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Article

Effect of Exogenous Glucose at Different Concentrations on the Formation of Dark-Brown Humic-like Substances in the Maillard Reaction Pathway Based on the Abiotic Condensation of Precursors Involving δ-MnO2

by
Nan Wang
1,
Qi Zhang
2,
Wanhong Li
3,
Chengxin Bai
1,
Yan Song
1,
Shuai Wang
1,* and
Zhijiang Liu
4
1
College of Agriculture, Jilin Agricultural Science and Technology University, Jilin 132101, China
2
College of Agricultural Resources and Environment, Jilin Agricultural University, Changchun 130118, China
3
College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China
4
Animal Husbandry Station in Jilin City, Jilin 132013, China
*
Author to whom correspondence should be addressed.
Sustainability 2022, 14(18), 11603; https://doi.org/10.3390/su141811603
Submission received: 16 August 2022 / Revised: 9 September 2022 / Accepted: 11 September 2022 / Published: 15 September 2022
(This article belongs to the Topic Synthesis, Characterization and Performance of Materials for a Sustainable Future)
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Versions Notes

Abstract

:
The Maillard reaction is a type of nonenzymatic browning process and is an important pathway for the formation of humic-like substances (HLSs). Glucose is one of the three crucial precursors for the Maillard reaction, and a change in glucose concentration can inevitably affect the humification pathway, thereby regulating the composition and quality of HLSs. To verify the scientific hypothesis, the method of liquid shake-flask culture was adopted. Both catechol and glycine with fixed concentrations were added to a phosphate buffer including δ-MnO2, and only the concentration of glucose was adjusted in the sterile culture system. The obtained supernatant fluid and dark-brown residue were collected dynamically through the centrifugation method. The E4/E6 ratio and total organic C (TOC) of the supernatant fluid, the humus composition, and FTIR spectra for the dark-brown residue, and the elemental composition of humic-like acid (HLA) extracted from the dark-brown residue were analyzed to reveal the effect of varying glucose concentrations on the abiotic humification pathways for the Maillard reaction and the characteristics of relevant products under abiotic processes. The results reveal that (1) the exogenous addition of glucose at different concentrations simplifies the molecular structure in the supernatant fluid, and the TOC content is decreased to varying degrees, among which the addition of 0.24 mol/L glucose leads to the formation of simpler organic molecules in the supernatant compared to that for the other treatments, and the addition of 0.03 mol/L glucose shows the largest decrease in TOC content; (2) Under the coexistence of glycine and catechol, CHLA treated with the addition of glucose at different concentrations shows an upward trend in the course of the culture, which is significantly higher than that obtained for the CK control. The addition of 0.12 mol/L glucose results in the largest increase in CHLA. During the culture period, the structure of HLA molecules from each treatment first become complex and then gradually become simpler. Finally, the molecular structure of HLA treated with different concentrations of glucose becomes more complex, but the structure of HLA molecules from the CK control tends to be simplified. The addition of glucose can improve the condensation degree of HLA molecules, among which the addition of 0.12 mol/L glucose shows the most significant effect. With increasing exogenous glucose concentration, the number of N-containing compounds in the HLA molecules further decreases, while the number of O-containing functional groups increases. (3) The greater the concentration of glucose added, the higher the proportion of aromatic C structures in the dark-brown residue. During this process, the Mn-O bond lattice vibration of the δ-MnO2 layered structure is greatly enhanced. The organic molecules in the dark-brown residue and δ-MnO2 are bound to each other through intermolecular hydrogen bonding. The CHLA/CFLA ratio for each treatment increases to varying degrees after the culture period, indicating that the addition of glucose is more conducive to the improvement of humus quality than the CK control, among which the addition of 0.12 mol/L glucose shows the best effect.

