Separation and characterization of the constituents of compost and soil humic acids by two-dimensional electrophoresis

To investigate the chemical heterogeneity of humic acids (HAs), we applied two-dimensional (2-D) electrophoresis to HAs from a compost and two types of soils. In this method, HAs are first separated by isoelectric focusing (IEF) and then separated by polyacrylamide gel electrophoresis (PAGE). IEF and PAGE were carried out in the presence of 7?M urea. Upon 2-D electrophoresis of HAs, dark-colored substances were spread out across the gel mainly in the isoelectric point (pI) range of 3.0–4.5. Green fluorescence was observed in the smaller molecular size region of the gel, especially in the pI range of 3.0–4.5, and the most intense fluorescence was found at the moving front. The gels were divided into 36 sections, and then HA constituents were extracted from the individual sections and recovered by precipitation with acid. The distribution of organic carbon (C) among the gel sections coincided with that of the dark-colored substances on the gel. The total C recoveries were only 43–50%, suggesting that a considerable amount of HA constituents was lost during the extraction from the gels and purification. High-performance size-exclusion chromatography confirmed that the constituents of HAs were separated based on their molecular sizes by PAGE. The measurement of diffuse reflectance infrared Fourier transform (DRIFT) spectra indicated that the chemical properties of the HA constituents differed depending on the position on the gels and were affected by the molecular size rather than the pI. The fractions of the compost HA were characterized by higher proportions of aliphatic, proteinous and polysaccharide moieties and by the presence of lignin-derived structures. For the soil HAs, the fractions were characterized by a high proportion of the carboxyl group and a low proportion of aliphatic moieties. The proportion of proteinous and polysaccharide moieties in the fractions of soil HAs decreased with decreasing molecular size. The chemical properties of the green fluorescent substances remained unclear, since there was not enough of the substances to measure the DRIFT spectra. The present study showed that 2-D electrophoresis in the presence of concentrated urea offers an effective method for fractionating and isolating the constituents of HAs.     

Tandem size exclusion chromatography-Polyacrylamide gel electrophoresis of humic acids

Humic acids (HAs) from four soils were fractionated by size exclusion chromatography (SEC) on Sephadex G-75. Three fractions were obtained in all humic acids, collected and assayed by Polyacrylamide gel electrophoresis (PAGE). The unfractionated HA from each soil was used as reference. Each chromatographic fraction formed one electrophoretic zone corresponding closely to one band of the reference sample with some admixture of the fractions preceding or following. The results indicate that fractionation of HAs by tandem SEC-PAGE can be successfully used for obtaining fractions of reduced polydispersity and different electrophoretic mobilities. Pyrolysis/methylation-gas chromatography-mass spectrometry of the full size preparations of HA and fractions with exactly defined molecular size and electrophoretic mobility showed a different distribution in humic components, mainly lipids, lignin derivatives and N-containing compounds.     

The characterization of Nigerian varieties of pepper, Capsicum annuum and Capsicum frutescens by SDS- polyacrylamide gel electrophoresis of seed proteins

The possibility of using electrophoresis to characterize varieties of pepper, Capsicum annuum and Capsicum frutescens cultivated in Nigeria was investigated. The SDS- polyacrylamide gel electropherogram of extracted total seed proteins of 10 breeding lines in each of the 6 varieties investigated, revealed a pattern in which 12 polypeptide bands with apparent molecular weight range of 22 to 98 kilodaltons could be distinguished. The result showed that the six varieties could be characterized on the basis of presence/absence and staining intensities of 7 polypeptide bands. It is suggested that SDS- polyacrylamide gel electrophoresis of seed proteins provides a useful analytical technique for the characterization of varieties of pepper and there may be genotype duplicates in the collection of Nigerian Capsicum germplasm.     

