Bacterial community isolated from different units of a Drinking Water Treatment Plant (DWTP) including pre-ozonation unit (POU), the effluent-sludge mixture of the sedimentation unit (ESSU) and top-sand layer water sample from the filtration unit (TSFU) were acclimatized separately in the microcystin-leucine arginine (MC-LR)-rich environment to evaluate MC-LR biodegradation. Maximum biodegradation efficiency of 97.2 ± 8.7% was achieved by the acclimatized-TSFU bacterial community followed by 72.1 ± 6.4% and 86.2 ± 7.3% by acclimatized-POU and acclimatized-ESSU bacterial community, respectively. Likewise, the non-acclimatized bacterial community showed similar biodegradation efficiency of 71.1 ± 7.37%, 86.7 ± 3.19% and 94.35 ± 10.63% for TSFU, ESSU and POU, respectively, when compared to the acclimatized ones. However, the biodegradation rate increased 1.5-folds for acclimatized versus non-acclimatized conditions. The mass spectrometry studies on MC-LR degradation depicted hydrolytic linearization of cyclic MC-LR along with the formation of small peptide fragments including Adda molecule that is linked to the reduced toxicity (qualitative toxicity analysis). This was further confirmed quantitatively by using Rhizobium meliloti as a bioindicator. The acclimatized-TSFU bacterial community comprised of novel MC-LR degrading strains, Chryseobacterium sp. and Pseudomonas fragi as confirmed by 16S rRNA sequencing.

The study aims to explore microbial lipid production using an abundant and low-cost lignocellulosic biomass derived from forestry residues. Sugar-rich undetoxified hydrolysate was prepared using hardwood and softwood sawdust and used for lipid production as a carbon source from an oleaginous yeast, Rhodosporidium toruloides-1588. The maximum biomass obtained was 17.09 and 19.56 g/L in hardwood and softwood hydrolysate, respectively. Sugar consumption in both hydrolysates was >95%, with a maximum lipid accumulation of 36.68% at 104 h and 35.24% at 96 h. Moreover, R. toruloides-1588 exhibited tolerance to several toxic compounds such as phenols, organic acids and furans present in hydrolysates. The lipid characterization showed several monosaturated and polyunsaturated fatty acids, making it a potential feedstock for biofuels and oleochemicals production. This study confirms the credibility of R. toruloides-1588 as a suitable lipid producer using hydrolysates from forestry residues as a substrate. Additionally, lipids obtained from R. toruloides-1588 could be a potential feedstock for advanced biofuel production as well as for food and pharmaceutical applications.

Aims: To clone and express, the gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3). Further, the work is also comprised of purification and detailed biochemical characterization of L-asparaginase II. Place and Duration of Study: Biochemical Engineering laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiments conducted as a part of project and a PhD thesis from December 2010 to January 2014. Methodology: The gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 was cloned and expressed in E. coli BL21 (DE3). Affinity chromatography was employed to purify the enzyme to homogeneity. Detailed biochemical characterization, such as substrate specificity, operational stability in various effect or molecules, effect of pH and temperature, kinetic parameters were investigated. Results: The enzyme was found to be highly specific towards its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II was activated by various effector compounds, such as mono cations, L-cystine, L-histidine, 2-mercaptoethanol and glutathione. However, considerable inhibitory effect was observed with divalent cations and iodoactamide. Kinetic parameters (V_max, K_m, kc_at and K_cat/K_m) of purified recombinant L-asparaginase II were found to be 0.656 mM, 312.50 IU mg^-1, 1.38×10² s^-1 and 2.11×10⁵ M^-1s^-1, respectively. Optimum range of pH and temperature for the hydrolysis of L-asparagine by purified recombinant L-asparaginase II were found to be 6.5-9.5 and 47-52ºC, respectively. Under optimal levels of medium components and physical process parameters (pH, inoculum size, agitation), the production of recombinant L-asparaginase II was increased by 1.95 fold. The purified recombinant L-asparaginase II has shown no glutaminase activity. Conclusion: The present characterization experiments of the L-asparaginase II from Erwinia carotovora subsp. atroseptica SCRI 1043 showed very high specificity for its natural substrate, L-asparagine and shown no glutaminase activity which makes it a better alternative in therapeutic applications like an anticancer drug.

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588.