Comparative evaluation of compost humic acids and their effects on the germination of switchgrass (Panicum vigatum L.)

Purpose

This study aimed to investigate comparatively the main chemical and physico-chemical properties of the humic acid (HA) fraction of three different composts and to evaluate the bioactive effects of these HAs on the germination and early growth of four populations of switchgrass.

Materials and methods

Three compost HAs isolated from a green compost (HA_GC), a mixed compost (HA_MC), and a coffee compost (HA_CC) were characterized for some chemical and physico-chemical properties, such as ash content, elemental composition, total acidity, carboxylic and phenolic OH group contents, E₄/E₆ ratio, Fourier Transform infrared (FT IR), and fluorescence spectroscopies. In subsequent experiments conducted in vitro in a climatic chamber under controlled conditions, the bioactive effects of the three HAs at concentrations of 10, 50, and 200 mg?L^?1 were tested on the germination and early growth of four switchgrass (Panicum virgatum L.) populations, the octaploids Shelter, Shawnee and Dacotah, and the tetraploid Alamo.

Results and discussion

The ash content and the E₄/E₆ ratio were, respectively, much higher or slightly higher for HA_CC than for the other two HAs. HA_MC showed the lowest C and H contents and the highest O content, whereas HA_GC had the highest N content. The total acidity and phenolic OH group content followed the order: HA_MC?>?HA_CC?>?HA_GC. The fluorescence analysis of the three HAs evidenced a common fluorophore unit possibly associated to simple aromatic structures, such as phenolic-like, hydroxy-substituted benzoic and cinnamic acid derivatives. The FT IR spectra of all HA samples indicated the presence of aromatic phenolic structures. Significant beneficial effects were produced by any HA on switchgrass germination and early growth as a function of the population tested and the HA dose.

Conclusions

Results of this study demonstrated that the addition of compost HAs to the germination medium of four switchgrass populations positively influenced the germination process and the growth of primary root and shoot. Significant correlations were found between HA bioactivity and some HA properties. These results suggest a possible use of compost as soil amendment in areas where switchgrass grows naturally or is cultivated.     

Chemical and spectroscopic characterization of Humic acids from a soil toposequence in venezuela

Abstract

An investigation was conducted on physico‐chemical properties of humic acids (HAs) in Venezuelan soils. The HAs were extracted by the NaOH method from a Banco‐Bajio‐Estero soil toposequence (local names for soils located at high, intermediate and low topographic levels), in the Venezuelan plains (Mantecal, Apure State). The extracted HAs were analyzed for elemental composition and characterized by fluorescence, Fourier transform infrared (FT‐IR) and electron spin resonance (ESR) spectroscopies. The results showed that free radical concentration of HAs increased from soils at the highest to soils at the lowest topographic position. High carbon (C), nitrogen (N), and carboxyl group contents, E₄/E₆ ratio, aliphatic character and concentration of free radicals, and low oxygen (O) and phenolic hydroxyl group contents and total acidity were typical of HA from soils at the lower relief position. The FT‐IR spectra indicated that the HA from the soil at the lowest topographic position tended to have a slightly higher content of carboxyl groups than the HAs from soils at higher topographic levels. The observed fluorescence was attributed to the presence of condensed aromatic moieties and/or conjugated unsaturated systems of various complexity in the HA macromolecules.     

Analysis of polynuclear aromatic and aliphatic components in soil humic acids using ruthenium tetroxide oxidation

