Multielemental speciation analysis of fungi porcini (Boletus edulis) mushroom by size exclusion liquid chromatography with sequential on-line UV-ICP-MS detection

An analytical methodology to determine the molecular weight (MW) distribution patterns of several elements among different compounds present in commonly consumed edible mushrooms is presented in this work. A hyphenated technique based on size exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used. The association of the elements to high and low MW fractions was confirmed with sequential detection by UV and ICP-MS. Separation of the fractions was performed by injecting a 100 microL sample volume to a Superdex 75 column. The effect of different mobile phases on the separation was evaluated. Additionally, three different extraction conditions including 0.05 mol L(-1) NaOH, 0.05 mol L(-1) HCl, and hot water at 60 degrees C were applied to extract the elemental species from the mushroom samples. Significant differences were observed in the chromatograms depending on the extraction conditions utilized. Optimization of the experimental variables involved in the SEC-UV-ICP-MS coupling was carried out. The method was applied to investigate the fractionation patterns of Bi, Co, Cu, Fe, I, Mo, Ni, Se, and Zn in fungi porcini (Boletus edulis) mushroom. The results obtained in this work indicate an important association of most of the elements to high MW fractions.     

Se-enriched mycelia of Pleurotus ostreatus: distribution of selenium in cell walls and cell membranes/cytosol

The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.     

Studies of various elements of nutritional and toxicological interest associated with different molecular weight fractions in Brazil nuts

On-line hyphenation of size exclusion chromatography (SEC), UV, and inductively coupled plasma mass spectrometry (ICP-MS) was used to study the molecular weight distribution patterns of several elements in Brazil nuts (Bertholletia excelsa). This technique was used for the elemental speciation of different elements of nutritional and toxicological interests such as Mg, Fe, Co, Mo, Ag, Hg, and Pb. Elemental fractionation in Brazil nuts was studied using a Superdex peptide column with resolving capacity in the range of 14 to 0.18 kDa. Three different mobile phases, Tris buffer solution (pH 8.0), phosphate buffer (pH 7.5), and CAPS buffer solution (pH 10.0), were tried for the SEC fractionation. Size exclusion fractionation of all the extracted solutions was performed using a 50 mmol L(-)(1) Tris buffer (pH 8) as the mobile phase at a flow rate of 0.6 mL min(-)(1). Three different extractions, 0.05 mol L(-)(1) NaOH, 0.05 mol L(-)(1) HCl, and hot water at 60 degrees C, were performed, and the association of elements with various molecular weight fractions was evaluated. Total elemental concentrations in the extracted samples were determined and compared with the values obtained after total digestion to calculate the recovery values. Generally, high extraction efficiency was obtained with the NaOH solution as compared with HCl and hot water except in the case of magnesium, for which HCl was found to be a good extractant. Chromatographic elution profiles for these extractions were quite distinct from each other in most cases. Most of the elemental species were found to be associated with high molecular weight fractions. To study the differences obtained during the sample-processing step, the results obtained for nuts with shell were treated differently from those obtained for nuts purchased without shell and were compared.     

Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms

The aim of the study was to determine the contents of mineral elements (Ca, K, Mg, Na, P, Cu, Fe, Mn, Cd, Pb, and Se), vitamins (B(1), B(2), B(12), C, D, folates, and niacin), and certain phenolic compounds (flavonoids, lignans, and phenolic acids) in the cultivated mushrooms Agaricus bisporus/white, Agaricus bisporus/brown, Lentinus edodes, and Pleurotus ostreatus. Selenium, toxic heavy metals (Cd, Pb), and other mineral elements were analyzed by ETAAS, ICP-MS, and ICP methods, respectively; vitamins were detected by microbiological methods (folates, niacin, and vitamin B(12)) or HPLC methods (other vitamins), and phenolic compounds were analyzed by HPLC (flavonoids) or GC--MS methods (lignans and phenolic acids). Cultivated mushrooms were found to be good sources of vitamin B(2), niacin, and folates, with contents varying in the ranges 1.8--5.1, 31--65, and 0.30--0.64 mg/100 g dry weight (dw), respectively. Compared with vegetables, mushrooms proved to be a good source of many mineral elements, e.g., the contents of K, P, Zn, and Cu varied in the ranges 26.7--47.3 g/kg, 8.7--13.9 g/kg, 47--92 mg/kg, and 5.2--35 mg/kg dw, respectively. A. bisporus/brown contained large amounts of Se (3.2 mg/kg dw) and the levels of Cd were quite high in L. edodes (1.2 mg/kg dw). No flavonoids or lignans were found in the mushrooms analyzed. In addition, the phenolic acid contents were very low.     

