Understanding Physical and Biological Processes for Pesticide Removal by Woodchip Bioreactors, with Insight Employing Microbial Enzymes and Communities
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Understanding Physical and Biological Processes for Pesticide Removal by Woodchip Bioreactors, with Insight Employing Microbial Enzymes and Communities

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Contamination of surface and groundwater resources by conventional agricultural practices is becoming increasingly prevalent. In recent years, development of small footprint, low-maintenance, onsite woodchip bioreactors have helped to curb nitrate discharge from tile-drainage collection to downstream receiving waterways. Determining best practices to jumpstart microbial degradation of pesticides in similar reactors could help address pesticide pollution from agricultural areas. Chapter 1 of my dissertation provides the motivation for the following chapters, outlining the respective research objectives. Chapter 2 of my dissertation assesses pesticide removal in woodchip bioreactor systems at three scales: field-scale, bench-scale, and micro-scale batch reactors. I constructed a bench-scale continuous-flow woodchip bioreactor and operated the reactor under field-like conditions to evaluate joint pesticide and nitrate removal. The continuous-flow reactor achieved 83.5 ± 8% diuron removal and 61.6 ± 11.9% imidacloprid removal with a 24 h hydraulic retention time (HRT). I then designed a sequencing-batch reactor configuration to evaluate the impact of an aerobic phase on denitrification and pesticide removal performance. The sequencing-batch reactor achieved 89.2 ± 8.8% nitrate removal with an HRT of 12 h, while the continuous-flow design achieved 55.6 ± 9.1% nitrate removal with a 12 h HRT. There was no significant difference between pesticide removal between sequencing-batch and continuous-flow reactor types. Kinetic batch tests revealed sorption, not microbial degradation as the main mechanisms of removal for both diuron and imidacloprid under denitrifying conditions. Chapter 3 of my dissertation describes a combined ex-vivo and in silico screening approach I developed to understand and predict the catalytic competency of esterases for ester hydrolysis of pyrethroid pesticides and structurally related compounds. I hypothesized that esterases active toward pyrethroids would also interact with structurally similar esters. I screened fourteen representative, wild-type microbial esterases against four pyrethroids, four alternative esters, and a suite of designed/known esterase and lipase substrates. I assayed each enzyme/substrate pair at different concentrations and analyzed assay extracts for specific esterase transformation products to confirm esterase activity. Phenyl salicylate, an ester compound with low aquatic toxicity, was found to be degradable by some of the same enzymes that were found to degrade pyrethroids. In silico induced fit docking of target compounds with esterases of interest highlighted amino acid residues important to substrate binding. For some enzymes, interaction fingerprint data revealed residue interaction patterns that are indicative of catalytic competency (i.e., enzymatic activity). Findings from these in silico experiments could be used as constraints in future molecular docking simulations. Finally, Chapter 4 investigates the biodegradation potential of autochthonous microbial communities enriched on target pyrethroids as well as non-toxic, structurally similar substrates. I hypothesized that phenyl salicylate (identified in Chapter 3 as sharing enzyme activity with pyrethroids) stimulates enzymatic activity effective towards degradation of the two pyrethroids, bifenthrin and cypermethrin. I isolated a native microbial community from a functional woodchip bioreactor, and grew aliquots on phenyl salicylate, bifenthrin, cypermethrin, or glucose as the sole carbon substrate. I compared the growth patterns and metabolic characteristics. I found that the microbial consortia grew similarly on phenyl salicylate and glucose, while the growth curves of the bifenthrin and cypermethrin enrichments were characteristic of a more recalcitrant substrate (i.e., longer lag phase). I also employed a suspect screening approach to track the formation of commonly found metabolites of bifenthrin, cypermethrin, and phenyl salicylate. Results demonstrated that esterase activity occurred in each of these enrichments using the respective substrates. Results also provided evidence that phenyl salicylate-enriched communities may share enzyme profiles with bifenthrin- or cypermethrin-enriched communities and suggest that phenyl salicylate may act as a low-toxicity biostimulant for pyrethroid degradation.

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This item is under embargo until April 4, 2025.