Isolation and seasonal effects on characteristics of fulvic acid isolated from an Australian floodplain river and billabong

Fulvic acids from an Australian floodplain river and billabong were isolated using DEAE and DAX-8 resins, and characterised with the use of size exclusion chromatography and solid-state CP-MAS (13)C NMR spectroscopy. Differences between the two resin isolates were evident. Fulvic acids isolated using DEAE-cellulose had higher apparent M(n) and M(w) values, while the DAX-8 resin showed a slight preference for aliphatic components. Fulvic acids from the river and billabong had the same functional groups present, however, the river fulvic acids had higher apparent M(n) (number average molecular weight) and M(w) values (weight average molecular weight), and were more polydisperse than the billabong fulvic acid. There were no significant changes in the characteristics of the fulvic acid isolated from the river at four sampling times: summer, autumn, winter and spring. In contrast, fulvic acids isolated from a billabong displayed seasonal variation in molecular weights. This work emphasizes the importance in ecological studies of isolation procedure for the operationally defined fulvic acids.     

Fractionation analysis of trace metals in humic substances of soils irrigated with wastewater in Central Mexico by particle induced X-ray emission

The Mezquital Valley in Central Mexico has received wastewater from Mexico City for nearly 100 years. Wastewater brings in organic matter and nutrients but also trace metals. Humic substances, the main components of organic soil matter, are responsible for retaining and regulating the mobility of trace metals in soils. In this study, humic substances were extracted from the soil and separated into distinct fractions (humic acids, fulvic acids and humins). The particle induced X-ray emission (PIXE) technique was applied to determine the metal content in bulk soil as well as in humic acids and fulvic acids not soluble in H₃PO₄. In order to assess whether the long-term input of organic matter and metals modifies the metal association with these humic substances, parcels irrigated for three time periods (5, 47 and 89 years) were selected for this study. It was observed that metals such as Zn and Cu are mainly associated with the humic acids. Fulvic acids retain mainly Cr while Pb is distributed among humic and fulvic acids. It was also observed that in general, metal retention by humic substances increases with irrigation time. Depth also affects metal association with the humic substances.     

Studies on the sea surface microlayer. II. The layer of sudden change of physical and chemical properties

Seawater samples of the sea surface microlayer were obtained from the ocean, coastal waters, and laboratory imitation experiments adopting glass plate, rotating drum, screen, and funnel samplers. The result was that surface microlayer samples of thickness 50 microm could be taken, not by the popular screen and funnel techniques, but by glass plate or rotating drum techniques. The layer of sudden change of physical and chemical properties in the surface microlayer was found at 50 microm below the sea-air interface. These physical and chemical properties included tens of physical and chemical parameters, such as concentrations of organic matter, nutrients, and dissolved trace metals, biochemical oxygen demand, chemical oxygen demand chlorophyll-a, surface tensions, and pH. Therefore, it was suggested that the layer of sudden change of physical and chemical properties in surface seawater should serve as a basis for defining the sea surface microlayer should, and be regarded as the practical operational thickness of the sea surface microlayer. The apparent sampling thickness of the sea surface microlayer from surface seawater should be 50+/-10 microm.     

A new analytical approach for humin determination in sediments and soils

In this work a new analytical approach is proposed for the recovery of humin present in soil and sediments. The procedure is based on microwave oven treatment for humin deashing. In this way both the treatment time and the concentration of the HCl/HF mixture are significantly reduced (minutes rather than hours, 10% rather than concentrated). By means of the proposed scheme organic matter present in sediment and soil samples can be subdivided into the different fractions (hydrophobic and hydrophilic compounds, fulvic and humic acids, humin) making up the balance of organic carbon. Results obtained for samples characterised by different organic carbon content showed a loss of carbon ranging between 20% and 30%, consistent with previous reports about humin deashing.     

