Comparative studies of pyrolysis-mass spectra of melanins,model phenolic polymers,and humic acids

Curie-point pyrolysis in direct combination with a fast-scanning, quadrupole mass-spectrometer was used for the characterization of humic acids from soils, peats, and composted straw, fungal humic acid-type polymers (melanins), model phenolic polymers and lignins. All the humic acids, most of the fungal melanins and the model polymer with linked peptone yielded complex but highly similar spectra. Prominent homologous ion series were: m/e 34, 48 and 62 (“sulfides”); m/e 67, 81 and 95 (“pyrroles”); m/e 78, 92 and 106 (“benzenes”); m/e 94, 108 and 122 (“phenols”); and m/e 117, 131 and 145 (“indoles”). “Humic acid” fractions from fresh straw and straw composted for various periods of time yielded relatively strong but gradually diminishing lignin series peaks at m/e 94, 108, 120 and 122 (phenols); m/e 124, 138, 150 and 164 (methoxyphenols); and at m/e 154 and 168 (dimethoxyphenols). Weak signals for the methoxy and dimethoxyphenol series were also present in the soil humic acid spectra. In addition to the series peaks for the other fungal melanins, the S. chartarum spectrum showed a pronounced ion series at m/e 68, 82, 86 and 96 which is indicative of polysaccharides and at m/e 136, 150 and 164 which suggests the presence of alkylphenols.     

Chemical composition of the organic matter in forest soils: The humus layer

Decomposition and humification were studied within three types of forest humus (mull, moder, and mor) by means of CPMAS ¹³C NMR spectroscopy combined with degradative methods. The NMR data show that O-alkyl carbon decreases in all soils, and alkyl as well as carboxyl carbon increase as depth and decomposition increase; the percentage of aromatic carbon remains constant at about 25%. With increasing depth the amount of carbon that can be identified as belonging to specific compound classes by wet chemical methods decreases from 60% to 40%. Microbial polysaccharides and the proportion of non polysaccharide O-alkyl carbon increase with depth. A selective preservation of recalcitrant, condensed lignin structural units is also observed. In order to relate the spectroscopic and chemical data from investigations of whole soils with studies of humification, samples were fractionated into fulvic acid, humic acid, and humin fractions. The fulvic acid fraction contains large concentrations of carbohydrates irrespective of the soil horizon. The humic acid fraction contains less polysaccharides, but high amounts of alkyl carbon and aromatic structures. The percentage of aromatic carbon existing in the humic acid fraction increases with depth, probably reflecting the amount and degree of oxidative decomposition of lignin. A loss of methoxyl and phenolic groups is evident in the ¹³C NMR spectra of the humic acid fraction. The humin fraction resembles relatively unchanged plant-derived materials as evident from the lignin parameters and carbohydrate contents. All the observed data seem to indicate that humic acids originate form oxidative degradation of humin or plant litter.     

Contribution of lignin and polysaccharides to the refractory carbon pool in C-depleted arable soils

