1. Hydrometallurgy Conference - 20091 Indigenous microorganism strains as bio- extractants of Ca, Fe and Mg from metallurgical and mine drainages By E. Fosso Kankeu A.F. Mulaba-Bafubiandi B.B. Mamba T.G. Barnard

2. Hydrometallurgy Conference - 20092 Introduction  Metal enriched soils brought to the surface by mining activities could release metals into the environment following natural oxidation by water or air (Wang and Chen, 2006).  An excess of calcium and iron in water could cause aesthetic or operational problems while excess of magnesium in water could cause aesthetic and health problems (SABS, 2005).

3. Hydrometallurgy Conference - 20093 Introduction continues  Remediation of higher level of metals in surface water by physico-chemical techniques has been reported to be ineffective and costly, hence the need to shift to biological techniques which are cheaper and ecofriendly (Alluri et al., 2007; Cohen, 2006).  Microorganisms possess inherent abilities suitable for the removal of metals from solutions (Nies, 1999)

4. Hydrometallurgy Conference - 20094 Introduction continues  Indigenous microorganisms adapted to conditions in-situ are suitable for use in the removal of metals from water.

5. Hydrometallurgy Conference - 20095 Principles of microbial activities Metal removal It consists to take the metal out of the solution. Microorganisms perform this task through absorption (by physico- chemical interaction between cell membrane and metal) or accumulation (active transport of metal into the cytoplasm).

6. Hydrometallurgy Conference - 20096 Figure 1Absorption of copper on the cell surface of bacteria Principles of microbial activities

7. Hydrometallurgy Conference - 20097 Figure 2 Active transport of Arsenate into bacterial cytoplasm (accumulation) Principles of microbial activities

8. Hydrometallurgy Conference - 20098 Methodology  Water and soil samples collected around mine areas were analysed for their metal content using Inductively Coupled Plasma Spectrometer (ICP)  Microorganisms were isolated from soil and water samples using selective nutrient agar and strains were identified by gene sequencing at Inqaba Biotechnical Industries (Pty) Ltd-South Africa  Synthetic solutions of metals were prepared by diluting crystal or powder form into sterile distilled water to make a stock solution of 1000ppm.

9. Hydrometallurgy Conference - 20099 Methodology continues  To prepare biomass of microorganisms, strains initially isolated on site were inoculated in nutrient broth (Merck SA) and incubated with shaking (150 rpm) at 37oC overnight. Cells were concentrated by centrifugation for 15 minutes at a speed of 8000 rpm. Non-living biomass was prepared by autoclaving lyophilized cells.

10. Hydrometallurgy Conference - 200910 Methodology continues  Metal removal experiment was done by mixing microorganisms (100 mg wet cell) with 30ppm and 50ppm synthetic metal solution in 250 ml Erlenmeyer flask, the mixture was then incubated under the above conditions and 5 ml of solution was taken after one, two and twenty- four hours.  The amount of metal removed was determined by measuring the concentration of metal after exposure using ICP

11. Hydrometallurgy Conference - 200911 Results and discussions  Gene sequencing allows to identify isolated microorganisms as Bacillus subtilis, Shewanella sp and Brevundimonas sp.  Using these microorganisms to remove metal from synthetic solution, we found that Bacillus subtilis and Shewanella sp absorbed the higher amount of each of the three metal from solution (Figs 3,4 & 5)  Calcium was the metal easily removed

12. Hydrometallurgy Conference - 200912 Results and discussions continue  At higher concentration (50ppm) of metals in solution there was a decrease in the removal of metal by microorganisms.  Combining the metals in solution contributed to affect microorganisms affinity for the metals therefore reducing the removal efficiency.

13. Hydrometallurgy Conference - 200913 Results and discussions continue  In many circumstances microorganisms have the tendency to release the metals in solution after the second hour of incubation.

14. Hydrometallurgy Conference - 200914 Results and discussions continue Figure 3 Separate removal of Fe, Ca and Mg by Bacillus subtilis

15. Hydrometallurgy Conference - 200915 Results and discussions continue Figure 4 Separate removal of Fe, Ca and Mg by Shewanella sp

16. Hydrometallurgy Conference - 200916 Results and discussions continue Figure 5 Separate removal of Fe, Ca and Mg by Brevundimonas sp

17. Hydrometallurgy Conference - 200917 Results and discussions continue  Microbial affinity seemed to influence the removal efficiency of metals as cell wall composition (active group) could determine the binding of metal.  The decrease of percentage removal of metal after the second hour was certainly due to an efflux transport system developed by microorganism to reject the excess metal from their body or breakage of bonds between cell wall and metals.

18. Hydrometallurgy Conference - 200918 Results and discussions continue  The inhibition of microorganisms could have affected the removal of metals as we recorded higher removal in solution containing 30ppm of metal than in solution containing 50ppm of metal.  There was a decrease in the performance of microorganisms when removing simultaneously the three metals from solution.

19. Hydrometallurgy Conference - 200919 Results and discussions continue  Experiment on the metal tolerance by microorganisms in solution of mixed metal showed that biomass of Shewanella sp and Brevundimonas sp was adversely affected after one hour, while Bacillus subtilis resisted the presence of metal.  Relatively low metals’ removal was recorded in general, certainly due to a combination of factors such as: inhibition, efflux transport and use of small biomass (metal uptake is directly proportional to microbial biomass). Use of non-living biomass did not improve the results.

20. Hydrometallurgy Conference - 200920 Conclusion  Metal removal in this study is largely dependent on the affinity between microbial cell wall and the metal.  Regarding the removal ability, the resistance to metal and the retention (efflux mechanism minimised) of metal, Bacillus subtilis appeared to be the microorganism that performed the best.  Possible recovery of metals from microorganisms could be done by ion exchange mechanism after concentration of microorganisms through filtration.

21. Hydrometallurgy Conference - 2009 21 Your opinion count… Intelligence is relative But Discovery is objective E Fosso Kankeu