Determination of molecular formulas of natural organic matter molecules by (ultra-) high-resolution mass spectrometry: Status and needs

Electrospray ionization (ESI) combined with ultra-high-resolution mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer has been shown to be a very powerful tool for the analysis of fulvic and humic acids and of natural organic matter (NOM) at the molecular level. With this technique thousands of ions can be separated from each other and their m/z ratio determined with sufficient accuracy to allow molecular formula calculation. Organic biogeochemistry, water chemistry, and atmospheric chemistry greatly benefit from this technique. Methodical aspects concerning the application of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to NOM isolated from surface water, groundwater, marine waters, and soils as well as from secondary organic aerosol in the atmospheric are reviewed. Enrichment of NOM and its chromatographic separation as well as possible influences of the ionization process on the appearance of the mass spectra are discussed. These steps of the analytical process require more systematic investigations. A basic drawback, however, is the lack of well defined single reference compounds of NOM or fulvic acids. Approaches of molecular formula calculation from the mass spectrometric data are reviewed and available graphical presentation methods are summarized. Finally, unsolved issues that limit the quality of data generated by FTICR-MS analysis of NOM are elaborated. It is concluded that further development in NOM enrichment and chromatographic separation is required and that tools for data analysis, data comparison and data visualization ought to be improved to make full use of FTICR-MS in NOM analysis.     

Ultrasound-Assisted Extraction of Humic Substances from Peat: Assessment of Process Efficiency and Products’ Quality

Results of efficiency of obtaining humic substances (HSs) from peat in traditional alkaline extraction (TAE) and ultrasound-assisted alkaline extraction (UAAE) are presented. The influence of the duration of the process and ultrasound intensity on the efficiency of extraction of humic acids (HAs) and fulvic acids (FAs) extraction was determined. The composition of the fulvic acid fraction was examined depending on the type of eluent used. Fulvic acids were divided into fractions using columns packed with DAX-8 resin. For this process, 0.1 M NaOH and 0.5 M NH₃∙H₂O were used as eluents. For the quality assessment of specific fulvic acids fractions, spectroscopic methods (UV-Vis and FTIR) were used. Ultrasound had a positive effect on HS extraction efficiency, especially in increasing the amount of a desired hydrophobic fraction of fulvic acids (HPO). However, a negative effect of the excessive prolongation and ultrasound intensity (approximately 400 mW∙cm⁻²) on the extraction efficiency of HPO eluted with 0.1 M NaOH solution was observed. Using peat as a raw carbon material for the HS extraction process can be used as an alternative industrial application of peat. UAAE may be considered as an alternative method to TAE, which provides a higher efficiency in HS isolation from peat.     

Calorimetric investigation of complex formation of some humic compounds

In the present paper we studied the complexation capacity towards copper ion of fulvic acids extracted from Arno River and Lake Como sediments, as well as Antarctic Sea water at different pHs in order to evaluate the interaction of different complexing groups of fulvic acids with copper ions. The binding capacity studies were carried out by means of titration with a copper-selective electrode and calorimetric measurements. For the same purpose, the heat of reaction in the complexation of copper ions with small molecules containing functional groups similar to fulvic acid was also determined. Titration results indicated that the fraction of bound metal increases with increasing pH (from 5 to 7). This may be accounted for by the increase in the ionisation of the fulvic acid molecule. Results obtained from calorimetric measurements show that the quantity of total heat involved in the metal ion-fulvate interactions determines a decreasing exothermic response with increasing pH values. This revised version was published online in July 2006 with corrections to the Cover Date.     

全氟羧酸盐海洋微表层富集与影响因素分析

通过环境调查和表面张力测定实验,观察了全氟辛酸盐（PFOA）和全氟寅酸盐（PFNA）在海洋微表层中的富集现象,探讨了污染物浓度、温度、盐度等热力学因素对PFOA海水表面富集的影响趋势．利用钢丝网法采集的黄海北部开阔海域微表层中（厚约200μm）,PFOA和PFNA的浓度分别为1．92～17．66ng／L和0．40～9．30ng／L,几何均值为4-27和1．38ng／L;微表层富集系数的几何均值为2．5（1．0～17）和8．2（2．1-42）．微表层中PFNA与PFOA浓度比值几何均值为0．33,显著不同于次表层海水中的组成比例（0．10）．海洋表层水温度和盐度是影响全氟表面活性剂微表层富集状态的主要热力学因素．温度增高时,微表层富集系数随之降低;盐度增加时,富集系数随之增加．     

Dynamic behaviour of river colloidal and dissolved organic matter through cross-flow ultrafiltration system