Temporal changes in humic acids in cultivated soils with continuous manure application

Abstract

Although the application of manure to upland fields is believed to induce changes in the quality of humic substances in soil as well as the quantity, the direction and extent of these changes have not been elucidated. To understand temporal variations in humic acids, periodically collected soil samples from two fields, a Typic Hapludult (Togo) and a Pachic Melanudand (Kuriyagawa), with cattle manure and chemical fertilizer (CF) were examined. The content and degree of humification (darkening) of the humic acids were distinctly greater in Kuriyagawa than in Togo soil. Corresponding to the difference in the degree of humification, molecular size distribution, elemental composition, infrared (IR) spectra, and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra of humic acids differed between the two soils. Manure application at 40 Mg ha^?1 year^?1 for 16 years (Togo) and at 80 or 160 Mg ha^?1 year^?1 for 19 years (Kuriyagawa) resulted in greater humic acid content compared with plots with CF only because of its increase in the manured plots and/or decrease in the CF plots. Manure application at an extremely high rate (160 Mg ha^?1 year^?1) resulted in higher H content and greater signal intensities of alkyl C, O-alkyl C and amide C=O in the ¹³C CPMAS NMR and/or IR spectra. Although humic acids with larger molecule sizes increased in all the manured plots, differences between the humic acids from the plots with and without manure applied at practical levels in the elemental and spectroscopic analyses were small or scarce. These results were considered to be because of the similarity between the indigenous soil humic acids and the manure-derived ones in Togo soil (a low degree of humification) and because of the abundance of highly-humified humic acids in Kuriyagawa soil.     

腐植酸与尿素结合工艺对尿素在潮土中转化的影响

通过研究腐植酸与尿素结合工艺对尿素在土壤中转化的影响,为腐植酸尿素生产工艺的选择提供科学依据.在石灰性潮土上进行土壤培养试验,设置8个处理:不施尿素(CK)、普通熔融尿素(U)、0.5％添加量的腐植酸(HA0.5)、5％添加量的腐植酸(HA5)、腐植酸添加量为0.5％的掺混腐植酸尿素(HA+U0.5)、腐植酸添加量为0...     

Isoelectric focusing of soil humic substances in the presence of 8M urea

Summary The isoelectric focusing (IEF) of different-molecular-size humic fractions, either separately or recombined, was carried out in the presence and absence of 8M urea. In the presence of urea, the fractions were recognizable from both their characteristic microheterogeneity and ranges of focalization. Urea caused a slight shift of the bands towards lower pH values. The refocusing of characteristic band of one fraction produced a slight discrepancy in the isoelectric points (0.2–0.3 pH units). However, the maintenance of the banding in 8M urea and the homogeneity of the band under refocusing demonstrated the reliability of the IEF in fractionating structurally different humic substances. Such experiments should provide useful information on the association/dissociation properties of soil organic matter under controlled conditions.Joint program of C.N.R. (Italy) and C.S.I.C. (Spain), no. 7     

Chemical and spectroscopic characteristics of humic acid from a clay loam soil in Ontario after 52 years of consistent fertilization and crop rotation

Long-term fertilization and crop rotation can influence both organic C sequestration as well as the C composition of soils and the more resistant organic C compounds contained in humic acid(HA). This study examined the effects of fertilization and cropping type(monoculture corn(MC) and Kentucky bluegrass sod(KBS) and corn-oat-alfalfa-alfalfa rotation(RC)) on the HA composition of soil from a 52-year field study in southern Ontario, Canada. Humic acid samples were extracted from soil, and elemental analysis, infrared spectroscopy, solid state 13C nuclear magnetic resonance spectra, and electron paramagnetic resonance methods were used to determine the influence of the cropping type on the characteristics of HA. Both fertilization and cropping type affected the chemical characteristics of HA. Fertilization led to a 5.9% increase in C, a 7.6% decrease in O, and lower O/C and(N + O)/C ratios in HA as compared to the corresponding non-fertilized treatments. Rotation resulted in a lower proportion of C(48.1%) and a greater(N + O)/C ratio(0.7) relative to monoculture cropping. Infrared spectroscopy analysis showed that HA contained more C-O groups in fertilized soil than in non-fertilized soil under MC and KBS. Fertilization increased the O-alkyl-C, phenolic-C, and free radical contents of HA relative to non-fertilization treatments. Rotation decreased the aliphatic and carboxyl groups and increased the O-alkyl, carbohydrate, aryl, and phenolic groups and free radicals, relative to MC and KBS. Both long-term crop rotation and fertilization dramatically modified the soil HA composition. Significant relationships were observed between the molecular composition of HA and soil organic C. Hence, humic acid characterization could be used as an indicator of the long-term sustainability of crop management practices.     

