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  • sitagliptin phosphate Quantifying diffusion is greatly compl

    2020-01-21

    Quantifying sitagliptin phosphate is greatly complicated by the ubiquitous unevenness of soil surfaces and related water films, which introduce a large uncertainty into size (area) and quality of contact between the soil surface and the membrane, the problem being particularly substantial in well aggregated and coarse textured soils. Due to differences in sizes and shapes of soil particles and aggregates, presence of soil pores and incompletely decomposed plant residues, a soil surface is never perfectly flat, and thus the actual area in direct physical contact between the membrane and the soil may be relatively small (Fig. 1). Its size can be difficult to measure, though theoretically, on an ideally sliced dry soil surface, the contact area should be equivalent to the volumetric fraction of solid substances in soil, i.e. = [1.0 - soil porosity]. However, in moist soil the indirect contact areas, e.g. via water films, can be much larger in size than the direct contact areas (Fig. 1), but the quality of this type of contact is inferior to direct contact. By quality of the contact here we refer to spatial accuracy with which the fluorescent signal on the membrane can reflect activity of the enzyme. Due to tortuosity of diffusion pathways, such spatial accuracy resolution is expected to be much lower for indirect contact areas. The size of indirect contact depends on the soil water content and the soil water retention properties. Knowing the positions of the direct and indirect contacts on the membrane is an important prerequisite for soil zymogram\'s interpretation. Indeed, an absence of a fluorescent signal in a location that is in contact with the membrane indicates absence of enzymes, while absence of a signal in a location that had no or poor contact cannot be unequivocally interpreted. Here we addressed several methodological aspects improving the method for its use in rhizosphere experiments, and importantly for zymography expansion to a wider range of micro and macro scale studies in soil ecology. Since relatively little is known about transport processes involved in enzyme activities in soil, e.g., travel-distances of enzyme from producing microbial cells to substrate, amounts of product that can return to the enzyme producing cell, etc. (Allison et al., 2011; Burns et al., 2013), we used experimentation combined with modeling to better understand and interpret the results of 2D soil zymography. The specific objectives of the study were: 1) to quantify the diffusion pathways of substrates, enzymes and produced MUF involved in zymography analysis and enzymatic reactions; 2) to compare the methods estimating the area of the soil surface in contact with zymography membranes; and 3) quantitative estimation of in situ enzyme activity using zymograms and process modeling.
    Materials and methods
    Results
    Discussion
    Conclusions Based on visualization and diffusion experiments, the following general conclusions regarding enzymatic and diffusion processes during zymography are suggested:
    Acknowledgement This research was partly funded by the National Science Foundation\'s Long-Term Ecological Research Program (DEB 1027253), by the National Science Foundation\'s Geobiology and Low Temperature Geochemistry Program (Award no. 1630399), by the Department of Energy Great Lakes Bioenergy Research Center (DOE O_ce of Science BER DE-FC02-07ER64494), by Michigan State University\'s AgBioResearch (Project GREEEN), and by Michigan State University\'s Discretionary Funding Initiative. The work was supported by DAAD- German Academic Exchange Service\' program \"Research Stays for University Academics and Scientists, 2017\" (57314018) and by the Research Award from Alexander von Humboldt Foundation. Contributions of BSR and EB were motivated and supported within the framework of the priority program 2089, funded by the DFG- Projects Nr. 403670038 and Nr. 403664478, respectively. This work has also been conducted in part as a preparatory study for the recently launched DFG priority program 2089 Rhizosphere spatiotemporal organization—a key to rhizosphere functions PE 1523/10–1.