Selenium treatment alters the effects of UV radiation on chemical and production parameters in hybrid buckwheat

Ambient UV radiation is recognised as an important environmental factor in the regulation of plant growth and development, and selenium (Se) as a beneficial nutrient that can increase plant tolerance to different environmental constraints. The effects on hybrid buckwheat plants of full (+UV) and reduced (?UV) ambient UV radiation without (?Se) and with (+Se) foliar Se treatment (10?mg?L^?1 sodium selenate) provided the four conditions of Se?UV–, Se–+UV, +Se?UV and +Se+UV. Plant morphological, biochemical and physiological properties were examined, along with leaf elemental composition and content, efficiency of Se enrichment, and production parameters. Leaf anatomical parameters under all conditions were mainly affected by UV radiation, and less so by Se. +Se+UV plants showed a trade-off between primary and secondary metabolism, which resulted in high levels of protective substances (e.g. anthocyanins, UV absorbing compounds), and low levels of photosynthetic pigments. All +UV plants were significantly shorter in comparison to those under the reduced ?UV, while biomass production was highest for ?Se+UV plants and lowest for +Se+UV plants. +Se plants accumulated ～20-fold greater Se content compared to ?Se plants, and full ambient UV radiation partly reduced this effect. +UV plants showed increased phosphorus content in leaves, independent of Se status, while +Se plants showed increased K content. Si content was increased by Se treatment and decreased by the full UV exposure. Se treatment and the ambient UV radiation, separately have positive effects on growth and production of this hybrid buckwheat, while the combination of the Se treatment and ambient UV resulted in lower yields. However, under these conditions (+Se+UV), the buckwheat plants established good protection against the different environmental constraints that are becoming more and more frequent due to changes to our climate.     

Quantification of seed ionome variation in 90 diverse soybean (Glycine max) lines

Climate change and rising carbon dioxide (CO₂) levels are expected to reduce the mineral nutrient content of soybean seeds. The main objective of this study was to survey diverse soybean germplasm for variation in seed elemental concentrations and their relationships between elements, protein content, and individual seed weight. Seeds from 90 soybean genotypes were weighed and subjected to inductively coupled plasma-mass spectrometry (ICP-MS) ionomics analysis and Carbon/Nitrogen (C/N) analysis to determine protein. The results demonstrated substantial variation with the possibility of significantly improving most mineral nutrients, especially selenium (Se), copper (Cu), iron (Fe), and manganese (Mn). This diverse survey identifies genotypes that can complement existing soybean breeding programs for improving seed nutritional quality. Correlation analysis identified two clusters of co-variant elements: zinc (Zn), phosphorus (P), and sulfur (S) as well as Zn, Cu, Se, and rubidium (Rb) were positively correlated with each other. Tolerable upper limits of Rb intake are not defined for humans illustrating the need to monitor trace elements along with desirable nutrients.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

Effect of Cooking and Storage on the Amount and Molecular Weight of (1→3)(1→4)-β-d-Glucan Extracted from Oat Products by an In Vitro Digestion System

The extractability and molecular weight of β-glucan in oat bran, oat bran muffins, and oat porridge and the changes taking place during processing and storage were studied. The β-glucan was extracted using hot water and a thermostable α-amylase and by an in vitro system that simulated human digestion. Molecular weight (MW) of the extracted β-glucan was determined using high-performance size-exclusion chromatography. Hot-water treatment extracted 50–70% of total β-glucan in oat bran samples and rolled oats. The chromatographic peak MW of extracted β-glucan was in the 1.4–1.8 × 10⁶ range. Using the in vitro digestion system, 12–33% of total β-glucan in bran and rolled oats was solubilized, and peak MW was in the same range as β-glucan extracted by hot-water treatment. In muffins, 30–85% of total β-glucan was solubilized by in vitro digestion, with a major difference in extractability among muffins from different recipes. Peak MW of extracted β-glucan was lower in all muffins when compared to original bran. During frozen storage, extractable β-glucan decreased by >50% in all muffins, but no change in peak MW of extracted β-glucan was detected.     

Nutritional and toxicological importance of macro, trace, and ultra-trace elements in algae food products

The content of 5 macro elements (Na, K, Ca, Mg, and P), 6 trace elements (Fe, Mn, Zn, Cu, Se, and I), and 4 ultra-trace elements (As, Pb, Cd, and Hg) in 34 edible dried seaweed products of brown algae (Laminaria sp., Undaria pinnatifida, and Hizikia fusiforme) and red algae (Porphyra sp.) originated from China, Japan, and Korea and bought by retail in Germany was determined. The content of these elements was analyzed by spectrometric methods (ICP-AES, ICP-MS, HGAAS, and CVAAS). Assuming a daily intake with 5 g FM of algae, the contribution of the essential elements to the diet is low, with the exception of I. Brown algae contained as much as 1316 +/- 1669 mg of I/kg FM. More than 4000 mg of I/kg FM were found in several Laminaria sp. Moreover, some brown algae, such as Hizikia fusiforme, had high contents of total As (87.7 +/- 8.2 mg/kg FM).     