Although condensed aromatic components are considered to be one of the major structural units of soil humic acids (HAs) and to be responsible for the dark colour of HAs, their amount and composition remain largely unknown. In ruthenium tetroxide oxidation (RTO), condensed aromatic components are detectable as their degradation products, mainly benzenepolycarboxylic acids (BPCAs). We applied this technique to soil HAs with various degrees of humification (darkening). The yields of water‐ and dichloromethane‐soluble products from HAs upon RTO after methylation ranged from 210 to 430 mg g⁻¹ and 10–40 mg g⁻¹, respectively. Eight kinds of BPCAs with two to six carboxyl groups, and seven kinds of BPCAs with additional side chains (tentative assignment) were obtained as methylated counterparts. The yield of each BPCA and the sum of the yields of BPCAs (12–85 mg g⁻¹ HAs) increased with increasing degree of humification and aromatic C content. The compositions of BPCAs indicated that the degree of condensation was greater in the HAs with greater degrees of humification. The sum of the yields of aliphatic compounds ranged from 0.1 to 6.5 mg g⁻¹, and decreased with increasing degree of humification. The C₁₂ to C₃₀ monocarboxylic acid methyl esters accounted for > 56% of the aliphatic compounds assigned, which may be present mainly as end alkyl groups in the HA molecules. We also obtained the methylated counterparts of C₁₄ to C₂₄ dicarboxylic acids; these were possibly derived from polymethylene bridges between adjacent aromatic rings.     

Transformation of catechol in the presence of a laccase and birnessite

The transformation of naturally occurring phenols to humic polymers through oxidative coupling reactions may involve oxidoreductive enzymes and soil minerals as catalysts. There is limited information on the possible inhibitory or synergistic interactions between oxidoreductases and mineral catalysts as they participate in oxidative coupling of phenolic substrates. In this study, a ternary system was investigated, in which a fungal enzyme (Trametes villosa laccase), birnessite (δ-MnO₂), and a naturally occurring phenolic compound (catechol) were reacted together to model soil processes. Binary systems (catechol/laccase and catechol/birnessite) were included for comparison. In the absence of the mineral, T. villosa laccase (950 katal ml⁻¹) transformed 31% of catechol, whereas birnessite (1 mg ml⁻¹) in the absence of the enzyme showed a 24% catechol transformation. The percentages of catechol transformation in the binary systems did not accumulate in the ternary system; instead, birnessite and laccase tested together transformed only 36% of catechol. This suggested that birnessite had an inhibitory effect on substrate transformation by laccase catalysis. Enzyme assays indicated that inhibition was a result of enzyme deactivation by humic-like polymers produced by birnessite, and by Mn²⁺ ions released from the mineral. These observations underscore the importance of considering enzyme-soil mineral-organic matter interactions in studies of humus formation and contaminant removal.     

Influence of natural and synthetic humic substances on the activity of urease

The activity of a purified urease, obtained from Bacillus pasteurii, was inhibited by humic and fulvic acids obtained from an agricultural soil. Enzyme kinetic studies showed that the humic substances affected the affinity of the enzyme for its substrate (K_m) and the maximum velocity of the reaction (V_max). The V_max was inhibited to the same extent by both humic (HA) and fulvic (FA) acids, the precise effect depending on the pH and concentration of humic substance. At pH 4.0, HA concentrations of 25 pg cm^?3 and 10 μg cm^?3 inhibited the V_max by 38.5% and 20% respectively. HA and FA had similar effects on the K_m but in this case the lowering of the affinity of the enzyme for its substrate was not concentration dependent in the range 0–25 μg cm^?3 of humic substance. Typically, the affinity was decreased from a K_M of 50 mM in the control to 67 mM in the presence of HA and FA. The effects were not due primarily to the ash or N contents of the humic substances because de-ashed humic acid and synthetic model humic (made from catechol, guaiacol, pyrogallol, resorcinol and protocatechuic acid) and fulvic acid (made from polymaleic acid), containing virtually no ash or N, were equally as effective. The effect was not related to the phenolic monomers which, before polymerization, had no effect on urease activity.     

