Electrophoretic subfractionation of low and high molecular weight humic acids fractions

Summary Soil humic acid was fractionated on a molecular weight basis either using Sephadex gel filtration or electrophoresis on a discontinuous polyacrylamide gel. Low and high molecular weight fractions obtained by these two methods were choosen for subsequent subfractionation using electrophoretic methods. The high and low molecular weight fractions yielded several subfractions after separation by isotachophoresis or isoelectric focusing. Components of the high molecular weight fractions occupied the upper portion of the mobility train; components of the low molecular weight fractions lead the mobility train. Adsorption by Sephadex was avoided by using 4M urea as an eluent. The elution of the humic substances adsorbed to the polyacrylamide gel matrix was achieved by using a 0.1M Tris –0.025M EDTA solution.     

Viscosimetric studies on molecular weight fractions of fulvic and humic acids of aquatic,terrestrial and microbial origin

Summary Viscosities were investigated of solutions of fulvic and humic acid molecular weight fractions of aquatic, terrestrial and microbial origin. Aquatic fulvic and humic acid molecules were, at pH 7, more voluminous than other types of humic compounds of similar molecular weight. It would appear that in low molecular weight non-aquatic humic matter, more inter- than intra-molecular bonding is present, with increasing molecular weight the bonding becomes more intra-molecular. Differences between average molecular weight values as obtained by an ultrafiltration method (Amicon) and by viscosimetry ranged from –18.7 to 18.5%. The largest deviations were in the low molecular weight range (<5,500 daltons). Higher molecular weight humics (in particular humic acids) appeared to have a more elongated structure than lower molecular weight material (in particular fulvic acids). Indications were obtained that on hydration humic moleculars become more elongated.     

The fractional precipitation of soil humic acid by ammonium sulphate

Summary A number of fractions have been obtained from a soil humic acid by salting-out and by leaching with ammonium sulphate. The fractions obtained are characterised by infra-red and ultra-violet spectroscopy and osmometry. The fractions removed at relatively low saturation with ammonium sulphate are less highly charged and contain more aliphatic hydrocarbon groups per unit weight than the fractions precipitated at high concentrations of salt. In addition, the number average molecular weight of successive fractions precipitated with ammonium sulphate decreases significantly.     

Humic-like substances of bacterial origin. I. Some aspects of the formation and nature of humic-like substances produced by Pseudomonas.

Two bacterial strains Pseudomonas acidovorans No 26 and Pseudomonas sp. No 4 grown in Conn and yeast extract-glucose media, or in the media enriched with tyrosine, were found to produce dark brown pigment. It was shown that in the bacterial cultures numerous phenolic and quinone-type compounds were formed and transformed to humic-like polymers. Formation of humic-like substances started in the bacterial cells and was accompanied by the presence of phenyloxidases in the bacterial cultures. The bacterial "humic acids" were obtained from the supernatants in amounts varing from 0.05 to 0.865 mg/1 mg of dry weight of cells and from the cells in amounts of 0.02 to 0.165 mg/1 mg of dry weight of cells, depending on the medium used and time of incubation. The IR spectra of the bacterial "humic acids" appeared to be very similar to IR spectrum of the synthetic humic acids (Fluka A.G.) and contained the same chemical groups as the soil humic acids. The culture medium after growth of the strain No 26 was fractionated into "fulvic, hymatomelanic and humic acid" fractions. The hydrolysates from the obtained fractions contained amino acids and uronic acids. The amino acid composition appeared to be very similar to that of soil humic acids.     

