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Biodegradation of cyanide and subsequent nitrification-aerobic denitrification in cyanide containing watewater
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Environmental legislation focusing on wastewater disposal in industries that utilise cyanide and/or cyanide-related compounds has become increasingly stringent worldwide, with many companies that utilise cyanide products required to abide by the Cyanide International Code associated with the approval of process certifications and management of industries which utilise cyanide. This code enforces the treatment or recycling of cyanide-contaminated wastewater. Industries such as those involved in mineral processing, photo finishing, metal plating, coal processing, synthetic fibre production, and extraction of precious metals, that is, gold and silver, contribute significantly to cyanide contamination in the environment through wastewater. As fresh water reserves throughout the world are low, cyanide contamination in water reserves threatens not only the economy, but also endangers the lives of living organisms that feed from these sources, including humans. In the mining industry, dilute cyanide solutions are utilised for the recovery of base (e.g. Cu, Zn, Ni, etc.) and precious metals (e.g. Au, Ag, etc.). However, for technical reasons, the water utilised for these processes cannot be recycled upstream of the mineral bioleaching circuit as the microorganisms employed in mineral bioleaching are sensitive to cyanide and its complexes, and thus the presence of such compounds would inhibit microbial activity, resulting in poor mineral oxidation. The inability to recycle the water has negative implications for water conservation and re-use, especially in arid regions. A number of treatment methods have been developed to remediate cyanide containing wastewaters. However, these chemical and physical methods are capital intensive and produce excess sludge which requires additional treatment. Furthermore, the by-products that are produced through these methods are hazardous. Therefore, there is a need for the development of alternative methods that are robust and economically viable for the bioremediation of cyanide-contaminated wastewater. Biological treatment of free cyanide in industrial wastewaters has been proved a viable and robust method for treatment of wastewaters containing cyanide. Several bacterial species, including Bacillus sp., can degrade cyanide to less toxic products, as these microorganisms are able to use the cyanide as a nitrogen source, producing ammonia and carbon dioxide. These bacterial species secrete enzymes that catalyse the degradation of cyanide into several end-products. The end-products of biodegradation can then be utilised by the microorganisms as nutrient sources. This study focused on the isolation and identification of bacterial species in wastewater containing elevated concentrations of cyanide, and the assessment of the cyanide biodegradation ability of the isolates. Thirteen bacterial isolates were isolated from electroplating wastewater by suppressing the growth of fungal organisms and these species were identified as species belonging to the Bacillus genus using the 16S rDNA gene. A mixed culture of the isolates was cultured in nutrient broth for 48 hours at 37°C, to which FCN as KCN was added to evaluate the species‟ ability to tolerate and biodegrade cyanide in batch bioreactors. Subsequently, cultures were supplemented solely with agro-waste extracts, that is, Ananas comosus extract (1% v/v), Beta vulgaris extract (1% v/v), Ipomea batatas extract (1% v/v), spent brewer‟s yeast (1% v/v) and whey (0.5% w/v), as the primary carbon sources. Owing to the formation of high ammonium concentration from the cyanide biodegradation process, the nitrification and aerobic denitrification ability of the isolates, classified as cyanide-degrading bacteria (CDB) was evaluated in a batch and pneumatic bioreactor in comparison with ammonia-oxidising bacteria (AOB). Furthermore, the effects of F-CN on the nitrification and aerobic denitrification was evaluated assess the impact of F-CN presence on nitrification. Additionally, optimisation of culture conditions with reference to temperature, pH and substrate concentration was evaluated using response surface methodology. Using the optimised data, a continuous biodegradation process was carried out in a dual-stage packed- bed reactor combined with a pneumatic bioreactor for the biodegradation of F-CN and subsequent nitrification and aerobic denitrification of the formed ammonium and nitrates. The isolated bacterial species were found to be gram positive and were able to produce endospores that were centrally located; using the 16S rDNA gene, the species were found to belong to the Bacillus genus. The species were able to degrade high cyanide concentration in nutrient broth with degradation efficiencies of 87.6%, 65.4%, 57.0% and 43.6% from 100 mg F-CN/L, 200 mg F-CN/L, 300 mg F-CN/L, 400 mg F-CN/L and 500 mg F-CN/L respectively over a period of 8 days. Additionally, the isolates were able to degrade cyanide in an agro-waste supported medium, especially in a medium that was supplemented with whey which achieved a degradation efficiency of 90% and 60% from 200 mg F-CN/L and 400 mg F-CN/L, respectively over a period of 5 days. The nitrification ability of the isolates was evaluated and the removal of NH4 +/NO3 - by the CDB and AOB in both shake flasks and pneumatic bioreactor was determined to be pH dependent. The maximum NH4 +/NO3 - removal evaluated over a period of 8 days for CDB and 15 days for AOB, observed at pH 7.7 in shake flasks, was 75% and 88%, respectively, in the absence of F-CN. Similarly, the removal of NH4 +/NO3 - in a pneumatic bioreactor was found to be 97.31% for CDB and 92% for AOB, thus demonstrating the importance of aeration in the designed process. The nitrification by CDB was not inhibited by cyanide loading up to a concentration of 8 mg FCN/ L, while the AOB were inhibited at cyanide loading concentration of 1 mg F-CN/L. The CDB removed the NH4 +/NO3 - in PBSs operated in a fed-batch mode, obtaining efficiencies >99% (NH4 +) and 76 to 98% (NO3 -) in repeated cycles (n = 3) under F-CN (≤8 mg F-CN/L). The input variables, that is, pH, temperature and whey-waste concentration, were optimised using a numerical optimisation technique where the optimum conditions were found to be: pH 9.88, temperature 33.60 °C and whey-waste concentration 14.27 g/L, under which 206.53 mg CN-/L in 96 h can be biodegraded by the microbial species from an initial cyanide concentration of 500 mg F-CN/L. Furthermore, using the optimised data, cyanide biodegradation in a continuous mode was evaluated in a dual-stage packed-bed bioreactor connected in series to a pneumatic bioreactor system used for simultaneous nitrification including aerobic denitrification. The whey-supported Bacillus sp. culture was not inhibited by the free cyanide concentration of up to 500 mg F-CN/L, with an overall degradation efficiency of ≥99% with subsequent nitrification and aerobic denitrification of the formed ammoniu and nitrates over a period of 80 days.