Isolation, identification, and characterisation of fungi from a platinum mine by Kiara Haripersad (1645281) Dissertation Submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa Supervisor: Prof Karl Rumbold Co-supervisor: Dr Sanchia Moodley July 2022 ii DECLARATION I, Kiara Haripersad (1645281), am a student registered for the degree of Master of Science (Dissertation) in the academic year 2022. I hereby declare the following: • I am aware that plagiarism (the use of someone else’s work without their permission and/or without acknowledging the original source) is wrong. • I confirm that the dissertation submitted for assessment for the above degree is my own unaided work except where explicitly indicated otherwise and acknowledged. • I have followed the required conventions in referencing the thoughts and ideas of others. • I understand that the University of the Witwatersrand may take disciplinary action against me if there is a belief that this is not my own unaided work or that I have failed to acknowledge the source of the ideas or words in my writing. Signature: Date: 11 July 2022 iii ABSTRACT Mycohydrometallurgy is the application of fungi and their metabolites such as organic acids (OAs) for the extraction and recovery of metals through mechanisms such as bioleaching. The purpose of this study was to identify and characterise fungal isolates from a platinum mine to determine potential fungal candidates for bioleaching of a platinum concentrate. Fungi were isolated from platinum mining samples using traditional microbiological techniques and sixty-six isolates were identified based on their internal transcribed spacer (ITS) regions using Sanger sequencing, as members of either Ascomycota, Basidiomycota or Mucoromycota. Qualitative OA screening was conducted by inoculating isolates into Potato Dextrose Agar containing a pH indicator to select OA producing isolates. These isolates were quantitatively screened using high-performance liquid chromatography and showed the production of acetic, butyric, formic, lactic, propionic, and indole-3-acetic acid. Based on their OA production, Penicillium sp., Rhizopus microsporus, and Aspergillus terreus underwent tolerance tests to copper, chromium, and nickel. Their overall tolerance was low, suggesting that they are promising candidates for two-step direct bioleaching as it involves the growth of fungi prior to their exposure to the metal-containing solid material. iv Dedicated to my parents and sister, for their love and support. DEDICATION v ACKNOWLEDGEMENTS I would like to thank my supervisor, Prof Karl Rumbold for his support and guidance, as well as the opportunity to do my MSc under his supervision. I am highly grateful to my co-supervisor, Dr Sanchia Moodley for her help, guidance, encouragement, and suggestions throughout my project. I would also like to thank my BIORECOVER project colleagues at the School of Chemical and Metallurgical Engineering for their support in completing my project. A special thank you to the lab technicians Michael Tobin, Naledi Nkabinde, Motlatsi Phali, and Matshidiso Tsotetsi for their advice and assistance with reagents and equipment. My sincere appreciation goes to my fellow Industrial Microbiology and Biotechnology Laboratory members for their help, advice, and support, especially Koketso Sodi for all her help, motivation, and friendship. I am truly grateful I underwent this journey alongside her. Thank you to my friends, especially Akeel and Venisha for always being there for me whenever I needed advice, help, words of encouragement and someone to listen. To my parents, thank you for your love and endless support which allowed me to reach the point where I was able to do my MSc. None of this would have been possible without the two of you. Finally, I’d like to acknowledge the National Research Foundation (NRF) for funding my project. vi TABLE OF CONTENTS DECLARATION........................................................................................................... ii ABSTRACT ................................................................................................................ iii DEDICATION ............................................................................................................ iv ACKNOWLEDGEMENTS ............................................................................................ v LIST OF FIGURES ...................................................................................................... ix LIST OF TABLES ......................................................................................................... x NOMENCLATURE ..................................................................................................... xi 1 INTRODUCTION .................................................................................................... 1 1.1 Introduction ........................................................................................................ 2 1.2 Research Problem ............................................................................................... 3 1.3 Hypothesis, Aims and Objectives ........................................................................ 4 2 LITERATURE REVIEW ............................................................................................ 5 2.1 General Introduction ................................................................................................. 6 2.2 Mining of Platinum Group Metals (PGMs) ................................................................ 6 2.2.1 PGM reserves ..................................................................................................... 6 2.2.2 Processing of PGMs ............................................................................................ 7 2.2.3 Challenges of mining PGMs ............................................................................... 8 2.3 Mycohydrometallurgy ............................................................................................... 9 2.3.1 Introduction ....................................................................................................... 9 2.3.2 Fungi in mining environments ......................................................................... 10 2.3.3 Fungal bioleaching ........................................................................................... 13 2.3.3.1 Acidolysis ................................................................................................... 13 2.3.3.2 Complexolysis ........................................................................................... 13 2.3.3.3 Biosorption ................................................................................................ 14 2.3.3.4 Bioaccumulation ....................................................................................... 14 2.3.4 Indirect and direct bioleaching ........................................................................ 15 2.3.5 Biohydrometallurgical methods for base metal removal from PGM sources . 16 2.3.6 Biohydrometallurgical methods for platinum recovery .................................. 16 2.3.7 Microorganisms isolated from a platinum mine .............................................. 17 2.4 Summary ................................................................................................................. 18 3 ISOLATION AND IDENTIFICATION OF FUNGI ...................................................... 19 3.1 Introduction ............................................................................................................ 20 vii 3.2 Materials and Methods ........................................................................................... 21 3.2.1 Sample collection ............................................................................................. 21 3.2.2 Analysis of pH and redox potential .................................................................. 21 3.2.3 Microbiological characterisation ..................................................................... 22 3.2.4 Genomic DNA extraction ................................................................................. 22 3.2.5 Quantification of DNA ...................................................................................... 23 3.2.6 Sanger sequencing ........................................................................................... 23 3.3 Results and Discussion ............................................................................................ 24 3.3.1 Physicochemical characteristics and fungal counts ......................................... 24 3.3.2 Evaluation of DNA extraction method ............................................................. 29 3.3.3 Evaluation of identification method ................................................................ 30 3.3.4 Identification of fungal isolates........................................................................ 34 3.3.4.1 Ascomycota ............................................................................................... 36 3.3.4.2 Basidiomycota ........................................................................................... 41 3.3.4.3 Mucoromycota .......................................................................................... 41 3.4 Summary and Conclusions ...................................................................................... 42 4 CHARACTERISATION OF FUNGI .......................................................................... 44 4.1 Introduction ............................................................................................................ 45 4.2 Materials and Methods ........................................................................................... 45 4.2.1 Qualitative screening ....................................................................................... 45 4.2.2 Quantitative screening ..................................................................................... 46 4.2.3 Heavy metal tolerance test .............................................................................. 46 4.2.4 Statistical analysis ............................................................................................ 47 4.3 Results and Discussion ............................................................................................ 47 4.3.1 pH indicator test .............................................................................................. 47 4.3.1.1 Ascomycota ............................................................................................... 49 4.3.1.2 Basidiomycota ........................................................................................... 51 4.3.1.3 Mucoromycota .......................................................................................... 51 4.3.2 High-performance liquid chromatography ...................................................... 53 4.3.3 Morphological changes and heavy metal tolerance index (TI) ........................ 59 4.3.3.1 Morphology ............................................................................................... 59 4.3.3.2 Tolerance index (TI) .................................................................................. 61 4.4 Summary and Conclusions ...................................................................................... 64 5 CONCLUSIONS AND RECOMMENDATIONS ........................................................ 66 viii 5.1 Conclusions ............................................................................................................. 67 5.1.1 Introduction ..................................................................................................... 