High and low molecular weight fractions of humic and fulvic acids

Summary The present investigation was initiated to study molecular weight fractionation of humic compounds isolated from surface samples of an Ultisol (Red Yellow Pc lzolic soil) and a Spodosol (Podzol), using fine (mol. wt. cutoff 3500) and coarse (mol. wt. cutoff 12000) dialysis membranes and sephadex gel filtration. Characterization of the humic fractions was conducted by elemental analysis and infrared spectroscopy. The results confirmed that fulvic acid (FA) was higher in ash and elemental content than humic acid (HA). With careful purification the amount of ash was found to be reduced to a minimum, but not to zero. Sephadex gel filtration revealed that fine HA (obtained with fine membranes) was composed of smaller amounts of HA-I (high mol. wt.) and large amounts of HA-II (low mol. wt. fraction). Coarse HA (obtained with coarse membranes) had almost equal amounts of HA-I and HA-II. Fine or coarse FA yielded only low molecular weight components after elution through sephadex. Infrared spectra of the humic fractions were indicative for the presence of phosphoglyceric acid as a possible constituent of the low molecular weight fraction of humic compounds.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.     

Humic-like substances of bacterial origin. II. Fractionation of the bacterial humic-like substances by gel filtration on sephadex gels.

Humic-like substances obtained from cells of Pseudomonas acidovorans were separated on Sephadex G-25 into two groups of substances of different molecular weight. The substances of the molecular weight greater than 5000 were successively separated on Sephadex gels G-50, G-75, G-100. Five fractions of different molecular weight were obtained, the percentage of which varied depending on the media used and time of incubation of the bacteria. Most (38%--46%) of the compounds contained in the bacterial humic acids were of approximate molecular weight of 40 000--50 000. The distribution of the fractions in the bacterial "humic-acids" was compared with those of the humic acid made by Fluka A. G. The synthetic humic acid contained most (approximately 40%) of the compounds of approximate molecular weight of 8000--10 000. In the bacterial and synthetic material the content of the compounds with the molecular weight above 100 000 was very similar (8%--12%).     

Properties of poultry litter humic acid fractions and their metal-complexes

Summary Molecularly homogenous fractions of humic acid extracted from poultry litter were characterized by elemental and functional group analysis, molecular weight determination, U.V. and infrared spectroscopy. The divalent and trivalent metal complexes prepared from different fractions of humic acid were characterized by infrared spectroscopy. The molecular weight of molecularly homogenous fractions of poultry litter humic acid ranged from 2545 to 40219. High amounts of functional groups in low molecular weight of humic acid fraction has been indicated by infrared spectra and by chemical analysis. The presence of chromophores C=C and C=O and auxochromes C−OH, C−NH were indicated by infrared and U.V. spectra of these humic acid fractions. Stable complex formation of Fe³⁺, Cu²⁺, and Zn²⁺ with −OH, −NH₂ and −COOH ligands of humic acid fractions involved electrovalent and coordinate-covalent bonds. Intensity of absorption bands of molecularly homogenous fractions of humic acid in I.R. spectra is differing depending upon the functional groups content of humic acid fractions. Journal paper No. 5. Department of Soil Science, R.A.U., T.C.A., Pusa-Dholi Campus, Dholi-843121, Muzaffarpur, Bihar, India.     

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HAs130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.     

不同腐解期玉米秸秆对塿土胡敏酸基本性质及级分变异的影响

采用田间腐解试验,在研究不同腐解期玉米秸秆对土壤胡敏酸性质影响的基础上,利用酒精分级沉淀法对土壤胡敏酸进行分级,研究了不同腐解期土壤胡敏酸级分组成及性质变化.结果表明,在整个腐解过程中土壤胡敏酸由A型转化为P型又转化为A型,呈现由复杂到简单又到复杂的变化趋势.不同腐解期土壤胡敏酸的级分组成不同.玉米秸秆更新土壤胡敏酸过程是一个双向过程,一方面使胡敏酸中结构复杂成分(级分1、2、3)向简单化发展,另一方面一些小分子胡敏酸(级分6、7)随时间推移按Rp→P→A途径逐渐缩合.     

The effect of humic acids and their different molecular mass fractions on germination in sunflower

Low molecular mass humic acid fractions caused a decrease in the germination rate. Only the highest concentration of humic acid solution decreases the soaking of fruits. Neither sodium content nor water and osmotic potential can account for that decrease. Seed respiration was increased in the presence of humic acids and their medium molecular mass fraction. This increase did not produce an increment in embryonic growth possibly as a result of interference in the respiration chain or decoupling in the oxidative phosphorylation.     

