Comparative Evaluation of Humic Substances: Effect at Cell Level and Chlorophyll Retention during Accelerated Senescence

The influence of humic substances (HS) formulations derived from sedimentary and compost sources was studied on plant growth at cell level and chlorophyll retention during accelerated senescence of leaf tissue. The direct effect of HS formulations was studied on cell expansion using cucumber and radish cotyledon expansion test. The cucumber hypocotyl elongation test was used to study the effect on cell elongation. Chlorophyll pigment retention in excised leaf tissue incubated in dark with high temperature was assessed to study the effect on leaf senescence. Explant tissues were incubated directly in the solutions of the formulations at the concentration recommended for foliar application to the crop plants. HS formulations showed significant variations in their direct bio-stimulatory effects. Formulations derived from compost sources were found superior in terms of inducing a direct stimulatory effect on cell expansion and cell elongation and in maintaining chlorophyll pigment retention during accelerated senescence. HS from sedimentary sources stimulated cell expansion and delayed chlorophyll degradation to a lesser extent compared to HS from compost. However, HS formulations derived from sedimentary sources used in this study were not effective in inducing cell elongation in the cucumber hypocotyl elongation test. The direct bio-stimulatory effect of HS formulations differed significantly between the formulations that were evaluated.

THE OVERLOOKED ECOSYSTEM DRIVING FORCE IN NON-EUTROPHICATED FRESHWATER SYSTEMS:DISSOLVED HUMIC SUBSTANCES-A SHORT REVIEW AND OUTLOOK

This review starts with the description of the quantitative significance of dissolved organic material in general and dissolved humic substances (HS) in particular in various ecosystems. Despite their high quantities, the knowledge about the role of HS is still very low and full of old, but still recycled paradigms. HS are thought to be inert or at least refractory and too large to be taken up by aquatic organisms. Instead, I present evidence that dissolved HS that mainly derives from the terrestrial environment, are taken up and directly and/or indirectly interfere with freshwater organisms and, thus, structure biocenoses. Relatively well known is in the meantime the fuelling function of allochthonous HS, which, upon irradiation, release fatty acids, which serve as substrates for microbial growth. This is an indirect effect of HS. Microbes, in turn, are food for mixotrophic algae and (heterotrophic) zooplankton. Thus, non-eutrophicated freshwaters are net-heterotrophic, meaning that respiration exceeds primary production. Furthermore, model calculations exemplify that only a very small portion of the terrestrial production is sufficient to cause net-heterotrophy in these freshwater bodies. But, recent papers show also that due to different stoichiometries the maximal plankton biomass production with algae or mixotrophs is higher than with bacteria. Very recently, several direct effects of HS have been elucidated. Among them are:induction of chaperons (stress shock proteins), induction and modulation of biotransformation enzymes, modulation (mainly inhibition) of photosynthetic oxygen release of aquatic plants, production of an internal oxidative stress, modulation of the offspring numbers in the nematode Caenorhabditis elegans[WTBZ], feminization of fish and amphibs, interference within the thyroid system, and action as chemical attractant to C. elegans. We are still in the phase of identifying the various physiological, biochemical, and molecular-biological effects. Hence, the ecological and ecophysiological significance of these HS-mediated effects still remain somewhat obscure. Nevertheless, HS appear generally to have an impact on the individual as well as on the community and even ecosystem level comparable to that of, for instance, nutrients.

Effect of humic substances on the precipitation of calcium phosphate

For phosphorus （P） recovery from wastewater, the effect of humic substances （HS） on the precipitation of calcium phosphate was studied. Batch experiments of calcium phosphate precipitation were undertaken with synthetic water that contained 20 mg/L phosphate （as P） and 20 mg/L HS （as dissolved organic carbon, DOC） at a constant pH value in the range of 8.0-10.0. The concentration variations of phosphate, calcium （Ca） and HS were measured in the precipitation process; the crystalline state and compositions of the precipitates were analysed by powder X-ray diffraction （XRD） and chemical methods, respectively. It showed that at solution pH 8.0, the precipitation rate and removal efficiency of phosphate were greatly reduced by HS, but at solution pH ≥9.0, the effect of HS was very small. The Ca consumption for the precipitation of phosphate increased when HS was added; HS was also removed from solution with the precipitation of calcium phosphate. At solution pH 8.0 and HS concentrations ≤3.5 mg/L, and at pH ≥ 9.0 and HS concentrations ≤ 10 mg/L, the final precipitates were proved to be hydroxyapatite （HAP） by XRD. The increases of solution pH value and initial Ca/P ratio helped reduce the influence of HS on the precipitation of phosphate.

