1. Introduction
Humic substances (HS) are complex mixtures, mainly composed of fulvic acid (FA) and humic acid (HA). FA is soluble in both acidic and alkaline solutions, while HA is soluble in alkaline solutions but insoluble in acidic solutions [
1]. HS are formed from animal and plant waste. Through a protracted period of microbial decomposition–transformation and geophysical chemical action, oligomers or polymers are formed with several polar functional groups, such as -COOH, -OH, and C = O [
2]. The presence of these groups endows HS with many capabilities, such as acidity, hydrophilia, complexation, adsorptivity, and ion-exchange selectivity [
3], which makes HS have great potential in the pharmaceutical [
4], agricultural [
5,
6], industrial [
7], and environmental protection [
8] applications. Much research has been conducted to transform HS into realistic and technological productivity by the extraction, processing, and modification of HS. For instance, Volikov et al. [
9] enhanced surface activity of natural HS with respect to mineral surfaces by functionalization with organosilanes, which was conducted in water. Silanized HS to the soil was contributed to create aggregates, increase soil porosity, and enhance nutrient and water storage capacity. Qin et al. [
4] found that in the oxygen-containing functional groups, especially phenolic hydroxyl groups, molecular weight distribution, colloidal properties, and astringency were the material basis of the antidiarrheal activity.
Different composition and storage environments of raw materials cause the variability in HA’s composition, structure, and physical and chemical properties [
10]. Therefore, owing to the differences of raw materials and development methods, HS are mainly divided into two types: mineral HS and biochemical HS. Although mineral HS is readily available, the incomplete HS extraction and high HA molecular weight are still problems [
5]. In addition to mineral-HS, biochemical-HS also has attracted world-wide attention since the raw material (biomass) of biochemical-HS are renewable and abundant in China. Therefore, this paper proposed that: biomass and lignite were mixed as raw materials and co-thermally oxidated to prepare composite HS, which has high activity and high yield. It is not only an important method for the preparation of biochemical HS but also the highlight of agricultural resources and functionalization.
The chemical pretreatment of raw materials is able to reduce the molecular weight of HS and increase the content of its active functional groups, which can improve the biological activity of HS. Chemical oxidation pretreatment includes hydrogen peroxide activation [
11], nitric acid oxidation [
12], hydrothermal method [
13], and so on. Huang et al. [
14] found that nitric acid oxidation was the most effective pretreatment for increasing coal oxidation. This pretreatment method encouraged microbial solubilization in coal, thus increasing the HA yield and the number of oxygen and nitrogen functional groups [
15]. Fan et al. [
16] used KOH as an activator to extract natural HS and artificial HS from black soil under atmospheric pressure and from tulip leaves/wood chips in a hydrothermal reactor, respectively. The two kinds of HS showed high similarity on chemical structure (abundant aromatic frameworks) and ultimate contents (e.g., N and S elements). L. Aranganathan [
17] found that different extractants and extraction conditions would affect the structure and yield of HA. Based on the results, the high yield of HA was obtained from NaOH and KOH treatments and the high elemental “C” level in HA was extracted from NaOH and KOH, whereas high elemental “O” in HA was extracted with Na
4P
2O
7. It can be speculated that the extraction or synthesis conditions have great effects on the physical and chemical properties of the produced HS. Herein, the parametric optimization is vital for the synthesis of HS and its industrialization.
As a common process optimization method, single factor method can only obtain the influence trend of each factor on HS, which cannot reflect the interaction between several variables. Response surface methodology (RSM) is an effective statistical design tool, which generates a set of consecutive experiments to establish the relationships between independent factors and responses. In addition, the function variance and residual and response surface can be calculated directly through software analysis, which has the advantages of cost saving and high credibility. For example, Li [
18] used the RSM to optimize the extraction conditions of HA from lignite using Penicillium Ortum MJ51 cell-free filtrate. According to the research [
19], RSM was used to determine the optimal conditions, and a mathematical model was established to accurately predict the changes in sludge water content and the extraction rate of HA.
Considering the low yield of biochemical HS, large molecular weight, and low activity of mineral humic acid, this paper presents an effective method for the preparation of composite HS. The mixture of lignite and rice straw was used as raw material, which were pretreated by the co-thermal oxidation method using HNO3 solution, then extracted using KOH solution to obtain HS. The pretreatment conditions (including material ratio, liquid–solid ratio, nitric acid concentration, oxidation time, and oxidation temperature were optimized through single factor method and RSM. A mathematical model was established and verified to find the optimal process parameters for preparing HS. The group characteristics of FA and HA products were compared by elemental analysis and functional group analysis. It is expected to provide reference for HS application in industry and agriculture.
4. Conclusions
In this study, RSM was used to optimize the co-thermal oxidation conditions for obtaining FA and HA products. The BBD model took HS content as an output response to obtain the process conditions, and then the morphology, composition, and structure of FA and HA prepared under the optimized conditions were analyzed. The results are as follows:
(1) According to the results of the single factor experiment, the ideal pretreatment conditions for the synthesis of HS were as follows: the material ratio was 0.5, HNO3 concentration was 15%, liquid-to-solid ratio was 1:20, oxidation temperature was 80 °C, and oxidation time was 60 min. Under these conditions, HS content was 62.04%. HS contents were clearly influenced by these three parameters of material ratio, oxidation time, and oxidation temperature, which were subsequently chosen as the variables to RSM.
(2) According to the BBD model, TE, TI, and RA all had a significant influence on HS content. The order of significance of the three factors was: TE (F = 85.77) > RA (F = 42.75) > TI (F = 2.35). As can be seen from the 3D response surface diagram, the images were all raised, demonstrating that there was significant interaction between the two variables. The co-thermal oxidation conditions were optimized using RSM as follows: RA was 0.53, TE was 75.63 ℃, TI was 59.50 min, and the experimental HS content under repeated optimum conditions was 62.37%, which was almost equal to the expected value (62.27%) with a low relative inaccuracy of 0.16%, illustrating that the process model was trustworthy.
(3) Under optimized conditions, the obtained HA had a tightly packed block structure, and FA had a loosely spherical shape. The molecular weight of FA was 2487 Da and HA was 20,904 Da, meaning both had smaller molecular weight than that reported in the literature. FT-IR spectra showed that FA had strong bands at around 1720 cm−1, thus confirming the presence of more oxygen-containing functional groups than HA. The appearance of double peaks at 2900~2980 cm−1 indicated that HA contains more aliphatic chains.