Extraction Of Soil Humic Substances And Their Biogeochemical Characteristics In Forest,Agriculture,Riverside And Degraded Soils

Soil organic matter（SOM）is mostly composed of soil humic substances（HS）.Soil HS studied in this research mainly includes humic acid（HA）,fulvic acid（FA）and newly defined protein-like substances（PLS）,which plays many biogeochemical processes and functions such as agricultural productivity,retains nutrients and pollutants,complexation properties with trace elements,sorption and desorption properties,and the occurrence of redox reactions in soil matrixes.Soil pollution and its amendments to the immobilization of potentially toxic elements and organic pollutants are extensively crucial for the assessment and characteristics of soil HS.Their potential mobilization/transportation behaviors via water discharge/rainwater runoff could export nutrients,trace elements,toxic metals,HS components,organic and inorganic carbon are vitally not only important for soil fertility,agricultural productivity and all biogeochemical properties in soil environments,but also enormously important to ambient aquatic systems,thereby maintaining the water quality and aquatic microbial food webs.Stability characteristics of individual soil HA,FA and PLS during the water discharge/rainwater runoff are the prime determining factors for soil management,amendments and composts to ascertain their maximum efficiency under diverse soil conditions.This is the first aim of this study.In addition to the influence of p H,different soils HS in different environments are usually affected by existing environmental conditions,and thus to assess such biogeochemical transformation of HS compositions under existing soil environmental conditions,how does HS compositions could link with each other in diverse soils,such as forest,agriculture,riverside and degraded soils.This is the second aim of this study.HS in this study is operationally separated as two fractions:labile state of HS that can soluble relatively low molecular weight humic substances and alkaline extracted complexed state of HS that can dissolute relatively larger molecular weight humic substances.The first aim of this study focused on p H influences on the stability characteristics of soil HA,FA,and PLS.The functional groups bound to HS compositions are primarily responsible for occurrences of all such biogeochemical processes and functions by regulating the physical,chemical,and biological properties in soil and water systems,predominantly controlled by the media’s p H.p H influences on soil HA,FA and PLS of labile state and complexed state,which are the determining factors for each substrate’s stability characteristics and their mechanisms,are unclear to date.This study thus aimed to ascertain the individual changes of extracted labile and complexed states of HS compositions in two soils（paddy and maize）under different p H conditions（p H 1 to p H 13）for the first time using fluorescence（excitation-emission matrix,EEM）spectroscopy combined with parallel factor（PARAFAC）modeling.EEM-PARAFAC is widely used to characterize fluorophores/functional groups of individual HA,FA and PLS in terms of fluorescence properties（peaks and intensities）.Correspondingly,this study was measured absorption properties,dissolved organic carbon（DOC）,and nutrients（NH₄⁺,NO₂^-,NO₃^-and dissolved organic nitrogen（DON））.（1）The EEM-PARAFAC results demonstrated that the fluorescence peaks of HA（C and A）,FA（M and A）and PLS(humic part:M and A;protein part:T and T_UV),as well as their intensities,were wildly varied under different p H conditions.It is shown that the fluorescence peaks for the labile state of each substrate were commonly detected at shorter excitation-emission wavelength regions than in complexed state ones.At p H 7-8,all three substrates in the labile state showed its originality,remaining in existing soils.The results also showed that the labile state of soil HA was not detected entirely at acidic p H condition from p H 6 to p H 1,suggesting that labile state HA in acidic soil is constantly remained as a stable state in the soil layer due to their insolubility nature during any natural/artificial water discharge/runoff.But complexed PLS state HA was detected until p H 3 after which it was not detected at p H 2 to p H 1by EEM-PARAFAC,suggesting that complexed state HA is labile for acidic p H up to3 and is completely precipitated at p H 1-2.The mechanism behind such insolubility/solidification of labile and complexed state HA could be stated that the net intermolecular force among the functional groups in HA is higher compared to their net external force that is derived from the hydrogen-bonding of the functional groups of HA with its aqueous phase water molecules and its dissolved trace metals.Conversely,labile and complexed state FA has weakly appeared at p H 7-8,which is the originality that existed in soils.But different p H conditions have extensively