Contamination of humic acid by silica gel and sodium bicarbonate

Summary The extent of contamination of soil humic fractions by silica gel, sodium bicarbonate and sodium carbonate was investigated by infrared spectroscopy. Humic fractions were extracted with 0.1 N NaOH from a surface sample of a Davidson soil (Rhodic Paleudult, Reddish-brown Lateritic soil). After careful purification, the humic preparations were characterized by scanning with the Beckman IR-18A spectrophotometer using the micro KBr pellet technique. The spectra of the various humic fractions corresponded to the respective spectra reported in the literature. No indications were obtained for the presence of silica gel, sodium bicarbonate or sodium carbonate. Strong absorption at 1000 cm^-1, as exhibited by non-purified humic acid, could be caused by Si-O vibrations. However, this band disappeared after washing humic acid with HCl-HF.Contribution of the University of Georgia, College of Agric. Expt. Sta., College Sta., Athens, Georgia, USA.Contribution of the University of Georgia, College of Agric. Expt. Sta., College Sta., Athens, Georgia, USA.     

有机物质对土壤镉有效性的影响研究综述

土壤中的低分子有机酸和腐殖酸对镉的有效性有重要作用。根系分泌的低分子量有机酸能提高土壤镉的可提取率、移动性和生物有效性,但是更大分子量的有机酸EDTA却抑制植物对镉的吸收,腐殖酸促进土壤镉的溶解性;因腐殖酸组分和环境条件,腐殖酸能够促进也能抑制土壤镉活性,因此有必要深入研究影响腐殖酸固定镉的因素,以达到利用有机质抑制土壤镉活性的目的。     

Identification of Pt and Pd bound to humic acid species in spiked soils and street dusts by size exclusion chromatography and ICP-MS

Abstract

The formation of PGE-humic acid (HA) complexes in soil and street dust samples was investigated. In order to assess the distribution of Pt and Pd among molecular weight fractions of humic substances, the HA extracts (extracted by 0.1 mol L^?1 sodium pyrophosphate) were analysed by size exclusion chromato-graphy coupled on-line with UV-Vis detection. Similar chromatograms were obtained for soils and street dust samples (254 nm, 280 nm) and two size fractions were operationally defined as high (1600–5000 Da) and low molecular (< 1600 Da) HA fractions. The concentration of Pt and Pd in the separated extracts was determined by ICP-MS. The results indicate that up to 43% of Pt is in the high molecular and up to 52% in the low molecular HA fraction. In both type of soil samples, Pd is preferentially bound to the low molecular HA fraction. Dependence of Pd–HA and Pt–HA formation on the sample type both in the soils and in the street dust sample as well as on Pt oxidation state was established. Metallic Pt shows a tendency for complexation with the fractions of HA higher than 5000 Da. In the street dust samples, the distribution of Pt and Pd is similar and is strongly dependent on the sample type, being bound mainly to the fractions: higher than 5000 Da, 1600–5000 Da and the fraction lower than 1600 Da.     

Chemical and structural evolution of humic acids during organic waste composting

The effects of the composting process on the chemical and structural properties of humic acids have been studied in seven different organic waste mixtures from different origin. Only slight changes in elemental composition have been found in the humic acids after the composting process pointing to a more aromatic structure with higher proportions of oxygen and nitrogen. Functional groups were the most sensitive to the changes caused by the composting process showing a marked increase in the total acidity and phenolic, carboxyl and carbonyl groups. Gel permeation chromatography showed a slight increase in the average molecular weight of the humic acids. Infrared spectroscopy did not show important differences in humic acid structure but a clear decrease in the intensity of the bands in the region 3000–2850 cm^-1 corresponding to the aliphatic fractions. As a general result, the composting process yields humic acids in which the elemental and functional composition are closer to that of the more humified soil humic acids.     

