Methods for estimating Pg content in P type humic acid and for calculating Δlog K of its Pb fraction

Based on the assumption that P type humic acid is composed of green fraction (Pg) and brown fraction (Pb),. methods for estimating Pg content and calculatlng Δlog K of Pb by analyzing the absorption curve of P type humic acid were presented. A green fraction of P type humic acid obtained from an alpine meadow soil by gel filtration was used as the Pg standard (λmax 450, 575, and 615 nm, E 1% 290, 126, and 168).

Alkaline solutions of standard Pg obeyed the Beer-Lambert law within the concentration range of 1.6 to 31 ppm. To verify the methods, Pg content in the mixed solutions of Pg and A, B, or Rp type humic acid was estimated. It was found that the recovery of Pg would be 95±14 (2S) per cent with a probability of 0.99. Also, the calculation of Δlog K of humic acid in the mixed solutions gave satisfactory results.

It was distinctly shown that the shape of absorption spectra of P type humic acid did not only depend on Pg content, but also on Δlog K of Pb.     

The chemical nature of the green fraction of P type humic acid

P type humic acid has absorption bands near 615, 570 and 450mµ in the visible region, and is a common component of many soils in north temperate regions (1, 2, 3). Its unusual spectral characteristics are due to a green fraction (Pg), which can be separated from the humic acid dissolved in aqueous alkali by chromatography on Sephadex or cellulose powder, and is precipitated as a reddish-brown substance on acidification (2). In aqueous alkali, it has λmax at 613, 568, 448, 430 (infl.) and 281mµ. The distribution of Pg in different soils is probably related to the presence of fungal sclerotia. The highest content of Pg in p type humic acid is about 10%. We have now obtained evidence that the characteristic colour of Pg is due to the presence of a derivative of 4,9 - dihydroxyperylene - 3,10 - quinone.     

Abstracts of Nippon Nojohiryogaku Zasshi

Abstract

P type humic acid which has absorption bands near 615, 570, and 450 run due to the presence of dihydroxyperylenequinone (DHPQ) was investigated by gel-chromatography. Humic acid samples were fractionated using Sephadex G-50 as gel material and 0.1 n NaOH, with or without 1.5 m urea, as eluent. The absorption spectra of the effluent fractions were measured, and elution curves were drawn on the basis of the optical density at 600 nm.

In general, the intensity of the absorption bands due to DHPQ increased with increasing fraction number, accompanied by a change in effluent color from brown, greenish brown to green. Based on the elution curves, the effluent fractions of a humic acid containing a large amount of the green fraction were divided into six groups: B₁, B₂, BG, G₁, G₂, and G₃. These fraction groups, especially the G groups and cercosporin, a dihydroxyperylenequinone pig ment, were compared by absorption spectroscopy, ultrafiltration, ultimate analysis, TLC, and gel-chromatography.

The green fraction was composed of a non-humic, relatively lower-molecular weight polymer containing a considerable amount of DHPQ nucleus, while the brown humic fraction still maintained the characteristic light absorption due to DHPQ. It may be tentatively con cluded that the green fraction is not only a co-extracted impurity, but is also firmly associated with dark-colored humic substances.     

Studies on the humus forms of forest soils

The results of humus composition analysis of each fraction separated physically from each horizon of the five forest soils used in the last paper (3) were presented.

Regular changes in humus composition were observed with the gradation of horizons and With the decrease in particle size. Most of the humic acids from plant residual fractions belonged to the Rp type; hurnic acids from the L layers showed a tannin-like character which disappeared with the progress of decomposition, and humic acids from the smaller particle fractions of the layers contained a Pg fraction.

As for humic acids from the A horizon, they all belonged to the P or B type and contained a Pg fraction. In addition, it was concluded that the humification process in the mineral layer Was clearly different from that in the organic layer.     

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.     

Chromatographic Separation of Green Humic Acid from Podzol Humus

One of the authors, Kumada ¹⁾ has found that absorption spectra of podzol humic acids, remarkably different from the other soil humic acids, have fairly distinct absorption bands near 620, 570 and 450 mµ, and he tentatively named them. P type humic acid. P type humic acid seems specific not only in its visible absorption spectra, but also in infra. red absorption spectra, in susceptible to electrolytes, etc. The authors are now engaged in the study of the nature and properties of P type humic acid, and recently they have found that it is composed of at least two main fractions, the one is reddish brown, and the other green, in alkaline solution. The experimental results are briefly presented.     