Effects of pig slurry application on soils and soil humic acids

The effect of three annually consecutive additions of pig slurry at two rates (90 and 150 m3 x ha(-1) x year(-1) on soils and soil humic acids (HAs) was investigated in a field experiment under semiarid conditions. Soils and pig slurries were analyzed by standard methods. The HAs were isolated from soils and pig slurry by a conventional procedure based on alkaline extraction, acidic precipitation to pH 1, purification by repeated alkaline dissolutions and acidic precipitations, water washing, dialysis, and final freeze-drying. The HAs obtained were analyzed for elemental (C, H, N, S, and O) and acidic functional group (carboxylic and phenolic) composition, and by UV-vis, FT-IR, fluorescence, and ESR spectroscopies. With respect to the control soil, the pig slurry amended soils had greater pH and electrical conductivity, slightly larger total N content, and smaller values of C/N ratio. A decrease of total organic C was observed only in soils amended for 2 and 3 years at the higher slurry rate. With respect to control soil HA, pig slurry HA was characterized by larger contents of S- and N-containing groups, smaller acidic functional group and organic free radical contents, a prevalent aliphatic character, extended molecular heterogeneity, and smaller aromatic polycondensation and humification degrees. Amendment with pig slurry HA determines a number of modifications in soil HAs, including increase of C, S, and COOH contents, C/N ratios, and aliphaticity and decrease of extraction yields and N, O, phenolic OH, and organic free radical contents. These effects are generally more evident after the first year of slurry application and tend to disappear with increasing number of treatments. Most probably, over the years the slightly humified slurry HA is mineralized through extended microbial oxidation, whereas only the most recalcitrant components, such as S-containing, phenolic, and aliphatic structures, are partially accumulated by incorporation into soil HA.     

Selective effects of forest fires on the structural domains of soil humic acids as shown by dipolar dephasing <Superscript>13</Superscript>C NMR and graphical-statistical analysis of pyrolysis compounds

Purpose

Data management strategies of pyrolysis results and NMR acquisition modes were examined in humic acids (HAs) from control soils and fire-affected soils. The information supplied by dipolar dephasing (DD) ¹³C NMR spectroscopy and Curie-point pyrolysis were used to assess chemical structures hardly recognizable and measurable, or of unclear interpretation, when using ¹³C NMR under standard acquisition pulses (cross-polarization/magic angle spinning, CPMAS).

Materials and methods

The HAs were isolated from two forest soils under Pinus halepensis and Pinus sylvestris in control and burned sites affected by medium or severe-intensity wildfires. For NMR analyses, during DD acquisition conditions, a 180° ¹³C pulse was inserted to minimize phase shifts. Curie-Point pyrolysis was carried out at 510 °C for 5 s, and the pyrolysis fragments were analyzed by GC/MS. The total abundances of the major pyrolysis products were compared by an update of the classical Van Krevelen’s graphical-statistical approach, i.e., as surface density values in the space defined by the compound-specific H/C and O/C atomic ratios.

Results and discussion

The DD ¹³C NMR experiments displayed significant differences in the HA spectral profiles as regards to the standard CPMAS ¹³C NMR acquisition conditions, mainly in the chemical shift region of alkyl structures as well as for tannin- or carbohydrate-like O-alkyl structures. In fact, the comparison between DD and CPMAS solid-state NMR suggested shortening of alkyl chains and generation of carbohydrate-derived, unsaturated structures—viz. furans—which adds to the aromatic domain. Pyrolytic results showed fire-induced specific changes in HAs chemical structure and its molecular diversity. The changes were evident in the location and sizes of the different clusters of pyrolysis compounds defined by their atomic ratios.

Conclusions

The DD ¹³C NMR provided specific information on the fate of aliphatic structures and the origin of unsaturated HA structures, which could be helpful in differentiating “inherited” from “pyrogenic” aromatic structures. This is further confirmed by the analysis of the molecular assemblages of pyrolytic products, which showed accumulation of condensed polyaromatic domains in the HAs after the high-intensity fire, accompanied by a recalcitrant alkyl hydrocarbon domain. Medium-intensity fire led to aromaticity increase due to a selective accumulation of lignin-derived phenols concomitant to the depletion of aliphatic hydrocarbon constituents.