Composition and metal ion complexation behavour of humic fractions derived from corn tissue

Decomposition of fresh plant residues produces humic fractions with different molecular size and composition. It was hypothesized that the functional group-type and content of humic fractions depended on molecular size, which was expected to influence heavy-metal complexation behavior. In this study, corn (Zea maysL.) stalks and leaves were collected from the field and decomposed for an 8-month period to produce humic substances which were separated into three water soluble fractions, HF1, HF2 and HF3, from highest to lowest relative molecular size. Functional group determination showed that total, carboxylic and phenolic OH acidity increased as relative molecular size of humic fractions decreased. Furthermore, C/O ratios decreased, whereas N/C and H/C ratios remained relatively unaffected as relative molecular size of humic fractions decreased. Formation of Ca²⁺, Cd²⁺ and Cu²⁺ -humic fraction complexes and how these complexes were affected by pH and relative (humic fraction) molecular size were studied using potentiometric titration. Metal-humic complexes exhibited at least two types of sites with respect to Ca²⁺, Cd²⁺ and Cu²⁺ complexation. Relative molecular size had a large significant influence on total metal-ion complexation, but it had a relatively small influence on complex stability at low levels of metal-ion complexation. Strength of metal-ion humic complexes followed the order Cu²⁺ > Cd²⁺ > Ca²⁺ and was affected by pH, especially for low affinity sites. Carboxylic and phenolic OH groups were most likely involved in complex formation. Magnitude of the metal-humic formation constants at the lowest equilibrium metal-ion concentration, under the various pH values tested, varied from 5.39 to 5.90 for Ca²⁺, from 5.36 to 6.01 for Cd²⁺ and from 6.93 to 7.71 for Cu²⁺. Furthermore, the formation constants appeared to be positively influenced by decreasing molecular size of water-soluble humic fraction, and increasing pH. These results inferred that soil management practices causing build-up of humic substances would affect mobility and bioavailability of metal-ions.     

Association of Chernobyl-derived 239+240Pu, 241Am, 90Sr and 137Cs with different molecular size fractions of organic matter in the soil solution of two grassland soils

Radiocesium is normally bound only rather weakly and unspecifically by humic substances, in contrast to the actinides Pu and Am. Recently, however, it was observed that fallout ¹³⁷Cs in the soil solution from an Of-horizon of a podzol forest soil (slightly decomposed plant material) was associated essentially only with one single size fraction of the humic substances. In deeper soil layers with well humified material (AOh-horizon), radiocesium was associated with all size fractions of the dissolved organic matter (DOM). To examine whether this unexpected behaviour is also observable for DOM isolated from other soils, we determined the association of fallout ¹³⁷Cs,⁹⁰Sr,²³⁸Pu, ^239+240Pu and ²⁴¹Am with various size fractions of DOM from in situ soil solutions isolated from two layers (0–2 cm and 2–5 cm) of two grassland soils (a soddy podzolic soil and a peat soil) within the 10 km zone of the nuclear reactor at Chernobyl (Ukraine). The four size fractions of DOM as obtained by gel filtration of the soil solution were (mean nominal molecular weight in daltons): fraction I: ≥2000, fraction II: 1300; fraction III: 560, fraction IV: inorganic compounds. The results for the well humified DOM (humus accumulation horizon of podzol, deeper layer of peat soil) showed that Pu and Am are essentially associated with the high molecular weight fractions, while Sr is present only in the `inorganic' fraction. Radiocesium is found in all the size fractions separated. A quite similar pattern was also found for Pu, Am, and Sr in the soil solution from only slightly decomposed plant material (0–2 cm of peat soil), but not for radiocesium. This radionuclide was again essentially only observable in one single low molecular weight fraction of DOM. The above results thus support our recent observations in the different horizons of a forest podzol mentioned above, even though no reason for the different binding of radiocesium by well humified soil organic matter and by only slightly decomposed plant material can be given at present. The data demonstrate, however, that information on only the total amount of a radionuclide in the soil solution will not be sufficient to interpret or predict its fate adequately in the soil. Received: 13 February 1998 / Accepted in revised form: 14 July 1998     