Humus composition of soils under forest, coffee and arable cultivation in hilly areas of south Sumatra, Indonesia

To understand the effect of land use changes on the composition of humus in tropical soils, samples from land under primary forest, secondary forest, coffee plantation, and arable crops were investigated at three sites in south Sumatra, Indonesia. Total carbon and total nitrogen contents were 1.7 to 4.3 times and 1.1 to 2.8 times greater in the topsoil under primary forest than under the other types of land use. Following change from primary forest to other uses, the proportion of humic acids in the organic matter of the topsoils decreased while that of the fulvic acid fraction increased. Within the range of land uses, differences in the yields of humic acids and fulvic acid fractions were, respectively, larger and smaller than those in total carbon content. The humic acids were classified into the low and middle classes in the degree of humification. Absorption due to the green fraction of humic acids, Pg, was detected in the UV‐visible spectra of almost all the humic acids. No relation was observed between the degree of humification of humic acids or the strength of Pg absorption in their spectra and land use change. The fulvic acid fractions were fractionated on insoluble polyvinylpyrrolidone (PVP) into the adsorbed fractions consisting of humic substances and the non‐adsorbed fractions consisting of non‐humic substances. A positive correlation between the amount of the fulvic acid fraction and the percentage of the PVP‐adsorbed fraction within it indicated that the variation in the amount of the fulvic acid fraction was attributable to acid‐soluble humic substances. The ionization difference spectra of solutions between pH 12 and pH 7 suggested that the chemical structures of the PVP‐adsorbed fulvic acids have been altered by land use change.     

The determination of 1,2-dicarboxylate structures in humic acid by fluorescein formation

To detect and quantify 1,2-dicarboxylate structures in humic acid, a chemical reaction forming fluoresceins from 1,2-dicarboxylates and resorcinol was tested. Resorcinol reacted with humic acid to form products having absorption maxima at about 490 nm. A procedure was then developed for quantitative analysis of the radicals. Eight humic acid samples tested contained 0.4–1.0 mmol/g of 1,2-dicarboxylate structures. The 1,2-Dicarboxylates detected in humic acid were considered as ophthalate homologs.     

Characterization of the perylenequinone pigments in Japanese Andosols and Cambisol

The green fraction of humic acid (Pg) and the chloroform-extractable green fraction (CEGF) are characteristic soil organic matter (SOM) components. These alkaline solutions are green-colored due to the presence of 4,9-dihydroxyperylene-3,10-quinone (DHPQ) chromophore. While both of which are potential indicators for the effect of land use and paleoclimatic environment in the fields of soil science as well as geochemistry, CEGF as well as its relationship with Pg in soils are not yet fully documented. In this study, we firstly investigated the chemical properties of soil CEGF fractions by ultraviolet–visible (UV–Vis) and infrared (IR) method. Two CEGF components were separated by sequential liquid-liquid extraction using aqueous ammonium hydroxide (NH₄OH) followed by aqueous sodium hydroxide (NaOH). Results showed that the UV–Vis spectral shape of NH₄OH-extractable component is very similar to that of DHPQ, except that it is red-shifted. The solubility and UV–Vis spectrum of the NaOH-extractable fraction were completely identical with those of synthesized DHPQ. Their IR spectral shapes were also almost the same. Subsequently, the distribution of CEGF in humic acid (HA), fulvic acid (FA) and humin (HN) from Japanese Andosols and Cambisol was quantitatively evaluated by sequential extraction. Most of CEGF was detected in the HA (60–78%) and HN (22–40%), but not in the FA. While the UV–Vis spectral shape of CEGF extracted from Andosols HAs showed a relatively higher proportion of DHPQ than its derivative, the opposite was observed in Cambisol HA, whose CEGF is similar to that of sclerotium grain (one of the possible origin of CEGF). These results suggest the diversity of CEGF-producing soil fungi. Quantitative data also indicated that 35–49% of Pg consisted of a chloroform-soluble fraction (i.e., CEGF) and the remaining 51–65% of Pg was chloroform-insoluble. Based on these results, we propose that CEGF is composed of DHPQ and DHPQ-derivatives and that CEGF is one of the major fractions of Pg.     

