A synthetic biology and process engineering Approach to bioremediation

Bioremediation is a promising and environment-friendly strategy for emerging pollutant removal from wastewaters. In this work, we aimed at combining the approaches of synthetic biology and process engineering with bioremediation for the removal and degradation of water pollutants by using sustainable and environment friendly approaches. Here we focused on 2 water pollutants 1st glyphosate broad-spectrum herbicide and 2nd PFAS (per- and poly-fluoroalkyl substances). For glyphosate, based on previous work, a combined process including preconcentration of the molecules by adsorption, followed by biological reactor, is proposed. The adsorption step was characterized for equilibrium and kinetics to assess the efficiency of pollutant removal. The type of adsorbent (activated carbon and nanoparticles, and desorption techniques were investigated to understand the compatibility of the solution on the consortium growth. For this 10 mM ammonia and 40 mM CaCl2 were tested for their compatibility with microbial components of consortium. Concerning the microorganisms, two different approaches were adopted for the two pollutants. For glyphosate, a previously designed (bacteria-microalgae) consortium was validated and optimized with different microalgae named Scenedesmus obliquus. Further experiments were performed for scaling up the system. For PFAS, starting by a preliminary wet-lab analysis where 5 different microalgal species including Chlorella, Scenedesmus, Nostoc, Synechocystis and Galderia sulphuraria were tested to assess their resistance to long chain PFAS molecules. Along with already identified Synechocystis, Nostoc and Galderia sulphuraria were also found resistant for 1mg/L concentration of PFAS. To further identify candidate enzymes involved in PFAS degradation/ transformation in the microbes are being investigated in silico by means of proteome-wide searches, molecular modeling and docking simulations. We are focused on dehalogenases and laccases as some out of such enzymes are able (or likely to be) to cut the very strong carbon-fluoride bond that make PFAS so recalcitrant to biodegradation and hence dangerous because of bioaccumulation. In this study, a detailed comparative analysis of bacterial, algal and fungal laccases was carried out. That includes classification based on conserved copper binding motifs, difference in redox potential of among different groups and identification of functional fingerprints for thermostability through sequence and structure analysis. For identification of functional fingerprints several parameters including compact packing, Aliphatic index, salt bridges, disulphide bonds and prolines were examined. The combined sequence and structure analysis revealed conserved positions of salt bridges and prolines that can play their role in enhanced thermostability of bacterial laccases. To experimentally validate this, dextran modification of a fungal laccase was performed to access whether surface modification could enhance thermal tolerance and catalytic function in laccases. Starting with the characterization of Trametes versicolor laccase for optimum temperature, PH, concentration, activity and free amino group dextran modification was performed. Later laccase modification with oxidized dextran was performed with an optimized protocol and differences in stability and catalytic activity evaluated.