Hydrolysis of glyphosate and other organic phosphorus compounds and associated isotope effects
Date
2023
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Publisher
University of Delaware
Abstract
Phosphorus (P) is an essential element of all life forms. Understanding the complex P cycling in the environment requires novel techniques that are capable of connecting sources and identifying the transfer and transformation of different P forms. Among them, isotope tracers are ideal because distinct isotope effects are imprinted during the hydrolysis of organic P compounds and reactions at biotic and abiotic interfaces. The overarching goal of this dissertation project was to characterize the isotope effects during substrate-specific hydrolysis and exchange reactions of glyphosate (the most common herbicide) and ATP (the most common energy storage and signaling molecule in cells), and orthophosphate. It integrated isotopes with spectroscopic (NMR, Raman, and IR) and spectrometric (MS/MS) characterization techniques and combined microbial genetics to determine the degradation pathway, selectivity, and fate of these molecules in the environment. This dissertation includes four different projects with their specific objectives. The objective of the first project (Chapter 2) was to develop a novel method to quantify the exchange among oxygen isotopologues of orthophosphate (Pi) and pyrophosphate (PPi). It employed Raman and 31P NMR spectroscopic methods to quantify the kinetics of isotope exchange. Both methods were found to be able to monitor the reversible hydrolysis–condensation reaction between PPi and Pi in the inorganic pyrophosphatase (PPase) catalyzed oxygen isotope exchange reaction between Pi and ambient water. The spectroscopic methods were able to detect as low as 0.2% 18O abundance at the PPi and Pi concentration ≥1 mM. The second project (Chapter 3) investigated the isotope effects during the hydrolysis of ATP by cell-free alkaline phosphatase and adenosine triphosphatase and synthesis by bacterial cells (Escherichia coli). The results revealed that enzyme- and substrate-specific reactions imprint distinct isotope effects, which enabled connecting products and identifying the sources of oxygen during the synthesis and hydrolysis reactions. The objective of the third project (Chapter 4) was to investigate bond–dissociation mechanisms in glyphosate in the presence of manganese oxide so that the feasibility of bias toward less persistent products could be tested. The HPLC and multi-nuclei (1H, 13C, and 31P) NMR allowed accurate quantitation of residual glyphosate and intermediate products formed during degradation. Further kinetic modeling and molecular dynamic simulations allowed to identify the free energy barrier for specific products. The results showed that both AMPA and glycine are dominant, but the glycine pathway became increasingly preferred with increasing reaction time and temperature. While the free energy barrier was comparable for both products, a high entropic energy barrier for AMPA caused this pathway to be less prevalent. The objective of the fourth project (Chapter 5) was to develop an improved understanding of the environmental fate of glyphosate in Delaware. This project replicated the state’s protocol of glyphosate application for weed (Phragmites australis) control in wetlands near Chesapeake and Delaware Canal. The Orbitrap MS/MS-based results revealed that glyphosate and AMPA could be detected more than 100 d after field application. The P mass balance calculation suggested that AMPA was a minor product- indicating glyphosate degradation likely dominated through the glycine pathway. This result was corroborated by targeted gene abundance analysis which showed that the expression of phnJ gene (which codes C-P lyase enzyme) was dominant over gox gene (which codes oxidoreductase enzyme). The prevalence of C-P bond cleavage, which means AMPA is a minor pathway of degradation, is a unique and safer way to degrade glyphosate. Overall, this dissertation research generated novel methods for isotope analyses, identified distinct isotope effects during hydrolysis and synthesis of glyphosate and AMPA, and identified possible routes to decrease the environmental toxicity of glyphosate. These findings are expected to contribute to the environmental chemistry of P as well as the technology to mitigate the exposure of P-based herbicides.
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Keywords
ATP, Glyphosate, Hydrolysis, Isotope effects, Orthophosphate, Phosphorus