Indigenous microorganism strains as bio-extractants of Ca, Fe, and Mg from metallurgical and mine drainages

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1 MULABA-BAFUBIANDI, FOSSO-KANKEU, E., and MAMBA, B.B. Indigenous microorganism strains as bio-extractants of Ca, Fe, and Mg from metallurgical and mine drainages. Hydrometallurgy Conference 2009, The Southern African Institute of Mining and Metallurgy, Indigenous microorganism strains as bio-extractants of Ca, Fe, and Mg from metallurgical and mine drainages A.F. MULABA-BAFUBIANDI*, E. FOSSO-KANKEU*, and B.B. MAMBA *Department of Extraction Metallurgy, Faculty of Engineering and the Built Environment, University of Johannesburg, South Africa Department of Chemical Technology, Faculty of Science, University of Johannesburg, South Africa Attempts to remediate the high concentration of calcium, iron and magnesium in surface waters from metallurgical areas by experimenting at laboratory scale on the removal of these metals in synthetic solutions (30 and 50 ppm), using indigenous strains of Shewanella sp, Bacillus subtilis sp and Brevundimonas sp, revealed variable abilities of these microorganisms in the removal process. Bacillus subtilis sp and Shewanella sp absorbed the higher amount of each of the three metals from solution, and calcium was the metal most easily removed. Metals removal from solution decreased when their concentrations were at 50 ppm. It was found that when metals were combined in the same solution they contribute to inhibit microorganisms, change the microbial affinity for metal and affect the removal efficiency. Depending on the metal, there was a tendency for microorganisms to release the absorbed metal into the solution after a certain time, likely due to an efflux transport system previously demonstrated by some authors. Use of non-living biomass did not improve the removal efficiency. Keywords: indigenous strains, biosorption, bioaccumulation, living biomass, non-living biomass, metal removal Introduction For decades concerned companies and local authorities have demonstrated interest in managing water from metallurgical and mining areas. A general tendency is to shift from the ineffective and costly physico-chemical techniques to the biological techniques found to be cheap and eco-friendly (Alluri et al., 2007; Cohen, 2006; Kefala et al., 1999). Although some of the bench-scale experiments have been successful, the challenge remains to implement the technique at industrial level. This results from the fact that the concept is not entirely understood and therefore requires more effort in improving the removal process on operational level (Wang and Chen, 2006). INDIGENOUS MICROORGANISM STRAINS AS BIO-EXTRACTANTS 93

2 The presence of residual metals in effluent water represents primarily a substantial loss in revenue for metallurgical companies. Secondly, it could be the cause of legal liability vis a vis environmental safety as these metals are potential pollutants of the water system. According to the South African National Standard (2005) an excess of calcium and iron in water could cause aesthetic or operational problems while excess of magnesium in water could cause esthetic and health problems. Microorganisms possess inherent abilities suitable for the removal of metals from solutions (Nies, 1999; Langley and Beveridge, 1999; Beveridge and Murray, 1976). These abilities have been identified as passive or active for accumulation and biosorption respectively (Brandl and Faramarzi, 2006). However, there are always challenges that contribute to undermine the benefit of this process: the high concentration of metals could be toxic for microorganisms and acidic conditions are unfavourable for the development of most of microorganisms which are neutrophiles (Kim et al., 2006; Sandrin and Maier, 2003). To overcome these challenges, one needs to use indigenous microorganisms adapted to conditions in situ, or alternatively use either genetically modified microorganisms or non-living biomass that could cope with existing conditions. Bacillus strains have been widely reported in literature to be effective in the removal of metals (Pb, Cd, Cu, Ni, Co, Mn, Cr, Zn) from waste waters (Kim et al., 2007; Srinath et al., 2003; Philip and Venkobachr, 2001), but other strains (Brevundimonas and Shewanella) also identified during this study at mining sites, have not been tested for the treatment of metallurgical and mine drainages. In this study, living and non-living biomass of all these strains will be tested for their effectiveness to clean out Ca, Mg and Fe predominant in surface water around mining areas in Nigel. Methodology Isolation and identification of microorganisms Water and soil samples collected in sterile glass bottles around mining areas in Nigel were preserved at 4 C during transportation to the laboratory. Soil samples were suspended in sterile distilled water vortexed, and the supernatant as well as the water samples diluted in sterile phosphate buffer prior to inoculation in chromogenic media for coliforms and Escherichia coli, and in nutrient agar. After overnight incubation of cultured media, colonies of microorganisms were isolated from the plate and subcultured in fresh media. Unknown microorganisms were subsequently identified by gene sequencing at Inqaba Biotechnical Industries (Pty) Ltd South Africa. Preparation of synthetic solutions Synthetic metal sulphates obtained in powder or crystal form were weighed and diluted in sterile distilled water to make a stock solution of ppm. From the stock solution various volumes were removed into the final solution to obtain concentrations between 30 and 50 ppm. Microorganisms growth and biomass preparation Microorganisms were inoculated into the nutrient broth ( Lab-Lemco powder 0.1%, yeast extract 0.2% and NaCl 0.5%), incubated at 37ºC in the incubator with shaker (150 rpm) for twenty-four hours. The culture was centrifuged for 15 minutes at a speed of rpm, the supernatant was discarded and the pellet washed several times with sterile distilled water then suspended in a sterile flask. Concentrated cells were lyophilized and autoclaved at 121ºC for 15 minutes to prepare non-living biomass. 94 HYDROMETALLURGY CONFERENCE 2009

