S1 Supporting Information - Environmental Science & Technology ...
S1 Supporting Information - Environmental Science & Technology Ultrahigh Resolution Mass Spectrometry and Indicator Species Analysis to Identify Marker Components of Soil- and Plant Biomass-Derived Organic Matter Fractions Tsutomu Ohno,*, †, Zhongqi He, ‡, Rachel L. Sleighter, ¶, C. Wayne Honeycutt, ‡, Patrick G. Hatcher, ¶ Department of Plant, Soil, and Environmental Sciences, 5722 Deering Hall, University of Maine, Orono, ME 04469-5722; New England Plant, Soil, and Water Laboratory, USDA-ARS, Orono, ME 04469; and Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529 *Corresponding author phone: 207-581-2975; e-mail: [email protected]. †
University of Maine, ‡ USDA-ARS, ¶ Old Dominion University
Number of pages: 19 Number of Figures: 1 Number of Tables: 6 Contents: Soil and Plant Biomass Analysis Methods.
S2
EEM/PARAFAC Analysis Methods.
S3
Soil Chemical Properties.
S4
Climate and selected physical and chemical properties of the soils used for the extraction of the water-extractable and humic substances fractions of the soil organic matter. (Table S1)
S5
Average percentage distribution of lipid, protein, lignin, carbohydrate, unsaturated hydrocarbon, and condensed aromatic structure class organic matter components for plant biomass water extractable organic matter, soil water extractable organic matter, mobile humic acid, and calcium humic acid fractions. (Table S2)
S6
Number of components found in n number of shared samples. (Fig. S1)
S7
Indicator components for the calcium bound humic acid (CaHA) fraction of soil organic matter using an indicator value threshold of 50 (Table S3)
S8
Indicator components for the mobile humic acid (MHA) fraction of soil organic matter using an indicator value threshold of 50. (Table S4)
S11
Indicator components for the water-extractable organic matter (WEOM) fraction of the soils using an indicator value threshold of 50. (Table S5)
S12
Indicator components for the water-extractable organic matter fraction of plant biomass using an indicator value threshold of 50. (Table S6)
S14
Literature Cited
S19
S2 Soil and Plant Biomass Analysis. The humic substances extraction procedure used in this work was that reported previously (S1). Briefly, 35 g of soil was shaken in 350 mL of 0.25 M NaOH under N2 gas for 20 h. After centrifuging, the supernatant of the soils was decanted and acidified to pH 2.0 to precipitate the MHA fraction. Then, the extraction residues were repeatedly washed with 0.1 M HCl followed by centrifuging and disposal of the supernatant, until the pH of the supernatant remained below 1.3. The residues were similarly washed again in water to remove excess HCl until the supernatant pH rose above 2.0. The recalcitrant humic acid fraction that had been bound to Ca was then extracted by NaOH using the above extraction procedure to obtain the CaHA fraction. The fractions were dialyzed for 2 d against successively weaker HCl solutions and for a third day against water to H-saturate the fractions. The fractions were freeze-dried and stored in desiccators under dark conditions until use. Soil WEOM was extracted by adding 10.0 mL of deionized-H2O to 1.00 g of soil in a 15-mL centrifuge tube. The suspensions were shaken on an orbital shaker for 30 min at room temperature (22 ± 1 ºC), centrifuged at 900×g for 30 min, and filtered through 0.45 µm Acrodisk syringe filters. The extraction period was selected to minimize microbial WEOM alteration during extraction (S2). The plant-derived WEOM was extracted with a 40:1 (v/w) water to sample ratio using cold water and refrigerating (4°C) the suspension for 18 h with periodic shaking by hand (S3). The suspensions were then centrifuged (900×g) for 30 min prior to vacuum filtering through 0.4-μm pore size polycarbonate filters. Each extraction for the MHA, CaHA, soil WEOM, and plant WEOM was performed in triplicate for analysis by EEM/PARAFAC.