Through cross-flow filtration (CFF) with a 1-kDa regenerated cellulose Pellicon 2 module, the ultrafiltration characteristics of river organic matter from Longford Stream, UK, were investigated. The concentration of organic carbon (OC) in the retentate in the Longford Stream samples increased substantially with the concentration factor (cf), reaching approximately 40 mg/L at cf 15. The results of dissolved organic carbon (DOC) and colloidal organic carbon (COC) analysis, tracking the isolation of colloids from river waters, show that 2 mg/L of COC was present in those samples and good OC mass balance (77-101%) was achieved. Fluorescence measurements were carried out for the investigation of retentate and permeate behaviour of coloured dissolved organic materials (CDOM). The concentrations of CDOM in both the retentate and permeate increased with increasing cf, although CDOM were significantly more concentrated in the retentate. The permeation model expressing the correlation between log[CDOM] in the permeate and logcf was able to describe the permeation behaviour of CDOM in the river water with regression coefficients (r(2)) of 0.94 and 0.98. Dry weight analysis indicated that the levels of organic colloidal particles were from 49 to 71%, and between 29 and 51% of colloidal particles present were inorganic. COC as a percentage of DOC was found to be 10-16% for Longford Stream samples.     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.

Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.

Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.

In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

Functional speciation of metal-dissolved organic matter complexes by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry and deconvolution analysis

High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (HP-SEC–ICP-MS), in combination with deconvolution analysis, has been used to obtain multielemental qualitative and quantitative information about the distributions of metal complexes with different forms of natural dissolved organic matter (DOM). High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry chromatograms only provide continuous distributions of metals with respect to molecular masses, due to the high heterogeneity of dissolved organic matter, which consists of humic substances as well as biomolecules and other organic compounds. A functional speciation approach, based on the determination of the metals associated to different groups of homologous compounds, has been followed. Dissolved organic matter groups of homologous compounds are isolated from the aqueous samples under study and their high performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry elution profiles fitted to model Gaussian peaks, characterized by their respective retention times and peak widths. High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry chromatograms of the samples are deconvoluted with respect to these model Gaussian peaks. This methodology has been applied to the characterization of metal–dissolved organic matter complexes in compost leachates. The most significant groups of homologous compounds involved in the complexation of metals in the compost leachates studied have been hydrophobic acids (humic and fulvic acids) and low molecular mass hydrophilic compounds. The environmental significance of these compounds is related to the higher biodegradability of the low molecular mass hydrophilic compounds and the lower mobility of humic acids. In general, the hydrophilic compounds accounted for the complexation of around 50% of the leached metals, with variable contributions of humic and fulvic acids, depending on the nature of the samples and the metals.     

Rapid automated screening, identification and quantification of organic micro-contaminants and their main transformation products in wastewater and river waters using liquid chromatography-quadrupole-time-of-flight mass spectrometry with an accurate-mass database

In this study we have developed and evaluated an analytical method for a rapid automated screening and confirmation of a large number of organic micro-contaminants (almost 400) and also the quantification of the positive findings in water samples of different types (surface and wastewaters) using liquid chromatography-electrospray quadrupole-time-of-flight mass spectrometry (LC-QTOFMS) based on the use of an accurate-mass database. The created database includes data not only on the accurate masses of the target ions but also on the characteristic in-source fragment ions, isotopic pattern and retention time data. This customized database was linked to commercially available software which extracted all the potential compounds of interest from the LC-QTOFMS raw data of each sample and matched them against the database to search for targeted compounds in the sample. The detailed fragmentation information has also been used as a powerful tool for the automatic identification of unknown compounds and/or transformation products with similar structures to those of known organic contaminants included in the database. The database can be continually enlarged. To confirm identification of compounds which have no fragment ions (or fragments with low intensity/relative abundance) from in-source CID fragmentation or isomers which are not distinguished within full single mass spectra, a "Targeted MS/MS" method is developed. Thereafter, these compounds can be further analyzed using the collision energy (CE) in QTOF-MS/MS mode. Linearity and limits of detection were studied. Method detection limits (MDLs) in effluent wastewater and river waters were, in most cases, lowers or equal to 5 and 2 ng/L, respectively. Only 15 compounds had MDLs between 5 and 50 ng/L in effluent wastewater matrix. We obtained a linearity of the calibration curves over two orders of magnitude. The method has been applied to real samples and the results obtained reveal that most of the pharmaceutically active compounds contained in the created database were present in the water samples with concentrations in the range of ng/L and μg/L levels and in most of the samples between 2 and 15 pesticides of the 300 contained in the database were also detected. In addition to the compounds included in the database, some degradation products were found, thus revealing the method as a useful tool for the analysis of organic micro-contaminants in waters.     