3 Metal removal The ability of microorganisms to remove metal from solution depends on two mechanisms called biosorption and bioaccumulation, which are passive or active respectively: Biosorption is described as a chemical interaction between an anionic group (amino acids, hydroxyl, phosphate, etc.) on the bacterial cell membrane and the positively charged metal in solution. There is no net energy required during this process. (Figure 1.) Bioaccumulation is a reaction whereby the metal is sequestered through the bacterial membrane into the cytoplasm of the cell; during this process microorganisms use energy (ATP hydrolysis) to catalyse the reaction. This process could also contribute to the supply of cofactor for enzymatic reactions; however, an excess of metal could be excreted from the cell by an efflux transport system (Nies, 1999). (Figure 2.) For the experimentation of metal removal in this study, synthetic metal solution was mixed with microorganisms (100 mg wet cell) in a 250 ml Erlenmeyer flask, and sterile distilled water added up to a final volume of 100 ml. Microorganisms were exposed to 30 ppm and 50 Figure 1. Adsorption of copper on the cell surface of bacteria (from Kim et al., 2007) Figure 2. Active transport of arsenate into bacterial cytoplasm (from Nies, 1999) INDIGENOUS MICROORGANISM STRAINS AS BIO-EXTRACTANTS 95

4 ppm concentrations of each metal separately as well as with a mixture of metal ions. The mixture was then incubated at 37ºC in the incubator with shaker (150 rpm) and 5 ml of sample solution was taken after one, two and twenty-four hours, as previous authors obtained maximum metal removal at these times (Kefala et al., 1999; Kim et al., 2007). The solution was then centrifuged at rpm for five minutes and the supernatant collected for quantification of the amount of metal. Metal quantification and experimental procedure Determination of the amount of metal in solution was done using inductively coupled plasma optical emission spectrometry (ICP-OES). All the experiments were done in triplicate with a control; the difference between the replicate was less than 10%. The average value of the triplicate was considered when drawing the graph. Strains of Bacillus subtilis, Shewanella sp and Brevundimonas sp were identified in soil and water samples from mining areas by gene sequencing. These strains were tested for their abilities to remove Fe, Ca and Mg from synthetic solution containing 30 and 50 ppm of the above metal. Metal tolerance To determine whether microorganisms were alive during the whole removal experiment, at each time sample was collected for analysis; one ml was simultaneously inoculated in phosphate buffer, diluted five times (10-1, 10-2, 10-3, 10-4 and 10-5 ) then plated on agar media (chromogenic media for coliforms and Escherichia coli) and incubated at 37ºC overnight. Microorganism survival was then assessed as the occurrence and number of typical colonies in the agar media. Results and discussions Removal of individual metal The ability of Bacillus subtilis to remove metal ions varied depending on the metal specie; there was a greater affinity for calcium than for other metals (Figure 3). In fact calcium removal was around 14% while iron and magnesium removal was less than 10%. It was also noticed that the removal efficiency was lower at 50 ppm concentration of metal after 24 hours. % metal removed Figure 3. Separate removal of Fe, Ca and Mg by Bacillus subtilis sp 96 HYDROMETALLURGY CONFERENCE 2009