1. Introduction

Humic substances (HS) are considered partially decomposed or microbially altered polymers from animal and plant residues formed by polymerization reactions, which are an important material basis for soil fertility in nature and sustainable agricultural development, and there are many classical hypotheses about the formation of HS. The HS are formed by different polymerization pathways of the precursors which includes polyphenols, quinonyl compounds, reducing sugars, saccharides, and nitrogenous compounds. The HS are proposed to be formed by biotic and abiotic pathways, the products formed during abiotic processes, we called humic-like substances (HLSs). The abiotic pathways were often neglected in existed composting or theoretical studies on HLSs formation. As an indispensable abiotic pathway for the formation of HLSs in nature, the Maillard reaction is a type of nonenzymatic browning process that involves the reaction of carbonyl compounds, particularly reducing sugars, with compounds that possess a free amino group, such as amino acids, amines, and proteins [1]. The browning process involves the polycondensation of sugar and amino acids [2]. During the formation of HS, biomacromolecules such as proteins, polysaccharides, lignin, and polyphenols are hydrolyzed into low-molecular-weight mixtures such as amino acids, sugars, and quinones, which are called humic precursors [3]. These precursors can be polymerized via oxidative and nucleophilic reactions to form dark polymers, whose structure is similar to HS [4]. Polycondensation reactions between low-molecular-weight compounds, such as catechol, glycine and glucose, are crucially important processes in the formation of HLSs [5].
Many studies have attached importance to the relationship between the Maillard reaction and the formation of HLSs. Jokic et al. [6] studied the humification process for glucose, glycine, and catechol catalyzed by δ-MnO2 and pointed out that the catalysis is closely related to the adsorption and condensation of the mineral surface. The literature reveals that the glucose-glycine-bimessite system involves a redox reaction, with the reduction in Mn(IV) to Mn(II), which then enters the supernatant fluid and the concomitant oxidation of glucose, possibly forming α-dicarbonyl compounds that then couple with glycine [7]. Hardie et al. [8] demonstrated the important role of sugars in affecting abiotic humification pathways and related products in natural environments and also pointed out that increasing the molar ratio of poly-phenols to Maillard reagents substantially enhances humification reactions and promotes the formation of HS with a stronger aromatic character. Zhang et al. [9] investigated the mechanism for abiotic humification by tracking the fate of the precursors in systems containing glucose, glycine, and various catechol concentrations, and the results showed that the N-containing organic molecules can significantly contribute to controlling the darkening effect. Increasing the concentration of catechol can promote the formation of fulvic-like acid (FLA) and humic-like acid (HLA) and can be more helpful to increase the degree of unsaturation in the HLA molecules. The addition of exogenous precursors can greatly improve the conversion efficiency of fulvic acid (FA) to humic acid (HA) during composting. Adding more precursors can promote maturation and increase the HS content of the compost. The humification process can be promoted by increasing the number of precursors, and the maturity of composting and the generation of HS during composting can be enhanced [10]. In comparison with biotic processes, abiotic processes have attracted extensive attention since they are faster and more efficient reactions, and the structural features of HLA can be manipulated by changing the operating conditions [11,12]. Although humic-like polymers can also be formed in the absence of microbial life [13], abiotic pathways have often been more easily ignored in existing composting studies [14]. In contrast, few available reports have addressed the effect of exogenous glucose at different concentrations on the formation of dark-brown HLSs in the Maillard reaction pathway based on the abiotic condensation of precursors.
It was originally thought that sugars control the formation of HS by affecting microbial activity in biotic processes. However, under the abiotic condensation reaction (Maillard reaction), changes in sugar concentration can promote the humification process and enhance the production of HLSs. This is a topic that has always puzzled and fascinated us. In view of this, the method of liquid shake-flask culture was adopted. Both catechol and glycine with fixed concentrations were added into the phosphate buffer containing δ-MnO2, and only the concentration of glucose was adjusted in the sterile culture system. The obtained supernatant fluid and dark-brown residue were collected dynamically through centrifugation, and the E4/E6 ratio and TOC for the supernatant fluid, the humus composition and FTIR spectra for the dark-brown residue, and the elemental composition of HLA extracted from the dark-brown residue were analyzed to reveal the effect of different glucose concentrations on the abiotic humification pathways for the Maillard reaction and to reveal the characteristics of related products under abiotic processes.

2. Materials and Methods

2.1. Materials

δ-MnO2 was synthesized with reference to the method of Parida et al. [15]: 180 g of MnSO4·H2O was dissolved into 1500 mL of H2SO4 solution with a concentration of 29 mmol·L−1. Another 120 g of KMnO4 was dissolved into 1500 mL of distilled water, and the solution of KMnO4 was slowly added into the MnSO4 solution while stirring. The formed precipitate was collected by centrifugation, dried at 55 °C, and ground and passed through a 0.01 mm sieve for use.
Analytical grade catechol (C6H6O2), glucose (C6H12O6), and glycine (C2H5NO2) were obtained from Sinopharm Chemical Reagent Co., Ltd. in Shanghai, China.
Preparation of 0.2 mol/L (pH 8.0) phosphate buffer: 5.3 mL of 0.2 mol/L NaH2PO4 and 94.7 mL of 0.2 mol/L Na2HPO4 were mixed thoroughly, and 0.02% thimerosal (C9H9HgNaO2S) was added.