不同分子量风化煤腐殖酸对玉米植株主要代谢物的影响

【目的】腐殖酸的分子量决定了其功能特性,研究不同分子量腐殖酸对玉米生长发育的影响,可为腐殖酸资源的高效利用提供理论依据。【方法】选用‘郑单958’为供试玉米品种,以霍格兰营养液为基础溶液进行水培试验。选择分子量为> 50 kDa (HA_H)、10 kDa～50 kDa (HA_M)和<10 kDa (HA_L)三种腐殖酸,添加量均为碳10 mg/L,以不添加腐殖酸为对照(CK),共四个处理。幼苗生长20 d后收获,分为根、茎和叶样品,烘干称重,采用傅里叶变换红外光谱仪(型号VERTEX 70,德国Bruker公司)检测玉米植株根、茎和叶的光谱特征。【结果】腐殖酸处理玉米根、茎和叶的干物质重较对照分别显著提高了91.07%、89.27%和88.53%。三种腐殖酸处理,以小于10 kDa分子量腐殖酸(HAL)对玉米植株生长的促进作用最大,根、茎和叶的干物质重分别提高了143.14%、123.41%和150.54%,其次为10～50 kDa分子量腐殖酸(HA_M),大于50 kDa分子量腐殖酸(HAH)效果又次之。腐殖酸处理的玉米根系FTIR光谱在3420 cm^–1和1655 cm^–1波数附近的透射率均低于对照,茎FTIR光谱在3420 cm^–1和1655 cm^–1波数附近的透射率均高于对照,表明腐殖酸处理能增加玉米根系碳水化合物、蛋白质、多肽和氨基酸类物质的含量,减少其在茎中的积累,腐殖酸各处理玉米叶片在3420、2920、1735、1655、1518、1380、1250和1050 cm^–1波数附近的透射率均低于对照,小于10 kDa分子量腐殖酸表现尤为明显。这表明不同分子量(尤其是小于10 kDa分子量)腐殖酸能够增加玉米叶片碳水化合物、脂类物质、蛋白质、多肽、氨基酸类物质和核酸等的含量。【结论】外源添加腐殖酸能够增加玉米干物质重,引起玉米植株不同器官中碳水化合物、脂类物质、蛋白质、多肽、氨基酸类物质及核酸发生变化,小分子量(<10 kDa)的腐殖酸对玉米植株生长的促进作用大于大分子量的,在生产中应尽量选择小分子腐殖酸产品,以充分发挥其促生作用。     

Influence of plant growth regulators and humic acid on the phytoremediation of lead by maize in a Pb-polluted calcareous soil

Phytoremediation is a cleanup technology for the removal of contaminants from polluted soils. The influences of humic acid (HA) and four plant growth regulators, i.e. gibberellic acid, salicylic acid, 3-indoleacetic acid, and 6-benzylaminopurine on the phytoremediation of Pb by maize were investigated in a pot trial on a Pb-contaminated soil. Foliar spray of 6-benzylaminopurine and salicylic acid significantly increased mean root dry matter but none of growth regulators had a significant effect on that of shoot. Addition of HA decreased root dry matter and had no significant effect on that of shoots. Application of all growth regulators except gibberellic acid significantly increased root Pb uptake. Application of salicylic acid significantly increased mean uptake efficiency and shoot Pb concentration and uptake. Although addition of HA significantly increased mean phytoextraction and translocation efficiencies, these responses were mainly attributed to the role of HA in the reduction of both root dry weight and Pb uptake, meaning that HA is not promising in Pb phytoremediation by maize. Among the four plant growth regulators evaluated, salicylic acid was the most effective in enhancing phytoremediation through phytostabilization of Pb.     

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.