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Bioremediation of gold mine wastewater using fusarium oxysporum
Akinpelu, Enoch Akinbiyi
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The legislative requirements for handling cyanide containing wastewater have become stringent internationally. Cyanide properties make it indispensable in the mining industry especially for gold recovery. The resultant wastewater generated is discarded to tailing ponds. Any leakages or total collapse of tailing ponds can result in the contamination of surface water bodies; endangering aquatic organisms’ and humans’ alike. The over reliance on physical and/or chemical treatment methods for cyanide wastewater treatment is not sustainable due to high input costs and the generation of by-products. A feasible alternative treatment method for cyanide contaminated wastewater is the biodegradation method, as a wide range of microorganisms can degrade cyanide. In this study, the cyanide biodegradation ability of Fusarium oxysporum was assessed in two stages. Firstly, optimal operating conditions for maximum cyanide biodegradation were determined using a central composite design (CCD) at an elevated cyanide concentration, i.e. 500 mg F-CN/L. Thereafter, using the optimum conditions obtained, (i.e temperature 22°C and pH 11), cyanide biodegradation kinetics and microbial growth kinetics in the cultures at lower cyanide concentrations of 100, 200 and 300 mg F-CN/L were assessed. This was followed by the assessment of cyanide biodegradation at a temperature of 5°C, which was used to simulate winter conditions. In general, lower cyanide concentrations are used in the extraction of gold, therefore, the resultant wastewater will contain free cyanide concentration less than 300 mg F-CN/L. For the first stage of experiments, an isolate, Fusarium oxysporum from cyanide containing pesticides was cultured on potato dextrose agar (PDA) plates, followed by incubation at 25°C for 5 days. A response surface methodology (RSM) was used to evaluate design parameters for the biodegradation of cyanide by this fungus. The temperature evaluated at this stage ranged from 9°C to 30°C and pH range of 6 to 11 in cultures solely supplemented with agrowaste, i.e Beta vulgaris waste. Beta vulgaris is commonly known as Beetroot. The Fusarium oxysporum inoculum (2% v/v) was grown on a Beta vulgaris waste solution (20% v/v), as the sole carbon source in a synthetic gold mine wastewater (39% v/v) containing heavy metals; arsenic (7.1 mg/L), iron (4.5 mg/L), copper (8 mg/L), lead (0.2 mg/L) and zinc (0.2 mg/L), for 48 hours using a rotary shaker at 70 rpm. Thereafter, free cyanide as a potassium cyanide solution (39% v/v), was added to the cultures to make a final cyanide concentration of 500 mg F-CN/L in the culture medium which was incubated for a further 72 hours at 70 rpm. Optimal operating conditions for the biodegradation of cyanide were then determined using a numerical option in the Design-Expert® software version 6.0.8 (Stat-Ease Inc., USA). Subsequently, using the optimal pH obtained (pH =11) and a preselected temperature of 5°C (to represent winter conditions), cyanide biodegradation rates and microbial growth kinetic studies were carried out using Beta vulgaris waste containing a Fusarium oxysporum (0.7% v/v; grown overnight) inoculum in wastewater (32.7% v/v) and potassium cyanide in phosphate buffer (53.7% v/v). The cultures contained 100, 200 and 300 mg F-CN/L. The cultures were incubated in an orbital shaker at 70 rpm for 144 hours and samples taking every 24 hours. An Ordinary Differential Equation (ODE) solver (Polymath) was used for modelling cyanide degradation kinetics while the Monod’s growth kinetic model was used to monitor the microbial growth parameters of the cultures. For the first stage, the optimum operating conditions were determined as a temperature of 22°C and a pH of 11 for maximum cyanide biodegradation of 277 mg F-CN/L from an initial cyanide concentration of 500 mg F-CN/L over a 72 hour period, with residual ammonium-nitrogen and nitrate-nitrogen of 150 mg NH4+-N/L and 37 mg NO3--N/L, respectively. Although, the residual ammonium-nitrogen inhibited cyanide biodegradation, it was consumed as a nitrogen source for microbial growth. The Beta vulgaris waste was determined to be a suitable substrate for cyanide degradation. From the biodegradation response quadratic model, temperature was determined to influence cyanide biodegradation. For the cyanide degradation kinetics, at an optimum temperature of 22°C, the biodegradation efficiency was 77%, 58% and 62% with the corresponding maximum microbial population of 1.56 x 107, 1.55 x 107 and 1.57 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L, being achieved. An indication that the F. oxysporum cultures were efficient at lower cyanide concentration. Furthermore, at a temperature of 5°C, the biodegradation efficiency, although slightly lower, was 51%, 43% and 44% with the corresponding maximum microbial population of 1.21 x107, 1.11 x 107 and 1.12 x 107 CFU/mL for 100, 200 and 300 mg F-CN/L cultures, respectively, with minimal differences observed for cultures with 200 and 300 mg F-CN/L. The cyanide biodegradation rates increased with temperature increases and varied with different cyanide concentrations below 500 mg F-CN/L. The estimated energy of activation for cyanide degradation for a change in temperature from 5°C to 22°C using the Arrhenius model was 19.6, 12.7 and 14.9 kJ/mol for 100, 200 and 300 mg F-CN/L, respectively. The means and standard deviations for rate of degradation of cyanide at 5°C and 22°C for the ODE models was 0.0052 (± 0.0011) h-1 and 0.0084 (± 0.0027) h-1, respectively. The inhibitory effect of the cyanide was quantitatively pronounced under cold temperature as the heavy metals, residual ammonium-nitrogen and nitrate-nitrogen hindered the cyanide degradation. Similarly, microbial growth rates increased with a temperature rise (from 5°C to 22°C), resulting with a reduction in the microbial populations’ doubling time. When compared with the simulated winter conditions, the specific population growth rate increased 4-fold, 5-fold and 6-fold in 100, 200 and 300 mg F-CN/L, respectively, for higher temperatures; an indication that the Fusarium oxysporum isolate prefers higher temperature. The estimated energy of activation for cellular respiration was 44.9, 54 and 63.5 kJ/mol for 100, 200 and 300 mg F-CN/L cultures, respectively, for the change in temperature from 5°C to 22°C. The means and standard deviations of microbial growth rate at 5°C and 22°C were: 0.0033 (± 0.0013) h-1 and 0.0151 (± 0.0027) h-1, respectively. The difference in error (standard deviation) of the cyanide biodegradation rate and microbial growth rate was insignificant (0.02% at 5°C) especially at temperature 22°C where there were minimal differences, indicating the reliability and reproducibility of this biodegradation system in batch operated bioreactors.