We assessed the contribution of polysaccharides and lignin, major components of plant residues, to the refractory pool of soil organic carbon (SOC) in arable soils. Soil samples from two contrasting treatment types of European long-term agroecosystem experiments, i.e. conventionally managed (fertilized) and C-depleted plots, enriched in refractory compounds, were compared. Bulk samples from eight experimental sites and particle-size fractions of two of the sites were investigated. The CuO oxidation technique was used as a relative measure of lignin and its degree of structural alteration. The contents and composition of polysaccharides were determined following hydrolysis with trifluoroacetic acid (TFA). For the bulk samples, the amount of lignin phenols declined more than the total OC in the course of C-depletion. The contribution of lignin phenols to total OC was thus lower in the C-depleted versus the fertilized plots. A greater lignin biodegradation was found in the bulk samples of the depleted plots compared with the fertilized plots. The analysis of size fractions revealed lower OC-normalized contents of lignin phenols and a higher degree of lignin alteration in fractions <63 μm of the depleted versus the fertilized plots. These findings indicate that lignin does not accumulate within the refractory C pool of arable soils. The refractory SOC pool shows a lower contribution of lignin as compared with more labile fractions of SOC. If lignin-derived carbon is present in the stable pool it has been extensively modified so that it can no longer be identified as phenolic CuO oxidation products. OC-normalized contents of polysaccharides (neutral sugars and galacturonic acid) were similar in bulk samples of the C-depleted and fertilized plots. The contrasting treatments showed similar polysaccharide contents especially in separates <6 μm. The separates <6 μm in the C-depleted plots retained between 50 and 100% of the polysaccharide amounts in the fertilized plots. The mass ratio of (galactose+mannose)-to-(arabinose+xylose) (GM/AX) was higher in bulk samples of the C-depleted versus the fertilized plots, indicating a higher relative contribution of microbial sugars. Within a particular soil, the fine separates were those with the highest GM/AX ratio. These results indicate that the refractory C pool has a similar proportion of polysaccharides as the labile C pool, but refractory polysaccharides are mainly associated with fine separates and show a dominant contribution of microbial sugars. Our results provide evidence that polysaccharides, mainly those of microbial origin, are stabilized over the long-term within fine separates of arable soils. In contrast, CuO lignin is associated mainly with the coarse fractions and does not contribute to the refractory C pool.     

The [13C]nuclear magnetic resonance spectrum of a methylated humic acid

A humic acid has been investigated by [¹³C]NMR spectroscopy using the methylated humic acid dissolved in chloroform which gave a relatively simple spectrum.Estimates of different types of C show that 21% of the C is aromatic, 35% consists of methylene chains and methyl groups, 3%, as the carbonyl C of methyl esters, and 13% as the methoxyl C of methylated phenols and alcohols. The rest, 28%, is not accounted for and may be mostly C bound to O as in polysaccharides or in peptides. No ketonic or quinonic groups were detected.The [¹³C]NMR spectrum of the methylated humic acid is entirely different from those of lignin and its derivatives thus suggesting that lignin residues play no significant role in the structure of the humic acid.     

Abbau und Umwandlung von Pflanzenrückständen und ihren Inhaltsstoffen durch die Mikroflora des Bodens

Degradation and transformation of plant residues and their components by the microflora of the soil This review describes recent results of publications in this area. It condsiders the dynamics of degradation under field and laboratory conditions and indicates some of the problems of simulation models. The preponderant part of transformation processes apparently takes place in a relatively small fraction of soil organic matter which also includes the biomass. Methods for the quantitative measurements of the biomass have been considerably improved. The turnover rates of plant residues are effectively influenced by their chemical composition, for instance by their C/N ratio and their content of lignin or polysaccharides. C/N ratios also seem to influence the socalled priming effect and the transformation of increasing amounts of plant residues added to the soil. Some progress has been also made in the transformation of plant, residues under different climatic conditions. Experiments with polysaccharides and glucose have indicated that a major portion of the residual carbon residues in soil are contained in the biomass or N-containing microbial metabolites. A small part is also present in the phenolic constituents of humic compounds. Root excretions also contribute carbohydrates. Recent experiments indicated the transportation of a considerable amount of photosynthetic products from sprouts through roots into the soil. They cause intensive microbial turnover processes in the root zone. N-containing compounds are stabilized in the soil biomass. Proteins and aminopolysaccharides are furthermore stabilized by sorption on humic compounds or clay particles. They are even more effectively stabilized by linkage into humic compounds. Plant residues contain appreciable amounts of free and polymerized phenols which are degraded by microorganisms or incorporated into humic compounds. Easily oxidisable phenols are more slowly degraded in soil than the more stable ones. This is especially true for low concentrations of phenols added to the soil. The pH-values of the soil and its content of humic compounds influence essentially the degradation rate. Lignins belong to the most important natural biopolymers. Methods for their specific labelling with 14°C have essentially promoted studies about their degradation and transformation in soil and have enlarged the knowledge about lignin degrading organisms. Beside lignins and other plant polyphenols, melanins may play an important role in the formation of humic compounds. These melanins are synthesized form carbohydrates by some microscopic fungi through secondary metabolic reactions. They are formed from phenols, quinones, polyenes, aminoacid and aminosugar derivatives. Similar to humic acids they are relatively stabile against rapid microbial degradation. Analytical methods developed in the last years indicate that both groups of polymers contain similar building blocks.     