67 5.1.2 Traditional microbiological isolation of fungi .................................................. 67 5.1.3 Molecular identification of fungi .................................................................... 67 5.1.4 Qualitative organic acid characterisation ........................................................ 68 5.1.5 Quantitative organic acid characterisation ...................................................... 69 5.1.6 Heavy metal tolerance characterisation .......................................................... 69 5.1.7 Contribution to the research field ................................................................... 69 5.2 Recommendations .................................................................................................. 70 REFERENCES .......................................................................................................... 71 APPENDICES .......................................................................................................... 89 APPENDIX A ................................................................................................................... 90 APPENDIX B ................................................................................................................... 92 ix LIST OF FIGURES Figure 2.1 The world’s estimated reserves of platinum group metals (PGMs) 7 Figure 2.2 The traditional processing of platinum group metal (PGM) ore in South Africa 8 Figure 3.1 Spread plates of the mine samples 28 Figure 3.2 Percentage of sixty-three fungal isolates belonging to their respective families and phyla 35 Figure 4.1 Qualitative screening for organic acid (OA) production 48 Figure 4.2 Morphological changes of fungal isolates exposed to heavy metals 60 Figure 4.3 Heavy metal tolerance index (TI) of fungal isolates 63 x LIST OF TABLES Table 2.1 Fungi isolated from mining environments with acquired characteristics 11 Table 3.1 Physicochemical characteristics and fungal counts of samples obtained from the Mogalakwena platinum mine 25 Table 3.2 Identification and classification of fungal isolates from the Mogalakwena platinum mine 31 Table 4.1 Acid unitage (AU) values calculated for the fifteen organic acid- producing fungal isolates 52 Table 4.2 The pH and organic acid (OA) concentration (mM) of Potato Dextrose Broth (pH 4.8) inoculated with fungal isolates after 7 days of incubation at 25oC 55 Table 4.3 Studies involving the quantification of organic acids (OAs) produced by fungal isolates 58 xi NOMENCLATURE % percentage oC degree Celsius < less than ≥ greater than or equal to µB microbar µg/ml microgram per millilitre µl microlitre µm micrometre Al aluminium ANOVA analysis of variance As arsenic Au gold AU acid unitage BLAST Basic Local Alignment Search Tool C6H8O7 citric acid Cd cadmium CFU colony-forming unit CFU/g colony-forming unit per gram CFU/ml colony-forming unit per millilitre Co cobalt Cr chromium CTAB cetyltrimethylammonium bromide Cu copper CuCl2.2H2O copper (II) chloride dihydrate EDTA ethylenediaminetetraacetic acid Eh redox potential Fe iron FeO ferrous oxide xii FeS iron sulphide g gravities g gram g/l gram per litre H hydrogen H2SO4 sulphuric acid Hg mercury HNO3 nitric acid HPLC high-performance liquid chromatography IAA indole-3-acetic acid Ir iridium ITS internal transcribed spacer K2Cr2O7 potassium dichromate K2Pt(II)Cl4 potassium tetrachloroplatinate K2Pt(IV)Cl6 potassium hexachloroplatinate kg kilogram LMWOAs low molecular weight organic acids M molar mg/l milligram per litre mg/ml milligram per millilitre MIC minimum inhibitory concentration min minute ml millilitre ml/min millilitre per minute mM millimolar mm millimetre Mn manganese mV millivolt n number of replicates NaCl sodium chloride xiii NCBI National Center for Biotechnology Information ng/µl nanogram per microlitre Ni nickel NiCl2.6H2O nickel (II) chloride hexahydrate NiO nickel (II) oxide NiSO4 nickel sulphate nm nanometre O2 oxygen OA organic acid Os osmium Pb lead PCR polymerase chain reaction Pd palladium PDA Potato Dextrose Agar PDB Potato Dextrose Broth PGMs platinum group metals ppm parts per million Pt platinum PtCl42- tetrachloroplatinate PtCl62- hexachloroplatinate Pt(NH3)4Cl2- tetraamminedichloroplatinum(iv) Rh rhodium rpm revolutions per minute Ru ruthenium S sulphur SO4 -2 sulphate Sn tin SO2 sulphur dioxide TE tris - ethylenediaminetetraacetic acid TI tolerance index xiv UG2 Upper Group 2 Zn zinc 1 1 INTRODUCTION 2 1.1 Introduction Platinum (Pt) is one of the rarest and most valuable metals on earth and is therefore labelled as a precious metal. It is the most well-known metal in the six- member family of platinum group metals (PGMs), where the remaining five members are ruthenium (Ru), osmium (Os), rhodium (Rh), iridium (Ir), and palladium (Pd) (Vermaak, 1995). Platinum is the only competitor for gold (Au) in terms of investment and the manufacturing of jewellery due to its malleability and resistance to fading (Jones, 2005). However, PGMs are also of great importance in the chemical and industrial industry for their use as catalysts and catalytic converters, respectively (Rao and Reddi, 2000). They also have important applications as catalysts in the production of petroleum and other fuels and chemicals from crude oil (Rao and Reddi, 2000). In the industrial sector, their application as catalytic converters are important for exhaust control in transport vehicles (Rao and Reddi, 2000). South Africa accounts for the largest production of PGMs in the world and as a result, it contributes significantly to the gross domestic profit of the country (Ndeddy Aka and Babalola, 2017). However, the mining and production of these precious metals come with their own challenges, which include a lack of economic, environmentally friendly, and effective processing technologies for ore, concentrate, tailings, waste, and near-surface oxidised ores (Hedrich et al., 2020; Mudd, 2010; Thethwayo, 2018). Prior to the extraction of PGMs, the extraction of base metals such as copper (Cu), chromium (Cr), cobalt (Co), nickel (Ni), and iron (Fe) from PGM concentrate need to be considered; as these precious and base metals have similar geochemical behaviour and are usually concentrated together geologically (Hedrich et al., 2020; Jones, 2005; Thethwayo, 2018). Conventional technologies such as pyrometallurgy and hydrometallurgy, which have been used for the processing of PGM ore has led to ineffective extraction of base metals and PGMs and have high energy requirements (Mudd, 2010; Thethwayo, 2018). Therefore, where pyrometallurgy and hydrometallurgy are 3 unable to be efficiently applied, biohydrometallurgy could be the possible alternative. Biohydrometallurgy is the use of biological agents for the extraction and solubilisation of metals and is a more economic, effective, and environmentally-friendly removal and recovery process compared to conventional methods (Johnson, 2014). Bioleaching is a biohydrometallurgical process which has been used in previous studies to recover or remove base metals from solid material using microorganisms such as bacteria and fungi and the metabolites which they produce e.g. organic acids (OAs) (Johnson, 2014). Some studies have used acquired strains of microorganisms while others have isolated microorganisms from the mining environment or contaminated soil on which they intend to perform bioleaching experiments. Bacteria have been isolated from soil samples from a platinum mine tailings dam and have been considered as candidates for bioleaching of metal-contaminated soil (Ndeddy Aka and Babalola, 2017). However, studies on the isolation of fungi from a platinum mine have not been conducted. The studies which have been performed on the isolation of fungi involve their isolation from various other mining and metal-contaminated sites. The genera Aspergillus and Penicillium have often been isolated from such sites and were found to have adaptive qualities, such as their ability to produce extracellular metabolites, which aids in their tolerance to high concentrations of heavy metals (Deng et al., 2012; Ghosh and Paul, 2015). The tolerance to heavy metals is partially due to their production of OAs which solubilise and chelate metals from complexes (Din et al., 2020; Sazanova et al., 2015). As a result, Aspergillus niger has been extensively used in biohydrometallurgical studies due to its known ability to produce OAs (Din et al., 2020). 1.2 Research Problem Fungi and bacteria have been isolated from mining and metal-contaminated sites for their use in the bioleaching of base metals. However, only bacteria have been isolated from platinum mines specifically and there have not been any studies thus far involving the isolation of fungi from a platinum. There is also a need for the 4 identification of more fungal candidates which have a high base metal bioleaching efficiency and potential to bioleach PGMs from PGM materials. Therefore, the identification and characterisation of fungi isolated from a platinum mine is important to determine fungal candidates for the bioleaching of base metals and PGMs from PGM materials. 1.3 Hypothesis, Aims and Objectives This study hypothesised that fungi will be isolated from platinum mine samples, from the genera Aspergillus and Penicillium due to their frequent isolation from mining environments. Isolates from these genera have the ability to produce OAs which assists in their adaptation to the heavy metal concentrated environments present in a mine. Therefore, the second hypothesis was that the isolates identified in the study will produce OAs. This study aimed to identify and characterise fungal isolates from a platinum mine to determine potential fungal candidates for bioleaching of platinum concentrate. The objectives were: (i) To isolate and enumerate fungi from soil, slurry, and liquid samples retrieved from a platinum mine using traditional microbiological techniques. (ii) To identify the fungal isolates using molecular techniques such as genomic DNA extraction, polymerase chain reaction (PCR), agarose gel electrophoresis, and Sanger sequencing. (iii) To characterise the fungal isolates based on their OA production using qualitative and quantitative methods. (iv) To characterise selected isolates on their heavy metal tolerance by determining their heavy metal tolerance index for selected heavy metals. 5 2 LITERATURE REVIEW 6 2.1 General Introduction The literature review will first discuss the world’s PGM reserves, followed by traditional processing of PGM ore in South Africa and the challenges faced with the mining of PGMs in the country. Thereafter, the isolation of fungal isolates for mycohydrometallurgical processes and the mechanisms which these isolates use to carry out such processes will be reviewed. This will be followed by a review of the different steps involved in fungal bioleaching. Finally, the isolation of microorganisms from a platinum mine from previous studies will be discussed and the use of microorganisms for base metal and platinum bioleaching from platinum sources will be evaluated. 2.2 Mining of Platinum Group Metals (PGMs) 2.2.1 PGM reserves South Africa and Russia are the world leaders in platinum production, however, Russia produces less than a fifth of platinum compared to South Africa (Aleksandrova and О’Connor, 2020). Platinum is a member of the family of PGMs which also consists of Ru, Os, Rh, Ir, and Pd. Figure 2.1 summarises the world’s PGM reserves and shows that South Africa accounts for over 90% of the world’s reserves (Garside, 2021). The main deposits of PGM ore in South Africa are located in the north of the country, in the Bushveld Igneous Complex, which contains the Merensky Reef, Upper Group 2 (UG2) Reef, and Platreef (Thethwayo, 2018). Since 1925, the Merensky Reef has been the main source for PGMs, however, due to the depletion of the Merensky resources, the UG2 Reef is now responsible for most of the platinum-bearing ore in South Africa (Thethwayo, 2018). The Platreef is lower in PGM values but higher in base metal values and as a result, it was the last of the three ore bodies to be exploited (Thethwayo, 2018). 