Influence of molecular weight fractions isolated from roasted malt on the enzyme activities of NADPH-cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells.

In the present study, water-soluble nonenzymatic browning products (melanoidins) formed in roasted malt were separated, quantified, and investigated for their effects on detoxifying mechanisms in intestinal Caco-2 cells. The melanoidins were prepared from roasted malt by hot water extraction, and the water-soluble compounds were separated into different molecular weight (MW) fractions by gel filtration chromatography. By monitoring the effluent at 300 nm, seven molecular fractions I-VII were consecutively collected, revealing that approximately 2.3% of the water-soluble compounds had mean MWs between 10000 and 30000 Da. Thus, the bulk of water-soluble malt melanoidins consisted of MW > 30000 Da, among which approximately 58% showed mean MWs between 60000 Da and 100000 Da, whereas approximately 32% exhibited mean MWs of 200000 Da. Biotransformation enzyme activities of NADPH-cytochrome c-reductase (CCR) and glutathione-S-transferase (GST) were analyzed in Caco-2 Cells after 48 h of exposure to the different MW fractions. The low MW fraction of 10000 Da was most effective in activating the CCR and the GST activities (+122 and +33% vs control, respectively). The majority of the mid molecular weight compounds tested showed an activating effect on CCR activity and an inhibitory effect on GST activity. These effects were most pronounced for compounds of up to 70000 Da and >200000 Da but less distinct for fractions of an average molecular weight of 100000 Da.     

Distribution of carbon, nitrogen, phosphorus, sulphur, and acidity in two molecular weight fractions of organic matter in soil chronosequences

Ten different topsoils from three soil chronosequences were pretreated with 0.1 m HCl and 0.1 m HCl: 0.3 m HF, then extracted with neutral 0.1 m Na₄P₂0₇, followed by 0.5 m NaOH. Pretreatments and extracts were purified and fractionated into two nominal molecular weight (MW) fractions (> 50000 and < 50000) using gel filtration. The distribution between the two MW fractions of total carbon (C), nitrogen (N), phosphorus (P), sulphur (S), total acidity, and carboxyl (COOH) acidity and their ratios relative to C were determined. Organic matter in the > 50000 MW fraction contained lower N, P, S and acidity ratios relative to C than those of the < 50000 MW fraction. The chemical nature ofthe > 50000 MW fraction remained unaffected by soil development or vegetation, whereas that of the < 50000 MW fraction changed with soil age and appeared to be influenced by vegetation. The results strongly suggest that two chemically different extractable fractions of organic matter can be isolated from most soils, represented by the active fraction (< 50000 MW) and the relatively large (> 50000 MW) and less active (passive) fraction. A three-phase system of organic matter is proposed comprising the two extractable fractions and the non-extractable component.     

Analyses of glycolipids from fish, shellfish, and sea snake lipids by high-performance liquid chromatography.

To determine the existence of glycolipids (neutral glycosphingolipid and glycoglycerolipid) in sea snake, round frigate mackerel, sardine, sea urchin, and abalone, we performed silica gel chromatography and high-performance liquid chromatography (HPLC) using an Aquasil-SS column and a C(8)-reversed phase silica gel column. HPLC with a UV absorption detector was used to analyze neutral glycosphingolipid. These chromatograms showed typical peaks in round frigate mackerel lipid, in sea snake crude fat, in abalone intestine lipid, and in sea urchin intestine lipid. UV-HPLC was also used to analyze glycoglycerolipid. These chromatograms indicated a large peak in round frigate mackerel lipid and a small peak in purified sardine oil. In addition, we observed the same peaks in the glycolipid fraction of round frigate mackerel muscle lipids and sea snake crude fat using a differential refractometer detector. The results of this study suggest that the peaks are neutral glycosphingolipid or glycoglycerolipid and that neutral glycosphingolipid and glycoglycerolipid may have specific physiological functions in each living creature.     