2.2. Methods

Sterile conditions were maintained throughout the experiments to ensure that abiotic transformation dominated. All glassware, phosphate buffer, and other apparatuses were autoclaved prior to use. A certain number of 500 mL conical flasks were prepared. A total of 250 mL of 0.2 mol/L phosphate buffer was introduced into each flask, and δ-MnO2 was added to each system at 2 g per flask. Catechol and glycine were both added at a concentration of 0.06 mol/L and incubated with increasing glucose concentrations (0, 0.03, 0.06, 0.12, and 0.24 mol/L). The above treatments of different glucose concentrations are represented by Glu0, Glu0.03, Glu0.06, Glu0.12, and Glu0.24. The CK control was set as follows: 2 g of δ-MnO2 was added to the phosphate buffer only without the participation of glucose, catechol, or glycine. All reactions were conducted in duplicate.
The liquid shake-flask culture was initiated under sterile operating conditions, with the rotation speed kept at 150 rpm. During the culture process, a 2 mL aliquot of the supernatant fluid was drawn from each system at 0, 3, 6, 18, 28, 48, 76, 124, 172, 240, and 360 h and centrifuged at a high speed of 16,000 rpm for 5 min. A 1 mL aliquot of the supernatant fluid was withdrawn from it and diluted to 25 mL, and the absorbances at 465 and 665 nm (E4 and E6) were measured. Then, the E4/E6 ratio was further calculated. Its TOC content was determined with the total organic C analyzer vario TOC cube. Another 10 mL aliquot of the supernatant fluid was dynamically drawn from the conical flasks after culture for 0, 18, 48, 76, 124, 172, 240, and 360 h and then centrifuged at high speed (16,000 rpm) for 10 min. The obtained dark-brown residue was acidified to pH 1.0 with concentrated HCl (1.0 mol/L) and equilibrated for 24 h. After the appearance of a flocculation-like precipitate, high-speed centrifugation (16,000 rpm, 10 min) was performed again. The obtained supernatant was fulvic-like acid (FLA), the pH of which was adjusted to neutral, and the solution was diluted to 25 mL. The remaining precipitate was humic-like acid (HLA), which was dissolved with a 0.1 mol/L NaOH solution at 60 °C, adjusted to neutral and diluted to 25 mL (HLA liquid sample). The organic C contents of HLA and FLA (CHLA and CFLA) were both measured by the TOC analyzer, and the CHLA/CFLA ratio could be further calculated. After culture for 360 h, the remaining culture medium in the conical flask was centrifuged and adjusted to pH 1.0 to separate HLA and FLA. The solid phase was HLA, which was dissolved in 0.1 mol/L NaOH solution, rinsed with a 150 mL mixture of HCl (6%, v/v) and HF (6%, v/v) to remove its inorganic impurities, magnetically stirred for 24 h, and centrifuged at 16,000 rpm for 10 min. After removing impurities, the HLA was freeze-dried with a freeze dryer after electrodialysis to prepare a solid sample of HLA, which was ground and passed through a 0.01 mm sieve for use.

2.3. Analysis

The absorbances of diluted aliquots of the supernatant fluid and HLA-diluted liquid sample at 465 and 665 nm (E4 and E6) were measured by a UV–visible spectrophotometer (TU-1900, Beijing Purkinje General Instrument Co., Ltd., Beijing, China), and then the CHLA/CFLA ratio was calculated. The percentages of C, H, O, and N in the solid HLA samples were measured by an elemental analyzer (PerkinElmer PE 2400II CHNS/O). Structural characterization of the dark-brown residue was measured by FT-IR spectrophotometry (Model: FTIR-850, Tianjin Gangdong Sci & Tech Development Co., Ltd., Tianjin, China). FTIR spectra were taken in the wavelength region from 400 to 4000 cm−1, analyzed with FTIR 850 software and presented as graphs with Origin 8.0 software.

2.4. Statistical Analysis of the Data

All calculations and statistical tests were performed using Microsoft Excel 2003 and one-way univariate ANOVA (the Duncan test) in SPSS 18.0. FTIR spectra were analyzed using the ZWin software supplied with the FTIR-850 spectrometer. Set the measurement parameters as follows: 32 scans with 4 cm−1 resolution, data interval 1.93 cm−1. Choose the triangle as the apodization mode and the option of collecting background before collecting samples. Save the processed spectra with the transmittance (%) as Y-axis as a new CSV file and plot them using Origin 8.0.