Insights into colloid-mediated trace element release at the soil/water interface

Organic or inorganic colloids play a major role in the mobilization of trace elements in soils and waters. Environmental physicochemical parameters (pH, redox potential, temperature, pressure, ionic strength, etc.) are the controlling factors of the colloidal mobilization. This study was dedicated to follow the colloid-mediated mobilization of trace elements through time at the soil/water interface by means of an experimental approach. Soil column experiments were carried out using percolating synthetic solutions. The percolated solutions were ultrafiltrated with various decreasing cutoff thresholds to separate the different colloidal phases in which the dissolved organic carbon and trace element concentrations were measured. The major results which stem from this study are the following: (i) The data can be divided into different groups of organic compounds (microbial metabolites, fulvic acids, humic acids) with regard to their respective aromaticity and molecular weight. (ii) Three groups of elements can be distinguished based on their relationships with the colloidal phases: the first one corresponds to the so-called "truly" dissolved group (Li, B, K, Na, Rb, Si, Mg, Sr, Ca, Mn, Ba, and V). The second one can be considered as an intermediate group (Cu, Cd, Co, and Ni), while the third group gathers Al, Cr, U, Mo, Pb, Ti, Th, Fe, and rare earth elements (REE) carried by the organic colloidal pool. (iii) The data demonstrate that the fulvic acids seem to be a major organic carrier phase for trace elements such as Cu, Cd, Co, and Ni. By contrast, the trace elements belonging to the so-called colloidal pool were mostly mobilized by humic acids containing iron nanoparticles. Lead, Ti, and U were mobilized by iron nanoparticles bound to these humic acids. Thus, humic substances allowed directly or indirectly a colloidal transport of many insoluble trace elements either by binding trace elements or by stabilizing a ferric carrier phase. (iv) Finally, the results demonstrated also that REE were mostly mobilized by humic substances. The REE normalized patterns showed a middle REE downward concavity. Therefore, as previously shown elsewhere humic substances are a major control of REE speciation and REE fractionation patterns as well since the humic substance/metal ratio was the key parameter controlling the REE pattern shape.     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

Using a simple high-performance liquid chromatography separation and fraction collection methodology to achieve compound-specific isotopic analysis for dissolved organic compounds

A new application for the quantitative and isotopic analyses of dissolved inorganic and dissolved organic carbon compounds has been developed. Dissolved organic matter (DOM) in natural water samples can be separated on a high-performance liquid chromatography (HPLC) column and collected as fractions. Each discrete fraction can then be analyzed using the technique of St-Jean (Rapid Commun. Mass Spectrom. 2003; 17: 419-428) with a total inorganic carbon/total organic carbon (TIC/TOC) analyzer interfaced with a continuous-flow isotope ratio mass spectrometer. Experimental data using short-chain fatty acid standards (formic, acetic, and propionic acids) show that fraction recoveries of 100% are possible and that sample integrity is maintained. 13C-isotopic analyses of products prior to and subsequent to extraction and collection show no isotopic effects associated with the methodology, and errors are well within the accepted analytical uncertainty of the IRMS. Comparison of data from pure standards and organic-rich natural waters shows that quantitative analyses still need to be done with standards that more closely imitate the matrices of the samples, in order to acquire an appropriate calibration curve. Injections of organic-rich matrices on the HPLC column did not affect fraction recovery, nor did they create high background of partially retained organic compounds slowly released from the HPLC column, and hence 13C-isotopic results are relatively unaffected. The specific limitation on this methodology is the required use of carbon-free carrier solvents due to potential memory effects associated with the TIC/TOC analyzer. Further developments of this application could make routine compound-specific isotopic analyses (CSIA) for a wider range of organic materials possible.     

Segmented-flow analysis: On-line microwave sample processing for the analysis of natural waters

 A flow system including an on-line coated capillary column for the determination of traces of metal ions in natural waters is proposed, employing a manifold of flow microwave sample processing. This method eliminates the noise caused by the organic matrix, i.e. humic and fulvic acids. The system was applied to the determination of Pb, Co, Mn(II), and Fe(III) in swamp water. The proposed technique can be useful for routine analysis of natural and waste waters. Received: 29 January 1996/Revised: 17 July 1996/Accepted: 20 July 1996     

Fatty acids and amino acids of entomopathogenic fungus <Emphasis Type="Italic">Conidiobolus coronatus</Emphasis> grow on minimal and rich media