Sodium amalgam reduction of humic acid—II. application of the method

The nature and distribution of the phenolic compounds found in humic acid on sodium amalgam reduction resemble those reported to be found in microbial cultures grown on lignifiedplanttissue.lt has also been shown that the more humified and decayed fractions of soil organic matter yield humic acids that bear less resemblance to lignin than do the humic acids from less decayed plant remains. The aromatic portion of humic acid would seem to consist of biologically modified and transformed lignin, together with phenolic units synthesized by soil microbes. Sodium amalgam reduction can be employed to estimate the degree of transformation, but cannot, however, be used to “finger print” humic acids.     

Pyrolysis of a soil humic acid and of their hydrolyzed and methylated products

Pyrolysis of soil humic acid yields a variety of products arising from heterogeneous materials associated to the humic structure and from this self. After acid hydrolysis most of the compounds related to proteins, polysaccharides and lignin dissappear. Adsorbed compounds such as fatty and dicarboxylic acids are released after methylation and subsequent pyrolysis. In addition pyrolysis produces aromatic and substituted aromatic hydrocarbons. All the identified pyrolysis products have also been isolated by different complex and time-consuming chemical degradations and solvent extraction procedures. Pyrolysis provide some evidence of the humic acid structure.     

Changes of lignin phenols and neutral sugars in different soil types of a high-elevation forest ecosystem 25 years after forest dieback

Long-term effects of forest disturbance 25 yr ago on lignin and non-cellulosic polysaccharide pools in an unmanaged high-elevation Norway spruce (Picea abies L. [Karst.]) forest were investigated by comparing three dieback sites with three adjacent control sites with non-infested spruce on identical soils. Samples were taken from the forest floor and the mineral soil; one Ah horizon sample per site was physically fractionated into density and particle size fractions. Additionally, changes in the above- and belowground input of lignin and non-cellulosic polysaccharides after forest dieback were quantified. Lignin and its degree of structural alteration in plant and soil samples were assessed by CuO oxidation and subsequent analysis of the lignin phenols. Non-cellulosic polysaccharides were determined after hydrolysis with trifluoroacetic acid (TFA), derivatisation of their neutral sugar monomers by reduction to alditols, and subsequent acetylation. The total plant-derived input of lignin and non-cellulosic polysaccharides to the soil was similar for the dieback and the control sites. The chemical composition of the input has changed considerably after forest dieback, as shown by significantly higher syringyl/vanillyl (S/V) ratios and significantly lower (galactose+mannose)/(arabinose+xylose) (GM/AX) ratios. This indicates a changed plant input and a higher contribution of microbial sugars. Contents of lignin phenols in the forest floor and coarse particle size fractions of the A horizons were significantly smaller at the dieback sites (p<0.01). Moreover, larger acid-to-aldehyde ratios of vanillyl units (Ac/Al)_v indicated an increased degree of lignin phenol alteration. Also contents of neutral sugars were significantly (p<0.01) smaller in the forest floor, but not in the A horizons of the dieback sites. The GM/AX mass ratios as well as the (rhamnose+fucose)/(arabinose+xylose) (RF/AX) ratios in the forest floor and coarse particle size fractions of the mineral topsoil were significantly (p<0.01) larger after forest dieback, indicating a larger relative contribution of microbial sugars. In general, the lignin phenol and neutral sugar pools of all three soil types exhibited similar response patterns to the changed site conditions. Our results demonstrate that the lignin and neutral sugar pools of humic topsoil horizons are highly sensitive to forest disturbances. However, the two compounds show different patterns in the mineral soil, with the major neutral sugar pool being stabilized against changes whereas the lignin phenol pool decreases significantly.     