3. Results

3.1. E4/E6 Ratio and Total Organic Carbon (TOC) for the Supernatant Fluid

As shown in Figure 1, with the liquid shake-flask culture, the effect of exogenous addition of glucose at different concentrations on the E4/E6 ratio of the supernatant fluid is different. During the culture of 0~3 h, the E4/E6 ratio for Glu0 treatment decreases drastically from 2.58 to 2.04, remains stable at 28 h for the culture, and then increases significantly after 28 h until the end of the culture; the E4/E6 ratio for Glu0.03 treatment decreases greatly from 2.63 to 2.33 during the period of 0~3 h, then stabilizes, and shows a large increase at 76 h until the end of the culture; the E4/E6 ratio for Glu0.06 treatment decreases greatly from 3.37 to 2.56 during the culture time of 0~6 h, after that a smooth fluctuation is observed, with a significant increase at 48 h for the culture, followed by a gradual increase until the end; the E4/E6 ratio for Glu0.12 treatment shows a slight decrease from 2.88 to 2.69 during the culture time of 0~3 h, after that it remains stable, and shows a gradual upward trend at 28 h until the end; the E4/E6 ratio for Glu0.24 treatment shows a trend of first decreasing and then increasing; the E4/E6 ratio for CK control shows a decrease in the fluctuations. Compared to the results at 0 h, at the end of the culture (360 h), the E4/E6 ratios for the Glu0, Glu0.03, Glu0.06, Glu0.12, and Glu0.24 treatments are increased by 57.2%, 44.9%, 21.0%, 51.6%, and 4.3%, respectively, in which the lowest increase is obtained for the Glu0.24 treatment; nevertheless, the E4/E6 ratio for the CK control is decreased by 46.7%.
As shown in Figure 2, with the culture affected by the exogenous addition of glucose at different concentrations, the change law for TOC content in the supernatant fluid is different. The TOC content in the supernatant fluid for Glu0 treatment fluctuates between 4.7 and 5.0 g/L during the period of 0~124 h, reaches a maximum value at 124 h for the culture, and then gradually decreases from 5.0 to 4.2 g/L; the TOC content in the supernatant fluid for Glu0.03 treatment fluctuates between 6.1 and 7.2 during the period of 0~240 h, experiences a significant decrease from 6.4 to 4.3 g/L at a culture time of 360 h; for the Glu0.06 treatment, the TOC content of the supernatant fluid increases rapidly from 9.3 to 10.3 g/L during the period of 0~3 h, then decreases to 8.2 g/L at a culture time of 6 h, and fluctuates between 8.2 and 8.7 g/L until the culture is completed; the TOC content in the supernatant fluid obtained from Glu0.12 treatment is increased from 14.1 to 15.0 g/L during the period of 0~3 h, and then gradually decreases until the culture is completed; the TOC content in the supernatant fluid obtained from the Glu0.24 treatment shows a gradual decrease from 25.7 to 22.0 g/L for the whole culture process; the TOC content in the supernatant fluid obtained from the CK control shows a low level fluctuation for a concentration of 0.4~0.9 g/L. Compared to the results at 0 h, at the end of the culture (360 h), the TOC contents in the supernatant fluid obtained from the Glu0, Glu0.03, Glu0.06, Glu0.12, Glu0.24, and CK control treatments is decreased by 13.6%, 39.1%, 10.0%, 11.3%, 14.4%, and 34.3%, respectively, for which the TOC content of the Glu0.03 treatment shows the greatest decrease, which is higher than that of the CK control.

3.2. C Content of Humic-like Acid (CHLA), CHLA/CFLA Ratio and FTIR Spectra for the Dark-Brown Residue

As shown in Figure 3, as the culture proceeds, the change in the CHLA of CK is relatively smooth and fluctuates between 0.05 and 0.08 g/L. With the addition of glucose at different concentrations, all CHLA shows an upward trend. Compared to the result for 0 h, at the end of the culture (360 h), the CHLA for Glu0, Glu0.03, Glu0.06, Glu0.12, Glu0.24 treatments and the CK control is increased by 409.2%, 522.0%, 336.1%, 636.3%, 279.8%, and 72.8%, respectively. CHLA treated by the addition of Maillard reaction precursors is higher than that for the CK control, and the CHLA for Glu0.12 treatment shows the greatest increase.
Sustainability 14 11603 g003 550
Figure 3. Effects of changes in the glucose concentration in the Maillard precursors on CHLA extracted from the dark-brown residue.
Figure 3. Effects of changes in the glucose concentration in the Maillard precursors on CHLA extracted from the dark-brown residue.
Sustainability 14 11603 g003
The CHLA/CFLA ratio, as an important parameter, is widely used to describe the degree of condensation [16]. As shown in Figure 4, the CHLA/CFLA ratio determined for the different treatments follows different rules. The CHLA/CFLA ratios for the Glu0, Glu0.24, and CK control treatments show a gradually increasing tendency; whereas the CHLA/CFLA ratios for the Glu0.03, Glu0.06, and Glu0.12 treatments show an increasing trend first followed by a decreasing trend. Compared to the result at 0 h, at the end of the culture (360 h), the CHLA/CFLA ratios for the Glu0, Glu0.03, Glu0.06, Glu0.12, and Glu0.24 treatments and the CK control are increased by 102.1%, 395.1%, 720.9%, 1072.0%, 701.7%, and 84.6%, respectively, among which the Glu0.12 treatment shows the greatest increase in the CHLA/CFLA ratio. The increase in the CHLA/CFLA ratio obtained upon the addition of Maillard reaction precursors is higher than that obtained for the CK control, which is more conducive to improving the quality of the humus.
Combining Figure 5 and Table 1, it can be seen that the broad peak at 3419~3435 cm−1 can not only represent the stretching vibration of -OH of δ-MnO2 or interlayer water molecule -OH but also the hydroxyl group -OH stretching vibration for alcohol or phenolic compounds [17]; the peaks at 2922 cm−1 and 2850 cm−1 are attributed to the stretching vibrations of asymmetric aliphatic C-H and symmetric aliphatic C-H [18], respectively, which are only reflected in the CK control; the band observed at 1606~1633 cm−1 is characteristic of the bending vibration of surface adsorbed H2O of δ-MnO2 or the stretching vibration of the aromatic ring skeleton C=O; the absorption peak at 1369~1387 cm−1 is assigned to the phenolic OH, and C=O stretching of carboxylates [19]; the peak at 1101 cm−1 represents the stretching vibration of O-H [20], which is only reflected in the CK control; the sharp absorption peak located at 563~600 cm−1 is attributed to the lattice vibration of the layered Mn-O bond.
As summarized in Table 1, compared with the CK control, the absorption intensity of the peak at 3419~3435 cm−1 under the exogenous addition of Maillard precursors is increased to varying degrees. This is because each treatment with exogenous precursors shows both the stretching vibration from -OH of δ-MnO2 or the -OH of interlayer water molecules and the stretching vibration of hydroxyl -OH from alcohol or phenolic compounds. The superposition of the above peaks results in the intensity of the peak at 3419~3435 cm−1 being greater than that of the CK control. The peaks at 2922 cm−1 and 2850 cm−1 representing aliphatic C are merely observed in the CK control, owing to the addition of 0.02% thimerosal (C9H9HgNaO2S). The stronger intensity of the peak at 1606~1633 cm−1 and the weaker intensity of the peak at 1369~1387 cm−1 indicates the addition of a large concentration of glucose, the higher proportion of aromatic C structure in the dark-brown residue, and, at the same time, the lower proportion of carboxyl groups.

