Release of Dissolved Organic Matter from Sludge Biochar and Its Spectral Characteristics in Different Environmental Media

Abstract

The widespread use of biochar in soil remediation has heightened interest in the role of its derived dissolved organic matter (DOM) in soil nutrient dynamics. However, how pyrolysis temperature shapes the characteristics of DOM released from sludge biochar remains unclear. The study examined variations in the composition and properties of DOM extracted from sludge biochar under two different solutions—ultrapure water (UP) and artificial root exudates (ARE)—across a range of pyrolysis temperatures. Results indicate that the dissolved organic carbon (DOC) content did not differ significantly between extraction environments. In contrast, pyrolysis temperature markedly influenced both the content and composition of DOM. DOM in sludge biochar was primarily composed of humic-like (C1, C2, C3) and tyrosine-like (C4) components. Specifically, DOM from low-temperature biochar was dominated by C2, C3, and C4, whereas high-temperature biochar contained mainly C2 and C4.

1. Introduction

The widespread use of biochar in soil remediation has highlighted the role of its derived dissolved organic matter (DOM) in soil nutrient dynamics. As a key component, DOM is integral to carbon cycling, pollutant transport and transformation, and nutrient bioavailability [1,2]. In recent years, with the advancement of sludge resource recovery, sludge-derived biochar has attracted attention as a soil amendment and carbon sequestration vehicle. Biochar DOM is a critical determinant of environmental behavior, affecting the adsorption and desorption of heavy metals and organic contaminants, while also participating in microbial metabolism and regulating soil fertility [3,4,5].

Most current studies have focused on the effects of biochar preparation conditions, such as pyrolysis temperature and feedstock type, on its adsorption performance and stability. However, the release behavior and compositional evolution of biochar-derived dissolved organic matter (DOM) under different environmental conditions remain poorly understood. Critically, simple extractants like ultrapure water do not represent the complex biochemical conditions of the rhizosphere. In the rhizosphere, root exudates actively interact with soil amendments. This gap is especially relevant in natural soil systems, where active biochemical processes (e.g., root exudation of organic acids and sugars) interact with biochar surfaces, potentially altering DOM solubility and composition [6,7,8]. Furthermore, most research has focused on plant-derived biochar. As a result, the behavior of waste-derived biochar (e.g., sludge biochar) in root-influenced environments remains largely unexplored. Pyrolysis temperature, a key preparation parameter, controls the pore structure, surface functionality, and extent of organic transformation in biochar, thereby shaping the release potential and chemical nature of DOM [9,10,11]. Nevertheless, the response mechanisms of DOM from sludge biochar under root-simulated conditions at different pyrolysis temperatures are still poorly understood. Specifically, the micro-interface processes by which root exudates regulate DOM release, such as through ligand exchange or complexation, are not clear. Furthermore, additional research is needed to clarify the relationship between the evolution of DOM components and their environmental behavior.

To address these knowledge gaps, this study specifically investigates the medium-dependent release dynamics of DOM from sludge-derived biochar. This work hypothesizes that: (1) Artificial root exudates (ARE), compared to ultrapure water, will significantly alter the composition and chemical properties of released DOM by promoting the solubilization of specific components through complexation and ligand exchange. (2) The pyrolysis temperature will critically modulate this medium-dependent effect, with low-temperature biochar exhibiting greater compositional shifts in ARE due to its higher abundance of labile fractions and complexed metals. (3) Sludge biochar will demonstrate distinct DOM release patterns in ARE, characterized by enhanced humification and metal-complexing features, which are not evident in plant-derived biochar or in simple aqueous extracts.

Following these hypotheses, this study selected sludge-derived biochar as the research object. Biochar samples were prepared at two pyrolysis temperatures (300 °C and 700 °C), and DOM was extracted using both ultrapure water and an artificial root exudate (ARE) solution to simulate rhizosphere and non-rhizosphere conditions. The released DOM was characterized using ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy. These techniques were combined with two-dimensional correlation spectroscopy (2D-COS) and parallel factor analysis (PARAFAC), to determine the DOM’s chemical composition and structural features across different environmental media. This study aims to clarify the mechanisms by which pyrolysis temperature and environmental factors influence the properties of DOM in sludge biochar, with a specific focus on the medium-dependent release behavior and interface processes mediated by root exudates. The findings aim to provide a theoretical and empirical basis for assessing its practical application and potential ecological risks in real-world scenarios.