Chemical studies on soil humic acids

Seventeen samples of soil humic acids, two fractions of soil fulvic acid sample, and several related compounds such as lignin, tannin, flavonoid and artificial humic substances were decomposed in conc. KOH solution at 180°C. Succinic acid, glutaric acid, phloroglucin, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, 3,4-dihydroxy-5-methoxybenzoic acid, and gallic acid were detected in the degradation products of humic acids. The amounts of these degradation products were discussed in relation to the degree of humification or the sources of the humic acid samples. Succinic acid also resulted from glucose, polymaleic acid, and the humic acid and humin prepared from glucose, but glutaric acid resulted only from glucose humic acid and glucose humin but not from glucose and polymaleic acid. Succinic acid and glutaric acid were supposed to result from the same structural portions in humic acids because of the very significant positive linear correlation between their amounts. p-Hydroxybenzoic, vanillic, protocatechuic, and 3,4-dihydroxy-S-methoxybenzoic acids were presumed to result mainly from lignin structure in humic acids. Soil humic acids yielded small amounts of gallic acid although the yields by hydrolysable tannins were in large amounts. The yields of above-mentioned degradation products from humic acids decreased with increasing degree of humification. Phloroglucin resulting from ftavonoids including condensed tannins were also found in the degradation products of humic substances. Its yield showed no linear correlation with RF value of humic acid, and is presumed to be rather related to the vegetation at the sites of soil sampling.     

Decomposition of 14C-labeled glucose,plant and microbial products and phenols in volcanic ash-derived soils of chile

During 4 months from 70 to 79% of the carbon of added glucose, cellulose, and Leuconostoc dextranicus polysaccharide had evolved as CO₂ from normal agricultural soils of Chile and California. The presence or additions of allophanic material reduced losses of glucose C by about 25% and of the C of the polysaccharides by 36–65%. From wheat straw, the polysaccharide fraction of wheat straw, and protein, C losses were 60, 78 and 67%, respectively, in the normal soils. Reductions related to allophane were about 41–67%. For a number of microbial cells, C loss reductions due to allophanic materials ranged from 31 to 55%. Carbon losses from catechol and ferulic acid were more related to reactivities of the phenols, the soil pH, and the organic matter content of the soil than to the presence or absence of allophanic material.     

Impact of plant cell wall network on biodegradation in soil: Role of lignin composition and phenolic acids in roots from 16 maize genotypes

Residue quality is a key factor governing biodegradation and the fate of C in soil. Most investigations of relationships existing between crop residue quality and soil decomposition have been based on determining the relative proportions of soluble, cellulose, hemicellulose and lignin components. However, cell wall cohesion is increased by tight interconnections between polysaccharides and lignin that involve cross-linking agents (phenolic acids). The aim of this study was to determine the role of lignin composition and phenolic acids on short- to medium-term decomposition of maize roots in soil. Sixteen maize genotypes, presenting a range of chemical characteristics related to root lignin and phenolic acids, were used. The main components were characterized by Van Soest (VS) extraction and cell wall acid hydrolysis, and the non-condensed Syringyl and Guaicyl lignin monomers, esterified phenolic acids and etherified phenolic acids were determined. Maize roots were then incubated in soil under controlled conditions (15 °C, −80 kPa moisture) for 796 days. Results showed that VS extraction over-estimated the structural hemicellulose content and that VS lignin was more recalcitrant than Klason lignin. The tremendous effect of cell wall chemical characteristics was shown by marked variations (almost two-fold differences in C mineralization), between the 16 maize roots. Decomposition was controlled by soluble residue components in the short term whereas lignin and the interconnections between cell wall polymers were important in the long-term. Notably the cell wall domain rich in non-condensed lignin and esterified phenolic acids was prone to decomposition whereas the presence of etherified ferulic acids seemed to hamper cell wall decomposition.     

