Studies on Bioremediation of Cr (VI) using Indigenous Bacterial Strains Isolated from a Chromite Mine
Heavy metals are released into the environment either by natural processes or by anthropogenic activities. Industries such as leather tanning, textiles, metallurgical, electroplating and mining activities discharge the chromium along with other heavy metals, which causes water pollution and environmental degradation. There are many conventional methods to overcome this problem such as chemical precipitation, ion exchange, reverse osmosis, etc but, these methods have certain drawbacks like generation of secondary sludge, inefficient removal of metal ions of low concentration, high cost etc. To overcome these limitations by conventional methods, an environmental friendly method, namely bioremediation has been adopted. Bioremediation uses microorganisms, biodegradable industrial wastes, or plants to mitigate this problem. In this investigation, bacterial strains have been isolated from the soil and water samples collected from a chromite mine in Karnataka. The capability of these bacterial strains have been assessed to remediate Cr (VI) in batch experiments in order to achieve the prescribed standards of regulatory agencies, and to elucidate the mechanisms of bioremediation of Cr (VI). Additionally, using these bacterial strains, biosensors have been developed to detect Cr (VI) ions in the solution by electroanalytical techniques. The major objectives of this research investigation are: a) Isolation, characterization and identification of bacterial strains from water and soil samples obtained from a chromite mine in Karnataka. b) To study the ability of three isolated bacterial strains namely Arthrobacter sp, Exiguobacterium sp. and Micrococcus sp. to remediate Cr (VI) during growth in media, amended with different concentrations of Cr (VI) c) Delineation of the probable mechanisms of bioremediation of Cr (VI) by three bacterial strains with the aid of proteomic and metabolomic studies d) Optimization of factors influencing the bioremoval of Cr (VI) using the isolated bacterial strains as biosorbents in batch experiments. c) Elucidation of mechanisms of bioremoval of Cr (VI) at the microbe – metal interface for all three bacterial strains, adopting characterization techniques like FTIR, XPS, SEM – EDS and zeta potential measurements. d) Micrococcus sp. was chosen for the fabrication of biomodified carbon paste electrode (CPE) to sense the Cr (VI) ions using voltammetric techniques, namely cyclic voltammetry (CV) and differential pulse cathodic stripping voltammetry (DPCSV). The salient findings of this research work are highlighted as follows: Firstly, bioremediation experiments were carried out using the bacterial strains isolated from soil and water samples collected from the chromite mines of Mysore Minerals Limited, Hassan district, Karnataka, India. Initially, the characterisation of the isolated bacterial strains were carried out with respect to their biochemical aspects, antibiotic susceptibility, morphology using scanning electron microscopy and cell wall nature by Gram’s staining. The identification of the three isolated bacterial strains were accomplished by 16S rRNA method and the three bacterial strains have been identified as Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp.. The experiments were conducted to assess the potential of the isolated bacterial strains namely, Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., for the remediation at two different concentrations of 10 mg/L and 30 mg/L of Cr (VI) ions, during cell growth i.e. using metabolically active cells of bacteria. It was found that the three bacterial strains could bioreduce toxic Cr (VI) to the less toxic Cr (III) form, by 95% to 99%, within a time span of 12 h to 120 h. In the experiment with sulphate as the competitive ion in the growing mode of the bacterial strains Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., the percentage bioreduction of Cr (VI) to Cr (III) was not hampered. Scanning electron microscopic studies on the bacterial cells of Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp., before and after interaction with Cr (VI) showed the morphological changes after interaction with Cr (VI), as an adaptive strategy to counter the toxic effect of Cr (VI). Further, to elucidate the mechanisms of bioreduction of Cr (VI) to Cr (III) by the three bacterial strains, the proteins and metabolites were isolated from the pristine bacterial cells and Cr (VI) interacted bacterial cells. The proteins were isolated from different parts of the cells and assessed for the differential expression of proteins under Cr (VI) stress. It was found that, seven differentially expressed protein bands were observed on SDS PAGE profile of Arthrobacter sp. interacted with Cr (VI), from the soluble protein isolated from the crude extract, devoid of cell membrane. A single band of differentially expressed protein was observed in the extracellular secretion in Exiguobacterium sp. and in the case of Micrococcus sp. four differentially expressed proteins were observed in the membrane fraction of proteins. The mass spectrometry data of the differentially expressed proteins were used to identify the probable protein candidates using MASCOT search in NCBIr database. It was found that some of these proteins were a