Influence of the Extractant on the Complexing Capacity of Humic Substances from Peat for Macro and Micronutrients Using Continuous Flow : Agricultural Application and Environmental Impacts

O objetivo do presente trabalho foi otimizar o procedimento para extração de substâncias húmicas de turfa, utilizando fluxo contínuo, com diferentes extratores (NaOH, KOH e Na4P2O7). A influência do extrator na capacidade de complexação das substâncias húmicas por macro e micronutrientes também foi investigada. Os resultados mostraram que a extração por fluxo contínuo é eficaz, reduzindo o tempo necessário para o passo de extração e, além disso, indicando que as substâncias húmicas apresentaram elevada afinidade para os nutrientes essenciais Ca e Mg. Além disso, as maiores capacidades de complexação foram obtidas quando utilizou o extrator Na4P2O7, mostrando não só a eficiência desse composto como agente de extração, mas também a sua aplicação promissora no processo, uma vez que a sua elevada capacidade de complexação permite disponibilizar os nutrientes complexados, bem como diminuir a quantidade de estes nutrientes que podem ser lixiviados, contribuindo, assim, para evitar contaminação do meio ambiente.


Introduction
Peat are natural sinkholes of macro and micronutrients and carbon of the soil.The accumulation of these species in the peat is due to processes involving biochemical reactions and complexation with organic matter. 1 The fraction of this organic matter, with undefined chemical structure, is called "humic substances" (HS) and the study of their characteristics as complexing agents is of great environmental interest, because they act directly on the bioavailability of different species in the environment. 2ne of the first issues, when studying HS, is the extraction of humic material, taking into account that the extraction conditions may change their physicochemical properties.Thus, it is fundamentally important to extract them with minimal change into its original structure. 3onsidering the extraction of humic substances from soil, two classes of extractants are designated: -moderate extractants; -alkaline extractants.
4][5] These extractants seem to decrease the occurrence of structural changes into the organic material, in spite being less effective than alkaline extractants.
][8][9][10][11] Despite the higher efficiency, the use of alkaline solution for extracting HS can change the organic matter by hydrolysis or self-oxidation. 5,12Nevertheless, the most used procedure in such extractions is still based on the use of alkaline solution (NaOH), enabling comparisons among different works. 9,11,13owadays, fertilizers commercialized as "organics" and/or "natural", e.g., peat or humic substances extracted from peat, are enriched with macro and micronutrients which, according to the manufacturers, are released to the plant as it needs.This is a relatively new market, with great economic potential and, therefore, very attractive.However, there is still no proper regulation for this sector, mainly because there is no scientific data about the releasing processes of those nutrients by HS.Having in view a better yield and lower environmental impact, studies are necessary to analyze the interactions (for example, the complexation capacity) between metal species (essential macro and micronutrients) and humic substances. 14everal chemical models seek to evaluate the modeling of the interactions between the HS and metal species. 15,16The NICA-Donnan model is a combination of the non-ideal competitive adsorption (NICA) isotherm description of binding to a heterogeneous material, coupled with a Donnan electrostatic sub-model describing the electrostatic interactions between ions and the humic material.NICA-Donnan has been shown in several studies to be a versatile model capable of describing observed humic ion-binding behavior well over a wide range of conditions. 17,18herefore, the objective of this study was the optimization of the extraction process by continuous flow of humic substances from peat for commercial purposes, using different extractants (NaOH 0.1 mol L -1 , Na 4 P 2 O 7 0.1 mol L -1 and KOH 0.1 mol L -1 ) as well as the evaluation of the influence of the extraction on the complexation of nutrients by HS.In this way, these results may provide subsidies for the application of HS enriched with nutrients in agricultural soils, reducing environmental impacts.

Sample collection and preparation
The peat samples have been collected around the Mogi River region, near to Ribeirão Preto city, São Paulo State, Brazil.Figure 1 shows the sampling procedure, at a depth of 50 cm, so that single samples A 1 -A 5 formed the composite sample A.
The samples were, then, transferred to wood trays and, after air drying, passed in a plastic sieve with a 2 mm mesh.

Continuous flow
The extraction of HS by continuous flow was carried out by transferring a 5.00 g sample of peat into the extraction flask (EF), under magnetic stirring; the extractant solution (50.00 mL) (R 1 ) was added at a flow rate of 1.0 mL min -1 through the tube 1 (T 1 ).Nitrogen was injected into the EF at a flow rate of 100 mL min -1 to minimize the material oxidation.The extracted HS were suctioned to the tube 2 (T 2 ) at a flow rate of 1.0 mL min -1 , passing through the filter (F 1 ) in order to retain the insoluble part, called 'humin' and the supernatant was then dried in a greenhouse with air exchange for subsequent determination of the yield (Figure 2).In the extraction of HS by continuous flow, the addition of the extracting agent occurs at a rate of 1.0 mL min -1 , thus the overall extraction time decreases, being performed within 2 h.The extractants used were: NaOH, KOH and Na 4 P 2 O 7 , at the concentration of 0.1 mol L -1 .

