by M. Mwanza and Antoine F. Mulaba- Bafubiandi

1. An investigation into fungal strains bio-extraction of metal impurities from aqueous effluents emanating from Ekhuruleni (Gauteng, South Africa) metallurgical and mining operations by M. Mwanza and Antoine F. Mulaba- Bafubiandi Minerals Processing and Technology Research Group, Department of Extraction Metallurgy, Faculty of Engineering and the Built Environment

2. Fungi: what are they? • Fungi belongs to the kingdom of heterotrophic single-celled, multinucleated, or multicellular organisms, including yeasts, molds, and mushrooms. • The organisms live as parasites, symbionts, or saprobes • Previously classified in the plant kingdom, fungi are nonmotile, like plants, but lack the vascular tissues (phloem and xylem) that form the true roots, stems, and leaves of plants

3. The Importance of Fungi Food : Fungi are also important directly as food for humans. Many mushrooms are edible and different species are cultivated for sale worldwide. Medicines: Many other fungi also produce antibiotic substances, which are now widely used to control diseases in human and animal populations. The discovery of antibiotics revolutionized health care worldwide e.g. penicillium species Recycling: Fungi, together with bacteria, are responsible for most of the recycling which returns dead material to the soil in a form in which it can be reused.

4. The Importance of Fungi… Biocontrol: Fungi such as the Chinese caterpillar fungus, which parasitise insects, can be extremely useful for controlling insect pests of crops. E.g. Chinese caterpillar fungus Crop Diseases: Fungal parasites may be useful in bio-control, but they can also have enormous negative consequences for crop production. Some fungi are parasites of plants. E.g.: aspergillus species Food Spoilage: Fungal damage can be responsible for large losses of stored food, particularly food which contains any moisture Animal and Human Disease: A wide range of fungi also live on and in humans, but most coexist harmlessly. Athletes foot and Candida infections are examples of human fungal infections

5. The Importance of Fungi… Fertilizers: Fungi are vitally important for the good growth of most plants, including crops, through the development of mycorrhiza association. Mycorrhizae and plant growth Production of toxins: under certain conditions, fungi can produce toxins which can be harmful to both human and animal after a certain concentration of ingestion. Looking at the above list, it is clear that fungi play a role in just about every part of our daily lives!

6. Factors influencing fungal growth: -temperature:8-40ºC -water activity (aw) : aw < 0.75 -pH:(2-8) - atmospheric air (oxygen): - Chemical characteristic of the environment: Ca, Mg, Na, Fe, K,… (Moss, 1991,Ayerst, 1986; Russell et al., 1991)

7. Mines and environment effects • Pollution of the surrounding (water and soil): mines drainage and waste

8. Objectives • The main objectives of this study where: - To investigate on fungal strains surviving in the contaminated soil surrounding mines areas in Gauteng -To study their possible capacity to absorb or capture and reduce selected metal in soil and water

9. Sampling • Soil samples (21) from Nigel (Gold mine) in Gauteng (South Africa) were collected in sterile caped bottles and were transported to the laboratory in a cooler box and stored in the fridge.

10. Methodology • Sample culturing and fungal isolation : - 1 g soil + 9ml of sterile ringer solution - 1 ml of the solution milliliter from each McCartney bottle was added to a petri dish containing 20ml of Ohio Agricultural Experimental Station agar (OAESA) (Kaufman et al., 1963) and potato dextrose agar (PDA) - Incubation for 4-7 days at 28oC. The composition and preparation of OAESA and PDA are given in Appendix 1. On the fourth to the 7th day, plates were screened for different types of fungal colonies. These isolated fungal colonies were sub-cultured on PDA, Czapek 20 (CY20S), Czapek (CZ) and malt extract agar (MEA) (Klich, 2002) under aseptic conditions and incubated at 28ºC for 3 to 4 days • Fungal identification: - macroscopic, - microscopic - Polymerase Chain Reaction for confirmation (Inqaba Biotech.)

11. Methodology… • Metal experimentation: Aspergillus niger and A. fumigatus 200 mg of fungal cultures were inoculated in sterile medium composed of 97 ml of distilled water rand 30 ppm of metal or 95ml of distilled water and 50 ppm of metal. All experiments were run in triplicate and a control was also run without fungal. The mixture was incubated in a shaker incubator at 25º C and a speed of 150-200 rpm. Five ml of the solution were collected every 1hour, 2 hours and 24 hours for analysis. The collected samples were filtered using a filter paper and analysed for metal quantification using the ICP-MS.

12. Results and Discussion • Fungal contamination: Aspergillus niger and Aspergillus fumigatus were found to be the most prevalent fungal strains present in soil

13. Results and discussion… Aspersgillus fumigatusAspergillus niger (left)

14. Results and Discussion…. Fungal contamination in Nigel soil samples

15. Results and Discussion…Metal absorption by fungi Aspergillus niger Aspergillus fumigatus Metal Fe Ca Mg Fe Ca Mg ____________________________________________________________________ Conc. Exposure ppm Time % absorption ______________________________________________________________________ 30 1 hour 21.50 21.05 06.33 22.21 27.98 26.36 2 hours 27.3.9 29.50 17.63 33.50 35.29 30.25 24hours 81.80 85.50 46.60 85.23 88.65 42.26 50 1hour 5.50 7.45 05.89 06.91 07.31 07.02 2hours 16.56 15.36 13.52 21.06 29.05 40.25 24hours 65.34 75.24 57.97 70.50 84.06 61.13

17. Discussion and conclusion • Aspergillus fumigatus revealed an slightly higher absorption capacity as compare to aspergillus niger • Calcium was the highly absorbed by both fungi as compare to iron and magnesium • Reason of absorption: -fungal biology - environmental conditions

18. Discussion and Conclusion… • The current global environmental challenges are mainly due to pollution caused; to a large extend, by modern industries and mines. It is thus important to look on environment friendly techniques to reduce water and soil pollution. There is a necessity of metal detoxification in soil and probably in water running from mines. • The use of Fungi can be one of them. • How can this be made applicable???

19. Acknowledgment • Professor Mike Dutton: Food Environmental and Health Research Group, Faculty of Health Sciences, University of Johannesburg • Dr Herman Van Nierkek: RAUSPECTRA, University of Johannesburg. • The University of Johannesburg Research Council and National Research Foundation.