7 Figure 2.1: The world’s estimated reserves of platinum group metals (PGMs) (Garside, 2021). 2.2.2 Processing of PGMs The traditional processing of PGM ores in South Africa involves comminution, flotation, smelting, converting, base metal refinery, and precious metal refinery (Figure 2.2). Comminution results in the reduction of the ore size through the crushing and milling of the ore which is then treated using gravity separators (Safarzadeh et al., 2018). The produced fines are treated using flotation cells to separate it into tailings and sulphide-rich PGM concentrate (Thethwayo, 2018). Smelting is then used to separate the valuable (sulphide) minerals from the gangue (oxide and silicate) minerals (Jones, 1999). The gangue is discarded as slag and the sulphide minerals are further treated in the converting process where sulphur (S) and iron sulphide (FeS) are oxidised to sulphur dioxide (SO2) and ferrous oxide (FeO), respectively (Jones, 1999). SO2 and FeO are removed as an off-gas and slag, respectively (Thethwayo, 2018). The converter matte then undergoes base metal refinery which uses processes such as pressure oxidation which results in the separation of base metals from PGM residue (Bezuidenhout et al., 2013). The base metal-free concentrate then undergoes precious metal refinery where the 63 000 000 kg (90.9%) 3 900 000 kg (5.6%) 1 200 000 kg (1.7%) 900 000 kg (1.3%) 310 000 kg (0.5%) South Africa Russia Zimbabwe United States of America Canada 8 individual metals are separated using solution extraction and precipitation methods (Safarzadeh et al., 2018). Figure 2.2: The traditional processing of platinum group metal (PGM) ore in South Africa (Thethwayo, 2018). 2.2.3 Challenges of mining PGMs The processing of PGM ore results in high energy consumption and despite lower energy consumption by open-pit mines, such as the Mogalakwena mine (24°0' South, 28°55' East), the open-pit mine produces large volumes of waste rock (Mudd, 2010). The large volumes of the waste rock and tailings produced require management to prevent it from having a negative impact on the environment and communities near the mines (Mudd, 2010). Curtis (2008) highlighted how important the managing of mine waste is due to the study conducted on the impact of the Anglo Platinum mine on local communities. It was found that their 9 water resources were contaminated due to mining activities, and contained high concentrations of total dissolved salts, such as sulphate and nitrate (Curtis, 2008). Therefore, an environmentally friendly and effective remediation process needs to be developed. The same is required to process and further process the near- surface oxidised ore from the open-pit mines and slag wastes from smelters, respectively (Hedrich et al., 2020; Mudd, 2010). Without a feasible and effective method, the processing of this ore and waste will not take place and the possible extraction of valuable metals will not occur. As mentioned previously, there has been an increase in exploitation of the UG2 Reef due to the depletion of the Merensky Reef, however, problems arise with the use of ore from the UG2 Reef due to its high chromite content (60%) (Thethwayo, 2018). This is due to the concentrate produced after flotation having a high chrome content which smelters are not equipped to handle (Thethwayo, 2018). Therefore, an alternate method to smelting needs to be considered to ensure efficient processing of the concentrate. 2.3 Mycohydrometallurgy 2.3.1 Introduction Pyrohydrometallurgy and hydrometallurgy are two types of extractive metallurgical processes (Anderson, 2016). Pyrometallurgy involves using high temperatures to carry out smelting and refining operations to extract metals from their minerals, whereas hydrometallurgy uses aqueous solutions to separate the metals (Anderson, 2016). These processes result in low metal recovery, high operating costs, and have a negative environmental impact; therefore, alternative extractive metallurgical processes have been explored (Guezennec et al., 2014). Microorganisms such as bacteria and fungi have been studied for their use in biohydrometallurgy, which is a branch of extractive metallurgy which involves the use of biological systems and aqueous chemistry to recover metals from substances such as ores, concentrate, and waste materials (Ofori-Sarpong et al., 10 2010). Extensive research has been conducted on the use of bacteria in this field, however, there are fewer studies on the use of fungi. Mycology is the branch of biology which focuses on the study of fungi, therefore, the term “mycohydrometallurgy” describes the link between mycology and hydrometallurgy and is defined as the application of fungi for the extraction and recovery of metals (Ofori-Sarpong et al., 2010). The first step in developing a mycohydrometallurgical process is the selection of fungi. The fungi selected can either be acquired or indigenous to the mining environment from where the solid material, which will be used for bioleaching, is obtained. The advantage of isolating indigenous fungi is that they would have acquired traits which allow them to adapt to e.g., the mining or metal- contaminated environment they were isolated from (Din et al., 2020). 2.3.2 Fungi in mining environments Fungi are found in various environments and the fungal community vary depending on the pH, redox potential (Eh), moisture content, and temperature of the environment from which they are isolated (Brockett et al., 2012; Gagnon et al., 2020). Additionally, a parameter that also affects the fungal community is the presence of heavy metals (Gagnon et al., 2020). Table 2.1 lists studies that have been performed regarding the isolation and identification of fungi from mining environments, where fungal strains were isolated from ore, soil, tailings, or overburden samples. These strains were identified due to their ability to tolerate heavy metals, produce OAs or solubilise phosphates, as most studies screen fungal isolates for these characteristics prior to their identification. Therefore, although a greater number of fungi may be isolated from a sample, the strains which are usually identified are those which exhibit characteristics relevant to the specific study. From Table 2.1, the species which are commonly isolated belong to the genera of Aspergillus and Penicillium. This is due to the variety of heavy metals which they acquire tolerance to and due to the OAs which they produce, as these characteristics are important when considering the development of a 11 mycohydrometallurgical process. The studies in Table 2.1 have either isolated fungi for bioprocessing or bioremediation purposes. Bioremediation involves the use of microorganisms for the removal of heavy metals from contaminated water and soil (Iram et al., 2013). The development of bioremediation methods is important due to the metal contamination which results from many mining sites and the effect it has on communities around it, such as the contamination of water sources (Curtis, 2008). Bioprocessing involves the use of microorganisms to obtain a higher-grade concentrate (Hedrich et al., 2020). The use of bioprocessing methods is important in developing more environmentally friendly and feasible processing technologies. The mechanisms which fungi use to carry out bioremediation and bioprocessing will be discussed in Section 2.3.3. Table 2.1 lists fungi that have been isolated and identified from various mines and their tolerance to selected base metals. However, fungi have not been isolated from platinum mines before and therefore when Argumedo-Delira et al. (2020) conducted a study on the tolerance of fungi to platinum, they used strains isolated from samples surrounding a landfill. The strains of Aspergillus niger MX5, Trichoderma harzianum MX2, Fusarium oxysporum MX17, Hypocrea lixii MXPE12, and Fusarium solani MXPE15 were found to be tolerant to a platinum concentration as high as 300 mg/L with little morphological changes observed compared to the control (Argumedo-Delira et al., 2020). These findings allowed the authors to conclude that these strains could be used in the development of biotechnological processes for the recovery of platinum from primary and secondary sources. Table 2.1: Fungi isolated from mining environments with acquired characteristics. Fungal isolates Mining site source Characteristic Reference Trichoderma virens, T. harzianum, T. gamsii Mine tailings - wastewater samples Cr, Pb, Zn, Ni, Cu1 (Tansengco and Tejano, 2018) Trichoderma saturnisporum Mine tailings - wastewater samples Cr, Pb, Ni, Cu (Tansengco and Tejano, 2018) 12 Trichoderma ghanense, Rhizopus microsporus Gold and gemstone mine site - soil samples Cu, Pb, Fe, Cd, As (Oladipo et al., 2018) Fomitopsis meliae Gold mine site - soil samples Cu, Pb, Fe (Oladipo et al., 2018) Rhizopus oryzae Gold mine tailings Cr, Cu, As, Pb, Cd (Sey and Belford, 2021) Trametes versicolor Gold mine tailings Cr (Sey and Belford, 2021) Trichoderma viride, Aspergillus fumigatus, Trichophyton rubrum Gold mine tailings Cr, Cu (Sey and Belford, 2021) Penicillium chrysogenum Smelting industry - soil sample Cd, Cu, Pb, Zn (Deng et al., 2012) Aspergillus sclerotiorum, Komagataella phaffi, Trichoderma harzianum Nanjing lead-zinc mine - soil sample Cd, Cr, Pb (Liaquat et al., 2020) Aspergillus niger, A. aculeatus Nanjing lead-zinc mine - soil sample Cd, Cr (Liaquat et al., 2020) Trichoderma atroviride Nickle mining wastewater - soil and sediment samples Cr, Co, Ni (Hernahadini et al., 2014) Aspergillus niger, A. humicola, A. awamori, A. versicolor, A. phoenicus Chromite mine - overburden dumps Cr, Co, Ni, Fe (Ghosh and Paul, 2015) Penicillium Iron ore Phosphate solubiliser (Adeleke et al., 2010) Alternaria, Epicoccum Iron ore - (Adeleke et al., 2010) Aspergillus, Alternaria, Penicillium, Sterila, Trichoderma, Fusarium Chhattisgarh region in India - soil samples taken from coal, iron ore and limestone mining sites, as well as from a steel plant. Organic acid producers (Gupta and Khan, 2018) Aspergillus sp., Penicillium sp. Limonite ore (low- grade nickel ore) Ni (Handayani and Suratman, 2017) 1 – Heavy metals to which the fungal isolate is tolerant. 13 2.3.3 Fungal bioleaching There are four main mechanisms that fungi use for bioleaching namely, acidolysis, complexolysis, biosorption, and bioaccumulation (Dusengemungu et al., 2021). However, acidolysis is the most rapid and frequently used bioleaching mechanism (Dusengemungu et al., 2021). 