3.3. E4/E6 Ratio and Elemental Composition of the Humic-like Acid Extracted from the Dark-Brown Residue

As shown in Figure 6, with the culture, the E4/E6 ratios for HLA obtained from all the treatments and CK control show a trend of first decreasing and then increasing. This indicates that the molecular structure of HLA becomes complex at first and then gradually becomes simpler. The E4/E6 ratio for HLA from the Glu0 treatment decreases from 2.16 to 0.95 during the period of 0~76 h, and increases to 1.75 at the end; the E4/E6 ratio for HLA obtained from the Glu0.03 treatment decreases from 2.58 to 0.97 during the period of 0~48 h, and then gradually increases to 1.69 when the culture is completed; the E4/E6 ratio for HLA obtained from the Glu0.06 treatment decreases from 2.13 to 0.96 during the period of 0~48 h, and then gradually increases to 1.78 at the end of the culture; the E4/E6 ratio for HLA obtained from the Glu0.12 treatment decreases from 4.65 to 1.49 during the period of 0~124 h, and then gradually increases to 2.13 at the end of the culture; the E4/E6 ratio for HLA obtained from Glu0.24 treatment shows a decrease from 3.22 to 1.92 during the period of 0~240 h, and then attains a value of 2.13 at the end of the culture time; the E4/E6 ratio for HLA obtained from the CK control decreases from 2.13 to 0.99 at a culture time of 0~76 h, and then gradually increases until it reaches a value of 3.33 at the end of the culture. Compared to the result at 0 h, at the end of the culture (360 h), the E4/E6 ratios for HLA obtained from Glu0, Glu0.03, Glu0.06, Glu0.12, and Glu0.24 treatments are decreased by 19.0%, 34.5%, 16.4%, 54.2%, and 33.8%, respectively, indicating that the molecular structure of HLA becomes more complex. Among them, the E4/E6 ratio for HLA obtained from the Glu0.12 treatment shows the greatest decrease, indicating that the promotion effect on the molecular complexity of HLA is the greatest; however, the E4/E6 value for HLA obtained from the CK control is increased by 56.3%, indicating that its molecular structure tends to be simplified.
As shown in Table 2, the CK control was treated with the addition of δ-MnO2 and no other precursors in a phosphate buffer with a concentration of 0.2 mol/L, so HLA could not be extracted, and its corresponding elemental data could not be obtained. Here, Glu0 treatment served as the control. Under the addition of glucose at different concentrations, compared with the Glu0 treatment, the H/C ratio for HLA extracted from the dark-brown residue decreased by varying degrees, in which the H/C ratio of the Glu0.12 treatment was the smallest. With increasing glucose concentration, the C/N ratio of HLA gradually decreases, while the O/C ratio gradually increases. The above results show that under the coexistence of glycine and catechol, the addition of glucose can improve the degree of condensation of HLA molecules based on the abiotic condensation reaction catalyzed by δ-MnO2. Among them, the addition of 0.12 mol/L glucose shows the most significant effect on improving the condensation degree of HLA molecules. With increasing glucose concentration, the number of N-containing compounds in HLA molecules decreases, whereas the number of O-containing functional groups increases, resulting in a decrease in the C/N ratio and an increase in the O/C ratio.