土壤胡敏素研究进展

土壤有机质是土壤的重要组成部分,在土壤肥力和生态环境等方面都具有重要作用.作为土壤有机质主体的腐殖物质的研究,一直受到各国学者的普遍重视.在土壤腐殖物质3组分中,以往的研究主要集中于可溶性的胡敏酸和富里酸,而对不溶性的胡敏素则较少涉及.从分离、结构和作用3个方面,综述了土壤胡敏素的研究现状和最新进展,并对今后的研究方向进行了展望,以促进胡敏素研究的继续深入开展.提出近期的研究重点主要包括:(1)比较不同分离方法获得的土壤胡敏素的差异,以达到方法上的可比性和统一性,同时提出更适宜的分离方法;(2)通过多种分析测试手段的相互补充验证,以获得对土壤胡敏素结构本质更加明确、清晰和深入的认识;(3)加强土壤胡敏素肥力和环境作用的研究,尤其应重视从分子水平上探明其构效关系,为生产实践和应用提供理论依据.     

The influence of humic substances on the molecular weight distributions of phosphate and iron in epilimnetic lake waters

1. The influence of humic substances on the association of free inorganic iron and phosphate with material of larger molecular weight was investigated in epilimnetic samples from two humus‐rich lakes of contrasting ionic strength. After modification of the molecular weight distribution of the humic substances in the samples using dialysis and ultrafiltration, the molecular weight distribution of added radioisotopes ( Fe3+ and PO43?) was assessed using gel filtration chromatography.
2. The association of Fe3+ and PO43? with larger molecular weight fractions (>50 000 and 10 000–50 000 Da) was not in general related to the quantity of humic substances of the same molecular weight in the samples. However, the proportions of Fe3+ and PO43? observed in higher molecular weight peaks were strongly correlated to the quantity of humic substances of the same molecular weight in (a) the 10 000–50 000 Da peak in the sample of low ionic strength at pH 5.5 and pH 7.0, and (b) the> 50 000 Da peak in the sample of higher ionic strength at pH 4.0.
3. It was concluded that humic substances promote the association of Fe3+ and PO43? with higher molecular weight fractions primarily by acting as peptizing agents for inorganic colloids containing Fe and P. Association of Fe3+ and PO43? with material of higher molecular weight via the formation of humic substance‐Fe3+–PO43? complexes was identified but only at specific pH and within specific molecular weight ranges for each of the epilimnetic lake water samples studied.     

Comparative effects of soil organic matter fractions on phosphatase activities in wheat roots

Summary Soil organic matter was obtained from two agricultural soils using alkali extraction followed by acidification to produce humic and fulvic acids which were further fractionated by adsorption and gel chromatography.All the products inhibited the activity of phosphatase prepared from wheat roots, but to different extents. Humic acids produced a greater inhibition of enzyme activity than either the fulvic acids or water extracts of soil. Aspergillin, fromAspergillus niger, had a similar C, H and N content to humic acid and produced a similar inhibition of phosphatase activity.The inhibitions produced by corresponding fractions derived from the two soils were slightly different, but the trends between similar fractions from different soils were comparable. The lower mol. wt. components of humic acid inhibited phosphatase activity to a greater extent than higher mot. wt. fractions. Although fulvic acid comprised only low mol. wt. components it was less effective in inhibiting enzyme activity than those components of comparable mol. wt. present in the corresponding humic acid. Synthetic polymaleic acid, produced an inhibition of phosphatase activity similar to that caused by fulvic acid.     

Evaluation of extractants for soil humic substances

Summary An evaluation of alkaline extractants was done by isotachophoresis and compared with infrared, ultraviolet and visible spectroscopic analysis. The electrophoretograms show that the humic acid extracted. This result is partially corroborated by the E₄/E₆ ratio. Differences in the carboxyl content and TRIS. In the pyrophosphate humic acid three fractions of high molecular weight were not extracted. This resultis partially corroborated by the E₄/E₆ ratio. Differences in the carboxyl content found by ir spectra of the TRIS humic acid and the NaOH humic acid were not shown in the isotachophoretic separation.