2.3.3.1 Acidolysis The conversion of organic compounds (e.g., glucose or sucrose) by enzymatic reactions in the cytosol and mitochondrion of heterotrophic fungal cells results in the production of OAs such as gluconic, citric and lactic acids (Dusengemungu et al., 2021). Acidolysis is a process that utilises protons provided by these low molecular weight organic acids (LMWOAs) to protonate oxygen atoms of metal oxides (Bahafid et al., 2017). The attachment of protons to the metal surface reduces the strength of bonds and results in the removal of metal ions from the surface and the production of water molecules due to the reaction of protons with the oxygen atoms (Bahafid et al., 2017). An example of acidolysis can be seen in equation (1), which displays the reaction of protons with nickel (II) oxide (NiO) to produce nickel ions. NiO + 2H+ Ni2+ + H2O (Equation 1) 2.3.3.2 Complexolysis Complexolysis takes place through the chelation mechanism and allows for the formation of complexing agents which leads to metal extraction (Dusengemungu et al., 2021). It occurs due to the bond formed between metal ions and ligands (OAs), being stronger than the lattice bonds formed between the metal ions and solid particles (McKenzie et al., 1987). Therefore, allowing the solubilisation of metal ions and enhancing the bioleaching process. Complexolysis also allows for the stabilisation of metal ions produced during acidolysis and reduces their (the acidolysis 14 metal ions) toxic effect on the fungi (Srichandan et al., 2019). An example of complexation can be seen in equation (2), where nickel ions complex with citric acid. Ni2+ + C6H8O7 Ni(C6H5O7)- + 3H+ (Equation 2) 2.3.3.3 Biosorption During biosorption, the metal ions which occur in the solution due to the production of OAs which dissolve the metal from the solid material, are adsorbed by the fungal biomass (Dusengemungu et al., 2021). Thereby decreasing the quantity of metal in the solution. The reactions which are involved in the biosorption of metals are ion exchange, complexion, adsorption, and precipitation (Bahobil et al., 2017). With the use of a direct bioleaching method (Section 2.3.4), biosorption could take place and result in an increase in the bioleaching efficiency by the removal or recovery of base metals and PGMs. 2.3.3.4 Bioaccumulation Bioaccumulation is the uptake of metals by the biomass of living organisms (Dusengemungu et al., 2021). It takes place when metal ions accumulate or precipitate in vacuoles due to their transportation between the cell membrane and the cell of these organisms (Brandl et al., 2001). Unlike biosorption, bioaccumulation does not require the production of metabolites for the uptake of metals, however, the process is slower than biosorption. Fungal bioaccumulation is thought to take place due to the binding of metal ions to functional groups present in fungal mycelia, such as amine, phosphate, hydroxyl, carboxyl, and sulphate, which allows the metal ions to act as a cation exchanger (Dusengemungu et al., 2021). An example of such a reaction can be seen in equation (3). Ni2++ SO4 −2 NiSO4 (s) (Equation 3) complexolysis bioaccumulation 15 2.3.4 Indirect and direct bioleaching There are different methods for bioleaching namely indirect and direct bioleaching (Handayani and Suratman, 2017). Indirect bioleaching occurs when the solid bioleaching material is exposed to the metabolites produced by microorganisms, without the microorganisms present in the bioleaching setup (Handayani and Suratman, 2017). Whereas direct bioleaching occurs in the presence of microorganisms and can be sub-divided into one-step and two-step bioleaching procedures (Brandl et al., 2001). One-step bioleaching takes place when the microorganism is inoculated into the media containing the solid material to be bioleached (Brandl et al., 2001; Deng et al., 2012). Two-step bioleaching involves the growth of the microorganism in growth media, followed by the addition of the solid material after exponential biomass growth occurs (Brandl et al., 2001; Deng et al., 2012). This allows the microorganisms a chance to grow and produce metabolites prior to being exposed to the solid material (Deng et al., 2012). Handayani and Suratman (2017) compared indirect and direct bioleaching of low- grade nickel ore using species of Aspergillus and Penicillium. They found that direct bioleaching resulted in a 9% increase in the percentage of nickel extracted compared to indirect bioleaching for both Aspergillus sp. and Penicillium sp. (Handayani and Suratman, 2017). Penicillium sp. had a 9% lower bioleaching efficiency than Aspergillus sp. using the direct method, as they had a 48% and 57% bioleaching efficiency, respectively (Handayani and Suratman, 2017). Brandl et al. (2001) determined the efficiency of Thiobacillus thiooxidans, T. ferrooxidans, A. niger, and P. simplicissimum to bioleach Cu, Ni, tin (Sn), aluminium (Al), lead (Pb), and zinc (Zn) from electronic waste using a one-step procedure. However, they suggested that a more efficient method would be two-step bioleaching to allow biomass growth to be separated from metal leaching. A study performed by Deng et al. (2012) compared one-step and two-step bioleaching using P. chrysogenum to bioleach heavy metals from contaminated soil. They found that two-step bioleaching had a leaching efficiency of 13%, 21%, 5%, and 16 14% greater than the one-step procedure for cadmium (Cd), Cu, Pb and Zn, respectively. Therefore, the bioleaching method which could result in the greatest efficiency to process PGM ores may be two-step bioleaching. 2.3.5 Biohydrometallurgical methods for base metal removal from PGM sources The use of fungi in biohydrometallurgical processes for the removal of base metals from PGM sources has not been studied in previous research. However, the use of iron and sulphur oxidising bacteria has been investigated. Mwase et al. (2012) used a culture of Metallosphaera hakonensis to bioleach Cu (91.1%), Ni (98.5%) and Co (83.5%) from low-grade flotation concentrate obtained from Platreef ore. Due to the success of this experiment, they used the same culture to bioleach Cu, Ni and Co from crushed whole ore where 93%, 75% and 53% of each metal was removed, respectively (Mwase et al., 2014). Hedrich et al. (2020) conducted bioleaching experiments on oxidised PGM ore from stockpiles of the Mogalakwena mine using acidophilic iron and sulphur oxidising bacteria and reported an 86% total base metal recovery of metals such as Co, Cu, Ni, and manganese (Mn). 2.3.6 Biohydrometallurgical methods for platinum recovery Compared to the studies performed on the recovery or removal of base metals from ores, concentrate, waste materials, and contaminated soil, studies on the recovery of platinum using biohydrometallurgical methods are scarce. The studies which have been performed have used bacterial isolates which were acquired from culture collections or isolated from wastewater and not directly isolated from platinum mining sites or processes. The bioleaching experiment performed by Hedrich et al. (2020) described in Section 2.3.6, resulted in the recovery of base metals, however, it also resulted in the release of platinum group minerals. Therefore, bioleaching resulted in the enhancement of the following chemical leaching process, HNO3/NaCl and a 17 cyanide leach, which was used to extract PGMs. The direct extraction of platinum using bioleaching methods was shown in a study by Brandl et al. (2008) where cyanogenic bacteria were used to extract platinum (0.2%) from automobile catalytic converters. Maes et al. (2016) reported the extraction of 98% of Pt (II) and 97% of Pt (IV) from solutions of 100 mg/L of potassium tetrachloroplatinate (K2Pt(II)Cl4) and potassium hexachloroplatinate (K2Pt(IV)Cl6) using mixed cultures of Halomonasae, Bacillaceae, and Idiomarinaceae, which are families of halophilic bacteria. Therefore, this study demonstrated the platinum recovery potential of halophilic mixed cultures in acidic saline conditions (Maes et al., 2016). In 2017, Maes et al. (2017) studied the biosorption recovery of five Pt-complexes under acidic conditions using four Gram-positive species, Shewanella oneidensis, Cupriavidus metallidurans, Geobacter metallireducens, and Pseudomonas stutzeri and one Gram-negative species, Bacillus toyonensis. All species were found to completely recover platinum from PtCl4 2- and PtCl62- , whereas only S. oneidensis and C. metallidurans recovered 99% of platinum from cisplatin (Maes et al., 2017). Carboplatin was partially recovered with a maximum recovery of 25% and no platinum recovery was observed for Pt(NH3)4Cl2 (Maes et al., 2017). 2.3.7 Microorganisms isolated from a platinum mine As explained in Section 2.3.2, there are no studies involving the isolation of fungi from a platinum mine. However, there are a few studies which have been performed that involve the isolation of bacteria from a platinum mine. Neddy Aka and Babalola (2017), isolated bacteria belonging to the genera Pseudomonas, Bacillus, Alcaligenes and Proteus, which displayed multiple tolerance to the heavy metals, Cr, Cd, and Ni, from a platinum mine in South Africa. Bacteria were also isolated from a platinum mine tailings dam in South Africa, where Paenibacillus lautus and Stenotrophomonas maltophilia were found in high abundance (Rauwane et al., 2018). Therefore, the presence of these bacterial species in platinum mines may indicate their possible use for bioleaching, however, there are no studies which investigated the bioleaching efficiency of these species. 18 2.4 Summary This study is aimed at identifying and characterising fungi isolated from a platinum mine. The characterisation of fungi based on their OA producing ability and heavy metal tolerance allows for the selection of indigenous fungal isolates to be used in biohydrometallurgical processes. Fungal bioleaching is such a process that uses fungi to assist in the recovery of metals from ore, concentrate, and waste materials. The literature review in this chapter discussed the worlds PGM reserves and the traditional processing methods of PGMs, where a need for a more environmentally friendly, feasible and effective processing method was seen. Mycohydrometallurgy as a possible solution was then considered and previous literature which involved the identification of fungal isolates from mines and the use of fungi for mycohydrometallurgical processes to remove or recover base metals from solid material was reviewed. A need for the identification of fungi from a platinum mine for their possible use in bioleaching processes was seen as there have only been studies involving the isolation of bacteria from platinum mines and the use of bacteria for the bioleaching of platinum sources which have been conducted. These studies all occurred in South Africa, most likely due to the country containing the highest PGM reserves in the world. Therefore, the need for the identification of fungi from a platinum mine, especially from South Africa is apparent, in order for mycohydrometallurgical processes for the recovery of platinum to be developed. 19 3 ISOLATION AND IDENTIFICATION OF FUNGI 20 3.1 Introduction The isolation and identification of microorganisms from mine and heavy metal contaminated environments are important to understand the diversity of microorganisms present in these environments. This is due to the presence of microorganisms which have developed adaptive qualities that allow them to tolerate these harsh heavy metal concentrated environments (Din et al., 2020). The isolation and identification of such adapted microorganisms are important to determine which isolates can be used in biohydrometallurgical processes to extract and solubilise heavy metals from ore, concentrate, and waste materials for bioprocessing and bioremediation purposes (Hedrich et al., 2020; Iram et al., 2013). The possible identification of fungal isolates which have not previously been identified in mining or metal-contaminated environments is also of interest. Bacteria and fungi are examples of microorganisms which have been isolated from these environments, however, the studies involving the isolation of bacteria are far greater compared to fungi (Ofori-Sarpong et al., 2010). With regards to a platinum mine, bacteria have previously been isolated from such a mine, however, there are no known studies involving the isolation of fungi (Neddy Aka and Babalola, 2017; Rauwane et al., 2018). Additionally, the enumeration (total fungal count) of fungi from mine and metal-contaminated sites are rarely performed as most studies focus on the sole isolation of specific heavy metal tolerant fungi for their biosorption capabilities. However, the enumeration of fungi assists with obtaining a better understanding of the fungal diversity in the heavy metal concentrated environment. There are two methods which have been used for the identification of microorganisms, namely, morphological, and molecular techniques. Molecular methods are usually preferred as more accurate results are obtained due to the possible identification of isolates to their species level (Raja et al., 2017). When considering extracting DNA from fungi, the first step in performing molecular techniques is determining the use of an effective DNA extraction method due to 21 their strong cell wall being harder to lyse compared to other microorganisms (van Burik et al., 1998). Additionally, the use of a DNA extraction kit is expensive and at times ineffective when dealing with multiple different fungal isolates (Umesha et al., 2016). Therefore, this study aimed to isolate and identify fungi from samples retrieved from a platinum mine using microbiological techniques and a feasible and effective DNA extraction method in order to obtain pure DNA for downstream applications, respectively. 