Elemental,functional group and infrared spectroscopic analysis of actinomycete melanins from brazilian soils

Eleven actinomycete melanins were characterized by elemental and functional group and infrared analysis. A soil humic acid from a Brazilian topsoil, a darkred latosol under savanna grassland, analysed previously, was used for comparative purposes. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for soil humic acids and fungal melanins. Compared to the soil humic acid, the actinomycete melanins showed greater detail, indicative of higher aliphaticity. Most of these were, in varying degrees, similar to the type III IR spectra of humic acids, which are characteristically high in proteinaceous material, and with variable amounts of polysaccharides. The exceptions were two melanin spectra that showed more resemblance to the humic acid from the dark-red latosol, which belongs to the type I spectra of soil humic acids, a category that includes most soil humic acids. The probable participation of melanic actinomycetes in the formation of humic polymers in discussed.     

Reaction rates of phenolic humus constituents and anilines during cross-coupling

Oxidative coupling processes operative in soil during the formation of stable humic materials and bound residues involving anilines and soil organic matter were studied. We examined the relationship between chemical structure and reactivity for the peroxidase-mediated coupling of potential phenolic humus constituents of plant and microbial origin. The results demonstrated that the acrylic group significantly increased the reactivity of the naturally-occurring phenolic compounds tested. These results suggested that lignin-derived phenols containing the acrylic group, e.g. ferulic acid, may be utilized preferentially during the peroxidase-mediated synthesis of humic materials in soils. We also investigated the peroxidase-mediated cross-coupling of mono-substituted anilines with various phenolic compounds. Reactivity of aniline, nitroaniline and chloroaniline was greatly enhanced in the presence of a highly-reactive electron donor such as ferulic acid. The enhanced reactivity suggested that the aniline compounds were reacting by a different mechanism than when they are present as sole electron donors. The most plausible mechanism is a secondary chemical reaction between anilines and intermediates or products produced during the enzymatic oxidation of phenolic electron donors.     

Decomposition and distribution of residual activity of some 13C-microbial polysaccharides and cells,glucose, cellulose and wheat straw in soil

Studies were made to determine the rate of decomposition of some ¹⁴C-labeled microbial polysaccharides, microbial cells, glucose, cellulose and wheat straw in soil, the distribution of the residual ¹⁴C in various humic fractions and the influence of the microbial products on the decomposition of plant residues in soil. During 16 weeks from 32 to 86 per cent of the C of added bacterial polysaccharides had evolved as ¹⁴CO₂. Chromobacterium violaceum polysaccharide was most resistant and Leuconostoc dextranicus polysaccharide least resistant. In general the polysaccharides, microbial cells, and glucose exerted little effect on the decomposition of the plant products. Upon incubation the ¹⁴C-activity was quickly distributed in the humic. fulvic and extracted soil fractions. The pattern of distribution depended upon the amendment and the degree of decomposition. The distribution was most uniform in the highly decomposed amendments. After 16 weeks the bulk of the residual activity from Azotobacter indicus polysaccharide remained in the NaOH extracted soil. From C. violaceum polysaccharide both the extracted soil and the humic acid fraction contained high activity. About 50–80 per cent of the residual activity from the ¹⁴C-glucose, cellulose and wheat straw amended soils could be removed by hydrolysis with 6 n HCl. The greater part of this activity in the humic acid fraction was associated with the amino acids and that from the fulvic acids and residual soils after NaOH extraction with the carbohydrates. About 8 16 per cent of the activity of the humic acid fraction was present in substances (probably aromatic) extracted by ether after reductive or oxidative degradation.     