Interaction of humic substances and hematite:FTIR study

The present work extended the knowledge on the binding and complexation of humic substances(humic acid or fulvic acid) and hematite by Fourier transform infrared spectroscopy(FTIR). The FTIR data gained gave the consist evidences by two different sampling preparation methods that the interaction mechanism between humic substances and hematite was mainly conform to the ligand exchange involving carboxylic functional groups of humic substances and the surfaces sites of hematite. The present method, although associated with some uncertainties, provided an opportunity to increase the knowledge in this field.

Improving extraction yield of humic substances from lignite with anthraquinone in alkaline solution

Based on the characteristics of the lignite sample, effects of assistant anthraquinone （AQ） on extraction yield of humic substances （HS） and the action mechanisms of AQ in alkaline condition were studied by Fouvier transform infrared （FT-IR） spectroscopy. The results indicate that assistant AQ can not only increase the extraction yield of HS but also reduce the alkali dosage （NaOH） as well as the extraction temperature and extraction time. Under the optimal conditions of alkali dosage of 9%, AQ dosage of 0.75%, extraction temperature of 80 ℃, extraction time of 30 min, stirring speed of 600 r/min and solid-to-liquid ratio of 1：3, the extraction yield of HS reaches 80.08%, which is increased by more than 20% compared with the conventional extraction. FT-IR spectra show that AQ is able to prevent dissolved HS from being destroyed into undissolved substance by alkali and 1-IS obtained in the presence of AQ possesses more groups of COOR and --COOH than that obtained without AQ.

^(15)N NMR Spectroscopic Study on Nitrogen Forms in Humic Substances of Soils

Nitrogen forms of humic substances from a subalpine meadow soil, a latentic red soil and a weathered coal and the effect of acid hydrolysis on N structures of soil humic substances were studied by using 15N cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy. Of the detectable 15N-signal intensity in the spectra of soil humic substances 71%-79% may be attributed to amide groups, 10%-18% to aromatic/aliphatic amines and 6%~11% to indole- and pyrrole-like N. Whereas in the spectrum of the fulvic acid from weathered coal 46%, at least, of the total 15N-signal intensity might be assigned to pyrrole-like N, 14% to aromatic/aliphatic ammes, and the remaining intensities could not be assigned with certainty. Data on nonhydrolyzable residue of protein-sugar mixture and a 15N-labelled soil fulvic acid confirm the formation of nonhydrolyzable heterocyclic N during acid hydrolysis. Project (No. 39790100) supported by the National Natural Science Foundation of China.

Removal of humic substances by biosorption

Fungal pellets of Aspergillus niger 405,Aspergillus ustus 326,and Stachybotrys sp.1103 were used for the removal of humic substances from aqueous solutions.Batchwise biosorption,carried out at pH 6 and 25°C,was monitored spectrophotometrically and the process described with Freundlich’s model.Calculated sorption coeffcients Kf and n showed that A.niger exhibited the highest effciency.A good match between the model and experimental data and a high correlation coeffcient(R2)pointed out to judicious choice of ...

Partitioning of water soluble organic carbon in three sediment size fractions:Effect of the humic substances

Water soluble organic carbon （WSOC） in sediments plays an important role in transference and transformation of aquatic pollutants. This article investigated the inherent mechanisms of how sediemnt grain size affect the partitioning coeffcient （k） of WSOC. Influences of NaOH extracted humic substances were particularly focused on. Sediments were sampled from two cross-sections of the middle Yellow River and sieved into three size fractions （〈 63 μm, 63-100 μm, and 100-300 μm）. The total concentration of WSOC in sediments （Cwsoc） and k were estimated using multiple water-sediment ratio experiments. Results showed that Cwsoc ranged from 0.012 to 0.022 mg/g, while k ranged from 0.8 to 3.9 L/kg. Correlations between the spectrum characteristics of NaOH extracted humic substances and k were analyzed. Strong positive correlations are determined between k and the aromaticity indicators of NaOH extracted humic substances in different sediment size fractions. Comparing with finer fractions （〈 63 μm）, k is higher in larger size fractions （63- 100 μm and 100-300 μm） related to higher aromaticity degree of NaOH extracted humic substances mostly. While negative relationship between k and the area ratio of fourier transform infrared spectroscopy （FT-IR） at 3400 and 1430 cm^-1 implied that the lowest k was related to the highest concentration of acidic humic groups in particles 〈 63 μm. WSOC in finer fractions （〈 63 μm） is likely to enter into pore water, which may further accelerate the transportation of aquatic contaminants from sediment to water.