Selenium species in selenium-enriched and drought-exposed potatoes

The aim of this work was to study selenium (Se) speciation in the potato ( Solanum tuberosum L.) cultivar Desiree, enriched in Se by foliar spraying with a water solution containing 10 mg of Se/L in the form of sodium selenate. Four combinations of treatments were used: well-watered plants with and without Se foliar spraying and drought-exposed plants with and without Se foliar spraying. Water-soluble Se compounds were extracted from potato tubers by water or enzymatic hydrolysis with the enzyme protease XIV, amylase, or a combination of protease XIV and amylase. Extraction was performed using incubation at a constant temperature and stirring (37 degrees C at 200 rpm) or by ultrasound-assisted extraction (300 W), using different extraction times. Separation of soluble Se species (SeCys2, SeMet, SeMeSeCys, selenite, and selenate) was achieved by ion-exchange chromatography, and detection was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Results showed that the concentration of selenate extracted was independent of the enzymatic extraction technique (approximately 98 ng/g for drought-exposed and 308 ng/g for well-watered potato tubers), whereas the extraction yield of SeMet changed with the protocol used (10-36%). Selenate and SeMet were the main soluble Se species (representing 51-68% of total Se) in potato tubers, regardless of the growth conditions.     

Biodegradable PASP can effectively inhibit nitrification,moderate NH3 emission,and promote crop yield

Polyaspartic acid (PASP) is a low-cost, environmentally friendly, and multifunctional polymer material. The knowledge regarding the effects of PASPs, especially the PASPs with a different molecular weight (MW), on nitrogen use efficiency (NUE), ammonia (NH₃) volatilization and nitrous oxide (N₂O) emission in crop fields is scarce. In this study, maize pot experiments were conducted to evaluate three types of PASPs with different MW. Five treatments were designed: (1) application of chemical phosphorus (P) and potassium (K) fertilizer (PK), (2) PK plus urea (NPK), (3) NPK plus PASP-1 (PASPT1, MW: 5517), (4) NPK plus PASP-2 (PASPT2, MW: 6934), and (5) NPK plus PASP-3 (PASPT3, MW: 7568). The yield indicators of crop height, straw dry weight and 100-grain weight showed that PASP application improved the crop growth. In PASP3, NUE reached 46.1%, almost double of that in NPK (28.6%). Moreover, there were significantly less N losses in the forms of NH₃ volatilization and NO₂ emission following PASP amendment than regular urea application. Another positive impact revealed that PASP inhibited the transformation of NH₄⁺-N to NO₃^–N. Among the three PASPs, PASP-3 with the highest MW overall presented optimal effects, implying that MW was a major driving factor for PASP performance on maize production.     

Selenium species in aqueous extracts of alfalfa sprouts by two-dimensional liquid chromatography coupled to inductively coupled plasma mass spectrometry and electrospray mass spectrometry detection

The complementary use of two different liquid chromatographic mechanisms coupled to inductively coupled plasma mass spectrometry (ICP-MS) for selenium (Se) specific detection has permitted the screening of the most abundant Se-containing fractions in selenized alfalfa sprouts (Medicago sativa). Aqueous extracts of the sprouts were fractionated first by size exclusion chromatography (SEC) using a Superdex Peptide column and a mobile phase containing an ammonium acetate buffer (pH 7). Further purification of the individual SEC Se-containing fractions was carried out using two different chromatographic systems: a Shodex Ashaipack column, with a mixed mechanism of size exclusion and ion exchange, and a conventional reversed phase C8 using ion-pairing reagents. In both cases, the columns were coupled to an inductively coupled plasma mass spectrometer equipped with an octapole reaction system for Se specific detection. This system allowed the on-line monitoring of the most abundant Se isotopes (78Se, 80Se) by reducing the possible polytomic interferences affecting these ions by adding hydrogen (2 mL min(-1)) to the octapole reaction cell. The results obtained by both separation mechanisms were highly comparable, revealing the presence of Se-methionine and Se-methyl selenocysteine. Both compounds were then confirmed by analyzing the corresponding fractions by electrospray quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. Finally, an additional Se-containing species showing Se isotope distribution was detected at a molecular ion m/z 239 in the ESI-Q-TOF. The collision-induced dissociation of the m/z 239 and 237 ions (corresponding to 80Se and 78Se isotopes, respectively) revealed the possible presence as well of a derivative of the Se-2-propenyl selenocysteine.     