3.2 Materials and Methods 3.2.1 Sample collection A total of thirty-nine samples were collected from four regions at the Mogalakwena platinum mine (24°0' South, 28°55' East) namely: the concentrator plant, the pond outside of the concentrator plant, in-front of the crusher, and in- front of the concentrator. All the samples collected from the concentrator plant were liquid samples collected at different stages of the flotation process. The samples obtained from the remaining three regions were soil, slurry, or liquid samples taken from around the mine. The full list of sample sets is shown in Table 3.1. The samples were placed in either 50 ml sterile centrifuge tubes or a polyethylene bag and were transported in a polystyrene box, stored at 4oC, and were analysed at the Industrial Microbiology and Biotechnology Laboratory (University of the Witwatersrand) within 24 hours of sampling. 3.2.2 Analysis of pH and redox potential The pH and Eh in units of mV, for all samples, were measured (Crison - Model Basic 20- pH-Meter). To prevent contamination of the samples, liquid samples were analysed by aseptically transferring 2 ml of sample into a 15 ml centrifuge tube. Solid samples were analysed by aseptically transferring 1.0 g of sample into a 15 ml centrifuge tube and adding 10 ml of distilled water to the tube. 22 3.2.3 Microbiological characterisation For the isolation of fungi from the samples, a 10-1 to 10-3 dilution series was performed aseptically with sterile saline (0.9%) being used as the diluent. Thereafter, aliquots of 100 µl from each dilution were spread plated onto Potato Dextrose Agar (PDA) (pH 5.6 ± 0.2) plates containing 1% chloramphenicol and incubated at 25oC for 7 days. The antibiotic, chloramphenicol, was added to inhibit bacterial growth. The results are represented as colony-forming units (CFU) per ml or per g of sample. Distinct fungal colonies were then sub-cultured until pure cultures were obtained. The cultures were maintained on PDA plates at 4oC. 3.2.4 Genomic DNA extraction The genomic DNA extraction method was modified from protocols performed by Doyle and Doyle (1987) and van Burik et al. (1998). The sixty-six pure fungal isolates were inoculated into 10 ml of Sabouraud Dextrose Broth in a 15 ml centrifuge tube and incubated at 25oC for 7 days on a 200-rpm orbital shaker. The mycelia were transferred into 2 ml microcentrifuge tubes, a pellet of cells was obtained through centrifugation (MiniSpin microcentrifuge – Eppendorf) at 1 677 x g for 10 min and the supernatant was discarded. The tubes were then covered with parafilm, and tiny perforations were made. They were stored in the – 80oC freezer overnight and the pellets were lyophilised (VirTis BenchTop Pro Lyophilizer – SP Scientific) at 222 µB and – 80oC, for 24 hours. The lyophilised pellets were suspended in a 2 ml microcentrifuge tube containing; 500 µl extraction buffer [1 M Tris (pH 8.0), 5 M NaCl, 0.5 M EDTA and 2% CTAB in 1 L distilled H2O] which was pre-heated at 65oC (Waterbath WNB 7 – Memmert), 1 µl β-mercaptoethanol, and four sterile glass beads (5 mm). This was vortexed thoroughly and cells were lysed by sonication (Scientech Ultrasonic Cleaner – 702) for 1 hour, at 55oC, and the high- frequency setting. The mixture was then transferred to a new tube and incubated at 65oC for 60 min. Thereafter, all steps were performed on ice. After incubation, 500 µl chloroform: isoamyl alcohol (24:1) was added and mixed by vigorous inversion. This mixture was centrifuged at 12 045 x g, 4oC, and for 10 min. The top 23 aqueous layer was extracted and transferred to a new tube and the chloroform: isoamyl alcohol step was repeated. The nucleic acid was precipitated with an equal volume of isopropanol and the tube was gently inverted until DNA precipitated. It was then centrifuged at 12 045 x g, 4oC, and for 10 min and the supernatant was discarded. The pellet was washed in 500 µl ice-cold 70% ethanol, vortexed briefly and centrifuged at 12 045 x g, 4oC, and for 10 min and the wash step was repeated. The pellet was air-dried and resuspended in 50 µl 1 x TE buffer containing 20 µg/ml RNase A. 3.2.5 Quantification of DNA A Thermo Scientific™ NanoDrop™ 1000 Spectrophotometer was used to determine the concentration and purity of the extracted DNA at 260 nm. A concentration of ≥ 10 ng/µl was considered as acceptable. The ratio of absorbance of ≥ 1.8 at 260/280 nm was considered as acceptable and pure DNA. 3.2.6 Sanger sequencing PCR, agarose gel electrophoresis, and Sanger sequencing were carried out at Inqaba Biotech (Pretoria, South Africa). Amplification of the internal transcribed spacer (ITS) region was performed using OneTaq® Quick-Load® 2X Master Mix (NEB, Catalogue No. M0486) with the fungal universal primer set ITS1 (5’- TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC) (White et al., 1990). The PCR products were run on an agarose gel and the bands were extracted using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Catalogue No. D4001). The fragments were sequenced in the forward and reverse direction (Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000) and purified (Zymo Research, ZR-96 DNA Sequencing Clean-up Kit™, Catalogue No. D4050). The purified fragments were analysed (ABI 3500xl Genetic Analyzer (Applied Biosystems, ThermoFisher Scientific)) for all isolates. The .ab1 files generated by the ABI 3500XL Genetic Analyzer were analysed using the CLC Bio Main Workbench v7.6 and the results were analysed using the Basic Local 24 Alignment Search Tool (BLAST) (National Center for Biotechnology Information (NCBI)). 3.3 Results and Discussion 3.3.1 Physicochemical characteristics and fungal counts The pH of the samples obtained from the platinum mine ranged from slightly acidic (6.78) to alkaline (9.27) with the lowest pH being measured from sample 31 which was the liquid sample obtained from the pond outside of the concentrator plant and the highest pH was measured from sample 32 which was the soil sample taken from in-front of the crusher (Table 3.1). The Eh in the present study ranged from 26 to -103 mV (Table 3.1), with the lowest reducing environment being sample 31 and the highest reducing environment being sample 23 which was the dry soil sample obtained from the same region as sample 31. The CFU/ml or CFU/g of sample ranged from 0 to 1.2 x 104 among all the samples retrieved from the mine (Table 3.1). The highest fungal count (1.2 x 104) was from sample 33 which was a soil sample taken from in-front of the crusher. The low fungal counts were observed from all the liquid samples obtained from the concentrator plant, with most samples from this region having 0 CFU/ml and the highest count (2.0 x 102 CFU/ml) being from sample 17 which was obtained from cleaner scavenger tail B. The soil, slurry, and liquid samples obtained from the three remaining regions, namely, the pond outside of the concentrator plant, in- front of the crusher, and in-front of the concentrator resulted in higher fungal counts, apart from sample 31 which was the liquid sample obtained from the pond outside of the concentrator plant, as it had a count of 2.0 x 102 CFU/ml which was the same as sample 17. These low fungal counts were also observed for the other two liquid samples from the same region (samples 29 and 30). 25 Table 3.1: Physicochemical characteristics and fungal counts of samples obtained from the Mogalakwena platinum mine. Mine Sample no. Sample name Region in the mine where samples were obtained pH Eh (mV) CFU/ml or CFU/g of sample 1 Rougher Feed A1 8.69 -79 0 2 Rougher Feed A 8.61 -81 1.0 x 102 3 Rougher Tail A A 8.15 -47 0 4 Rougher Tail A A 8.17 -45 0 5 Rougher Tail B A 7.69 -27 0 6 Rougher Tail B A 7.68 -28 0 7 Scavenger Feed A 8.51 -71 0 8 Scavenger Feed A 8.51 -67 0 9 Scavenger Tail A A 7.35 -9 0 10 Scavenger Tail A A 7.40 -11 1.0 x 102 11 Scavenger Tail B A 8.55 -72 1.0 x 102 12 Scavenger Tail B A 8.53 -79 1.0 x 102 13 Cleaner Feed A 8.27 -59 1.0 x 102 14 Cleaner Feed A 8.40 -66 0 15 Cleaner Scavenger Tail A A 8.23 -55 0 16 Cleaner Scavenger Tail A A 8.45 -66 0 17 Cleaner Scavenger Tail B A 8.36 -63 2.0 x 102 18 Cleaner Scavenger Tail B A 8.42 -64 1.0 x 102 19 Dry soil B2 8.63 -99 1.2 x 103 20 Dry soil B 9.11 -103 4.0 x 102 21 Dry soil B 8.30 -64 1.2 x 103 22 Dry soil B 8.25 -77 8.0 x 102 23 Slurry B 7.27 -23 1.1 x 103 24 Slurry B 6.96 3 1.4 x 103 25 Slurry B 6.90 1 5.0 x 103 26 Slurry B 7.53 -32 1.0 x 103 27 Slurry B 6.87 8 1.1 x 103 28 Slurry B 7.39 -18 1.0 x 104 29 Liquid B 6.86 15 6.0 x 102 30 Liquid B 6.90 8 3.0 x 102 31 Liquid B 6.78 26 2.0 x 102 32 Soil C3 9.27 -99 7.0 x 102 33 Soil C 9.21 -100 1.2 x 104 34 Soil C 8.96 -73 4.0 x 103 35 Soil C 8.78 -60 2.1 x 103 36 Slurry C 7.90 -48 5.0 x 103 26 37 Slurry C 8.27 -65 3.0 x 103 38 Slurry C 8.32 -65 3.0 x 103 39 Soil D4 8.49 -47 1.6 x 103 There are only a few studies which have determined the fungal counts and pH of samples isolated from mining or metal-contaminated environments, with the Eh of samples not often being mentioned. However, pH and Eh are inversely correlated as explained by the oxidation of two molecules of water (losing four electrons) resulting in the production of O2 and 4H+, therefore leading to acidification (Husson et al., 2018). This is seen in the present study as the samples with pH values below 6 all had higher Eh values. Oladipo et al. (2014) determined the fungal counts of samples obtained from gold and gemstone mines and determined the counts to range between 2.4 x 104 to 2.5 x 105 CFU/g of soil, with the pH ranging between 3.5 and 5.3, respectively. pH values in the acidic range, as seen in the mentioned study, have been found to result in a decrease in microbial growth due to an increase in metal dissolution which results in greater toxicity to microbes (Nwuche and Ugoji, 2008). However, this was not the case in the study by Tansengco and Tejano (2018) where they isolated fungi from wastewater retrieved from mine tailings, as they determined that higher fungal counts were observed in samples with lower pH values. In the present study, a correlation between pH and fungal counts is difficult to make as higher fungal counts were not observed in all samples with a higher pH. This is seen when comparing the pH and fungal counts of sample 32 (pH 9.27) and sample 33 (pH 9.21), as they both have a pH above 9. However, sample 32 has a much lower fungal count (7.0 x 102 CFU/g) compared to sample 33 (1.2 x 104 CFU/g). Therefore, the difference in fungal counts may be due