5 Shewanella sp also showed greater affinity for Ca but there was a tendency to release the metal into solution from the second hour of the experiment (Figure 4). A higher removal rate was recorded at 30 ppm concentration of metal. Among all the bacteria, Brevundimonas sp has the lower performance as far as metal removal is concerned. However, it also has greater affinity for calcium and the highest removal was approximately 9% of Ca at 50 ppm concentration (Figure 5). It is observed that metal removal in this experiment is greatly influenced by the affinity of the microorganism for the metal, as during the first hour (contact time) metal binding rate to the microbial cell wall differed from one metal to the other. According to work done by Kefala et al. (1999) the composition of the cell wall (reactive groups) of microorganism play an important role in determining the attraction of the metal. However, other factors such as surface availability on the cell wall could affect the removal efficiency, knowing that saturation of the cell is quickly reached in solution containing high concentration of metal, decreasing the ratio of metal removed to total metal in solution. This explains why generally after the first hour the percentage of metal removed is higher in solution with 30 ppm of metal. % metal removed % metal removed Figure 4. Separate removal of Fe, Ca and Mg by Shewanella sp Figure 5. Separate removal of Fe, Ca and Mg by Brevundimonas sp INDIGENOUS MICROORGANISM STRAINS AS BIO-EXTRACTANTS 97

6 During the second and twenty-fourth hours it is likely that a bioaccumulation mechanism dominates over biosorption, since the second mechanism reaches the maximum after 5 minutes contact time (Sadowski et al., 1991). The removal rate could therefore increase, meaning that the metal is progressively transported into the cell or decrease when there is efflux transport or rupture of bounds between metal and reactive groups on the microorganism cell wall. The low rate of metal removed during this study is partly due to the fact that a smaller mass of cells was used compared to other studies. It is reported (Schiewer and Volesky, 1995a; Kefala et al., 1999; Wang, 2002a) that higher cell concentration could increase metal removal. Removal of mixed metals To simulate the conditions in mine areas, living biomass was exposed to solution containing all three metals, each present at concentration of 30 or 50 ppm, depending on the set of experiment. Results (not shown) revealed that the removal efficiency of the microorganisms decreased by almost half and the affinity shifted to iron. To enhance the removal efficiency by overcoming the limitations associated with living cells such as metal toxicity and inefficiency under adverse operating conditions (Tobin et al., 1994), it was decided to use non-living cells, which are reported to have higher metal uptake capacities (Gadd, 1990). No difference was found (results not shown) in the removal efficiency of mixed metal as compared to the use of living cells but the specificity of metal binding was affected as the heating process could contribute to destabilize reactive groups. Metal tolerance Evaluation of the survival of microorganisms after one, two and twenty-four hours of metal exposure showed little decrease of microbial number in solution of 50 ppm of nickel. In solution containing mixed metals, cells of Shewanella sp were all dead after the first hour while Brevundimonas sp biomass was seriously affected. However, Bacillus subtilis is not affected by the presence of metal, showing no significant change in the growth rate. This implies that in the presence of high concentrations of metals, microbial biomass can be reduced, therefore reducing the removal efficiency of metal from solution. Conclusion Attempts to remove metal from solution using indigenous microorganism isolated around mine areas, showed that this process is largely dependent on the affinity between the microbial cell wall and the metal. It was found that when using living cells, removal efficiency could be affected by factors such as inhibition and efflux transport system. However, use of non-living biomass did not bring any change in removal efficiency but has the advantage that it is simple. Further studies to determine optimal biomass efficiency through a maximum metal removal rate in the shortest possible time (the kinetics of the reaction), coupled with analysis of cost involved, will improve the understanding of the approach to the recovery of excess residual metals in process water discharged from metallurgical activities. Acknowledgements Dr Barnard of the Water and Health Research Unit (UJ) has opened the doors to their research facilities and the South African National Research Foundation (NRF) and the University of Johannesburg provided the research funds and the scholarship. 98 HYDROMETALLURGY CONFERENCE 2009