紫色水稻土胡敏酸的形成——稻草腐解试验

土壤腐植酸类物质的形成是土壤固碳的重要过程,但对腐植酸类物质形成过程的了解仍不甚清楚,为了丰富土壤腐植酸类物质形成理论,采用富立叶变换红外光谱和固态交叉极化-魔角旋转^13C-核磁共振光谱技术分析了紫色水稻土稻草腐解过程中胡敏酸的波谱学特征。结果表明,稻草腐解的前期,胡敏酸的红外光谱所有吸收峰（3364、2933、1653、1599、1508、1461、1421、1331、1225、1126、1033cm^-1）强度皆有明显减弱,核磁共振光谱的烷基、多羟基和芳基的共振峰明显减弱且甲氧基的共振峰显著增强,即表明提取的胡敏酸为类胡敏酸的木质素;随着腐解的进行,胡敏酸的红外光谱的吸收峰强度皆显著增强,核磁共振光谱的烷基、芳基和羰基的共振峰增强,即表明此时的胡敏酸已是以木质素残体为核心并结合烷基、酰胺以及糖类物质反应形成的高分子聚合体;稻草腐解的后期,胡敏酸的红外光谱的2933cm^-1处的吸收峰强度减弱,1651、1599、1508、1461、1422和1224cm^-1处的吸收峰小幅增强,核磁共振光谱的烷基共振峰减弱,甲氧基共振峰增强,表明此时的胡敏酸发生脱烷基（主要是甲基）过程。因此,红外光谱吸收峰强度与核磁共振光谱共振峰强度的规律性变化反映了稻草腐解过程紫色水稻土胡敏酸的形成过程具有阶段性,紫色水稻±胡敏酸的形成过程符合木质素学说。     

Initial formation of soil organic matter from grass residues in a long-term experiment

Summary The formation of soil organic matter from grass residues was studied using samples of a long-term experiment (34 years) on humus and soil formation at Rostock, Germany (Hu 3), by elemental analyses (C and N) and pyrolysis-field ionization mass spectrometry of grass residues, humus-free loamy marl, mixtures of this loamy marl with grass roots, and whole soil samples from the 2nd, 7th, 13th, 19th, 25th, 29th, and 34th year of the experiment. The pyrolysis-field ionization mass spectra of the two grass species Phleum pratense and Lolium multiflorum were similar insofar as signals characteristic of lignin dimers and phytosterols dominated at higher masses and for mono-and polysaccharides at lower masses. The most prominent differences between overand underground plant constituents were indicated by higher relative abundances of lignin dimers in the stems and leaves and of sugars and suberin-derived phytosterols in the roots. In the investigation of the influence of mineral to organic matter ratios, comparatively weak effects of the inorganic matrix were obtained: firstly, in the lower mass range (m/z<250), secondly, for organic matter concentrations between 1.0% and 2.0%, and thirdly, for certain classes of compounds such as phenols, alkanes/alkenes, N heterocycles and mono-and polysaccharides. The qualitative differences in the molecular composition of soil organic matter were clearly attributed to its rapid increase during the first 7 years of the experiment and largely originated from a relative enrichment of lignin dimers. Then, in the period of steady-state soil organic matter levels, dynamic changes were indicated by slight enrichments of mono-and polysaccharides, alkanes/alkenes, fatty acids, N heterocycles, and fluctuating data for phenols/lignin monomers, lignin dimers, and the sum of N compounds. Alkylaromatics showed a steep increase between the 13th and 19th years and remained then on a high level.     

Transformation of lignin in surface and buried soils of mountainous landscapes

The content and composition of the lignin phenols in plants and soils of vertical natural zones were studied in the Northern Caucasus region and Northwestern Tien Shan. Three types of lignin transformation were revealed: steppe, forest, and meadow ones. It was shown that the degree of oxidation of the biopolymer during the transformation of organic matter increased when going from the living plant tissues to humic acids in surface and buried soils. The portion of lignin fragments remained unchanged during the biopolymer transformation in the following series: plant tissues-falloff-litter-soil-humic acids-buried humic acids. It was also shown that the biochemical composition of the plants had a decisive effect on the structure of the humic acids in the soils. The quantitative analysis of the lignin phenols and the ¹³C NMR spectroscopy proved that the lignin in higher plants was involved in the formation of specific compounds of soil humus, including aliphatic and aromatic molecular fragments. The first analysis of the lignin content and composition in buried soils of different ages was performed, and an increase in the degree of oxidation of the lignin structures was revealed in the soil chronoseries. It was proposed to use the proportions of lignin phenols in surface and buried soils as diagnostic criteria of the vegetation types in different epochs.     