     

Humus Characterization of Humid Tropical Forest,Tea Garden,and Field Crop Soils of West Bengal,India: Chemical,Potentiometric, and Spectroscopic Methods

Humic acids (HA) and fulvic acids (FA) were extracted from tropical humid forest, tea garden, and field crop soils, and their chemical, potentiometric, and spectrophotometric properties were measured. There was less HA than FA in the cultivated soils. The HAs contained more carbon (C) and nitrogen (N) and had greater C/H ratios but lower O (oxygen)/H (hydrogen) ratios than FAs. Cultivated soils had greater total acidity than the forest soil generally because of both —COOH and phenolic-OH. Molecular weights of HA an FA, estimated from intrinsic viscosities, revealed that cultivation had reasonably reduced the molecular weights. The difference in pH (ΔpH) values, corresponding to three-fourths and one-fourth of the pH at final inflexion point showed that they were polyprotic. The longer time required for stability of greater pH of HAs was related to greater degree of stable coiling. The (E₄/E₆), in general, revealed a greater amount of aliphatic moiety rather than aromatic moiety.     

<Superscript>13</Superscript>C-NMR analysis of components of chernozem humic acids and their fractions with different molecular sizes and electrophoretic mobilities

Tandem size-exclusion chromatography and polyacrylamide gel electrophoresis were used for obtaining stable fractions of different molecular sizes and electrophoretic mobilities from chernozem humic acids (HAs). The obtained fractions were analyzed using solid-phase ¹³C NMR. The tendencies of the changes in the aromatic and aliphatic components of the HA fractions with different molecular sizes and electrophoretic mobilities were experimentally revealed. The aromatic-to-aliphatic carbon ratio C_arom (165–108 ppm)/C_aliph (108-0 ppm) was used for comparing the degrees of aliphaticity and aromaticity of the HA macromolecules. This ratio increased by more than five times when going from the high-molecular-weight to the low-molecular-weight fractions and largely determined their hydrophilic properties. The obtained results can be useful for the interpretation of the structural organization and the ecological functions of soil HAs and their fractions.     

Purification and characterization of Ocimum basilicum L. polyphenol oxidase

A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be approximately 54 kDa. The contents of total phenolic and protein of O. basilicum L. extracts were determined. The total phenolic content of O. basilicum L. was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg 100 g(-1) on a fresh weight basis. The protein content was determined according to the Bradford method. The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the highest V(max)/K(m) value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for 4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature of 20, 40, and 50 degrees C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that optimum temperature and pH were dependent on the substrates studied. The enzyme activity with increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates decreased due to heat denaturation of the enzyme.     

Structure of humic acids in zonal soils from <Superscript>13</Superscript>C NMR data

The structure of humic acids (HAs) in zonal soil types—soddy-podzolic soils (two samples), gray forest soil (one sample), and chernozems (two samples)—was quantitatively studied by ¹³C NMR spectros-copy. In the series considered, the content of unsubstituted carbon in the aromatic fragments of HAs increased, and the fraction of unsubstituted aliphatic structures decreased. HAs of soddy-podzolic soils were found to be enriched with carbohydrate fragments compared to HAs of chernozems and gray forest soil. The carbon skeleton of HAs from typical rich chernozem contained significantly more aliphatic and carbohydrate fragments compared to typical chernozem, which probably reflected the lower degree of HA transformation in rich chernozem.     

Paramagnetic Properties of Humic Substances in Taiga and Tundra Soils of the European Northeast of Russia

The study of paramagnetic activity of humic substances in taiga and tundra soils of the Komi Republic and the assessment of the influence of soil hydromorphism on concentrations of free radicals in the structure of humic acids (HAs) and fulvic acids (FAs) have been performed. The concentration of free radicals in HA specimens was up to 11 times higher than that in FA specimens due to a higher content of aromatic and other condensed structures in HA molecules. This fact attests to the high capacity of HAs to polymerization and complexation reactions with participation of radicals. The average value of g-factor is higher for FA specimens than for HA specimens, which attests to a greater electron density shift of unpaired electron to oxygen atom in the structure of FAs because of its spin-orbital interaction with oxygen-containing functional groups, the concentrations of which are significantly higher in FAs than in HAs. An increase in the concentration of free radicals in the molecular structure of HAs is observed in taiga soils with an increase in the degree of their hydromorphism (from automorphic to semihydromorphic soils), which is related to the biohydrothermal conditions of humus formation in bog-podzolic soils with retarded biochemical processes and low degree of plant litter humification. As a result, HAs with the high content of free radicals in their structure are formed. An opposite situation is observed for HAs in tundra soils with a decrease in the content of unpaired electrons under conditions of the increased hydromorphism. The difference in the character of changes in the paramagnetic activity of HAs in taiga and tundra soils with different degrees of hydromorphism may be related to different natures of plant residues participating in humification processes. A tendency for a decrease in the paramagnetic activity in both HAs and FAs from the south to the north is observed, which may be related to a general decrease in the content of poly-conjugated systems in the structure of humic substances in tundra soils.     