to other factors not relating to pH. Rath et al. (2010) isolated fungi from iron and chromite mine soil as well as from a control sample and determined the fungal counts to be 9.2, 9.9, and 18.3 CFU/g 1 – A: concentrator plant, 2 – B: pond outside of the concentrator plant, 3 – C: in-front of the crusher, 4 – D: in-front of the concentrator 27 of soil and the soil pH to be 5.1 ± 0.09, 5.4 ± 0.05, and 6.6 ± 0.04, respectively. They determined that these low counts could be due to low nutrients in the soil, more specifically, low levels of organic carbon which is essential for the growth of heterotrophs (Rath et al., 2010). They also concluded that the acidic pH and high concentration of heavy metals in the soil could have resulted in extremely low fungal counts. Therefore, in terms of the present study, the fungal counts of the samples retrieved from the concentrator plant could have been very low as they were retrieved from various processes that occur during flotation. During flotation, there is a severe lack of nutrients compared to the other three regions from which the samples were retrieved, and the fungi are exposed to high concentrations of heavy metals which could prevent or inhibit fungal growth. In terms of the fungal counts among the samples from the remaining three regions, the difference in fungal counts between the samples could be due to the difference in levels of organic carbon between the samples, the adaptation of the fungi to the heavy metal concentrations for the specific sample site, and the difference in the microbial community as a whole. The spread plates for samples 13, 24, 33 and 39 obtained from the four different regions of the mine can be seen in Figure 3.1. The decrease in the number of colonies in terms of fungi, yeast, as well as bacteria resistant to the antibiotic used can be clearly seen from the 10-1 to the 10-3 spread plates. For sample 13 taken from the concentrator plant, the low colony counts, in general, can be clearly seen with only one fungal isolate appearing as a fluffy white colony in the lowest dilution at the edge of the plate. Sample 24 and sample 33 taken from the pond outside of the concentrator plant and in-front of the crusher, respectively, had many different types of filamentous fungal growth on their 10-1 spread plates, however, the types of colonies between the plates differed. This was seen by the different types of white fungal colonies as well as a yellow colony on sample 24’s plate and the presence of black, white, brown, cream, and black sporulating fungal colonies on sample 33’s plate. 28 Figure 3.1: Spread plates of the mine samples. Dilutions 10-1 to 10-3 (left to right) for samples 13, 24, 33, and 39 obtained from the four different regions of the platinum mine. The 10-1 spread plate for sample 39 mostly consisted of the growth of a white fluffy colony surrounding the plate with a greenish sporulating colony at one edge of the plate. The 10-2 spread plate for sample 39 also shows the interaction between an antibiotic-resistant bacterial colony with a filamentous fungal colony as a clear zone of growth inhibition of the fungal colony is observed due to the presence of the bacterial colony. Therefore, this could explain the low fungal counts of samples 20, 22, 29, 30, 31, and 32, as the microbial community as a whole may affect fungal growth negatively or positively (Boer et al., 2005). The identification of microbial communities may be done by using a 16S ribosomal ribonucleic acid gene and ITS next-generation sequencing method to identify bacterial and fungal species present in the samples, respectively (Böhmer et al., 2020). After the repeated sub-culturing of distinct fungal colonies from the spread plates, sixty-six pure cultures of fungal isolates were obtained, and molecular techniques were used to identify these isolates. 29 3.3.2 Evaluation of DNA extraction method The DNA extraction method used resulted in high concentrations and relatively good purity ratios of DNA (Table A1 – Appendix A). All isolates’ DNA extracts resulted in a concentration between 39.8 and 1809.3 ng/µl, a A260/A280 ratio between 1.85 and 2.29 and a A260/A230 ratio between 1.15 and 2.78 (Table A1 – Appendix A). It’s important to analyse the purity of DNA extracts to determine if the extraction method is sufficient and if contaminants will affect downstream applications such as PCR. The acceptable range for purity ratios is 1.8 to 2.0 for A260/A280 and 1.8 to 2.2 for A260/A230 (Olson and Morrow, 2012). High A260/A280 ratios indicate that RNA was co-extracted, however, this does not affect downstream applications, but it does result in an overestimation of the concentration of DNA (Li et al., 2008). Therefore, the DNA concentration for some of the isolates in the present study e.g., isolate no. 30 and 57 which had the highest concentrations of DNA, might be lower than indicated due to RNA contamination since these two isolates also had the highest A260/A280 ratios (Table A1 – Appendix A). Low A260/A230 ratios are known to indicate protein and polysaccharide contamination which can affect the amplification of DNA during PCR. However, protein contamination would have resulted in a low A260/A280 ratio and this was not observed for any of the isolates (Olson and Morrow, 2012). Therefore, the contaminants were likely polysaccharides. Despite the purity ratios not remaining within the acceptable ranges, this did not affect the amplification of DNA during PCR and the identification of the isolates was possible, therefore suggesting that the DNA extraction method used was successful. 30 3.3.3 Evaluation of identification method The sixty-six fungal isolates were identified and are listed in Table 3.2. The identification of thirty-two of the isolates to their species level was possible with the primer set used, however, the identification of the remaining thirty-four isolates to their exact species level was not possible. For these thirty-four isolates, either the species name, genus, or family was not possible to deduce based on the sequencing results obtained. Raja et al. (2017), conducted an extensive review on the methods used for fungal identification and concluded that, when possible, identification should occur using a combination of micromorphological, cultural, and molecular techniques. However, morphological identification leads to a variety of limitations which include the limited amount of morphological characteristics which can be used for identification and the frequent inability to identify the isolates according to their species level (Ko Ko et al., 2011; Raja et al., 2017). With regards to molecular identification, the ITS region which was amplified in the present study is the most useful nuclear ribosomal gene for species-level identification (Raja et al., 2017). However, it has been found to be ineffective when dealing with highly specious genera such as Aspergillus, Cladosporium, Fusarium, Penicillium and Trichoderma as these genera have limited or no barcode gaps in this region (Raja et al., 2017; Samson et al., 2014)). From the results in the present study, the mentioned genera were all identified and at least one of the isolates identified from each genus was not able to be identified to their exact species (Table 3.2). Raja et al. (2017), suggested the use of protein-coding genes to be used with or to replace ITS regions in species identification via barcoding due to the presence of intron regions which can evolve faster than ITS regions. Therefore, this could be a possible solution to identify the fungal isolates which could be selected for further studies for bioleaching purposes, to their species-level. 31 Table 3.2: Identification and classification of fungal isolates from the Mogalakwena platinum mine. Phylum Current name Family Mine sample no. GenBank accession no. Isolate no. Asco- mycota Bartalinia sp., Bartalinia pondoensis Bartaliniaceae 33 MT477058.1, KJ767127.1 16 Bartalinia pondoensis Bartaliniaceae 34 JQ425386.1 23 Clonostachys epichloe Bionectriaceae 23 KJ780769.1 41 Neoscytalidium dimidiatum Botryosphaeriaceae 33 MF580799.1 33 Tiarosporella graminis Botryosphaeriaceae 23 KF531828.1 55 Chaetomella raphigera Chaetomellaceae 33 KF193633.1 62 Chaetomium sp., Chaetomium strumarium Chaetomiaceae 24 KX618205.1, KY558668.1 54 Pseudothielavia terricola, Chrysocorona lucknowensis, Acrophialophora jodhpurensis Chaetomiaceae 27 NG_069813.1, MH877835.1, MH873474.1 49 Pseudothielavia terricola, Chrysocorona lucknowensis, Acrophialophora jodhpurensis Chaetomiaceae 33 NG_069813.1, MH877835.1, MH873474.1 64 Cladosporium anthropophilum, Cladosporium cladosporioides Davidiellaceae 34 MN511353.1, MK813966.1 3 Cladosporium sp., Cladosporium cladosporioides, Cladosporium westerdijkieae Davidiellaceae 39 MK355726.1, MT466517.1, MF473314.1 11 Cladosporium sp., Cladosporium cladosporioides, Cladosporium westerdijkieae Davidiellaceae 29 MK355726.1, MT466517.1, MF473314.1 12 Cladosporium sp., Cladosporium cladosporioides, Cladosporium westerdijkieae Davidiellaceae 31 MK355726.1, MT466517.1, MF473314.1 28 32 Cladosporium tenuissimum Davidiellaceae 28 MF473304.1 37 Pseudocoleophoma polygonicola Dictyosporiaceae 35 MZ492974.1 63 Didymella sp., Phoma sp., Didymella americana Didymellaceae 28 MG967669.1, KY790596.1, MK646045.1 43 Ectophoma multirostrata Didymellaceae 34 MG897497.1 29 Epicoccum sorghinum Didymellaceae 25 MN215621.1 45 Phoma sp. Didymellaceae 27 JN207257.1 10 Phoma herbarum, Epicoccum sorghinum Didymellaceae 27 MT420621.1, MN944541.1 27 Phoma herbarum, Epicoccum sorghinum Didymellaceae, Didymellaceae 29 MT420621.1, MN944541.1 24 Phoma sp., Boeremia sp., Aspergillus niger Didymellaceae, Didymellaceae, Trichocomaceae 30 MK066907.1, MH931265.1, KT963790.1 9 Phoma sp., Didymella pinodella Didymellaceae 35 JN207257.1, MW784722.1 61 Phoma sp., Didymella sp., Didymella pinodella Didymellaceae 19 JN207257.1, MH257388.1, MW784722.1 47 Phoma sp., Epicoccum phragmospora Didymellaceae 28 JN207257.1, NR_165920.1 52 Stagonosporopsis cucurbitacearum Didymellaceae 10 GU045304.1 22 Trichoderma longibrachiatum Hypocreaceae 26 KT852813.1 46 Trichoderma sp., Trichoderma erinaceum, Trichoderma koningiopsis Hypocreaceae 22 MK870709.1, KC884817.1, FR670342.1 56 Pyrenochaeta sp. Incertae sedis 34 EU750693.1 4 Pyrenochaeta sp. Incertae sedis 36 EU750693.1 5 Fusarium chlamydosporum Nectriaceae 36 MG250446.1 20 Fusarium chlamydosporum Nectriaceae 23 KX421422.1 50 Fusarium chlamydosporum Nectriaceae 22 MG250446.1 51 Fusarium equiseti Nectriaceae 23 MT626672.1 13 Fusarium equiseti Nectriaceae 23 MK780235.1 21 Fusarium oxysporum Nectriaceae 39 MK249867.1 48 Fusarium sp., Fusarium chlamydosporum Nectriaceae 39 MH582472.1, KX421422.1 59 33 Fusarium sp., Fusarium solani, Fusarium phaseoli Nectriaceae 39 MK640565.1, KX583231.1, KF717534.1 31 Alternaria alternata Pleosporaceae 29 MK773579.1 35 Alternaria sp. Pleosporaceae 36 MW784825.1 60 Alternaria sp., Alternaria alternata Pleosporaceae 19 MK640569.1, MK311341.1 15 Bipolaris zeae Pleosporaceae 23 MT505870.1 1 Bipolaris sp., Helminthosporium bondarzewii, Curvularia spicifera Pleosporaceae, Massarinaceae, Pleosporaceae 21 MF590167.1, MH877988.1, MH875110.1 18 Curvularia beasleyi, Curvularia hawaiiensis Pleosporaceae 28 MH414893.1, KC999927.1 8 Curvularia petersonii Pleosporaceae 33 NR_158448.1 34 Exserohilum rostratum, Setosphaeria rostrata, Bipolaris maydis Pleosporaceae 22 MT524320.1, LT837845.1, MH197141.1 36 Exserohilum rostratum, Setosphaeria rostrata, Bipolaris maydis Pleosporaceae 20 MT524320.1, LT837845.1, MH197141.1 39 Periconia sp., Periconia macrospinosa Pleosporaceae 10 HQ607981.1, MT658103.1 6 Neopyrenochaeta telephoni Pleosporineae 35 KM516291.1 65 Westerdykella ornata Sporormiaceae 