S3 EEM/PARAFAC Analysis. Excitation-emission matrix (EEM) fluorescence spectroscopy has been extensively used for organic matter characterization (S4,S5). Parallel factor analysis (PARAFAC), a multi-way statistical method, decomposes a suite of EEM landscapes into chemically meaningful spectral components (S6). All organic matter solutions were diluted with DI-H2O to set absorbance at 240 nm to 0.10 to minimize inner-filtration effects. Fluorescence measurements were obtained using a Hitachi F4500 spectrofluorometer with the excitation range set from 240 to 400 nm and the emission range set from 300 to 500 nm in 3 nm increments. Instrumental parameters were excitation and emission slits, 5 nm; response time, 8 s; and scan speed 240 nm min-1. A DI-H2O blank EEM was subtracted from each sample EEM to remove Raman scatter effects. Rayleigh scatter lines and the region immediately adjacent to the region where the emission and excitation wavelengths are equal were removed by setting the fluorescence intensity values of these data points as missing as well as where the emission wavelength is less than the excitation wavelength (upper left hand corner) which is physically not possible, and these data pairs are set to zero. The PARAFAC modelling was conducted with MATLAB version 7.1, Release 14 (Mathworks, Natick, MA) using PLS_Toolbox version 3.5 (Eigenvector Research, Manson, WA). A non-negativity constraint was applied to the parameters to allow only chemically relevant results because negative concentrations and fluorescence intensities are chemically impossible, assuming that quenching and inner filter effects are negligible. PARAFAC models with two to eight components were fitted to the data in order to investigate the correct number of components. Each extraction, in triplicate, was analyzed by EEM fluorescence. The three replicates gave PARAFAC results, where the percent coefficient of variation was less than 10%, indicating that our reproducibility was well within the range expected for soil extractions. Because the EEM/PARAFAC analysis shows that the triplicate extractions are reproducible and representative, the three replicates were pooled together into a single sample for analysis by ESI-FT-ICR-MS.
S4 Soil Properties. The ten soils investigated span a range of climate regions and included six of the ten USDA soil textural classes that are factors in the SOM formation process. Climate data of the field sites and selected physical and chemical properties of the soils are shown in Table S1. Total soil C content ranged from 0.50% found in the sandy Valentine soil to 3.51% in the silty clay loam Catlin soil. Total soil C was significantly (p=0.05) positively correlated to clay content and negatively to sand content, indicating the prominence of clay surfaces for the stabilization of C in soils. Total soil C was also significantly rank correlated to mean annual precipitation, which supports the findings of Jenny (S7) on the importance of climatic factors in SOM levels. Soil cation exchange capacity (CEC) was strongly correlated with clay content (p=0.001) and to a lesser degree with total soil C (p=0.05). The soil set has a wide range of mineralogy representation (Table S1). In general, the soils which had >10 cmol kg-1 CEC contained greater amounts of the higher charge minerals such as montmorillonite and mica, while the soils with
University of Maine, ‡ USDA-ARS, ¶ Old Dominion University
Number of pages: 19 Number of Figures: 1 Number of Tables: 6 Contents: Soil and Plant Biomass Analysis Methods.
S2
EEM/PARAFAC Analysis Methods.
S3
Soil Chemical Properties.
S4
Climate and selected physical and chemical properties of the soils used for the extraction of the water-extractable and humic substances fractions of the soil organic matter. (Table S1)
S5
Average percentage distribution of lipid, protein, lignin, carbohydrate, unsaturated hydrocarbon, and condensed aromatic structure class organic matter components for plant biomass water extractable organic matter, soil water extractable organic matter, mobile humic acid, and calcium humic acid fractions. (Table S2)
S6
Number of components found in n number of shared samples. (Fig. S1)
S7
Indicator components for the calcium bound humic acid (CaHA) fraction of soil organic matter using an indicator value threshold of 50 (Table S3)
S8
Indicator components for the mobile humic acid (MHA) fraction of soil organic matter using an indicator value threshold of 50. (Table S4)
S11
Indicator components for the water-extractable organic matter (WEOM) fraction of the soils using an indicator value threshold of 50. (Table S5)
S12
Indicator components for the water-extractable organic matter fraction of plant biomass using an indicator value threshold of 50. (Table S6)
S14
Literature Cited
S19