Phenanthrene sorption by compost humic acids

Nonlinear isotherms were observed for sorption of polycyclic aromatic hydrocarbons (PAHs) to humic materials, but the exact sorption mechanism is not clearly understood. This study aimed to investigate the sorption of PAHs by compost humic materials. Humic acids (HAs) were progressively extracted from two compost samples by a 0.1-M sodium pyrophosphate solution. Sorption of phenanthrene by the extracted HAs was studied with a batch equilibration technique. Competitive sorption between pyrene and phenanthrene in the HAs was also examined. Elemental analysis and solid-state ¹³C NMR were used to characterize HAs. All sorption isotherms were nonlinear although these samples contained little black carbons if any. Isotherm linearity increased with increasing number of extractions. Isotherm nonlinearity was negatively related to HA aliphaticity. Addition of pyrene to the phenanthrene-HA system increased isotherm linearity. Competition between phenanthrene and pyrene sorption was more pronounced in the first fraction of HAs with a higher content of aromatic moieties. This study showed that humic materials alone could lead to nonideal sorption for PAHs.     

Plant Uptake of Iron Chelated by Humic Acids of Different Molecular Weights

Abstract

Mobilization of iron (Fe) chelated by humic acids (HA) of low (HA_10,000) and high molecular weight (HA_100,000) fractions and its uptake by plants were investigated in growth experiments with sunflower seedlings. The iron chelates (labeled with ⁵⁹Fe) contained in dialysis bags (mw. cutoff=3500) were placed in minus iron Hoagland solutions as the Fe source and at the same time fulvic acid (FA), EDTA, and low and high molecular weight HA fractions were added in the solutions as mobilizators. Characterization of FA, HA_10,000, and HA_100,000 were performed by infrared spectroscopy and chemical analysis, e.g., total acidity, COOH, and phenolic‐OH content. Roots and leaves were harvested, dried, and ground for Fe activity determination. Iron contents and pH in the nutrient solutions were measured before and after treatments. The supply of Fe to the plants was apparently sufficient, because no Fe deficiency has been detected in the test plants but during the whole absorption period, the pH of the nutrient solution was about 4.5. The Fe contents in leaves indicated that part of the Fe was rapidly transported from roots to leaves. Judging from the Fe contents in leaves, it was assumed that the small size HA_10,000 and EDTA were the most efficient in affecting transport of Fe from root to leaf tissue. FA, HA_10,000, and especially HA_100,000 were unable to penetrate the dialysis bags and, hence, were effective in Fe mobilization only after the Fe, dissociated from the Fe‐HA chelate, has passed the dialysis membrane into the nutrient solutions. In contrast, the small size EDTA was expected to have penetrated the dialysis bags, permitting mobilization of chelated Fe by ligand exchange inside the bags, and transporting the Fe to the roots. The results suggested that the humic substances used in this study were able to form with the Fe³⁺ ion complexes that maintained the iron available to the sunflower plants. In the chemical form of Fe.L, where L was FA o HA, the iron within the bags or in solution or in the roots free space, was available for exchange reactions with the natural sunflower plant chelators for its transport to the leaves.     