24 NR_103587.1 2 Westerdykella sp., Westerdykella centenaria Sporormiaceae 26 MG250473.1, NR_156002.1 44 Albifimbria viridis Stachybotryaceae 35 MN625931.1 19 Stachybotrys microspora Stachybotryaceae 32 MK956918.1 66 Aspergillus flavus Trichocomaceae 29 MT645322.1 38 Aspergillus sp., Aspergillus costaricensis, Aspergillus tubingensis Trichocomaceae 33 MT447519.1, MT558927.1, MN067891.1 40 Aspergillus terreus Trichocomaceae 36 MT316343.1 17 Aspergillus terreus Trichocomaceae 28 MT316343.1 58 Aspergillus welwitschiae, Aspergillus niger Trichocomaceae 22 MK450669.1, MT620753.1 26 Penicillium sp., Penicillium magnielliptisporum Trichocomaceae 39 MT424930.1, MK450730.1 14 Coprinellus radians Psathyrellaceae 21 JN943117.1 25 34 Basidio- mycota Fomitopsis meliae Fomitopsidaceae 13 KT718002.1 32 Peniophora sp. Peniophoraceae 19 HQ607800.1 42 Physisporinus sp., Vanderbylia fraxinea, Physisporinus vitreus Meripilaceae, Polyporaceae, Meripilaceae 20 KU958554.1, KX081102.1, KU194320.1 7 Terana caerulea Phanerochaetaceae 20 KP134980.1 53 Mucoro- mycota Mucor fragilis Mucoraceae 35 MK910073.1 30 Rhizopus microsporus Mucoraceae 22 KJ417579.1 57 3.3.4 Identification of fungal isolates Of the sixty-six isolates, 89.39% belonged to the phylum Ascomycota, 7.58% to Basidiomycota, and 3.03% to Mucoromycota. The pie chart in Figure 3.2 does not represent all sixty-six isolates, as only sixty-three isolates were used to represent the phyla and families of the isolates in the pie chart. This was due to the omission of isolates no. 7, 9, and 18 (Table 3.2), as the families to which they belong to were uncertain. The percentages of phyla in the present study are similar to the study performed by Gupta and Khan (2018), where the majority of isolates belonged to Ascomycota with a few belonging to Mucoromycota. In a study by Held et al. (2020), fungi were isolated from various samples from an underground iron ore mine where out of the 164 fungal isolates identified, 65.85% were Ascomycota, 18.90% were Mucoromycota, and 15.85% were Basidiomycota. Therefore, the findings in the present study regarding Ascomycota being the major group of fungi is similar to previous studies of fungi in mine and metal-contaminated sites (Al- Garni et al., 2009; Bahobil et al., 2017; Ezzouhri et al., 2009; Gupta and Khan, 2018; Held et al., 2020; Iram et al., 2012; Siham, 2007). This is likely due to their organic acid (OA) production, biosorption or bioaccumulation abilities which helps them tolerate these heavy metal-containing environments. 35 Figure 3.2: Percentage of sixty-three fungal isolates belonging to their respective families and phyla. There was more than one possibility for the families of isolates no. 7, 9, and 18, therefore these isolates were excluded from the pie chart. However, isolates no. 9 and 18 are Ascomycota and isolate 7 is a member of Basidiomycota. Therefore, when considering the phyla’s percentages regarding the sixty-six isolates, 89.39% belonged to the phylum Ascomycota, 7.58% to Basidiomycota, and 3.03% to Mucoromycota. The identified isolates were members of several different families of Ascomycota, Basidiomycota, and Mucoromycota (Table 3.2 and Figure 3.2). The genera of isolates no. 16, 23, 41, 33, 55, 62, 49, 64, 63, 29, 7, 53, 65, 2, 44, and 19 (Table 3.2) which are members of the families, Bartaliniaceae, Bartaliniaceae, Bionectriaceae, Botryosphaeriaceae, Botryosphaeriaceae, Chaetomellaceae, Chaetomiaceae, Chaetomiaceae, Dictyosporiaceae, Didymellaceae, Meripilaceae/Polyporaceae, Phanerochaetaceae, Pleosporineae, Sporormiaceae, Sporormiaceae, and Stachybotryaceae, respectively, have not been previously identified in a mine, metal-contaminated site, or in any relevant studies where fungal heavy metal tolerance or OA production was tested. Most of the isolates identified in the present study have been known to be endophytes or phytopathogens, therefore, these newly identified genera from the platinum mine implies that these isolates Bartaliniaceae; 3.2% Bionectriaceae; 1.6% Botryosphaeriaceae; 3.2% Chaetomellaceae; 1.6% Chaetomiaceae; 4.8% Davidiellaceae; 7.9% Dictyosporiaceae; 1.6% Didymellaceae; 15.9% Hypocreaceae; 3.2% Incertae sedis; 3.2%Nectriaceae; 12.7% Pleosporaceae; 14.3% Pleosporineae; 1.6% Sporormiaceae; 3.2% Stachybotryaceae; 3.2% Trichocomaceae; 9.5% Fomitopsidaceae; 1.6% Peniophoraceae; 1.6% Phanerochaetaceae; 1.6% Psathyrellaceae; 1.6% Mucoraceae; 3.2% Basidiomycota; 6.4% Mucoromycota; 3.2% Ascomycota; 90.5% 36 were able to develop adaptive qualities to survive in such an environment which has not previously been reported for such genera. 3.3.4.1 Ascomycota Isolate no. 54 (Table 3.2) is a member of the family Chaetomiaceae. Species of Chaetomium have been identified by Tulsiyan et al. (2017), where they isolated fungi from coal mine samples to determine fungal diversity and the optimum coal powder concentration for fungal growth. The amount of coal powder which the different species of Chaetomium could optimally grow in the presence of ranged between 5 g and 10 g per PDA plate. Shindia et al. (2006) isolated fungi from Egyptian soil and sugar cane waste samples to determine their gluconic acid production and found that Chaetomium sp. was not capable of producing the acid. Li et al. (2016) isolated fungi from plants taken from the wasteland and slag heap of a Pb-Zn mining site and determined fungal tolerance to Pb, Zn and Cd. In their study, Chaetomium globosum was identified from the wasteland and was found to only be tolerant to Pb and Cd (Li et al., 2016). There were five isolates identified as Cladosporium, namely isolates no. 3, 11, 12, 28 and 37 (Table 3.2), which are members of the family Davidiellaceae. Cladosporium spp. have also been isolated by Li et al. (2016) from the wasteland and slag heap and were found to be tolerant to Pb and Cd. Cladosporium cladosporioides and C. herbarum were isolated by Shindia et al. (2006) and were determined to not produce gluconic acid and Cladosporium spp. was identified by Tulsiyan et al. (2017) and its optimum growth was determined to be in the presence of 5 g per PDA plate of coal powder. Văcar et al. (2021) isolated and identified fungi from the rhizosphere of a plant located in a mercury (Hg) contaminated site and determined fungal tolerance to Cu, Cd, Hg, Pb, and Zn, to assess fungal Hg remediation capabilities. In their study, Cladosporium sp. was identified and determined to be tolerant to Cu and Hg (Văcar et al., 2021). Al-Garni et al. (2009) isolated fungi from soil contaminated with industrial waste and determined fungal tolerance to Cd to select highly tolerant species for biosorption 37 of Cd from an aqueous solution. In their study, C. cladosporioides was identified and was found to be tolerant to Cd (Al-Garni et al., 2009). Cladosporium cladosporioides was also isolated from samples from a sewage treatment plant, and it was determined to be tolerant to Cd and Hg (Bahobil et al., 2017). Bahobil et al. (2017) aimed to determine fungal tolerance to the mentioned heavy metals to identify isolates with the greatest potential for the biosorption of Cd and Hg. Shankar and Sivakumar (2016), isolated fungi from leaf litter soil samples to determine their citric acid production and determined that Cladosporium sp. could produce this OA. Ngo et al. (2021) identified Cladosporium tenuissimum from the eyepieces of binoculars obtained from a museum. The identification of fungi in their study was important to develop modern glasses resistant to harmful fungi due to fungal deterioration of non-metallic materials (Ngo et al., 2021). Eleven possible isolates were members of the family Didymellaceae, namely isolates no. 22, 29, 43, 45, 10, 27, 47, 52, 61, 24, and possibly isolate 9 (Table 3.2). Strains of isolate no. 22 have not been identified from any relevant studies, however Stagonosporopsis sp. was identified in the study by Văcar et al. (2021) where it was determined to be tolerant to Cd, Hg, Pb, and Zn. Phoma costaricensis and Didymella glomerata were also isolated by Văcar et al. (2021) and were determined to be tolerant to Hg, Pb, and Zn and P. costaricensis was also found to tolerate Cu. Gupta and Khan (2018) identified fungal isolates from soil samples retrieved from various mine sites and a steel plant to determine fungal isolates capable of being used in biohydrometallurgical processes based on their OA production. In their study, species of Phoma were identified, however, they were not found to produce OAs (Gupta and Khan, 2018). Strains of isolate no. 45 have not been identified in any relevant studies, however, Epicoccum nigram and Phoma sp. were isolated by Li et al. (2016) where E. nirgam was determined to be tolerant to Pb and Cd whereas Phoma sp. was only tolerant to Cd. Phoma herbarum and E. nigram were identified from soapstone sculptures where their ability to produce the OAs, oxalic, malic, citric, acetic, butyric, fumaric and succinic acid, was evaluated (Boniek et al., 2017). They were found to produce all the 38 aforementioned OAs which are associated with the deterioration of the sculptures (Boniek et al., 2017). Isolates no. 46 and 56 (Table 3.2) are members of the family Hypocreaceae and were both identified as part of the genus Trichoderma. This genus has often been isolated from mining sites. Species of Trichoderma have also been isolated in the previously mentioned studies (Al-Garni et al., 2009; Bahobil et al., 2017; Ngo et al., 2021; Shindia et al., 2006). In the study by Al-Garni et al. (2009), T. longibrachiatum was determined to be tolerant to Cd and Bahobil et al. (2017) determined T. viride to be tolerant to Hg. Ngo et al. (2021) isolated T. koningiopsis and Shindia et al. (2006) isolated species of Trichoderma which were determined to not produce gluconic acid. There were two strains of Pyrenochaeta sp. identified, isolates no. 4 and 5 (Table 3.2). However, the family to which this genus is a member is unknown and hence it is referred to as incertae sedis (Kowalski and Bilański, 2021). Pyrenochaeta sp. has previously been identified as an Mn(II)-oxidising fungus from passive coal mine drainage systems (Santelli et al., 2010). A total of eight isolates were identified as Fusarium, namely isolates no. 13, 20. 