S2 Soil and Plant Biomass Analysis. The humic substances extraction procedure used in this work was that reported previously (S1). Briefly, 35 g of soil was shaken in 350 mL of 0.25 M NaOH under N2 gas for 20 h. After centrifuging, the supernatant of the soils was decanted and acidified to pH 2.0 to precipitate the MHA fraction. Then, the extraction residues were repeatedly washed with 0.1 M HCl followed by centrifuging and disposal of the supernatant, until the pH of the supernatant remained below 1.3. The residues were similarly washed again in water to remove excess HCl until the supernatant pH rose above 2.0. The recalcitrant humic acid fraction that had been bound to Ca was then extracted by NaOH using the above extraction procedure to obtain the CaHA fraction. The fractions were dialyzed for 2 d against successively weaker HCl solutions and for a third day against water to H-saturate the fractions. The fractions were freeze-dried and stored in desiccators under dark conditions until use. Soil WEOM was extracted by adding 10.0 mL of deionized-H2O to 1.00 g of soil in a 15-mL centrifuge tube. The suspensions were shaken on an orbital shaker for 30 min at room temperature (22 ± 1 ºC), centrifuged at 900×g for 30 min, and filtered through 0.45 µm Acrodisk syringe filters. The extraction period was selected to minimize microbial WEOM alteration during extraction (S2). The plant-derived WEOM was extracted with a 40:1 (v/w) water to sample ratio using cold water and refrigerating (4°C) the suspension for 18 h with periodic shaking by hand (S3). The suspensions were then centrifuged (900×g) for 30 min prior to vacuum filtering through 0.4-μm pore size polycarbonate filters. Each extraction for the MHA, CaHA, soil WEOM, and plant WEOM was performed in triplicate for analysis by EEM/PARAFAC.
S3 EEM/PARAFAC Analysis. Excitation-emission matrix (EEM) fluorescence spectroscopy has been extensively used for organic matter characterization (S4,S5). Parallel factor analysis (PARAFAC), a multi-way statistical method, decomposes a suite of EEM landscapes into chemically meaningful spectral components (S6). All organic matter solutions were diluted with DI-H2O to set absorbance at 240 nm to 0.10 to minimize inner-filtration effects. Fluorescence measurements were obtained using a Hitachi F4500 spectrofluorometer with the excitation range set from 240 to 400 nm and the emission range set from 300 to 500 nm in 3 nm increments. Instrumental parameters were excitation and emission slits, 5 nm; response time, 8 s; and scan speed 240 nm min-1. A DI-H2O blank EEM was subtracted from each sample EEM to remove Raman scatter effects. Rayleigh scatter lines and the region immediately adjacent to the region where the emission and excitation wavelengths are equal were removed by setting the fluorescence intensity values of these data points as missing as well as where the emission wavelength is less than the excitation wavelength (upper left hand corner) which is physically not possible, and these data pairs are set to zero. The PARAFAC modelling was conducted with MATLAB version 7.1, Release 14 (Mathworks, Natick, MA) using PLS_Toolbox version 3.5 (Eigenvector Research, Manson, WA). A non-negativity constraint was applied to the parameters to allow only chemically relevant results because negative concentrations and fluorescence intensities are chemically impossible, assuming that quenching and inner filter effects are negligible. PARAFAC models with two to eight components were fitted to the data in order to investigate the correct number of components. Each extraction, in triplicate, was analyzed by EEM fluorescence. The three replicates gave PARAFAC results, where the percent coefficient of variation was less than 10%, indicating that our reproducibility was well within the range expected for soil extractions. Because the EEM/PARAFAC analysis shows that the triplicate extractions are reproducible and representative, the three replicates were pooled together into a single sample for analysis by ESI-FT-ICR-MS.
S4 Soil Properties. The ten soils investigated span a range of climate regions and included six of the ten USDA soil textural classes that are factors in the SOM formation process. Climate data of the field sites and selected physical and chemical properties of the soils are shown in Table S1. Total soil C content ranged from 0.50% found in the sandy Valentine soil to 3.51% in the silty clay loam Catlin soil. Total soil C was significantly (p=0.05) positively correlated to clay content and negatively to sand content, indicating the prominence of clay surfaces for the stabilization of C in soils. Total soil C was also significantly rank correlated to mean annual precipitation, which supports the findings of Jenny (S7) on the importance of climatic factors in SOM levels. Soil cation exchange capacity (CEC) was strongly correlated with clay content (p=0.001) and to a lesser degree with total soil C (p=0.05). The soil set has a wide range of mineralogy representation (Table S1). In general, the soils which had >10 cmol kg-1 CEC contained greater amounts of the higher charge minerals such as montmorillonite and mica, while the soils with