Comparison of carbon skeletal structures in black humic acids from different soil origins

Andosols are characterized by an abundance of black humic acids (HAs) belonging to Type A with a high content of aromatic carbon (C) in particular condensed aromatic C. Black HAs are also observed in other soils, such as Chernozems and the subsoil of paddy field, and extracted after washing with an acid or using chelating agent such as sodium pyrophosphate (Na₄P₂O₇). However, contribution of condensed aromatic structures to those soil HAs are unknown. To obtain the information about C skeletal structures of black HAs in soils other than Andosols, HAs were obtained from 2 Chinese Chernozem samples, 2 subsoil samples from Japanese paddy fields (Fulvisols), and a Rendzina-like soil (Cambisols) as well as an Andosol sample (reference) by successive extraction with 0.1 M NaOH (HAs₁) and 0.1 M Na₄P₂O₇ (HAs₂), and ¹³C nuclear magnetic resonance and X-ray diffraction 11-band profile analyses were applied. In the black HAs₂ from the non-Andosol samples, the proportion of C present as aromatic C, size of C layer planes, and relative C layer plane content ranged from 52 to 59%, 0.48 to 1.92 nm (mean size, 0.76–0.91 nm), and 58 to 100 AU (arbitrary unit) mg^?1, respectively, with a positive correlation between total C layer plane content and the degree of humification. Those ranges were similar to the distribution ranges of Andosols HAs₁ reported by our previous study.     

Fractionation of humic acids upon adsorption on montmorillonite and palygorskite

The adsorption of three humic acid (HA) preparations by clays—montmorillonite (Wyoming, USA) and palygorskite (Kolomenskoe district, Moscow oblast)—has been studied. The HA preparations were isolated from samples of the humus-accumulative horizons of a leached chernozem (Voronezh) and a chestnut soil (Volgograd), and a commercial preparation of sodium humate (Aldrich) was also used. The solid-state ¹³C NMR spectroscopy and IR spectroscopy revealed the selective adsorption of structural HA fragments (alkyls, O-alkyls (carbohydrates), and acetal groups) on these minerals. As a result, the aromaticity of the organic matter (OM) in the organic-mineral complexes (OMCs) and the degree of its humification have been found to be lower compared to the original HA preparations. The fractionation of HAs is controlled by the properties of the mineral surfaces. The predominant enrichment of OMCs with alkyls has been observed for montmorillonite, as well as an enrichment with O-alkyls (carbohydrates) for palygorskite. A decrease in the C : N ratio has been noted in the elemental composition of the OM in complexes, which reflected its more aromatic nature and (or) predominant sorption of N-containing structural components of HA molecules. The adsorption of HA preparations by montmorillonite predominantly occurs on the external surface of mineral particles, and the interaction of nonpolar alkyl groups of HAs with this mineral belongs to weak (van der Waals, hydrophobic) interactions. The adsorption of HA preparations by palygorskite is at least partly of chemical nature: Si-OH groups of minerals are involved in the adsorption process. The formation of strong bonds between the OM and palygorskite explains the long-term (over 300 million years) retention of fossil fulvate-type OM in its complex with palygorskite, which we revealed previously.     

Analysis of the green fraction of humic acids

The green fraction of humic acids (HAs), Pg, was fractionated by gel chromatography on Sephadex G-50. Repeated chromatography of the crude Pg obtained by the first chromatography of HA yielded a sharp peak (G₁) and two broad bands (G₂) of purified Pg. The recovery of Pg in G₁ and G₂ as percentages of the total Pg content of the HA were 16.2 and 14.5%, respectively. Distinct peaks were detected at 615, 570, 450 and 280 nm in the UV-visible spectra of both G₁ and G₂ in alkali solutions. Estimates of weight-average molecular weight were 9.7 * 10³ for G₁ and 1.23 * 10⁴ for G₂. In the ¹H-NMR spectra the percentage of hydroxyl groups of 4,9-dihydroxy-perylene-3, 10-quinone (DHPQ) nuclei, which are known to be the chromophore of Pg, was 4.1 and 4.5% of the total H, respectively. Based on these values and the H/C ratios, DHPQ-C was estimated as 28% of total C. ¹³C-NMR spectra obtained using inverse-gated decoupling yielded 40–41% aromatic C, which suggested the presence of aromatic rings other than DHPQ. All of the IR, ¹³C- and ¹H-NMR spectra indicated that the two purified Pg samples largely differed from each other in their contents of alkyl and polysaccharide components.     