4. Discussion

4.1. E4/E6 Ratio and TOC for the Supernatant Fluid

Maillard precursors are recognized as the key factor that regulates HS formation [10]. Amino acids, polysaccharides, and reducing sugars can participate in HS formation via the Maillard reaction [21]. Exogenous addition of precursors can strengthen the relationship among different precursors and further promote the humification process [10]. As one of the necessary precursors, glucose is the C source for microorganisms [22], which can promote the biotic humification process. Under abiotic conditions, the role of glucose, one of the Maillard precursors, is not fully understood.
Under the influence of exogenous glucose at different concentrations, after the liquid shake-flask culture, the molecular structure of the supernatant fluid tends to be simplified. The addition of 0.24 mol/L glucose leads to the formation of simple organic molecules in the supernatant fluid compared to the other treatments; in contrast, more complicated molecules are formed in the supernatant fluid of the CK control. Hardie et al. [8] showed that increasing the molar ratio of glucose to catechol and glycine in the integrated catechol-Maillard system can enhance the formation of low-molecular weight, strongly aliphatic carboxylic Maillard reaction products in the supernatant, which is similar to our results. The TOC content in the supernatant fluid for each treatment is decreased to varying degrees after the culture, among which the Glu0.03 treatment shows the largest decrease, but which is still higher than that of the CK control. The decrease in TOC content in the supernatant fluid results from the decarboxylation of oxidized glucose and/or aliphatic fragments derived from ring cleavage of catechol, and a proportion of the TOC is transformed and released as CO2 [23].

4.2. CHLA, CHLA/CFLA Ratio and FTIR Spectra for the Dark-Brown Residue

The CHLA treated with the addition of Maillard precursors is higher than that for the CK control. Under the influence of glucose at different concentrations, CHLA shows an upward trend with culture. Among the different treatments, the Glu0.12 treatment shows the largest increase in CHLA. Zhu, N. reported that aromatic compounds such as phenols and quinones originating from lignin degradation can polymerize with amino acids, reducing sugars and polysaccharides to form HS. Polysaccharides are recognized as an important precursor for HS formation [24]. In addition, the contribution of reducing sugars (i.e., glucose) to HS formation is also important due to its incorporation into the darkening mechanism [25]. HLA is the main part of the HLSs. δ-MnO2 can promote abiotic pathways and accelerate the condensation of low-molecular-weight precursors (glucose, glycine, and catechol) through chemical catalysis [26], which is more conducive to the formation of HLA and the accumulation of CHLA. This is consistent with the conclusion of Wu et al. [27], who found that the greater the amount of reducing sugar added, the more obvious the promoting effect on HS synthesis.
The CHLA/CFLA ratio for each treatment is increased to varying degrees at the end of the culture, among which the Gly0.12 treatment shows the largest increase in the CHLA/CFLA ratio. The increase in the CHLA/CFLA ratio in the treatment with the addition of Maillard precursors is higher than that in the CK control, which is more helpful for improving humus quality. Under the coexistence of glycine and catechol, the addition of glucose can improve the condensation degree of HLA molecules based on the abiotic condensation reaction catalyzed by δ-MnO2. Among the different treatments, the addition of 0.12 mol/L glucose shows the most significant effect on improving the condensation degree of HLA molecules. The Maillard reaction involves the cleavage, rearrangement, and polymerization of proteins and sugars to form N-containing heterocycles, known as HLA [28]. Increasing the content of precursors can promote the Maillard reaction and improve the humification degree. Hardie et al. [8] synthesized HLA from glycine and glucose solutions, demonstrating that various sugar concentrations have a great influence on the humification degree. In the catechol-glycine system, the addition of glucose results in the newly formed HLA holding a lower carboxyl content but a higher phenolic-OH content and total acidity [29]. The addition of glucose to catechol results in a substantial enhancement in browning in the presence of birnessite. This can be attributed to the reaction of catechol with glucose dehydration, oxidation, and aromatization derivatives catalyzed by birnessite. Likewise, the addition of glucose to the catechol-glycine system substantially enhances browning in the presence of birnessite [8].
As the concentration of exogenously added glucose is increased, the absorption intensity at 1606~1633 cm−1 is enhanced, while the absorption intensity at 1369~1387 cm−1 is weakened. The following rule was found to be obeyed: combined with the assignment of two peaks, the higher the concentration of added glucose, the greater the proportion of aromatic C structures in the dark-brown residue. The addition of exogenous precursors and the dark-brown residue produced from the abiotic condensation covered the surface of δ-MnO2 to mask the vibrational intensity of the peak at 1101 cm−1, which represents the O-H stretching vibration of δ-MnO2, merely reflected in the CK control. The absorption intensity at 619~669 cm−1 assigned to the Mn-O bond lattice vibration of the δ-MnO2 layered structure becomes stronger after the addition of precursors for abiotic condensation and is enhanced with increasing glucose concentration. The increase in the intensity of the band at 3419~3435 cm−1 suggests that the organic components of the dark-brown residue and δ-MnO2 are bound to each other through intermolecular hydrogen bonding, thus increasing the lattice vibration of the Mn-O bond [30]. Hardie et al. [8] found that glucose can promote the formation of carbonate (MnCO3) at the expense of the aliphatic carboxylic groups in the solid residues of the birnessite-catalyzed catechol–glucose and integrated catechol–Maillard reaction systems. Additionally, the absence of significant amounts of free COOH, probably the decarboxylation of oxidized glucose and/or aliphatic fragments derived from ring cleavage of catechol or the partial complexation of ionized carboxylate groups with Mn(II) originating from the reduction in Mn(IV), can explain the lack of strong adsorption bands in the 1740~1710 cm−1 region [31].