The distribution of PG (green fraction of P type humic acid) and the degree of humification of PB (brown fraction of P type humic acid) in soils of central Japan

Abstract

Pg contents of soils chosen from the major soil groups occurring in the mountain area of Central Japan were measured by the method presented by the author in a previous paper. The Pg contents of the soils ranged from 0 to 0.58 per cent of the dry soils. In podzolic soils, Pg contents were high in A horizons and gradually decreased with depth and in some cases of brown forest soils, Pg contents were highest in B horizons. The percentage of Pg in humic acid ranged from 0 to 8.6 per cent, and the highest value was observed in the C horizon of Dando Bo soil, Pg contents were high in humid and acidic soils containing much humic acid. Humidity, soil pH and humus content may affect the Pg accumulation by controlling the activity of Pg-producing fungi. ? log K values of Pb were calculated to estimate the degree of humification of P type humic acids and indicated that Pb's were relatively immature.     

不同施肥处理土壤胡敏酸及其级分与Fe2+络合特征Ⅱ.胡敏酸级分与Fe2+络合特征

采用酒精沉淀法对不同施肥处理胡敏酸进行分级 ,在研究胡敏酸级分组成变异以及各级分性质变化的基础上 ,研究了胡敏酸各级分与Fe2+的络合特征。结果表明 ,胡敏酸各级分随级分数的增大芳构化度逐渐降低 ,分子结构趋于简单。在所分离的 7个级分中 ,均以级分 3与Fe2+的络合能力最强。从级分 1到级分 3络合能力逐渐增强 ,级分 3到级分 7络合能力逐渐降低。胡敏酸A型级分的络合能力一般大于P型。但若A型级分芳构化度过高 ,也存在A型级分小于P型现象。Rp型级分的络合能力明显比A型、P型级分小。不同施肥处理胡敏酸原样与Fe2+络合能力差异与其级分组成变异以及各级分的络合能力有密切关系     

Method for Estimating the Content of the Chloroform-Extractable Green Fraction (CEGF) in HCl-DMSO Extract of Soils

Chloroform-extractable green fraction (CEGF) was detected in the supernatant obtained by alkali precipitation from the HCI-dimethylsulfoxide (DMSO) extract of Pg-rich soil. In the alkaline solution, the color of CEGF was green and CEGF showed strong Pg-like absorption bands. Ultraviolet and visible (UV-VIS) spectral analysis and gel chromatography on Sephadex G-50 were performed to compare several properties between CEGF and Pg. CEGF, which was purified by gel chromatography on Bio-Beads SX-1, displayed strong absorption bands at 609, 562, 445 and 280 inn in the alkaline solution. These absorption bands were almost similar to those of Pg. Furthermore, the UV-VIS spectrum of CEGF in the organic solvents showed a similar characteristic pattern of 4,9-dihydroxyperylene-3,10-quinone (DHPQ), which was considered to be a chromophore of Pg. Based on the results of gel chromatography on Sephadex G-50, CEGF mainly consisted of two fractions, corresponding to the G₂ and G₃ fractions of Pg. These results indicated that the method for extracting CEGF from Pg-rich soil in the present study was easy and selective and that CEGF was one of the components of, or a closely related substance to Pg. A colorimetric method for the estimation of the CEGF content in soils was developed. The calibration curve of CEGF was linear over a wide range of contents from 2.75 to 220 mg L⁻¹. The CEGF content in twelve samples of various soils was examined. CEGF was detected in all the soil sampled (5 orders) including three samples (3 orders) where Pg was not detected, and the content ranged from 0.07 to 1.66 g kg⁻¹ (dry soil). Therefore, the method for estimating the CEGF content in soils developed in the present study was found to be suitable for various soil orders and it was assumed that CEGF occurred in various soil orders.     

New phenolic compounds formed by evolution of (+)-catechin and glyoxylic acid in hydroalcoholic solution and their implication in color changes of grape-derived foods

The reaction between (+)-catechin and glyoxylic acid in model solution system was investigated by LC/DAD and LC/ESI-MS analyses. The formation of phenolic compounds exhibiting absorption maxima near 300 nm and presenting a shoulder around 350 nm was observed. Their structures consisted of a (+)-catechin unit with one or two aldehyde groups linked at positions 6, 8 or 6 and 8 of the A ring. In addition, new yellow pigments exhibiting UV-visible spectra similar to those of xanthylium salts with absorption maxima at 450 and 280 nm were also detected. The major yellow compound was isolated and identified by ESI-MS and 1D and 2D NMR spectroscopy. The implication of these compounds in color change and browning observed during aging of grape-derived beverages is also discussed.     