Humic acid composition and humification processes in wetland soils of a Mediterranean semiarid wetland

Purpose

The present study focuses on a compositional characterization of the humic acid (HA) fraction of several wetland soils using solid-state ¹³C NMR spectroscopy. The data were analysed using the molecular mixing model (MMM), based on an empirical approach by Nelson and Baldock. The compositional data from HAs obtained with this model were used to obtain a wider assessment of the process of humification from comparison of total soil wetland organic matter composition and HA composition.

Materials and methods

Twenty samples of humic acids (HAs) isolated from a Mediterranean semiarid wetland (‘Tablas de Daimiel’, central Spain) were studied using elemental analysis and cross polarization magic angle spinning (CPMAS)¹³C nuclear magnetic resonance (NMR) spectroscopy. The NMR data were analysed with the molecular mixing model (MMM) considering up to six generic components (carbohydrate, protein, lignin, lipid, char and ‘carbonyl’). HAs are considered a conceptual mixture of these model components, and the MMM determines the proportions of the characteristic biomolecules contributing to HA composition.

Results and discussion

The composition of the HAs under study depends on local factors such as site vegetation and occurrence of fire. Correlations between the proportions of the six generic components and further comparison with those determined for the unfractionated OM (whole sample, WS), gave information on HA origin and humification mechanisms. In particular, the proportions of char and carbohydrate (R ² 0.637) and contents of lignin and protein (R ² 0.471) in the HAs were negatively correlated (P < 0.05). Significant correlations (R ² 0.439) also existed for char contents in whole sample (WS) compared to HA, and for carbohydrates in WS compared to HA (R ² 0.558). Char proportion grew in HA with respect to the WS, and carbohydrates dropped to a half on average in HA compared to WS.

Conclusions

Two different humification mechanisms could be identified for no-fire and fire areas. In the former, HA-char was preserved selectively from char in the sample, whereas in the latter, char was newly formed by fire effect.

     

The carbon distribution among the functional groups of humic acids isolated by sequential alkaline extraction from gray forest soil

Preparations of humic acids (HAs) were isolated from a gray forest soil by sequential alkaline extraction. From a sample of 500 g, HA preparations of 2.24, 0.23, and 0.20 g were obtained from the first, second, and third alkaline extracts, respectively. The structure of the preparations was determined by ¹³C NMR spectroscopy. At each next extraction step, the portion of aliphatic fragments in the HA preparations increased and the content of aromatic structures decreased. The conclusion was drawn that a single extraction is sufficient for obtaining a representative HA sample.     

Quantitative precursor studies on di- and trihydroxybenzene formation during coffee roasting using "in bean" model experiments and stable isotope dilution analysis

The objective of this study was to investigate the potential of various raw bean components as precursors of pyrogallol (1), hydroxyhydroquinone (2), catechol (3), 4-ethylcatechol (4), 4-methylcatechol (5), and 3-methylcatechol (6) under quasi "natural" roasting conditions by using the recently developed "in bean" model roast experiments. Freeze-dried, fully extracted bean shells were loaded with aqueous solutions of either single coffee compounds or fractions isolated from the raw bean solubles. After freeze-drying, these reconstituted beans were roasted, aqueous coffee brews were prepared, and the target phenols were quantified by means of a stable isotope dilution assay with LC-MS/MS detection. On the basis of the quantitative data, it can be concluded that upon coffee bean roasting, catechol (3) is primarily formed by degradation of caffeoylquinic acids from both the caffeic acid and the quinic acid moiety of the molecule, as well as from Maillard-type reactions from carbohydrates and amino acids. In contrast, pyrogallol (1) and hydroxyhydroquinone (2) are efficiently generated from carbohydrates and amino acids and, in addition, from free or chlorogenic acid bound quinic acid moieties. 4-Ethylcatechol (4) is exclusively generated upon thermal breakdown of caffeic acid moieties. 3-Methylcatechol (6) is formed primarily from the Maillard reactions and, to a minor extent, also from various phenolic precursors, whereas 4-methylcatechol (5) is produced in trace amounts only from all of the different precursors investigated. On the basis of this precursor study, reaction routes explaining the formation of the target phenols are proposed.