Nine bacterial isolates were screened from different sites of East Calcutta Wetland, an ecosystem located at the eastern fringes of Calcutta. Other than being a biodiversity rich area, the important feature of this system is that it is a natural sewage treatment plant for the city of Calcutta. In addition to daily sewage including solid and soluble wastes, a considerable load of toxic metals are released into the water bodies from industries, tanneries, agriculture, household as well as health sectors. Screening out microbes from such an environment was done keeping in mind their multi functional application. These bacterial isolates were found to produce extracellular protease which is known to have vast applications in the commercial market of enzymes. The key area of this study is exploring the role of these isolates in heavy metal remediation. These isolates were found to tolerate heavy metals like Ag, Al, Cu, Cr, Co, Ni and Pb. Energy Dispersive X Ray Fluorescence (EDXRF) analysis and Transmission Electron Microscopy of the metal treated cells confirm the intracellular accumulation. Moreover, a preliminary demonstration of the effect of metal induced stress on cell surface features was determined by Scanning Electron Microscopy

Abstract:The present paper put forth the isolation and complete characterization of three bacterial isolates from different sites of East Calcutta Wetland (ECW). These isolates show tolerance to heavy metals like Ag, Al, Cu, Cr, Co, Ni, Pb and were found to be efficient metal accumulators as evident from Energy Dispersive X Ray Fluorescence (EDXRF) analysis and Transmission Electron Microscopy (TEM). Beyond intracellular accumulation, these isolates also show metal deposition in form of particles inside the cells which can further open up the area of microbe fabricated nanoparticle generation. The heavy metal accumulation in the cell can trigger different response mechanism such as change in cell morphology which was observed by scanning electron microscopy (SEM). The metal uptake property of these isolates can be applied for the heavy metal removal and recovery from industrial effluents.

Extracellular protease from a novel bacterial isolate showing maximum similarity of 98.22% with Microbacterium luteolum was obtained from East Calcutta Wetland, India. It showed compatibility with commercial detergents. The enzyme retains more than 60% of its activity between 6.0 to 10.5 pH. The maximum activity is at pH 7.5 with 71% activity at pH 10.0 and 10.5. The protease retained its activity between 4 to 60°C with maximum activity at 30°C and a residual activity of 74.4% at 60°C after overnight incubation. It was completely inhibited by 5mM PMSF pointing towards the presence of serine group of protease. Its inhibition by EDTA indicates the involvement of metal cations in its catalytic activity. It is not effected by Cu +2 , partially inhibited by Pb +2 and Ni +2 , while completely inhibited by Co +1 , Cr +6 , Zn +1 , Al +3 , Ag +3 and Hg +2 . Strong reducing agents like β-merceptoethanol and oxidants like bleach and hydrogen peroxide inactivate the enzyme. The enzyme retains 88% of its activity on being mixed with commercially available detergents while it is inactivated by non-ionic Triton X 100. Its efficiency as an additive with detergent in terms of cleaning stains like grease, burnt mobil, vegetable curry and blood was found to be satisfactory. It could enhance the quality of washing as additive in case of all the ten detergents that were tried. The protease alone was also capable of cleaning but the detergent additive mixture could work better. The enzyme was found to work efficiently on different colors as well as on fabric. On mixing with detergent it was found to retain activity up to 2 months and there after, there was a drop in efficiency of washing. The bacterial cells were immobilized in calcium alginate and the released enzyme was found to be equally effective. Market surveys were carried out and the satisfactory result prompted the use of another additive (extracellular lipase) obtained from yet another bacterial strain from East Calcutta Wetland. The lipase activity was confirmed through degradation of coconut oil analyzed by Gas Chromatography. Thus the combination was observed to be more successful as indicated through the market survey. These observations suggest the suitability of the protease and lipase combination as additive to commercially available detergents.

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