Role of Metallo-resistant Microorganisms in Remediation of Heavy Metals using Biofilter

Abstract

Heavy metals are non-degradable and highly toxic in low concentration. Heavy metals are intermittently released from industries into ground water and soil, leading to human and environmental health problems. It is evident from recent studies that heavy metal i.e. Chromium (Cr) and Lead (Pb) toxicity and concentration is increasing day by day due to increase in their use in different industries. Before it goes beyond control, there is an emergent need to develop effective strategies for control and removal of heavy metals (Cr and Pb) contamination. The aim of this study was to evaluate the role of metallo-resistant microorganisms in remediation of heavy metals such as Cr and Pb. Total of 53 strains were isolated from soil and sludge samples Korangi and Lyari, Karachi (24˚52ʹ46.0ʺN 66˚59ʹ25.7ʺE and 24˚48ʹ37.5ʺN 67˚06ʹ52.6ʺE) respectively. Out of these 53 strains, two of the bacterial isolates designated as S48 and S54 from Korangi sludge were selected on the basis of high metal tolerance against K2Cr2O7(1500 mg/L) and Pb(NO3)2 (1200mg/L).The optimum temperature and pH for both the strains S48 and S54 were found as 30˚C and 35˚C and while their optimum pH was observed 6.0 and 7.0, respectively. Both biochemical and molecular basis of identification indicated that strain S48 was related to genus Bacillus with 99% similarity with Bacillus paramycoides whereas strain S54 to genus Escherichia with 99% similarity with Escherichia fergusonii. Bacillus sp. strain S48 can effectively reduce toxic Cr(VI) to less toxic Cr(III) and maximum reduction achieved was up to 65% after 96 hours. Similarly, Escherichia sp. strain S54 has proved Pb biosorption potential and can affectively remove 60% of Pb. Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX) and Fourier-transform infrared (FT-IR) spectroscopy result showed clear morphological and functional group shifts on bacterial strains S48 and S54 cell surfaces treated with metals. The enzyme chromate reductase activity was found during Cr reduction experiment with strain S48. The physicochemical variables influencing chromate reductase production were identified by statistical tools, Plackett-Burman Design (PBD) and Central Composite Design (CCD). Maximum production of chromium reductase was achieved at temperature 35°C and pH 7.0. The chromate reductase was purified to homogeneity by DRSML QAU xi size exclusion column chromatography with specific activity of 1416.5 U/mg, 59.5% yield, and 6.6-fold increase in purity. The maximum activity of chromate reductase was observed at temperature 40˚C and pH 7.0. The Km and Vmax values of enzyme were 1.36 µM and 909.09 µM respectively, using K2Cr2O7 as a substrate. The activity of chromate reductase was strongly inhibited by Mercury (Hg), sodium dodecyl sulphate (SDS) and Cetyl-tri-methyl-ammonium Bromide (CTAB). Bacillus sp. strain S48 reduced 69% of chromium at concentration upto100 mg/L after 96 hrs. Furthermore, effluent containing 100 mg/L of K2Cr2O7 was treated with 1-10% of both crude and purified chromate reductase. The crude and purified chromate reductase (10% v/v) showed maximum reduction of 72% and 78% after 120 hours, respectively. Furthermore, the activity of Chromate reductase and Lead binding protein were enhanced by cloning and expression of gene for chromate reductase (ChrR) and Lead binding protein (PbR) into expression host. Both chromate reductase gene (BparChR) from Bacillus sp. strain S48 and Lead binding protein (EferPbR) from Escherichia sp. strain S54were successfully cloned into intracellular PET-28a and expressed into BL21 (DE3). Ni-Affinity ion exchange column chromatography was used for the purification of BparChR and EferPbR and fractions were collected for measuring enzyme activity. The purity and molecular size of protein was checked through SDS-PAGE and approximately 35 kDa size of chromate reductase (ChR) and 20 kDa of lead binding protein (PbR) were observed. Chromate reductase (ChR) exhibited specific activity of 1680 U/mg, 34.19% yield, and 5.73-fold increase in purity under optimum physicochemical conditions. The maximum chromate reductase activity was observed at temperature 35˚C and pH 7.0, while the activity was strongly inhibited by Hg+ . A Total of 90% chromium reduction was achieved after 96 hours by treating effluent with recombinant BparChR. Biofilter was designed and operated with recombinant PbREcoliBL21 strain carrying PbR from Escherichia sp. strain S54.Both the experiments, batch and continuous column results showed that recombinant PbREcoliBL21significantly improved the adsorption capacity of the ceramic balls. SEM results showed clear morphological changes on recombinant PbREcoliBL21 cell surfaces treated with metals. FTIR spectra showed that PbREcoliBL21 provides binding sites for the retention of the metal cations. A total of 95% biosorption in lead were achieved by PbREcoliBL21 in fix bed biofilter. Finally, it is DRSML QAU xii concluded from the results that both chromium and lead-resistant bacteria can be effectively utilized in chromium and lead removal from contaminated industrial effluents. Biological based techniques using bacteria will help to provide a cheaper and environmentally friendly method for heavy metals removal, recovery and detoxification.