Browsing by Author "Singh, Shweta"

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    Biochemical Treatment of Acid Mine Drainage by Constructed Wetlands in Northeast India
    (2022) Singh, Shweta
    India is one of the largest producers of coal and coal mines are the major backbone of India’s economy. Amongst various pollution concerns of coal mining, the incidence of acid mine drainage (AMD) is widespread and globally regarded as the most challenging wastewater. The management of AMD remains a great challenge as many remediation measures fail to attain a satisfactory economical and sustainable approach. AMD from the North Eastern Coalfield (NEC), Assam, India, is highly polluted and critical investigation of the AMD pollution for the potential remedial measures is scarce. The present study demonstrates a passive treatment strategy for AMD from the NEC using constructed wetlands (CWs). The influence of climatic variables on AMD characteristics and its deleterious impact on the surrounding soil was investigated. Elevated concentration (in mg L–1) of Fe (0.12–302), Al (6.73–32), Mn (0.10–16), Co (< 1.47), Ni (< 5.24) and Cr (< 0.54) were found in the AMD. The release of pollutants increased in the monsoon season, followed by highly concentrated pollutants in the post-monsoon season. The change in the season had a predominant effect on the oxidative leaching of sulfide minerals. Horizontal subsurface flow CWs (HSSF-CWs) were designed to treat simulated AMD. The treatment performance of HSSF-CWs (A and C) utilizing various organic wastes as the wetland media were compared. Partial to complete removal of metals with negligible Mn removal and moderate sulfate removal (57–62%) was obtained. Media accounted major retention (58–95%) and plants (Typha latifolia) exhibited negligible metal uptake. Toxicity Characteristic Leaching Procedure suggested safe media disposal. The release of weakly bound Mn, Al, Co and Ni was indicated under an adverse environmental state. The role of plants and the impact of hydraulic loading rates (HLRs) in the remediation of AMD and metal attenuation were studied in planted (PCW) and unplanted (CCW) CWs. Significant impairment of sulfate reduction was observed in PCW (24–90%) than CCW (37–93%). Effluent water quality deteriorated and metal removal efficiency reduced drastically on increasing HLR. HLR has an important role in the passive treatment of AMD than the presence of plants in CWs. The application of a chelating agent and organic acids revealed high metal extraction from wetland media. Organic acids achieved comparable extraction efficiency with ethylenediaminetetraacetic acid (EDTA) and further good metal recovery values were obtained. The influence of seasonality on the efficacy of CW microcosms was studied. Rainfall has a stronger influence in the northeastern region of India. The effect of rainfall on sheltered (SCW) and unsheltered (UCW) CWs was investigated under natural conditions. Findings revealed a significant difference in the sulfate reduction of SCW (60%) and UCW (51%). Heavy rainfall imposed a highly sensitive and poor treatment response in UCW, resulting in a significantly lower treatment efficiency for all constituents due to possible shortening of hydraulic retention time in UCW with minimal or insignificant dilution effect. In addition, the long-term treatment appraisal of HSSF-CW (B) comprising gravel media was evaluated under varying COD/ratios using lactate as a carbon source. The high average metal removal efficiency was attained for Fe (73%), Al (79%), Zn (98%), Co (95%), Ni (99%) and Cr (100%), but Mn (21%). As COD/ reduced from 0.67 to 0.33, sulfate reduction decreased from 74% to 44% and metal removal efficiency subsided. Dissimilatory sulfate reduction was identified as the major biological pathway. The formation of insoluble metal precipitates (oxides, hydroxides and sulfides) accounted for about 32–76% of metal removal. Major sulfate-reducing and iron-metabolizing microbial groups were identified to control the biochemical cycling of iron (oxidation or reduction) and its bioavailability.
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    Strain improvement of Bacillus amyloliquefaciens SS35 for enhanced endoglucanase catalytic efficiency and identification of mutation causing the structure changes by cloning, expression and purification of glycoside hydrolase family 5 endoglucanase and its application in saccharification of Sorghum durra
    (2020) Singh, Shweta
    In bioethanol production, the enzymatic saccharification of lignocellulosic biomass for release of reducing sugars is the cost limiting step. Therefore, for reducing the production cost of bioethanol, strain improvement of cellulase producing microorganisms is important. Random mutagenesis by UV in microorganism resembles the natural evolution process. Therefore, it can be used for improvement of biochemical properties in industrial enzymes (cellulase). Wild-type strain of Bacillus amyloliquefaciens SS35 was exposed to UV irradiations to develop the UV2 mutant strain with improved endoglucanase catalytic efficiency and wide range pH stability. The gene encoding endoglucanase, BaGH5-WT and BaGH5-UV2 were amplified from wild-type and UV2 mutant of Bacillus amyloliquefaciens SS35, respectively, using degenerate primers for family 5 glycoside hydrolase (GH5) and were cloned in pET-28a(+) vector and expressed in E. coli BL21(DE3) pLysS cells. The recombinant mutant BaGH5-UV2 showed 22-fold higher catalytic efficiency and wider range pH stability than recombinant wild-type BaGH5-WT. The mutant enzyme, BaGH5-UV2 showed substitution mutation of residue, Asp256 to Gly256. This mutation was in loop connecting the β6 to α6 of (β/α)8 TIM-barrel fold. Molecular dynamics simulation studies showed more stable 3-D structure for BaGH5-UV2 than BaGH5-WT. Molecular docking results showed that BaGH5-UV2 gave maximum increase in Gibb’s free energy (ΔG°) against cellotetraose. Application of BaGH5-UV2 in saccharification of acid or base pretreated Sorghum durra stalk in cocktail with CtCBH5A and CtGH1 was explored for pretreatment specific customization of enzymes in mixture design. This report provides the information for protein engineering in GH5 endoglucanases for improving their biochemical properties and pretreatment specific optimization of enzymes in mixture for enzymatic saccharification.