altered the FA,showing disappeared at p H 9-10 and then newly appeared at p H 11-12.But FA has strongly exhibited two strong peaks（M and A）at acidic p H 1-2,primarily responsible for solubility nature at all acidic conditions.PLS was shown to appear its originality at p H 7-8,the predominant form in two soils.But the humic part of PLS is dominant at low p H or acidic conditions whilst its protein fraction is dominant at acidic p H 5 to alkaline condition up to p H 10,which predominantly observed in alkaline extracted solution in both soils.But such a trend did not consistently observe in labile state because the protein part of PLS is highly degraded in soil matrixes under existing environmental conditions.The mechanism behind solubility of labile and complexed state FA and PLS is the opposite of the insolubility of HA.It stated that due to the existence of a low number of the functional groups in FA or PLS,the net external force that is derived from the hydrogen-bonding of the functional groups of FA or PLS with its aqueous phase water molecules and its dissolved trace metals is higher compared to those of the net intermolecular force among the functional groups in FA or PLS.（2）Under different p H conditions,both labile and complexed state HS exhibited highly variable in their absorbance spectra,apparently differed from 220 nm up to 500nm because of their high absorbance.Still,very low absorbance was observed in the longer wavelength region（500-800 nm）.The absorption co-efficient results demonstrated several characteristic features for labile and complexed state HS solutions in two soils,which can be summarized:As the p H value increased,theα（280）andα（350）values of the paddy soil HS samples extracted from the water first rose and then decreased.Both were reduced at p H 11,and their minimum values（7.46,3.63）all appeared at p H 1.With the increase of p H value,the values ofα（280）andα（350）of maize soil samples extracted from water gradually increased,and their maximum（20.37,9.24）appeared at p H 11.Due to the influence of the solution’s acidity and alkalinity,hydrogen bonds may be formed between the solvent molecules and the HS molecules,or the dipole of the polar solvent molecules may increase the polarity of the HS molecules,that is,the solvent effect occurs.With the increase of p H value,the SR values of paddy soil samples and maize soil samples extracted from water showed the first increase and decreased.SR of different samples showed different p H dependencies.This may be attributed to some unknown factors other than p H.The higher SR value indicated that the MW of HS was relatively low.（3）DOC concentration,DON and NO₂^-were significantly decreased under acidic p H conditions,but NO₃^-and NH₄⁺did not change considerably over p H conditions.These results,therefore,imply that p H influences are significantly occurred on three HS compositions,thereby controlling their biogeochemical alterations under different p H conditions in acidic and alkaline soils,which could be the determining factors to remain their contents in soils along with their biogeochemical leaching in soil depths and ambient waters.The second aim of this study focuses on biogeochemical characteristics and transformations of soil HS and nutrients in forest,agriculture,riverside and degraded soil.This study extracted the labile and complexed state of soil solution to assess these issues using a new extraction method from 60 soil samples individually,which collected from three provinces（Hubei,Shaanxi and Inner Mongolia）.Soil solutions were then used to analyze the EEM spectra,absorbance spectra,high-pressure size exclusion chromatography（HPSEC）,DOC,DIC,nutrients(NH₄⁺,NO₃^-,NO₂^-,TN,PO₄^3-,TP and Si O₃^2-).To identify each HS components from EEM spectra,this studied applied EEM-PARAFAC modeling.High-performance size-exclusion chromatography（HPSEC）was applied to assess the molecular weight（MW）of HS composition in both labile and complexed states in diverse soils.The main findings are as follows:（1）The soil is generally alkaline（7.97±0.01,8.45±0.00）in Shaanxi and Inner Mongolia and the soil is generally acidic（6.84±0.01）in Hubei,showing the overall acidity characteristics in southern China and alkaline in northern China.The forest soil has the lowest p H and EC values and the highest STN value among the three provinces.The soil humidity material order is as follows:Hubei>Shaanxi>Inner Mongolia.Shaanxi soil samples were composed mainly of sand,supplemented by silt and clay.Samples of Hubei soil were composed predominantly of silt,followed by sand and clay.Samples from Inner Mongolia were composed predominantly of sand,followed by silt and clay.