High-resolution study on degradation and isotope effects of inositol phosphates in soils
Date
2024
Authors
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Publisher
University of Delaware
Abstract
High phosphorus (P) loading to water bodies promotes the proliferation of harmful algae and subsequent decline in water quality. Organic P represents a significant, hitherto largely uncharacterized P pool in the environment. Inositol phosphates (IPx) are the most dominant form of organic P in the environment but are under-studied due to a lack of appropriate analytical tools. These gaps are major barriers to a fuller understanding of the sources, cycling, and impacts of different P forms in an ecosystem. In this dissertation research, soil IPx was analyzed using a combination of biological and chemical methods, including the newly developed Orbitrap isotope ratio mass spectrometry (Orbitrap IRMS) method, which is capable of measuring position-specific isotopes of IPx isomers. An ion chromatography method capable of separating different stereoisomers and regioisomers of IPx was established and used to isolate IP6 from environmental samples. To develop method for Orbitrap IRMS to analyze oxygen isotopes (δ18OP) of intact phosphate in IPx from environmental samples, agricultural soil from a farm in the East Creek tributary to the Chesapeake Bay was spiked with IP6 and incubated with 18O-labeled water. The IP6 in soil was extracted and separated using fraction collection in ion chromatography and δ18OP of intact phosphates in the ring was measured by using Orbitrap IRMS. A systematic investigation of Orbitrap IRMS parameters was also undertaken to determine how each impacts the measured isotope values. Among them, analyte concentration and matrix composition in samples and ionization, ion count, and mass window in Orbitrap MS were found to play significant roles in the measured isotope values. A pure IP6 incubation study revealed that phosphate moieties in the inositol ring do not exchange isotopes with water, analogous to what is known for dissolved phosphate. A conspicuous and clear trend of progressive enrichment of δ18OP values of IP6 was observed in soil incubation experiments. This means inorganic phosphate in soils — which underwent isotope exchange with 18O labeled water—eventually phosphorylated in inositol to form a new IP6. The analysis of two gene expressions, degradative β-propeller phytase and synthetic myo-inositol phosphate synthase, corroborated the presence of active, opposite reactions—degradation and synthesis of IP6—creating the cryptic cycling of IPx in soils. This rather uncommon finding has significant implications because the synthesis is not known, and the prevailing understanding is that the accumulated IP6 in agricultural soil undergoes degradation. In summary, the Orbitrap IRMS method developed has tremendous applications in agricultural soils to fill in critical knowledge gaps regarding the fate of inositol phosphates, and the method can be readily translated to other organic phosphorus compounds in the environment.
Description
Keywords
Inositol phosphates, Ion chromatography, Orbitrap, Phosphate synthase, Soils, Stable isotopes