilobasidium spp., Hanseniaspora spp., Meyerozyma spp., Sporidiobolus spp., and Wickerhamomyces spp.were also identified. The diversity and abundance of yeasts in the water sources demonstrated that opportunistic pathogens were present. This was supported by results that indicated some isolates could grow at 37°C and higher. In conclusion, our results provide preliminary information on the distribution and diversity of yeasts in water sources of the North West Province, South Africa. Keywords: Yeasts, physico-chemical parameters, water sources, pathogens ii DECLARATION I declare that the dissertation for the degree of Master of Science (M. Sc) at the North-West University Potchefstroom Campus hereby submitted, has never been submitted by me for a degree at this or another University, that it is my own work in design and execution and that all material contained herein has been duly acknowledged. ……………………………………………. ……………………………………. Deidré Van Wyk Date iii ACKNOWLEDGEMENTS Without the commitment, endurance, guidance and support of the following people, this dissertation would not have reached completion. It is to these people that I pay my deepest gratitude. First of all God. Professor Carlos Bezuidenhout and Mr Owen Rhode for their encouragement and great help. My Parents for their motivation. Dr Jaco Bezuidenhout for statistical assistance. Lesego Molale, for support, motivation and assistance. Everyone at the Microbiology Department. A very special friend who always encouraged, motivated, supported and assisted me. NRF for financial support. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................................... ii DECLARATION ............................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................................... iv LISTS OF TABLES ........................................................................................................................ viii LISTS OF FIGURES .......................................................................................................................... x CHAPTER 1 ....................................................................................................................................... 1 INTRODUCTION .............................................................................................................................. 1 1.1 GENERAL INTRODUCTION AND PROBLEM STATEMENT .......................................... 1 1.2 RESEARCH AIM ..................................................................................................................... 3 1.3 THE OBJECTIVES OF THIS STUDY WERE: ...................................................................... 3 CHAPTER 2 ....................................................................................................................................... 4 LITERATURE REVIEW ................................................................................................................... 4 2.1 GENERAL CHARACTERISTICS OF YEASTS .................................................................... 4 2.2 SIGNIFICANCE OF YEASTS ................................................................................................ 5 2.3 ISOLATION AND GROWTH OF YEASTS ........................................................................... 6 2.4 YEAST REPRODUCTION ...................................................................................................... 7 2.5 MEDICAL IMPLICATIONS OF YEASTS ............................................................................. 8 2.6 YEASTS IN WATER SOURCES .......................................................................................... 10 2.7. WATER QUALITY IN SOUTH AFRICA ........................................................................... 11 2.8. WATER PHYSICO-CHEMICAL PARAMETERS ............................................................. 12 2.8.1. pH of water ..................................................................................................................... 12 2.8.2. Water Temperature ......................................................................................................... 13 2.8.3 Total Dissolved Solids (TDS) and Electrical Conductivity (EC) of water ...................... 13 2.9 WATER SITUATION IN THE NORTH WEST PROVINCE .............................................. 14 2.10 GENERAL METHODS USED IN THE ISOLATION OF YEASTS FROM WATER SOURCES .................................................................................................................................... 15 2.10.1 Sampling methods for yeasts ......................................................................................... 15 2.10.2 Isolation and enumeration of yeasts in aquatic environments ....................................... 15 2.10.3 Morphological and biochemical methods for identification of yeasts ........................... 16 2.10.4 Molecular methods for identification of yeasts ............................................................. 17 v CHAPTER 3 ..................................................................................................................................... 19 MATERIALS AND METHODS ..................................................................................................... 19 3.1 STUDY AREA AND SAMPLING ........................................................................................ 19 3.2 ISOLATION AND ENUMERATION OF YEASTS ............................................................. 21 3.3 MORPHOLOGICAL AND BIOCHEMICAL IDENTIFICATION ...................................... 21 3.3.1 Diazonium blue B (DBB) ................................................................................................ 21 3.3.2 Biochemical identification ............................................................................................... 21 3.4 MOLECULAR IDENTIFICATION....................................................................................... 22 3.4.1 Yeast genomic DNA isolation ......................................................................................... 22 3.4.2 PCR amplification ........................................................................................................... 23 3.4.3 Agarose gel electrophoresis ............................................................................................. 23 3.4.4 Sequencing of the amplicons ........................................................................................... 24 3.5 GROWTH TEMPERATURES OF YEASTS ........................................................................ 24 3.6 STATISTICAL ANALYSES ................................................................................................. 24 CHAPTER 4 ..................................................................................................................................... 25 RESULTS ......................................................................................................................................... 25 4.1 INTRODUCTION