21, 31, 48, 50, 51, and 59 (Table 3.2). Fusarium was the only genera identified as a member of the family Nectriaceae. Previous studies have also isolated and identified Fusarium species (Al-Garni et al., 2009; Bahobil et al., 2017; Li et al., 2016; Văcar et al., 2021; Shindia et al., 2006; Gupta and Khan, 2018). Fusarium oxysporum and F. chlamydosporum were found to be tolerant to Cd (Al-Garni et al., 2009) and Hg (Bahobil et al., 2017), respectively. Fusarium sp. was determined to be tolerant to Pb, Zn, and Cd in the study by Li et al. (2016), and F. solani, F. equiseti, and F. oxysporum were determined to be tolerant to Cd, Cu, Hg, Pb, and Zn in the study by Văcar et al. (2021). Additionally, F. solani was identified by Shindia et al. (2006) and was found to not produce gluconic acid. In a study by Siham (2007), fungi were isolated from soil samples obtained from an Electric Meter manufacturing facility where fungal tolerance to Pb and Cu was 39 determined. Fusarium equiseti and F. solani were identified in the study and were determined to be tolerant to Pb and Cu (Siham, 2007). Ezzourhi et al. (2009) isolated fungi from heavy metal contaminated sites in the Moghogha river (Tangier, Morocco) and the isolates tolerance to Pb, Cr, Cu, Zn, and Cd was determined. Fusarium spp. were identified in their study and found to be tolerant to Pb, Cr, Cu, and Zn (Ezzouhri et al., 2009). Fungi were isolated from soil irrigated with industrial wastewater in the study by Iram et al. (2012) and the isolates tolerance to Cr and Pb was determined. From the study, Fusarium spp. were identified and were found to be tolerant to both the metals (Iram et al., 2012). There were ten isolates identified which are possibly members of the family Pleosporaceae, isolates no. 1, 6, 8, 15, 34, 35, 36, 39, 60, and possibly isolate 18 (Table 3.2), as isolate no. 18 could either be a member of Pleosporaceae or Massarinaceae. Species of Alternaria and Curvularia are often isolated from heavy metal contaminated sites. Species of Alternaria have been isolated from the previously mentioned studies (Bahobil et al., 2017; Ezzouhri et al., 2009; Gupta and Khan, 2018; Li et al., 2016; Shankar and Sivakumar, 2016; Shindia et al., 2006; Siham, 2007). The species of Curvularia which were identified in the present study have not previously been isolated from mine or metal-contaminated sites to the best of my knowledge, however, a commonly isolated species of Curvularia is C. lunata. This species has been isolated in the previously mentioned studies (Bahobil et al., 2017; Boniek et al., 2017; Ngo et al., 2021; Tulsiyan et al., 2017). Helminthosporium sp. has been isolated in the studies by Iram et al. (2012) and Shankar and Sivakumar (2016) where it was determined to be tolerant to Cr and Pb and to produce citric acid, respectively. Species from the genus Bipolaris have not been identified from mine or metal contaminated sites, however, B. sorokiniana was identified in the study by Boniek et al. (2017) and found to produce the OAs oxalic, malic, citric, acetic, butyric, fumaric, and succinic acid. Species that belong to the genus Periconia, including P. macrospinosa were also identified by Boniek et al. (2017) and were found to produce the mentioned OAs. 40 Setosphaeria rostrata was isolated in the study performed by Al-Garni et al. (2009) and was determined to be tolerant to Cd. Isolate no. 66 (Table 3.2) is a member of the family Stachybotryaceae. Strains of this isolate have not previously been identified from mine or metal-contaminated sites. However, Stachybotrys chartarum has been isolated from tin mine tailings soil samples (Ding et al., 2018). Trichocomaceae is the last family which will be discussed that is a part of the phylum Ascomycota. The isolates no. 14, 17, 26, 38, 40, 58, and possibly isolate no. 9 (Table 3.2), are members of this family. The genera Penicillium and Aspergillus are often isolated from mine and metal-contaminated sites. The exact identity of isolate no. 14 was not specified as it could be P. magnielliptisporum or another species of Penicillium. Penicillium magnielliptisporum has not been isolated from any mine or metal contaminated site, however other species from this genus have been isolated in the previously mentioned studies (Al-Garni et al., 2009; Bahobil et al., 2017; Boniek et al., 2017; Ezzouhri et al., 2009; Gupta and Khan, 2018; Li et al., 2016; Ngo et al., 2021; Shankar and Sivakumar, 2016; Shindia et al., 2006; Siham, 2007; Tulsiyan et al., 2017; Văcar et al., 2021). They were determined to be tolerant to a variety of different heavy metals and were found to produce several OAs. Aspergillus is also known for its heavy metal tolerance and production of OAs and with regards to the species of Aspergillus which were identified in the present study, A. welwitschiae and A. costaricensis have not been identified from any mine or metal-contaminated sites. Aspergillus niger was also identified in the previously mentioned studies (Al-Garni et al., 2009; Bahobil et al., 2017; Ezzhouri et al., 2009; Iram et al., 2012; Shankar and Sivakumar, 2016; Shindia et al., 2006; Siham, 2007). Aspergillus terreus was identified in the studies by Al- Garni et al. (2009) and Shindia et al. (2006), and A. tubingensis was isolated from metal contaminated soil in a study by Din et al. (2020) where it was determined to be tolerant to cobalt (Co), nickel (Ni) Cd, Cu, and Pb and to produce the OAs, oxalic, gluconic, and fumaric acid. Aspergillus flavus was isolated in the studies by Al- Garni et al. (2009), Iram et al. (2012), Shankar and Sivakumar (2016), and Shindia 41 et al. (2006). However, after a literature search on the isolate, certain strains were found to produce aflatoxins which are naturally occurring mycotoxins (Saito and Machida, 1999). Therefore, the use of isolate no. 38 in further experiments was halted due to the possible production of this mycotoxin. As a result, sixty-five isolates were used in further experiments, instead of sixty-six isolates. 3.3.4.2 Basidiomycota Five Basidiomycetes were identified, and each was a member of a different family. Isolates no. 32, 25, 42, and 53 (Table 3.2) are members of the families Fomitopsidaceae, Psathyrellaceae, Peniophoraceae, and Phanerochaetaceae, respectively. Isolate no. 7 (Table 3.2) is possibly a member of the family Meripilaceae or Polyporaceae. These five isolates are all wood-decaying fungi (Bakir et al., 2021; Cartabia et al., 2021; Mahawaththage Dona et al., 2019; Oliver et al., 2010; Patel et al., 2021; Wolfaardt et al., 2004). A strain of isolate no. 32 was previously identified from a gold mine site where it was determined to be tolerant to Cu, Pb, and Fe (Oladipo et al., 2018). A strain of isolate no. 42 has previously been isolated from soil contaminated with Pb from a shooting range and it was found to produce oxalic, citric, malonic, and formic acid and determined to solubilise Pb (Sullivan et al., 2012). A strain of isolate no. 25 has been identified in the study by Boniek et al., (2017) and determined to produce oxalic, malic, citric, acetic, butyric, fumaric, and succinic acid. It has also been identified in the study by Ngo et al. (2021). 3.3.4.3 Mucoromycota Isolates no. 30 and 57 (Table 3.2) are both members of the family Mucoraceae. Strains of isolate no. 30 have not previously been identified in mining environments, however, species from the genus Mucor have been isolated in the previously mentioned studies (Bahobil et al., 2017; Gupta and Khan, 2018; Li et al., 2016; Shindia et al., 2006; Siham, 2007). Mucor sp. was also isolated in a study by Zahoor et al. (2017) from plants grown on heavy metal contaminated soil and 42 was identified due to its tolerance to multiple heavy metals which include, Mn, Zn, Cr, Co, and Cu. A strain of isolate no. 57 was also identified by Oladipo et al. (2018) where it was isolated from a gemstone mining site and found to tolerate Cu, Pb, Fe, Cd, and arsenic (As). 3.4 Summary and Conclusions To determine the physicochemical and fungal characteristics of the samples obtained from a platinum mine, the pH and redox potential of all samples were measured, and fungi were enumerated. The pH of the samples ranged from slightly acidic (6.78) to alkaline (9.27) and the Eh ranged from 26 to -103 mV, implying that the sample environment was a highly reducing environment. The fungal counts among all the samples retrieved from the mine ranged between 0 to 1.2 x 104 CFU/ml or CFU/g of sample. It was concluded that the effect of pH on fungal growth was difficult to infer as similar pH’s of samples taken from the same region had drastically different fungal counts, therefore, the differences in fungal counts could be due to the variation of heavy metal concentrations, nutrient availability, and microbial communities among all the samples. To verify this, future work could involve determining the nutrient concentrations, heavy metal concentration and the microbial community of the samples from the mine. In terms of the identification of the pure fungal cultures obtained after repeated sub-culturing, the majority of isolates identified were members of the phylum Ascomycota which corresponded to previous studies involving the isolation of fungi from mine and metal-contaminated sites. The large number of Ascomycetes is likely due to their ability to tolerate heavy metals. Several isolates which were identified had not previously been identified in any mine, metal-contaminated site, or in any relevant studies where fungal heavy metal tolerance or OA production was tested. However, species from the genera Cladosporium, Fusarium, Trichoderma, Penicillium, and Aspergillus were identified in the present 43 study which corresponded to various studies involving fungi from mine and metal- contaminated sites. 44 4 CHARACTERISATION OF FUNGI 45 4.1 Introduction Bioleaching is a mycohydrometallurgical process which fungi use to extract and solubilise heavy metals from ore, concentrate, and waste materials (Ofori-Sarpong et al., 2010). The main mechanism of fungal bioleaching is acidolysis which takes place due to the production of LMWOAs by fungi (Dusengemungu et al., 2021). Fungi have been isolated from mine and metal-contaminated environments and have exhibited this OA-producing characteristic due to its role in fungal adaption to heavy metal concentrated environments (Din et al., 2020). Therefore, the characterisation of fungi isolated from a mine based on their OA production is important to determine which isolates have the potential to be used in bioleaching (Gupta and Khan, 2018). Additional mechanisms which fungi use for bioleaching are biosorption and bioaccumulation which require the presence of fungal biomass to occur (Dusengemungu et al., 2021). Therefore, understanding fungal heavy metal tolerance capabilities are important in determining the bioleaching method which should be considered as either an indirect or direct approach could be taken, where the latter requires fungal exposure to the solid material undergoing bioleaching (Valix and Loon, 2003). Due to the OA production by fungal isolates from a platinum mine not previously being studied, the present study aimed to qualitatively determine OA production of sixty-five fungal isolates from a platinum mine, to quantitatively determine the production of LMWOAs of selected isolates and to determine the heavy metal tolerance of high OA-producing isolates. 4.2 Materials and Methods 4.2.1 Qualitative screening Sixty-five fungal isolates from a platinum mine were identified and are listed in Table 3.2. To determine their capability of producing OAs, qualitative OA screening 46 was conducted. The isolates were inoculated in 39 g/L of PDA (pH 5.6 ± 0.2), which contained 0.2 g/L of the pH indicator bromocresol green and the plates were incubated at 25oC for 7 days. Bromocresol green allows for a colour change from blue to yellow when the pH changes from 5.4 to 4.0. Acid unitage (AU) values for each colony were calculated by dividing the diameter of the yellow zone formed, by the diameter of the colony (Shaikh and Qureshi, 2013). 4.2.2 Quantitative screening The thirteen isolates which resulted in an AU value of ≥ 1 were further characterised to identify and quantify the OAs which they produce. The isolates were inoculated in 20 ml of Potato Dextrose Broth (PDB) (24 g/L) at a pH of 4.8 and were incubated at 25oC for 7 days on an orbital shaker set at 150 rpm. After the incubation period, the culture was filtered through Whatman no.1 filter paper and 1 ml of the filtrate was centrifuged at 1 677 x g for 10 min (Shankar and Sivakumar, 2016). The supernatant was then filtered through a 0.45 µm syringe filter for high-performance liquid chromatography (HPLC) analysis (Din et al., 2020). OAs were analysed using an HPLC (Agilent-1200 series) equipped with a BioRad fermentation column and refractive index detector. Separation occurred at a column temperature of 65oC and the eluent (20% H2SO4) was delivered at a rate of 1 ml/min for 13 min. Standards of lactic acid, citric acid, acetic acid, butyric acid, formic acid, propionic acid, gluconic acid and indole-3-acetic acid (IAA) were prepared at concentrations ranging from 1 mM to 5 mM. 4.2.3 Heavy metal t