class of transport proteins and a few belong to the reactive oxygen species scavengers. These findings suggested that the bioreduction of Cr (VI) to Cr (III) involved the efflux mechanism and ROS scavenger production, to resist the toxicity of Cr (VI). The metabolite concentration profile was studied for the all three bacterial cells in the absence and presence of Cr (VI) using NMR spectroscopy. The results of this study showed an increase and decrease in the concentration of various metabolite components after interaction with Cr (VI), and this was observed in all the three bacterial strains. Some of the metabolites identified using Chenomx 8.1 metabolite library, were found to be osmoprotectants like betaine, proline etc, which combat the stress of Cr (VI). Therefore, the overall bioremediation of Cr (VI) by metabolically active bacterial cells is through bioreduction of toxic Cr (VI) to the less toxic Cr (III) form and the resistance mechanisms to overcome the toxic effect of Cr (VI) is by the efflux mechanism, production of osmoprotectants and expression of ROS scavengers. In the third part of investigation, the bioremoval of Cr (VI) ions in batch experiments using metabolically inactive cells as biosorbents, for all the three bacterial strains, were studied. The bioremediation efficiency of each bacterial strain was evaluated, considering the various parameters like effect of contact time of bacterial cells with the Cr (VI) ions, pH of Cr ion solution, biomass loading and initial concentration of Cr (VI) ion. The Cr (VI) biosorption efficiency obtained for the bacterial strains Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp. was found to be 93 %, 85 % and 100 % respectively. Apart from the biosorption of Cr (VI) by bacterial cells, the residual Cr was found to be in the form of Cr (III) ions. Therefore, complete bioremoval of Cr (VI) ions could be achieved as a combined process of biosorption and bioreduction, for all three bacterial strains, meeting the acceptable limits prescribed for Cr (VI) ion for drinking water, by regulatory agencies i.e. 0.05 mg/L of Cr (VI) ions. The biosorption of Cr ions by all the three bacterial strains were found to follow a typical Langmurian behaviour. The bioremediation process by the bacterial strains was also evaluated using suitable kinetic models and the results indicated that the bioremoval of Cr (VI) by Arthrobacter sp., Exiguobacterium sp. and Micrococcus sp. followed pseudo second order kinetics. The next aim was to ascertain the mechanism of bioremoval of Cr (VI) ions by the metabolically inactive cells. For this, different characterisation techniques were adopted that aided in the elucidation of reactions occurring at the interface of bacterial cell surface and Cr solution. The nature of interacting forces in bioremoval process was found out by desorption studies, and it was observed that only partial desorption of Cr ions was achieved from the biosorbed bacterial cells. This was further confirmed, by calculation of Gibbs free energy and the values were found to be in the range of – 25 to -32 kJ/mol, thus indicating that the process of bioremoval of Cr (VI) ions by the bacterial cells, is by chemisorption process. The variation in the charge of the bacterial cell surface, before and after interaction with chromium ions, was studied by performing zeta potential measurements as a function of pH. The surface charge of the bacterial cells alone was found to be negatively charged over a wide range of pH. Subsequent to interaction of the bacterial cells with the negatively charged oxyanions of Cr (VI) ions, the surface charge was observed to be less electronegative, which further confirmed the binding of the positively charged Cr (III) ions, formed via bioreduction on the bacterial cell surface. FTIR spectral studies revealed the functional groups involved, in bioremoval of Cr ions, present on bacterial cell surface. The functional group facilitating the bioremoval of Cr ions are –NH, -COOH and phosphate. EDS studies confirmed the Cr peak for the bacterial cells interacted with Cr ions. The oxidation state of Cr ion bound to the bacterial cell surface was determined with the help of XPS analysis. It was interesting to observe the Cr (III) peaks along with their Cr (VI) peaks. These studies provided evidence in support of the bioreduction of Cr (VI) to Cr (III) and biosorption of bioreduced Cr (III) ions onto the surface of bacterial cells, apart from the fraction present in bulk solution. The next objective was to assess the potential of Micrococcus sp. as sensor for the detection of Cr (VI) ions, using electroanalytical techniques such as, cyclic voltammetery (CV) and differential pulse cathodic stripping voltammetry (DPCSV). For this, Carbon Paste Electrode (CPE) was coated with the bacterial strain namely, Micrococcus sp and the modified electrode was used as the working electrode in a three electrode system. The developed biomodified electrode showed an approximately 3-fold increase in the sensing of Cr (VI) ion in comparison with the unmodified electrode CPE, which is attributed to the binding of Cr (VI) ions to functional groups present on the bacterial cell surface. The lower limit of detection obtained for Cr (VI) ions using CV was found to be 1 x10-4 M. The lower limit of detection was improved to 1 x 10-9 M of Cr (VI) using DPCSV.