Extraction And Characterization Of Extracellular Polymeric Substances From Soil And Their Selective Retention Mechanism On Ferrihydrite Surface

Extracellular polymeric substances（EPS）are high molecular weight biopolymers secreted into the extracellular environment during the growth and metabolism of microorganisms.EPS are microbially-derived organic matter containing biomolecules such as polysaccharides,proteins,DNA and lipids.The interaction between EPS and soil minerals affects a wide range of biogeochemical processes,such as the formation and development of biofilms on soil substrates,the aggregation and stability of soil mineral particles,and the occurrence and fate of soil organic and inorganic pollutants,etc.EPS can benefit soil functions through its own properties（e.g.adhesiveness,hydroscopicity,and complexation）,such as improving aggregate stability,enhancing water retention,and immobilizing soil pollutants.Extraction analysis of actual soil EPS is a key prerequisite for understanding the extracellular responses of soil microorganisms and the ecological functions of extracellular products,while systematic insights into the extraction steps and chemical properties of soil EPS are still relatively inadequate.The sequestration of EPS by soil minerals is an important process for the retention and stabilisation of microbially-derived organic matter in soils,and early studies mainly focused on the effect of soil mineral types on the degree of EPS adsorption.However,EPS can form mineral-organic associations through two formation pathways,adsorption and coprecipitation.The interaction process between EPS and iron oxides and the selective retention of EPS components under the two formation pathways are still unclear.This paper focuses on the interfacial process of EPS mediated by soil minerals.The EPS from actual soils and the EPS secreted by the model bacteria Bacillus subtilis are used as the research objects.These researches are carried out using traditional elemental composition determination,phase analysis,fourier transform infrared spectroscopy combined with two-dimensional correlation spectroscopy（2D-FTIR-COS）,confocal laser scanning microscope（CLSM）,X-ray photoelectron spectroscopy（XPS）,near edge X-ray absorption fine structure（NEXAFS）,excitation emission matrices fluorescence spectroscopy combined with parallel factor analysis（EEM-PARAFAC）,excitation emission matrices fluorescence spectroscopy combined with principal component analysis（EEM-PCA）,fourier transform infrared spectroscopy combined with principal component analysis（FTIR-PCA）and other modern technical methods.The extraction process characteristics and response sensitivity of soil EPS are analyzed,and the different binding pathways and chemical fractionation behaviors between bacterial EPS and ferrihydrite（Fh）are explored.The main results are as follows:（1）The process characteristics of soil EPS extraction by the cation exchange resin（CER）method and the response sensitivity of soil EPS are clarified.For the two-step extraction process of the CER method,it is observed that H₂O as the extractant not only causes lysis of microbial cells due to osmotic pressure,but also to lead to premature extraction of EPS.The use of H₂O instead of Ca Cl₂ as the extractant in Step 1 should be avoided.The indiscriminate exchange extraction of the PBS buffer solution in Step 2 results in the release of similar components from soil.The addition of CER is a vital inclusion in the PBS buffer to ensure maximum extraction of EPS,and thus identify the differences among treatments.Step 2 enables the release of more dissolved organic carbon and biopolymers（polysaccharides,proteins and uronic acids）from the soil.The extraction solutions from Step 2 exhibit higher aromaticity,molecular weight and humification degree than that from Step 1,as well as increase in four of the five fluorescent components identified based on the EEM-PARAFAC model.Different soil pre-treatment methods（wetting,drying,and incubation）alter the chemical properties of EPS components.The sensitive responses of EPS to soil pre-treatment are reflected in the EEM-PCA and FTIR-PCA analyses.Through the quantitative determination of element and biopolymer content combined with the qualitative analysis of its molecular spectroscopic properties and spectroscopic chemometrics,our investigation provides comprehensive information on the extraction dynamics and spectral characteristics of soil EPS,and also demonstrates that the extraction analysis of soil EPS can serve as a vital indicator of microbial extracellular functions and responses.（2）The formation pathways differences and interaction mechanisms of the binding between bacterial EPS and Fh are revealed.For the formation pathways of adsorption and coprecipitation,the chemical complexation of the carboxylate functional group in the interaction of EPS with Fh occurs prior to the reaction of the amide functional group.The P=O functional groups are adsorbed to the Fh surface faster than the carboxylate and amide functional groups,while it lags behind the carboxylate and amide functional groups during EPS coprecipitation with Fh.The coprecipitation of EPS does not change Fh as the main mineral phase in the associations,but it significantly increases the apparent particle size of the EPS-Fh associations.Although the EPS-Fh coprecipitates hold more negatively charged EPS,the Zeta potential values of the adsorption complexes are always lower than those of the coprecipitates,and the XRD patterns of the adsorption complexes show more obvious additional peaks from EPS.This implies that the acidic groups in EPS are more neutralized and embedded during EPS coprecipitation with Fe species.The results provide new insights into the mechanism of the interaction process between EPS and Fh under the formation pathways of adsorption and coprecipitation,emphasizing that the initial C/Fe molar ratio and formation pathway are the main factors affecting the EPS retention mechanism.（3）The differences in the chemical fractionation of EPS components by different formation pathways of the EPS-Fh associations are elucidated.The EPS-C/N/P elements are disproportionately retained during the adsorption and coprecipitation process,and the selective fractionation effect of the adsorption process on EPS elements is greater than that of the coprecipitation process.It was observed that the protein components of EPS are relatively enriched on the surface of the adsorption complexes,while the polysaccharide components accumulate on the surface of the coprecipitates.Compared with the unreacted EPS and polysaccharide C components,the aliphatic C components are preferentially adsorbed and co-precipitated with Fh.The amide C components increase during adsorption process and decrease during coprecipitation process.The chemical fractionation behaviors of the EPS-C species are further analysed by advanced spectroscopic techniques.It is found that the adsorption process selectively retains the carboxyl/amide C-containing components,while the coprecipitation process relatively enriches the alkyl C-and O-alkyl C-containing components.The results indicate that the association formation pathway plays an important role in the binding and selective retention of EPS components,which provides an important theoretical support for understanding the Fe-C coupling regulation of microbial-derived organic matter reactivity and cycling in the natural environment.