Acid Dissociation Constants of Functional Groups in Humic Substances:I.Affinity Spectrum Model Analysis of Potentiometric Data of Fulvic and Humic Acids Solutions from Weathered Coal and Dark Loessial Soil

Potentiometric experiments were carried out on the proton binding equilibria of FA extracted from a weathered coal and HA and Fa extracted from a dark loessial soil.The affinity spectrum model was employed to treat the experimental data.The affinity spectrum model technique could“magnify” the heterogeneity of the proton binding equilibria.so it was useful for comparing and studying the characteristics of humic substances with similar properties.According to the affinity spectra,we also found that the direction of the titration could affect the properties of the equilibria of FA from the weathered coal,and the acidic functional groups contained in FA from the weathered coal were larger in quantity than those contained in HA and FA from the dark loessial soil.

An XPS Study of Nitrogen Structures in Soil Humic Substances

X-ray photoelectron spectroscopy (XPS) was applied to examine the N structures of soil humic substances and some of their analogues. It was found that for soil humic substances XPS method gave similar results as those obtained by 15N CPMAS NMR (cross-polarization magic-angle spinning nuclear magnetic resonance) method. 70%～86% of total N in soil humic substances was in the form of amide, and 6%～13% was presented as amines, with the remaining part as heterocyclic N. There was no difference in the distribution of the forms of N between the humic substances from soils formed over hundreds or thousands of years and the newly formed ones. For fulvic acid from weathered coal and benzoqu inone- (N H-4 )-2 S O-4 polymer the XPS results deviated significantly from the 15N CPMAS NMR data.

Structure characteristics and adhesive property of humic substances extracted with different methods

The structure characteristics and adhesive property of humic substance(HS) extracted with different methods were mainly studied by terms of elementary analysis,visible spectrum,FT-IR spectroscopy,viscosity,adsorption and pelletizing experiments.The results show that HSs extracted with new method(HS-a) own higher degree of aromatization and polymerization,larger relative molecular mass and more polar functional groups than HS extracted with usual method(HS-b).The viscosity of HS-b is about 30-40 mPa·s lower than that of HS-a.The maximum adsorption amounts of HS-a and HS-b onto iron concentrates are 9.11 mg/g and 8.08 mg/g,respectively.Meanwhile,HS-a has a better performance than HS-b in the practical application for pelletizing of iron concentrates.The difference in agglomeration behaviors with iron concentrates lies in the differences of the structure characteristics of HSs.With higher content of polar functional groups,larger relative molecular mass and viscosity of HSs,the agglomeration behavior is improved.

CONTENTS OF DISSOLVED HUMIC SUBSTANCES IN THE RIVERS OF EASTERN CHINA

Concentrations of dissolved humic substances in the rivers of eastern China were found to be 0.9- 14.4 mg/L with a median value of 4.6.The contents of dissolved humic substances in river water generally decrease from north to south,a trend similar to that of aquatic sediments and soils.Results of multiple regression analysis and path analysis showed that the dissolved stream humic substances are of a combined autochthonous and allochthonous origin.

APPLICATION OF SURFACE-ENHANCED RAMAN SPECTORSCOPY FOR CHARACTERIZING HUMIC SUBSTANCES

Surface-enhanced Raman Spectroscopy(SERS)has been used to characterize hurnic substances.By using HNO_3 -roughened silver foils as the enhancing surface,both fluorescence quenching and strong SERS signals have been observed at the same time.The results obtained herein show that SERS is a particularly sensitive and highly selective technique for the characterization of humic substances in the environment. Humic substances axe receiving ever-inereasing attention because of their importance in the environment. Very recently,we have reported the first successful application of Raman spectroscopy for characterizing humic substances.In this present paper,the application of surface-enhanced Raman spectroscopy(SERS)for humic substances has further been investigated.To our knowledge,no such studies have previously been reported.The results obtained herein dearly demonstrate that SERS has a great deal of promise as an analytical method for“In Situ”characterizing humic substances in the environment.