7 References ALLURI, H.K. et al Biosorption: An eco-friendly alternative for heavy metal removal. African Journal of Biotechnology, vol. 6, no. 25, pp BEVERIDGE, T. and MURRAY, R.G.E Uptake and retention of metals by cell walls of Bacillus subtilis. Journal of Bacteriology, vol. 127, no. 3, pp BRANDL, H. and FARAMARZI, M.A. Microbe-metal-interactions for the biotechnological treatment of metal-containing solid waste. China Particuology, vol. 4, no. 2, pp COHEN, R.R.H Use of microbes for cost reduction of metal removal from metals and mining industry waste streams. Journal of Cleaner Production. vol. 14, no , pp GADD, G.M. Biosorption. Chemistry and Industry, vol. 2, 1990, pp KEFALA, M.I., ZOUBOULIS, A.I., and MATIS, KA. Biosorption of cadmium ions by Actinomycetes and separation by flotation. Environmental Pollution, vol. 104, no. 2, pp KIM, S.U., CHEONG, Y.H., and SEO, D.C. et al Characterisation of heavy metal tolerance and biosorption capacity of bacterium strain CPB4 (Bacillus spp). Water Science and Technology, vol. 55, no. 3, pp LANGLEY, S. and BEVERIDGE, T.J. Effect of O-side-chain-Lipopolysaccharide chemistry on metal binding. Applied and Environmental Microbiology, vol. 65, no. 2, pp NIES, D.H. Microbial heavy metal resistance: Molecular biology and utilisation for biotechnological processes, pp PHILIP, L. and VENKOBACHR, C. An insight into mechanism of biosorption of Cu by B. Polymyxa. Indian Journal of Environmental Pollution, vol. 15, pp SADOWSKI, Z., GOLAB, Z., and SMITH, R.W. Flotation of Streptomyces pilosus after lead accumulation. Biotechnology and Bioengineering, vol. 37, pp SANDRIN, T.R. and MAIER, R.M. Impact of metals on the biodegradation of organic pollutants. Environmental Health Perspectives, vol. 111, no. 8, pp SCHIEWER, S. and VOLESKY, B. Mathematical evaluation of the experimental and modeling errors in biosorption. Biotechnology Technology, vol. 9, 1995a. pp SOUTH AFRICAN NATIONAL STANDARD Drinking Water. SANS 241, edition 6; ISBN SRINATH, T., GARG, S.K., and RAMTEKE, P.W. Biosorption and elusion of Cr from immobilized Bacillus coagulens biomass. Indian J Exp. Biol. vol. 41, pp TOBIN, J.M., WHITE, E., and GADD, G.M. Metal accumulation by fungi: applications in environmental biotechnology. Journal of Industrial Microbiology, vol pp ; WANG, J. and CHEN, C. Biosorption of heavy metals by Saccharomyces cereviceae: A review. Biotechnology Advances, vol. 24, pp ; WANG, J.L. Immobilization techniques for biocatalysts and water pollution control. Beijing: Science Press. 2002a. INDIGENOUS MICROORGANISM STRAINS AS BIO-EXTRACTANTS 99

8 Elvis Fosso-Kankeu University of Johannesburg, South Africa Master in Biotechnology completed in 2006 at the University of Johannesburg; Research Associate at the University of Johannesburg ( ); Assistant consultant in the monitoring and improvement of plant water treatments (2007). One full article published in 2008 and two manuscripts submitted for publication in Registered for a doctorate in bioprocessing at the University of Johannesburg (Department of Extraction Metallurgy). 100 HYDROMETALLURGY CONFERENCE 2009