Studies on tile composition of aromatic nuclei of humus

X-ray diffraction analysis of humic acid (1) revealed that. among four types of humic acid. A. D. Rp and P type according to the classification system proposed by Kumada (2), only A type had the distinct diffraction band corresponding to (002) spacing of amorphous carbon. It was also reported that several artificial humic acids prepared from anthraquinone, hydroxyanthraquinone. charcoal. anthracite etc. showed high similarity to A type humic acid in their spectrophotometric properties and stability toward oxidizing reagent (3).     

不同磺化腐殖酸磷肥提高冬小麦产量和磷素吸收利用的效应研究

【目的】腐殖酸对磷肥增效的调控效应与其结构性密切相关。本文比较了不同磺化反应方法制备的腐殖酸磷肥对冬小麦磷素利用的影响,为制备调控磷肥专用的腐殖酸增效载体提供依据。【方法】采用磷酸与氢氧化钾反应法制备普通磷肥 (P)、普通腐殖酸磷肥 (HAP),并采用加双氧水、硝酸等方法制备了四种磺化腐殖酸磷肥 (HA1P、HA2P、HA3P和HA4P)。用田间土柱栽培试验方法,在等磷量基础上,设置普通磷肥 (P)、普通腐殖酸磷肥 (HAP)、磺甲基化腐殖酸磷肥 (HA1P)、双氧水+磺甲基化腐殖酸磷肥 (HA2P)、硝酸+磺甲基化腐殖酸磷肥 (HA3P)、双氧水+硝酸+磺甲基化腐殖酸磷肥 (HA4P) 6个处理,同时设置不施磷肥对照 (CK) 处理和施用等量腐殖酸处理 (C-HA、C-HA1、C-HA2、C-HA3、C-HA4)。调查了小麦产量和产量构成及经济效益,分析了0—80 cm土壤有效磷含量。【结果】1) 与CK相比,普通腐殖酸 (C-HA) 和磺化处理腐殖酸 (C-HA1、C-HA2、C-HA3、C-HA4) 对小麦籽粒产量无显著影响。与P处理比较,HAP、HA1P、HA2P、HA3P、HA4P处理的小麦籽粒产量分别提高了6.3%、17.8%、10.1%、17.5%、11.1%,4个腐殖酸磺化磷肥 (HA1P、HA2P、HA3P、HA4P) 处理均高于普通腐殖酸磷肥 (HAP) 处理。2)与HAP比较,磺化腐殖酸磷肥处理HA1P、HA2P、HA3P、HA4P分别提高小麦地上部磷吸收量12.3%、12.3%、9.2%、10.8%,其中HA1P和HA2P处理最高。3）与HAP比较,磺化腐殖酸磷肥处理HA1P、HA3P分别提高小麦磷肥农学效率23.6%和7.1%。4）与HAP比较,磺化腐殖酸磷肥处理HA1P、HA2P、HA4P可分别提高0—20 cm土层土壤速效磷含量17.5%、16.2%、17.2%。【结论】磺化腐殖酸磷肥比普通腐殖酸磷肥可以更有效地提高土壤中磷肥的有效性,提高冬小麦对磷素的吸收利用,进而提高冬小麦籽粒产量。四种磺化工艺中,以磺甲基化处理的腐殖酸磷肥 (HA1P) 效果最优。     

Multivariate statistical analysis of mass spectra as a tool for the classification of the main humic substances according to their structural and conformational features

The aim of this work is to explore the suitability of the complementary use of mass spectra and the corresponding statistical analysis (principal components-Pareto analysis (PCA) and discriminant analysis (DA)) of these spectra to differentiate diverse humic samples as a function of their structural and conformational features. To this end, the mass spectra of humic samples belonging to the main humic fraction types (gray humic acid, brown humic acid, and fulvic acid) were obtained by electrospray ionization mass spectrometry (ESI-MS). The results obtained showed that the application of PCA yielded a clear separation between blanks and humic samples. However, a clear differentiation among the humic fraction types was not achieved. The DA of PCA data, however, yielded a clear separation among the humic substances (HS) samples belonging to each HS fraction type considered: gray humic acids, brown humic acids, and fulvic acids. These results showed that the mass spectra of each humic sample include characteristic mass/charge (m/z) distribution values that can be considered as a "fingerprint" representative of its specific structural features. Our results also indicate that, although the m/z values principally corresponded to single-charged ions, we cannot identify these molecular weight distributions with those of humic samples, since sample molecular fragmentation, as well as partial molecular ionization, cannot be ruled out under our experimental and instrumental conditions.     