Investigations on the Thermodynamic Stability and Availability of Nutrients for Plants by Humic Substances Extracted from Peat Samples

This work evaluated the complexation capacity, exchange constants and availability of micronutrients for plants and humic substances extracted from peat samples. Samples of humic substances extracted from two tropical peats （HS-P1 and HS-P2） were enriched with the micronutrients Cu（II）, Co（II）, Fe（II）, Mn（II）, Ni（II） and Zn（II） and the parameters for formation of the complexes （HS-N） were evaluated at different pH. The Scatchard model was used to calculate the maximum complexation capacity and the nutrient availability was studied using exchange capacity experiments based on ultrafiltration procedure. The optimum pH for complexation was 4.5 and the order of affinity was： Fe（II） 〉 Cu（II） 〉 Co（II） 〉 Mn（II） = Ni（II） 〉 Zn（II）. The maximum complexation capacity reached 56.8 mg·g-1 Fe of HS-P1 （the highest） and 1.7 mg.g1 Zn of HS-P2 （the slightest）. The exchange experiments showed that HS-P-Fe complexes were formed preferentially. The least stable complex was formed with Zn, which was therefore, more easily available. The results contribute to understand the behavior and availability of some nutrients in soils.

Characterization of Compost and Soil Humic Substances by Fluorescence Spectroscopy

Quality of soil humic substances and compost quality was assessed using different techniques of fluorescence spectroscopy. Emission, excitation, synchronous and emission-excitation matrix help us to characterize different fluorophores in humic substances molecule. Content of stabile carbon forms in soil was assessed by humic substances fractionation. Content of labile water extractable carbon and nitrogen was determined by analyzer Shimadzu TOC-VCSH with chemo-luminescent detection in infrared spectral region. Results showed that compost amendment caused changes in both stabile （recalcitrant） and labile carbon content. Humic substances isolated from compost consist mainly of simple structural components of wide molecular heterogeneity and low molecular weight. Humification degree and content of conjugated fluorophores in compost was lower compared with stabile soil humic substances. The last contained more conjugated aromatic π-electron systems with electron-withdrawing functional groups, which are responsible for the fluorescence shift to lower energy levels or longer wavelengths.

Enhancement of Phosphorus Solubility by Humic Substances in Ferrosols

An investigation was conducted to study the effect of humic substance (HS) on the phosphorus (P) solubility in acidic soil. The soil (2.5 g), HS (0, 0.5, and 2.5 g), and P as monocalcium phosphate (0.31 and 1.25 g P kg-1 soil) were mixed with 50 mL distilled water and two different sequences of adding HS and P were used. The results indicated that the P concentration in water and 0.01 mol L-1 CaCl2 solution increased with increasing amounts of humic substance. The concentrations of Fe and Al were also increased. However, Olsen P decreased with increasing amount of humic substance. Water-soluble P concentrations from P rates at 0.31 and 1.25 g P kg-1 soil in the treatment with 0.5 g (2.5 g) humic substance addition were 360% and 70% (500% and 90%) higher, respectively, than those in the treatment with no humic substance addition. P extracted by 0.01 mol L-1 CaCl2 in the treatments with 0.5 and 2.5 g humic substance addition was increased by 400% and 540%, respectively, compared with that in the treatment without humic substance at the rate of 0.31 g P kg-1 soil, while the corresponding P concentrations were increased by 80% and 90% at the rate of 1.25 g P kg-1 soil. The order of mixing humic substance and phosphate did not significantly affect desorbed P and labile P extracted with CaCl2.

Competitive Complexation of Copper and Zinc by Sequentially Extracted Humic Substances from Manure Compost

Chicken manure with similar content of copper and zinc was changes of organic carbon and humus substance complexed chosen to conduct a composting experiment to investigate the copper （HS-Cu） and zinc （HS-Zn）, which were extracted by water （H2O）, sodium hydroxide （NaOH）, and sodium pyrophate-NaOH mixture （Na4P2O7-NaOH）, sequentially. Distributions of copper and zinc in fulvic acids （FA） and humic acids （HA） in the three extracts were studied. During manure composting, the concentrations of copper and zinc increased from about 500 mg kg^-1 in the raw material to 1 100 mg kg^-1 in the final products. HS-Cu in H2O, NaOH, and Na4P2O7-NaOH extracts occupied 6.7, 26.7, and 19% averagely of total copper and HS-Zn represented 2.7, 13.7, and 17% averagely of total zinc in compost, respectively. In water extracts, both HA and FA mainly complexed with Cu and the mole ratio of Cu to Zn was 2.8 in HA fractions and was 2.6 in FA fractions, respectively. HA mainly complexed with copper, so that the ratios of HA-Cu to HA-Zn averaged 3.4 in NaOH extracts. FA had a similar potential to complex with copper and zinc, so that the ratio of FA-Cu to FA-Zn was close to 1. In Na4P2O7-NaOH extracts, HA or FA had a similar potential to complex with copper and zinc. The ratio of HS-Cu to HS-Zn was close to 1. With manure composting, Na4P2O7-NaOH extractable HS-Zn increased to a level as high as HS-Cu. This indicated that more and more stable complexes of HS-Zn were formed in the late decomposition period. The competition between copper and zinc to be complexed with humic substance became weaker and weaker with the decomposition process.