Batch
The extraction of humic substances by batch was made through methodology, recommended by IHSS. 13he extractants solutions: NaOH, KOH and Na 4 P 2 O 7 (0.1 mol L -1 ) were mixed with 5.0 g of peat in the ratio of 1:10 (m/v) and the mixture was left under mechanical stirring for 4 hours in the Jar Test equipment -JT 102, under a nitrogen atmosphere.Afterwards, the supernatant was separated by letting it to dry in a greenhouse, with air renovation for subsequent determination of the yield.

Humic substances purification
Inorganic (for example, salts, metal species etc.) and organic (for example, carbohydrates, proteins, lipids etc.) impurities are usually present in extracted HS. 13 Thus, the material requires purification to avoid the influence of contaminants on results related to the properties and characteristics of the HS.

Preparation of dialysis membranes
The procedure used for the preparation of dialysis membranes (SAMLESS CELL 16 X 100 CLEAR) was carried out as described by Town et al.. 19 After treatment for 10 minutes with solutions of NaHCO 3 2% (m/v) and Na 2 EDTA 0.01 mol L -1 , the membranes were washed with water (at 65 °C) and stocked at -6 °C.

Dialysis of humic substances
The humic extract was purified using the dialysis membrane against deionized water, until it has given negative test for chlorides (with silver nitrate solution 0.10 mol L -1 ).In addition, measurements of the deionized water conductivity were made until it has reached the initial value, i.e., before the contact with the dialysis membrane.

Determination of extraction yield
Extraction yield was calculated as the ratio between the mass of organic material extracted by the system (using continuous flow or batch) and the mass of dry peat submitted to extraction.

Determination of the complexing capacity of humic substances for macro and micronutrients by continuous flow with different extractants
The tangential ultrafiltration system, proposed by Burba et al., 20 was used to determine the complexing capacity of HS extracted from peat samples by flow, for macronutrients (Ca 2+ and Mg 2+ ) and micronutrients (Zn 2+ , Co 2+ , Mn 2+ , Cu 2+ and Mo 2+ ), individually.The titrations were carried out in volumes of 250 mL of HS solution 100 mg L -1 , extracted with NaOH 0.1 mol L -1 , KOH 0.1 mol L -1 and Na 4 P 2 O 7 0.1 mol L -1 (with the pH adjusted to about 5.5 with NaOH 0.1 mol L -1 ).Before the addition of the micro and macronutrients solutions, the system remained pumping during about five minutes for the membrane conditioning.Subsequently, the first aliquot was filtered (approximately 2 mL), corresponding to time zero, i.e., before the addition of nutrients solution.This aliquot includes a small amount of nutrients, corresponding to the free fraction (not complexed to HS) plus that fraction originally bound to the HS, with molecular size smaller than 1 kDa.Then, nutrients solution aliquots were added to the HS solution in order to achieve final concentrations of: 0.24, 0.48, 0.72, 1.32, 1.92, 2.52, 3.72, 4.92, 6.12 and 7.32 mg L -1 .After each addition, the system remained under stirring for 10 minutes to reach the complexation equilibrium.Aliquots were collected (about 2 mL) and the concentrations of free macro and micronutrients (not complexed by humic substances) determined by ICP-OES. 14,21termination of macro and micronutrients The concentrations of macro and micronutrients were determined by Atomic Emission Spectrometry with induced argon plasma (ICP-OES), Thermo Jarrell Ash, according to the manufacturer's guidelines. 17

Comparison between HS extraction by batch and by continuous flow
The extraction yields (Table 1) show that the extraction by continuous flow leads to better yields, when compared with the system by batch, decreasing about 2 hours the time required for extraction.These results may be attributed to the continuous renovation of the humic material and are in accordance with those obtained by Rosa et al., 22 which evaluated the continuous flow extraction of humic substances from soils with NaOH.Considering the different extractants, the highest yields for both extraction systems were obtained with NaOH 0.1 mol L -1 .Among the different extractants, Na 4 P 2 O 7 has been shown to be a strategic extractant, aiming agricultural applications of humic material, because the extraction in this case may occur simultaneously with the enrichment of the HS with phosphorus, an essential macronutrient.The literature has been suggesting the use of a mixture of NaOH and Na 4 P 2 O 7 as extractant, due to the little modifications in the chemical structure of extracted humic material. 9,10However, it is important to emphasize that there should be a control on the amount of extractant added, especially of Na 4 P 2 O 7 and the phosphorus amount retained in HS, because an excess of phosphorus might cause high environmental impact.Thus, parameters for controlling the amount of phosphorus released are of great environmental concern and the determination of the complexing ability of HS with macronutrients has shown to be a promising parameter to this purpose.