A comparative study on the chemical composition of humic acids from forest soil,agricultural soil and lignite deposit: Bound lipid,carbohydrate and amino acid distributions

Base- and acid-hydrolysable fractions of humic acids (HAs) isolated from a forest soil, an agricultural soil and a lignite deposit were analysed, and comparisons were made between the base hydrolysable lipid (bound lipid), carbohydrate and amino acid signatures of the different humic acids.Bound lipids differ depending on the humic acid origin. Their composition were rather similar for the two soil humic acids, with three main lipid classes identified: (i) aliphatic components, (ii) aromatic components and (iii) sterols and triterpenols. The aliphatic subfraction was the most abundant and consisted predominantly of cutin- and suberin-derived moieties some of which could be clearly related to the vegetation. A minor bacterial input was indicated by the presence of short chain α- and β-hydroxyalkanoic acids. Aromatic subfraction contributed to a low amount to the total base hydrolysates and consisted mainly of lignin-derived methoxyphenols. Present in trace amounts, sterols and triterpenols are mainly of higher plant origin. The base hydrolysate from lignite humic acid markedly differs. Bound lipids released from lignite HA comprised almost exclusively aliphatic components, largely dominated by long chain alkanoic acids. Lignin-derived moieties, hardly detected, consisted solely of vanillic and 4-hydroxybenzoic acids indicating a much higher degree of lignin alteration in lignite humic acid. Sterols and triterpenols were absent.Although the composition of monosaccharides released upon acid hydrolysis was rather uniform irrespective of the humic acid origin, the distribution changed with the degree of humification of the HAs. Ratios of (Galactose+Mannose) to (Xylose+Arabinose) increased from soil to lignite humic acids. The high values of the ratios indicate that carbohydrates are primarily of microbial origin.In all humic acids neutral and acidic protein amino acids dominated. Non protein amino acids were only minor components consisting mainly of hydroxy proline and ornithine. The amino acid distributions of both soil HAs were similar. The amino acid distribution of lignite HA resembled that of soil HAs except for the following differences: (1) the absence of hydroxy proline and the greater abundance of ornithine suggesting a higher microbial contribution to the amino acids as the degree of humification increases, (2) the higher contribution of polar amino acids suggesting a preferential preservation of these amino acids possibly by interaction with the humic acid surface through hydrogen bonds.     

The significance of solid‐state 13C NMR spectroscopy of whole soil in the characterization of humic matter

Abstract

Differences in characteristics of humic matter were investigated by solid‐state CP/MAS ¹³C NMR spectroscopy of whole (nontreated) materials and their extracted humic fractions. Samples used in the analysis were lignite, a commercial humate AG, and the Bh horizons of a Mascotte and a Lawnwood soil. Humic fractions were extracted by the 0.1 M NaOH or Na₄P₂O₇ (pH 9.8) method. The humic (HA) and fulvic acid (FA) obtained were weighed and analysed for total acidity, carboxyl and phenolic‐OH group contents. Whole lignite, humate AG and soil samples, and the HA and FA fractions were analyzed by solid state CP MAS ¹³C NMR and infrared spectroscopy. Carbon, H, and N contents were determined by chemical analysis. NMR spectra of the combined HA+FA extracts resembled the spectra of the whole materials. No additional signals were detected, indicating that alien compounds were not produced during the extraction. The best spectra were obtained with HA samples produced by the NaOH method. These spectra closely resembled those of the untreated materials. Spectral and chemical differences noticed between the HA (or FA) fractions were attributed more to differences in origin than to the extraction procedure. Aliphatic, aromatic and carboxyl groups were the major components of HA from lignite and humate AG. In contrast, HA from the two Haplaquods were characterized by four major components: the aliphatic, polysaccharide, aromatic, and carboxyl groups. Regardless of origin, all the HA fractions contained similar functional groups, as indicated by their close similarities in infrared spectra.     

The origin of oxygen in soil humic substances

The ¹⁸O/¹⁶O ratios of a number of soil humic and fulvic acids were measured and compared with those of lignin and cellulose samples originating from the same area. The average enrichments above ground water were: cellulose 32%₀ humic and fulvic acid 29%₀ and lignin 14%₀, suggesting that the oxygen in humic and fulvic acid originates pricipally from cellulose or other plant carbohydrates and not lignin as has been suggested.