（2）The DOC and DIC content in the labile state is classified in the HS derived from the three provinces as Shaanxi>Inner Mongolia>Hubei,and the DOC and DIC content in the complexed state are classified as Hubei>Shaanxi>Inner Mongolia.In three provinces,the DOC content and concentrations of nitrogen-nutrients in forest soil are comparatively higher than in agriculture,riverside,and desert soils,which could derive from a significant amount of litterfall each year on the forest floor and their subsequent humification and leaching processes.The content of DOC and DIC was much higher than labile state in complexed state.The extracted DIN species followed the order of NO₃^->NH₄⁺>NO₂^-in water and the order of NH₄⁺>NO₃^->NO₂^-in complexed state.The content of DOP and silicon was much higher than labile state in complexed state.（3）To understand the relationship between p H effects and soil organic matter in three provinces,this study conducted the Pearson correlation between p H values and soil DOC concentration in each province.The results showed that a significant correlation between p H values and DOC was observed in labile state for only Hubei province,but other two provinces did not follow any significant correlation.But in complexed state,the p H was significantly correlated with soil DOC in each province.（4）At each soil sampling site in three provinces,the absorption spectrum of HS has adopted the law of exponential decay from the ultraviolet region into the visible region.To assess the representative absorption coefficients for HA,FA and PLS,it is essential to account for 350 nm,300 nm,280 nm based on the consideration of peak C of HA,peak M of FA,peak T of PLS plus peak A of HA,peak A of FA,and peak T_UVof PLS,respectively.The three values display the following changes in scale for all sites:α（280）>α（300）>α（350）.The HS in the complexed state from the three provinces and the threeα-values of the forest soil all had the highest values characteristics.The threeα-values of HS in the labile state of forest soils in Hubei and Inner Mongolia are all the smallest.（5）The results from EEM-PARAFAC modeling showed that for all soils,the HS using water and alkaline extracts featured a maximum of four HS components that could be ascribed to HA,FA C-type,FA M-type,and PLS.In labile state of Shaanxi soils,EEM-PARAFAC modeling has identified a maximum of three HS components,including HA,FA C-type,and PLS in forest soils.But only two components（HA and PLS）were detected in the desert,riverside and agriculture soils.Four HS components were detected during complexed state in forest,agriculture,and desert soils except for riverside soil that detected three HS components（HA,FA M-type,and PLS）.In labile state of Hubei soils,EEM-PARAFAC modeling was identified HA,FA M-type,and PLS in forest soil,but only two HS components（HA and PLS）were detected in the riverside and agriculture soils.In complexed state,EEM-PARAFAC analysis on the sample EEM spectra demonstrated four HS components in riverside and agricultural soils except from forest soils,which existed three HS components（HA,degraded FA M-type）.In labile state of Inner Mongolia soils,EEM-PARAFAC modeling was identified three components（HA,FA C-type,and PLS）in desert,forest and agricultural soils.In complexed state,it was identified all four HS components in desert and agricultural soils except forest soil,which identified three HS components（HA,FA M-type and PLS）.In Shaanxi province,the peak C fluorescence intensity of forest soil in labile state is the largest.The peak C and peak M fluorescence intensities of desert soil are the largest in complexed state.In Hubei province,the peak C fluorescence intensity of forest soil in labile state and complexed state is the largest.In Inner Mongolia,the peak C and peak T fluorescence intensity of forest soil in labile state are the largest.Undoubtedly,the unequivocal identification of soil HA,FA,and PLS is beneficial for understanding their biogeochemical transformations and related consequences in diverse soil environments.（6）HPSEC was effectively applied to ascertain the molecular weight（MW）of HS compositions in both labile and complexed states in all 60 soil samples.At the same time,it was estimated the weight average molecular weight（M_W）,the number average molecular weight（M_N）,and the polydispersity coefficient（ρ）representing the degree of dispersion of the DOM MW distribution.The HS elution curve shows similar properties,including a total of 2 to 5 peaks,and the M_W of HS ranged from 1000 to 35k Da,and the components with MW of 15-25 k Da account for more than 90%of the total DOM.In labile state,the components with MW ranging from 15 to 25 k Da were detected the highest contents,accounting for 34%to 100%,followed by components with MW greater than 25 k Da（1%-66%）,the content of parts with a molecular weight of less than 15 k Da is 1%to 26%.In complexed state,the components with MW ranging from 15 to 25 k Da detected the highest content,accounting for 76%to 100%,followed by those with MW less than 15 k Da ranging from 3%to 24%,and those with an MW greater than 25 k Da Component（1%-6%）.These results suggest that the HS in the soil is mainly composed of DOM with an MW of>15 to<25 k Da.