4.3. E4/E6 Ratio and Elemental Composition of the Humic-like Acid Extracted from the Dark-Brown Residue

According to the change in the E4/E6 ratio, it can be judged that the structure of HLA molecules for each treatment become complex at first and then gradually become simpler during the culture period. Eventually, the molecular structure of HLA treated with the addition of the Maillard precursors becomes more complicated; however, the structure of HLA molecules from the CK control tend to be simplified. This is contrary to the conclusion of Jokic. Jokic et al. [6] reported that the presence of glucose perturbs the polycondensation reactions between glycine and catechol, possibly leading to the formation of more aliphatic structures. In addition, he hypothesized that E4/E6 is related to the reaction temperature. Both environmental factors and HS precursors can act as regulating factors that promote HS formation [27]. The E4/E6 ratio in the glucose–glycine–catechol–δ-MnO2 system increases with increasing reaction temperature [6]. The reaction temperature in this experiment was 28 °C, while the reaction temperature in the literature was 45 °C, which might be the direct reason for the smaller E4/E6 ratio obtained in this experiment. Under the test conditions, the higher the concentration of exogenous glucose, the higher the proportion of aromatic C structures in the dark-brown residue.
With increasing glucose concentration, the number of N-containing compounds in HLA molecules decreases, while the number of O-containing functional groups increases, resulting in a decrease in the C/N ratio and an increase in the O/C ratio. There is a positive relationship between O and C aromatics but a negative relationship between O and C aliphatics during the composting of activated sludge-green waste [32]. This is consistent with our conclusion that the aromaticity correlates well with oxidation.

5. Conclusions

With the addition of glucose at different concentrations, after the liquid shake-flask culture period, the molecular structure of the supernatant fluid tends to become simplified. The TOC content in the supernatant fluid from each treatment decreases to varying degrees after the culture. CHLA extracted from the dark-brown residue shows an upward trend during the culture. Among the different treatments, the Glu0.12 treatment shows the largest increase in CHLA. During the whole culture period, the structure of HLA molecules obtained after each treatment first become complex and then gradually become simpler. Finally, the molecular structure of HLA treated with the addition of glucose at different concentrations becomes more complex. Under the coexistence of glycine and catechol, the addition of glucose can improve the condensation degree of HLA molecules based on the abiotic condensation reaction catalyzed by δ-MnO2. Among them, the best effect is the addition of 0.12 mol/L glucose. With increasing glucose concentration, the number of N-containing compounds in HLA decreases, while the number of O-containing functional groups increases. The higher the concentration of glucose added, the higher the proportion of aromatic C structures in the dark-brown residue. The Mn-O bond lattice vibration of the δ-MnO2 layered structure is greatly enhanced after the addition of Maillard precursors because of abiotic condensation and is increased with increasing glucose concentration. The organic molecules obtained from the dark-brown residue and δ-MnO2 are bound to each other through intermolecular hydrogen bonding. The addition of exogenous glucose is more helpful to improving the humus quality compared with the CK control, among which the Gly0.12 treatment shows the best effect.