ADSORPTION OF HUMIC SUBSTANCES BY MACRO WEAKLY BASIC IONEXCHANGE RESIN AND THEIR EFFECTS ON REMOVAL OF Cu^(2+) AND Pb^(2+)

Adsorption of humic, tannic and gallic acids by a macro weakly basic ion-exchange resin JN-01 was studied. The adsorption capacity of this resin for gallic and tannic acids is much higher than that for humic acid, which can be explained on the basis of both their molecular size and ionization degree. Furthermore, humic acid is separated into different components with molecular weight in the range from 2000 Da to 100000 Da by ultra-filter, and their adsorption isotherms on resin JN-01 indicate that humic acid＇s molecular weight is an important factor which makes significant influence on adsorption. Finally, changes in the amount of Cu^2＋ and Pb^2＋ adsorbed on resin JN-01 as a function of the concentration of each of these three acids were studied. A large increase in the heavy metal ions uptake is observed in the presence of humic substance, such advantages are due to the interactions between the heavy metal ions and the unbound functional groups of the adsorbed organic acids.

Comparative sorption and desorption behaviors of PFHx S and PFOS on sequentially extracted humic substances

The sorption and desorption behaviors of two perfluoroalkane sulfonates（PFSAs）, including perfluorohexane sulfonate（PFHx S） and perfluorooctane sulfonate（PFOS） on two humic acids（HAs） and humin（HM）, which were extracted from a peat soil, were investigated. The sorption kinetics and isotherms showed that the sorption of PFOS on the humic substances（HSs） was much higher than PFHx S. For the same PFSA compound, the sorption on HSs followed the order of HM 〉 HA2 〉 HA1. These suggest that hydrophobic interaction plays a key role in the sorption of PFSAs on HSs. The sorption capacities of PFSAs on HSs were significantly related to their aliphaticity, but negatively correlated to aromatic carbons,indicating the importance of aliphatic groups in the sorption of PFSAs. Compared to PFOS,PFHx S displayed distinct desorption hysteresis, probably due to irreversible pore deformation after sorption of PFHx S. The sorption of the two PFSAs on HSs decreased with an increase in p H in the solution. This is ascribed to the electrostatic interaction and hydrogen bonding at lower p H. Hydrophobic interaction might also be stronger at lower p H due to the aggregation of HSs.

Light-induced variation in environmentally persistent free radicals and the generation of reactive radical species in humic substances

Environmentally persistent free radicals(EPFRs)in humic substances play an essential role in soil geochemical processes.Light is known to induce EPFRs formation for dissolved organic matter in aquatic environments;however,the impacts of light irradiation on the variation of EPFRs in soil humic substances remain unclear.In this study,humic acid,fulvic acid,and humin were extracted from peat soil and then in situ irradiated using simulated sunlight.Electron paramagnetic resonance spectroscopy results showed that with the increasing irradiation time,the spin densities and g-factors of humic substances rapidly increased during the initial 20 min and then gradually reached a plateau.After irradiation for 2h,the maximum spin density levels were up to 1.63×10^17,2.06×10^17,and 1.77×10^×10^17 spins/g for the humic acid,fulvic acid,and humin,respectively.And the superoxide radicals increased to 1.05×10^l4-1.46×10^14spins/g while the alkyl radicals increased to 0.47×10^14-1.76×10^14 spins/g.The light-induced EPFRs were relatively unstable and readily returned back to their original state under dark and oxic conditions.Significant positive correlations were observed between the concentrations of EPFRs and reactive radical species(R2=0.65-0.98,/?<0.05),which suggested that the newly produced EPFRs contributed to the formation of reactive radical species.Our findings indicate that under the irradiation humic substances are likely to be more toxic and reactive in soil due to the formation of EPFRs.