Bioremediation of heavy metals polluted soil of active gold mines using bacteria biopolymers

Abstract

Mining activities have increased environmental pollution which has consequently resulted in the release of large quantities of heavy metals into the environmental media. These metal species tend to accumulate in the environmental media because they are non-degradable causing several toxic effects on biological systems. Hence, there is a need for appropriate treatment techniques for effective removal of heavy metals from contaminated media. This study was designed to screen for prospective biopolymer producing heavy metal resistant bacterial isolates that can be used for metal removal from polluted media. Soil samples were collected from gold mining sites in Vryburg, South Africa. The soil physicochemical properties and metal concentrations were determined. Each soil sample was digested and the metal concentration determined through inducible conductivity plasma-optical emission spectroscopy. The pH values of the soil samples ranged from 5.9 to 7.5 which is slightly acidic to slightly alkaline. The properties of the soil revealed that the soil samples are sandy in nature and deficient in nutrients for microbial activities. Heavy metal concentrations for the soil samples ranged between 0.03-0.36 mg/kg for Cd, 0.22-0.41 mg/kg for Cr, 0.12-0.64 mg/kg for Cu, 127.9-1213.2 mg/kg for Fe, 0.2-1.9 mg/kg for Pb and 0.07-1.2 mg/kg for Zn. These values fall within the recommended limit for South African soil and sediments, but still have potential impacts on biological systems. Ninety-eight heavy metal (Cd, Cr and Pb) resistant bacterial isolates were isolated from the soil samples using Luria-Bertani agar supplemented with each of the heavy metals. These isolates were screened for biopolymer production and a total of 20 isolates (20.4%), that were positive for biopolymer production were selected for further studies. The metal tolerance of the biopolymer producing isolates were determined and 55% of the isolates grew on 1000 mg/L of Pb. Few isolates tolerated up to 300 mg/L of cadmium and chromium. All isolates were characterized biochemically and molecularly and the results showed that the phylum Firmicutes were the dominant organisms. The isolates were further characterized by 16S rRNA gene sequence analysis and identified as belonging to the genera Acinetobacter, Agrobacterium, Bacillus, Citrobacter, Enterobacter, Pantoea and Pseudomonas. The isolates were screened for heavy metal resistance genes encoding for Cd (cadA, CzcA, CzcB and CzcD), Cr (chrA and chrB) and Pb (PbrA and PbrT). Multi- metal resistance was found in most of the isolates, notably, Bacillus cereus MH399230, B. toyonensis MH399231, Pseudomonas korensis MH399240 and Pantoea sp. MH399244. B. cereus MH399230 was positive for all the screening test for biosurfactant production and was selected for biosurfactant production and metal removal. B. cereus MH399230 has emulsification of 22%, 24%, 37% and 54% on hexadecane, vegetable oil, kerosene and engine oil respectively, and was able to reduce the surface tension of 39.5 mN/m. The biosurfactant produced by B. cereus MH399230 was identified as a lipopeptide with stability over a wide range of pH and temperature. The biosurfactant produced by B. cereus MH399230 was able to remove 78% of Pb, 56% of Cd and 35% of Cr from polluted soil after single washing with biosurfactant. The genome of B. cereus MH399230 was mined and revealed the presence of 44 gene clusters involved in antibiotic and secondary metabolite biosynthesis. These included non-ribosomal peptide synthetase (NRPS) gene clusters, lipoprotein, lipopolysaccharides, binding proteins, proteins related to degradation of toxic compounds and biofilm secretion genes. Genes responsible for resistance to toxic heavy metals such as arsenic, cadmium, chromium, cobalt, copper, lead, mercury and zinc were also detected in the genome of B. cereus MH399230. P. korensis MH399240 and Pantoea sp. MH399244 were further screened for bioflocculant production based on the high flocculating activity produced with kaolin clay. Maximum flocculating activity of 71.3% and 51.7% with glucose and yield of 2.98 g/L and 3.26 g/L was obatanied for Pantoea sp. and P. korensis respectively at optimum pH (7.5) and temperature (30?C). Characterization of the partially purified bioflocculant using FTIR revealed the presence of carboxyl, hydroxyl and amino groups. These groups are responsible for metal binding. Metal sorption by the partially purified bioflocculants of Pantoea sp. removed 51.2%, 52.5%and 80.5% of Cd, Cr and Pb respectively while that of P. korensis removed 48.5%, 42.5% and 73.7% of Cd, Cr and Pb respectively from aqueous solution. This study shows the potentials of these heavy metal resistant isolates for biopolymer production and removal of heavy metals from polluted media.