腐殖酸在黑云母表面的吸附动态和分布特征研究

采用原位原子力显微镜智能模式初步观察了弱酸性土壤腐殖酸分子在层状硅酸盐含钾矿物黑云母表面的吸附动态和形貌变化。采用纯化、干燥后的腐殖酸固体样品配制p H 6.0,浓度分别为0 mg L-1、100 mg L-1和1000 mg L-1的腐殖酸溶液。原位观察所用的矿物薄片为土壤中常见的2∶1型含钾矿物—黑云母,其化学结构式为K(Mg2.46Fe0.45Ti0.09)[Al Si3O10](OH)2。结果表明,时间尺度是影响界面上腐殖酸形貌结构的一个重要因素。在通入p H 6.0的腐殖酸溶液作用下,原位反应0~180min内,腐殖酸分子与黑云母(001)面的相互作用以吸附为主并伴随脱附现象,吸附的腐殖酸呈球状,为单个或多个腐殖酸分子的聚集体,高度介于1~5 nm之间;原位反应180~300 min内,黑云母(001)面上吸附的腐殖酸仍以颗粒态为主,高度介于3~5 nm之间,平均为4 nm;而黑云母(001)面上的台阶和边缘位置则分布有较多的片状结构以及黑云母线型边缘诱导产生的链状结构腐殖酸聚集体,其高度介于6~8 nm,较颗粒态腐殖酸的聚集体平均高出约2.5 nm。此外,腐殖酸分子不仅能够在黑云母表面进行吸附和脱附作用,还能够溶解黑云母的表面结构,促进层间K及结构单元内Al和Si的释放。因此,腐殖酸与云母类矿物的界面反应是一个溶解—吸附—聚集并伴随着脱附和离子释放的过程。     

The humus status of brown forest soils and its changes upon intensive tea growing in the subtropical zone of Russia

Changes in the humus status of acid brown forest soils used for tea growing have been studied in a long-term stationary experiment after 20 years of the application of mineral fertilizers in increasing doses. It is shown that long-term tea growing has resulted in a reliable increase in the humus content within the upper cultivated horizon (Ap, 0–40 cm) at the expense of the nonhydrolyzable fraction of humus (humin) and the first fractions of humic and fulvic acids. The fractional composition of humus has changed against the background of a stable fulvate type of humus (Cha/Cfa < 0.5). The portion of the first fraction of humic and fulvic acids in the total amount of humus has increased, as well as the portion of the first fraction of humic acids in the total amount of humic acids. These changes are due to the enhanced humification of plant residues with a parallel decrease in the contents of the second (Ca-bound) and third (bound with clay and stable sesquioxides) fractions of humic substances.     

Effects of prolonged irrigation on the humus of steppe soils in southwest Siberia

Changes in humus resulting from up to 20 years' irrigation with slightly saline water were studied in dry steppe soils in southwest Siberia. The amounts of brown humic acids and of the Pg fraction increased with the period of irrigation, and their maximum amounts occurred at greater depths in the profile. Other fractions of humus and humic acids fluctuated within the limits typical of dry steppe soils. The humus therefore remained essentially stable under irrigation for up to 20 years.     

The infrared identification of a humo‐polysaccharide ester in soil humic acid

Abstract

Studies involving methylation, dissolution, infrared spectroscopy and gas‐liquid chromatography were conducted with humic compounds extracted from a Greenville loam and a Cecil sandy loam.

The results indicated that methylated humic acid and non‐methylated hymatomelanic acid have similar infrared characteristics, with intense C‐H absorption at 2980 ‐ 2920 cm^‐1, and strong C=0 stretching but with very weak C00^‐ stretching at 1720 and 1650 cm^‐1 respectively. Gas‐liquid chromatography yielded chromatograms, having the same number of components, appearing at similar retention times. Dissolution separated hymatomelanic acid into fraction A, with humic acid characteristics; and fraction B, a polysaccharide. This separation released the infrared carboxyl peak at 1650 cm^‐1 in fraction A.

It was concluded that hymatomelanic acid was a naturally occurring ester compound, composed of humic material in ester linkages with polysaccharide.