Determination of the conditional complexing capacity of HS of peat extracted by flow with different extractants for macro and micronutrients.
For the commercialization of humic substances as organic fertilizer, beyond the extraction step, the enrichment of the extracted material with macro and micronutrients is a common practice, providing to consumers a better product from the agronomic viewpoint.However, little is known about the ability of extracted HS to complex those nutrients.Therefore, the complexing capacity (CC) data are useful, giving the maximum concentrations of nutrients that HS can complex, preventing waste as well as possible environmental contaminations due to the excess of nutrients lixiviated into springs, which could lead to eutrophication.
The determination of the conditional complexing ability of HS at pH 5.5, extracted from peat samples by flow for macronutrients (Ca 2+ and Mg 2+ ) and micronutrients (Zn 2+ , Co 2+ , Mn 2+ , Cu 2+ and Mo 2+ ) was made by titration, using the tangential ultrafiltration system.During titration, firstly occurs the saturation of the stronger binding ligands and, after that, the saturation of the weaker.Thus, a typical curve used in the determination of the complexing ability of HS with those nutrients is obtained by plotting the concentration of the free nutrient against the total nutrient concentration (mmol L -1 ) (Figure 3).The curve shows an inflection point and the complexing ability is obtained by the intersection of the two linear sections of the graphic (Figure 3).
The CC values determined for the HS bring important quantitative data, such as the complexed amount of each metal as well as information about the affinity of the HS for different metal species.Thereby, according to the Table 2, the following increasing order of affinity between the studied metal ions and the humic substances from peat, extracted with different extractants, can be established: Analyzing the conditional complexing capacity of humic substances extracted from peat samples with different extractants, it was observed that the greater affinity is related to magnesium and calcium.This higher affinity, in turn, may be related to the great amount of oxygenated groups present in the HS samples.The elements Mo 2+ , Co 2+ and Cu 2+ showed low affinity for humic substances, independently of the extractant used.
The results from Table 2 also show that the studied elements (Ca 2+ , Mg 2+ and Mn 2+ ) presented higher conditional complexing capacity for the humic substances extracted with sodium pyrophosphate, when compared to other extractants.Furthermore, several studies show that humic substances extracted with sodium pyrophosphate have higher concentrations of oxidized molecules. 23,24ccording to Gieguzynska et al., 24 Ca 2+ and Mg 2+ are able to establish bonds between the local charges of negative surface and anionic humic macromolecules.Studies proposed by Milne et al. 16 using the NICA-Donnan model showed that in interactions between fulvic acid (FA) and calcium ions, the metal ions are almost entirely connected with the first distribution sites (carboxylic groups present in the structure of HS), with a greater contribution to the sites of the second distribution (smaller groups present in the structure of HS) for metal-FA.Thus, it might be inferred that the greater affinity of Ca 2+ and Mg 2+ for HS extracted with pyrophosphate may be related to the large amount of oxygenated groups present in the HS structures.
The interactions between the metal species and HS strongly depend on the metal ion and pH.At pH 5.5 (experiments in this work) the contribution of electrostatic interactions will be through the Donnan effect. 16n determination of the complexing ability of HS occurs the saturation of binding sites.In this case, the observed differences in the conditional complexing capacity shows the differences between the HS materials come from the extraction procedures and there are differences in the total number of sites available for binding metals.
The highest conditional complexing ability of the extracted HS for the macronutrients calcium and magnesium was observed with all extractants, indicating a high affinity of HS for Ca 2+ and Mg 2+ .Moreover, the complexation of these macronutrients by HS is also favored when using sodium pyrophosphate as extractant.Studies proposed by Rosa et al., 22 showed that the use of different extractants influences directly the performance of the HS, however, they might not act, depending on the functional groups present in its structure.Accordingly, the greater affinity of HS extracted with pyrophosphate for nutrients is due to possible molecular rearrangements occurred during the extraction process.These results show that the use of sodium pyrophosphate as humic extractant is interesting, since it has high affinity for macro and micronutrients, compared to other extractants.Thus, the HS extracted with sodium pyrophosphate, can be enriched with nutrients, presenting interesting characteristics for commercial purposes.
Once applied to the soil, these HS can provide the complexed nutrients and, thanks to a high complexing capacity, reduce the amount of these nutrients that could be leached.Therefore, our results provide a manner for an appropriate enrichment of HS extracted from peat for commercial purposes, preventing environmental impacts as well as adding commercial value to the product.

Conclusions
The continuous flow showed to be adequate and efficient, reducing the time required for extraction and providing higher yields when compared with the batch procedure, saving work in the steps of filtration, centrifugation, pH adjustment and handling of samples.
The conditional complexing capacities showed that, independently of the extractant, the HS presented higher affinity for Ca and Mg, indicating that, even if a change in the chemical structure of humic molecules occurs during the extraction, it does not affect the complexing capacity.However, the molecular rearrangement during the extraction process, using sodium pyrophosphate, favored complexation with essential macronutrients.Thus, the use of sodium pyrophosphate as extractant, despite not presenting the highest yield, increases the complexation capacity of humic material with essential macronutrients, demonstrating to be very promising in the extraction of HS, aiming their agricultural application.

Figure 1 .
Figure 1.Diagram of the procedure used to collect peat samples.

Table 1 .Figure 3 .
Figure 3. Graph for determination of the conditional complexing capacity of extracted humic substances from peat samples with NaOH 0.1 mol L -1 by Cu 2+ ions at pH ≈ 5.5 and [HS] = 100 mg L -1 .