Author Contributions

N.W. wrote the paper; N.W. and S.W. conceived and designed the experiments; N.W., Q.Z., Y.S. and S.W. analyzed the data; Q.Z., W.L., Z.L. and C.B. performed the experiments and collected the data. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Jilin Province (No. YDZJ202101ZYTS100), Overseas Students Science & Technology Innovation and Entrepreneurship Project of Jilin Province in 2021, and Science & Technology Innovation Development Plan Project of Jilin City (No.20210103074).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effects of changes in the glucose concentration in the Maillard precursors on the E4/E6 ratio of the supernatant fluid. Note: The treatments with different glucose concentrations (0, 0.03, 0.06, 0.12, and 0.24 mol/L) are represented by Gly0, Gly0.03, Gly0.06, Gly0.12, and Gly0.24, respectively. The CK control is only 2 g of δ-MnO2 in phosphate buffer without the participation of glucose, catechol, or glycine. The error bars in the scatter plots represent the standard deviation for each data point. The same applies Figure 2 and Figure 3 below.
Figure 1. Effects of changes in the glucose concentration in the Maillard precursors on the E4/E6 ratio of the supernatant fluid. Note: The treatments with different glucose concentrations (0, 0.03, 0.06, 0.12, and 0.24 mol/L) are represented by Gly0, Gly0.03, Gly0.06, Gly0.12, and Gly0.24, respectively. The CK control is only 2 g of δ-MnO2 in phosphate buffer without the participation of glucose, catechol, or glycine. The error bars in the scatter plots represent the standard deviation for each data point. The same applies Figure 2 and Figure 3 below.
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Figure 2. Effects of change in the glucose concentration in the Maillard precursors on the TOC of the supernatant fluid.
Figure 2. Effects of change in the glucose concentration in the Maillard precursors on the TOC of the supernatant fluid.
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Figure 4. Effects of changes in the glucose concentration in the Maillard precursors on the CHLA/CFLA ratio of the dark-brown residue. Note: Different capital letters indicated significant differences among the different cultural times within the same treatment (p < 0.05); Different lower-case letters indicated significant differences among the different treatments under the same cultural time (p < 0.05).
Figure 4. Effects of changes in the glucose concentration in the Maillard precursors on the CHLA/CFLA ratio of the dark-brown residue. Note: Different capital letters indicated significant differences among the different cultural times within the same treatment (p < 0.05); Different lower-case letters indicated significant differences among the different treatments under the same cultural time (p < 0.05).
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Figure 5. Effects of changes in the glucose concentration in the Maillard precursors on the FTIR spectra measured for the dark-brown residue.
Figure 5. Effects of changes in the glucose concentration in the Maillard precursors on the FTIR spectra measured for the dark-brown residue.
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Figure 6. Effects of changes in the glucose concentration in the Maillard precursors on the E4/E6 ratio of a diluted liquid sample of humic-like acid extracted from the dark-brown residue. Note: The treatments with different glucose concentrations (0, 0.03, 0.06, 0.12, and 0.24 mol/L) are represented by Gly0, Gly0.03, Gly0.06, Gly0.12, and Gly0.24, respectively. The CK control is only 2 g of δ-MnO2 in phosphate buffer without the participation of glucose, catechol, or glycine. The error bars in the scatter plots represent the standard deviation for each data point.
Figure 6. Effects of changes in the glucose concentration in the Maillard precursors on the E4/E6 ratio of a diluted liquid sample of humic-like acid extracted from the dark-brown residue. Note: The treatments with different glucose concentrations (0, 0.03, 0.06, 0.12, and 0.24 mol/L) are represented by Gly0, Gly0.03, Gly0.06, Gly0.12, and Gly0.24, respectively. The CK control is only 2 g of δ-MnO2 in phosphate buffer without the participation of glucose, catechol, or glycine. The error bars in the scatter plots represent the standard deviation for each data point.
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Table
Table 1. FTIR relative intensities (% of total area) for the dark-brown residue under different glucose concentrations in the Maillard precursors.
Table 1. FTIR relative intensities (% of total area) for the dark-brown residue under different glucose concentrations in the Maillard precursors.
Wavenumbers (cm−1)3419~3435292228501606~16331369~13871101563~600
Treatments
Glu076.90013.31.408.3
Glu0.0376.20013.51.508.2
Glu0.0675.60013.61.309.3
Glu0.1275.00014.21.509.0
Glu0.2474.40013.91.109.7
CK control62.20.80.210.60.522.52.4
Table
Table 2. Elemental composition of the humic-like acid extracted from the dark-brown residue under different glucose concentrations in the Maillard precursors. The data in the table are expressed in the following format: mean value ± standard deviation, different lowercase letters following the data indicate significant differences among the different treatments (p < 0.05).
Table 2. Elemental composition of the humic-like acid extracted from the dark-brown residue under different glucose concentrations in the Maillard precursors. The data in the table are expressed in the following format: mean value ± standard deviation, different lowercase letters following the data indicate significant differences among the different treatments (p < 0.05).
TreatmentsH/C RatioC/N RatioO/C Ratio
Glu01.07 ± 0.03 a7.3 ± 0.2 e0.73 ± 0.02 e
Glu0.031.00 ± 0.01 b8.7 ± 0.2 d0.78 ± 0.01 d
Glu0.060.97 ± 0.02 c9.7 ± 0.4 c0.87 ± 0.02 c
Glu0.120.92 ± 0.01 d 13.0 ± 0.4 b0.90 ± 0.03 b
Glu0.240.96 ± 0.02 c 13.9 ± 0.6 a0.98 ± 0.04 a
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Wang, N.; Zhang, Q.; Li, W.; Bai, C.; Song, Y.; Wang, S.; Liu, Z. Effect of Exogenous Glucose at Different Concentrations on the Formation of Dark-Brown Humic-like Substances in the Maillard Reaction Pathway Based on the Abiotic Condensation of Precursors Involving δ-MnO2. Sustainability 2022, 14, 11603. https://doi.org/10.3390/su141811603

AMA Style

Wang N, Zhang Q, Li W, Bai C, Song Y, Wang S, Liu Z. Effect of Exogenous Glucose at Different Concentrations on the Formation of Dark-Brown Humic-like Substances in the Maillard Reaction Pathway Based on the Abiotic Condensation of Precursors Involving δ-MnO2. Sustainability. 2022; 14(18):11603. https://doi.org/10.3390/su141811603

Chicago/Turabian Style

Wang, Nan, Qi Zhang, Wanhong Li, Chengxin Bai, Yan Song, Shuai Wang, and Zhijiang Liu. 2022. "Effect of Exogenous Glucose at Different Concentrations on the Formation of Dark-Brown Humic-like Substances in the Maillard Reaction Pathway Based on the Abiotic Condensation of Precursors Involving δ-MnO2" Sustainability 14, no. 18: 11603. https://doi.org/10.3390/su141811603

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