Assessment of selenium bioavailability from high-selenium spirulina subfractions in selenium-deficient rats

It was previously found that the bioavailability of Se from Se-rich spirulina (SeSp) was lower than that from selenite or selenomethionine when fed to Se-deficient rats. The present study examined the bioavailability of Se from SeSp subfractions: a pellet (P) issuing from the centrifugation of a suspension of broken SeSp and a retentate (R) resulting from ultrafiltration of the supernatant through a 30 kDa exclusion membrane. Animals were fed a torula yeast based diet with no Se (deficients) or supplemented with 75 microg of Se/kg of diet as sodium selenite (controls) for 42 days. Se-deficient rats were then repleted for 56 days with Se (75 microg/kg of diet) supplied as sodium selenite, SeSp, P, or R. During this period, controls continued to receive sodium selenite. Speciation of Se in subfractions showed that the majority was present in the form of high molecular weight compounds; free selenomethionine was only a minor constituent. Gross absorption of Se from sodium selenite, P, and R was not different and was higher than from SeSp. Only retentate allowed full replenishment of Se concentration in liver and kidney (as did sodium selenite) and glutathione peroxidase (GSHPx) activity in liver, kidney, plasma, and erythrocytes. The bioavailabilities of Se in retentate, as assessed by slope ratio analysis using selenite as a reference Se, were 89 and 112% in the tissue Se content and 106-133% in the GSHPx activities. SeSp and P exhibited a gross bioavailability of <100%. These results indicate that Se in retentate is highly bioavailable and represents an interesting source of Se for food supplementation.     

A Rapid Two-Step Bioremediation of the Anthraquinone Dye, Reactive Blue 4 by a Marine-Derived Fungus

A rapid two-step technique for bioremediation of the anthraquinone dye, the Reactive Blue 4 (RB4) by a marine-derived fungus is reported here. In the first step, 1,000?mg?l^?1 of this dye treated with partially purified laccase of this fungus resulted in 61?% color removal and twofold decrease in chemical oxygen demand by 12?h. The metabolites formed during the enzymatic degradation were characterized by mass spectrometry, ultra performance liquid chromatography, and UV/visible spectroscopy. These analyses confirmed changes in the aromatic character of the parent dye and formation of low molecular weight phenolic compounds as the final products of the enzymatic degradation. Based on these results, the probable degradation products of RB4 were 2-formylbenzoic acid, 1,2,4,5-tetrahydroxy-3-benzoic acid, 2,3,4-trihydroxybenzenesulfonic acid, and 1,2,3,4-pentahydroxybenzene. In the second step, the enzyme-transformed dye solution subjected to sorption on the powdered fungal biomass resulted in a further reduction in color up to 93?% within 10?min. Sorption of the degraded dye was confirmed by the changes in the pattern of Fourier transform infrared spectroscopy spectrum. The two-step treatment resulted in a decrease of 29?% in total carbon accompanied by twofold decrease in toxicity. This is the first report on decolorization, detoxification, and mineralization of RB4 by laccase from a marine-derived fungus.     

Molecular size and molecular size distribution affecting traditional balsamic vinegar aging

A first attempt at a semiquantitative study of molecular weight (MW) and molecular weight distribution (MWD) in cooked grape must and traditional balsamic vinegar (TBV) with increasing well-defined age was performed by high-performance liquid size exclusion chromatography (SEC) using dual detection, that is, differential refractive index (DRI) and absorbance (UV-vis) based detectors. With this aim, MW and MWD, including number- and weight-average MW and polydispersity, were determined with respect to a secondary standard and then analyzed. All investigated vinegar samples were recognized as compositionally and structurally heterogeneous blends of copolymers (melanoidins) spreading over a wide range of molecular sizes: the relative MW ranged from 2 to >2000 kDa. The extent of the polymerization reactions was in agreement with the TBV browning kinetics. MWD parameters varied asymptotically toward either upper or lower limits during aging, reflecting a nonequilibrium status of the balance between polymerization and depolymerization reactions in TBV. MWD parameters were proposed as potential aging markers of TBV.     

Limited effects of land use on soil dissolved organic matter chemistry as assessed by excitation–emission fluorescence spectroscopy and molecular weight fractionation

Dissolved organic matter (DOM) in soil solution represents a complex mixture of organic molecules and plays a central role in carbon and nitrogen cycling in plant–microbial–soil systems. We tested whether excitation–emission matrix (EEM) fluorescence spectroscopy can be used to characterize DOM and support previous findings that the majority of DOM is of high molecular weight (MW). EEM fluorescence spectroscopy was used in conjunction with MW fractionation to characterize DOM in soil solution from a grassland soil land management gradient in North Wales, UK. Data analysis suggested that three distinct fluorescence components could be separated and identified from the EEM data. These components were identified as being of humic‐like or fulvic‐like origin. Contrary to expectations, the majority of the fluorescence signal occurred in the small MW (<1 kDa) fraction, although differences between soils from the differently managed grasslands were more apparent in larger MW fractions. We conclude that following further characterization of the chemical composition of the fluorophores, EEM has potential as a sensitive technique for characterizing the small MW phenolic fraction of DOM in soils.