Entomopathogenic fungi are referred to as potential candidates as insect pest control agents. The objective of the study was to identify fatty acids and amino acids from Conidiobolus coronatus cultured on two different media. Each medium was extracted with ethyl acetate and its mixtures with isopropanol, acetonitrile and methanol. Analyses of fatty acids and amino acids of entomopathogenic fungus C. coronatus were performed by means of gas chromatography coupled with mass spectrometry. The analysis showed that the fungus C. coronatus produces the following groups of compounds: fatty acids and amino acids; α- and β-glucopyranose were also identified. The identified fatty acids included 12–20, 22 and 24 carbon atoms per chain. The highest content of fatty acids was detected in a mycelium sample cultured in a liquid minimal medium extracted with ethyl acetate. The lowest content of these organic compounds was identified in mycelium cultured in a liquid nutrient-rich medium extracted with ethyl acetate–methanol mixture. Fatty acids were found to account for 62.0 mass % to 94.4 mass % of all organic compounds in the analyzed mycelia. C18:1 acids were detected in the highest amounts when ethyl acetate was used as the extracting agent. The identified amino acids accounted for 4 mass % to 21 mass % of all organic compounds. Upon extraction of C. coronatus mycelium samples with the ethyl acetate—methanol mixture, two anomeric forms of glucose were also identified. An analysis of the studied material confirmed, that the entomopathogenic fungus C. coronatus is a very rich source of organic compounds, which might encourage its further research so as to identify an even larger number of compounds being produced by this species.     

Partitioned dispersive liquid-liquid microextraction: an approach for polar organic compounds extraction from aqueous samples

Partitioned dispersive liquid-liquid microextraction (PDLLME) efficiency was demonstrated for the extraction of polar organic compounds (chlorophenoxyacetic acids) prior to high performance liquid chromatography (HPLC). The method was based on the formation of tiny droplets of an organic extractant in an aqueous sample (river water) by injecting a mixture of a water-immiscible organic solvent [tetrachloroethylene (TCE)] as extractant dissolved in a water-miscible organic dispersive solvent [tetrahydrofuran (THF)]. Based on their partition coefficients, polar compounds were extracted into the dispersed TCE droplets as well as into THF. Different parameters affecting the extraction efficiency were evaluated and precision, linearity, detection limit and an enrichment factor were determined.     

Key factors in electromembrane microextraction systems for metals analysis in natural waters

ABSTRACT

Application of an electric potential to hollow fibre liquid phase microextraction (HF-LPME) systems previously optimised for metals preconcentration from natural waters could improve their analytical performance increasing enrichment factors and reducing operation times. Nevertheless, the effectiveness of the direct application of an electric potential to these systems may be limited due to the effects produced by other operational parameters.

In this work, the effect of a variable electric potential on the enrichment factors of four HF-LPME systems used for the analysis of trace metals in natural waters (Cd, Ni, Ag and Cu) has been studied. In addition, the effect of organic phase composition, distance between electrodes and electrodes diameter has been also studied. From the results obtained, composition of organic phase can be considered as the key factor in electromembrane (EME) systems, since its polarity determine the operational range of the applied electric potential and consequently the enrichment factor that could be achieved.

EMEs have demonstrated to be a real alternative to preconcentrate Cd, Ag and Cu from natural water samples in very short times (30 min). In fact, enrichment factors increased up to one order of magnitude if compared with HF-LPME methodology without application of an electrical potential.     

Effects of acid rain on soil humic compounds

The modifications induced by acid rain on the solubility, molecular configuration and molecular weight distribution of humic (HA) and fulvic (FA) acids were studied. A natural soil was subjected to simulated acid rain until a soil pH of 4 was obtained; HA and FA acids were then extracted and characterised. The results obtained were compared both with those of natural soil and with those of a soil subjected to acid rain. Elute analysis indicates the continuous release of soluble organic compounds as a consequence of acid rain simulation, although no relationship was found with the process of soil acidification. The yields of HA and FA show that HA values are the same while FA amount is higher in the natural soil; in acid soils their water solubility increases. The molecular weight distribution shows that HA consist of a mixture of compounds of different molecular weights; they are molecules for the most part larger than 100 kDa and their distribution is not changed by soil acidification. FA can be considered to form a much more homogeneous system; in natural soil, the molecules are larger than 50 kDa, while in acidified soil they are for the most part smaller than 3 kDa.