2. Materials and Methods

2.1. Sludge Source and Biochar Preparation

The sludge used in this study was obtained from a wastewater treatment plant in Zhongshan, China. After collection, the sludge was washed, air-dried at room temperature, and stored. A portion of the dried sludge was ground mechanically and sieved through a 100-mesh sieve to ensure homogeneity. Pyrolysis was performed in a muffle furnace under a nitrogen atmosphere at 300 °C (Low-temperature pyrolysis) and 700 °C (High-temperature pyrolysis). Once the target temperature was reached, it was held for 2 h before allowing the furnace to cool to room temperature. The resulting biochar was stored in brown glass bottles for subsequent use. For comparative purposes, the original, non-pyrolyzed raw sludge (designated as WB0) was included as a reference material.

2.2. Preparation of Extraction Solutions

As the content and composition of dissolved organic matter (DOM) released from biochar can vary depending on the environmental conditions, this study simulated DOM release under two scenarios: soil pore water and the soil root zone, represented by ultrapure water (UP) and an artificial root exudate (ARE) solution, respectively. The ARE solution was prepared to contain (in g/L): glucose (3.31 g/L), fructose (3.31 g/L), sucrose (3.15 g/L), citric acid (1.77 g/L), lactic acid (1.66 g/L), succinic acid (1.63 g/L), alanine (0.74 g/L), serine (0.97 g/L), and glutamic acid (0.81 g/L). The prepared ARE solution had a total organic carbon (TOC) concentration of 6 mg/L and a pH of 4.4. The used analytical grade chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China without any purification.

2.3. DOM Extraction Experiment

DOM was extracted from biochar using a method adapted from Li et al. [1]. Briefly, biochar was mixed with the extraction solution at a solid-to-liquid ratio of 1:20 (w/v) and reacted for 2 h. Specifically, 1.5 g of sludge biochar was added to a 40 mL brown glass bottle containing 30 mL of extraction solution. The mixture was shaken in the dark at 150 rpm for 2 h and then centrifuged at 3000 rpm for 10 min. The supernatant was collected and filtered through a 0.45 μm membrane filter (Millipore, Billerica, MA, USA) to obtain the DOM extract. All extracts were stored in 30 mL brown bottles at 4 °C prior to analysis. All experiments were conducted in quintuplicate.

2.4. Analytical Method

The dissolved organic carbon (DOC) content of the extracts, representing the concentration of released DOM, was measured using a total organic carbon analyzer (TOC-L CPN, Shimadzu, Kyoto, Japan). UV-visible absorption spectra of the DOM samples were recorded using a UV-visible spectrophotometer. Two-dimensional correlation spectroscopy (2D-COS) enhances spectral resolution by extending spectral variables into a second dimension. The signs and presence of cross-peaks in synchronous and asynchronous spectra facilitate the identification of functional groups. In this study, 2D-COS analysis was performed on the FTIR spectra of DOM, with pyrolysis temperature as the external perturbation. Calculations were performed using 2D-shige software Version 1.3 (Waseda University, Tokyo, Japan) [12]. Fluorescence measurements were conducted using a fluorescence spectrophotometer (RF5301PC, Shimadzu, Japan). Excitation-emission matrix (EEM) spectra were collected by scanning emission wavelengths from 250 nm to 600 nm at 1 nm intervals, with excitation wavelengths ranging from 220 nm to 400 nm at 5 nm increments. To minimize inner-filter effects, all samples were adjusted to a standard DOC concentration of 1 mg/L prior to EEM analysis [13]. Parallel factor (PARAFAC) analysis was performed in MATLAB R2010b using the DOM Fluor toolbox [14]. The number of components was validated through residual analysis, split-half validation, and core consistency diagnostics. The model was run under non-negativity constraints, and no outliers were detected. Several optical indices were derived: the specific UV absorbance at 254 nm (SUVA254) was calculated as the ratio of the absorbance at 254 nm (a254) to the DOC concentration; the spectral slope ratio (SR) was determined by dividing the spectral slope in the 275–295 nm range by that in the 350–400 nm range; the humification index (HIX), fluorescence index (FI), and biological index (BIX) were calculated from the EEM data. The relative concentration of each PARAFAC component was assessed using its maximum fluorescence intensity (Fmax) [15].

2.5. Statistical Analysis

Statistical analysis was performed using SPSS software Version 25.0, with results presented as means and standard deviations. Data were analyzed by one-way ANOVA. Significant differences were further examined using the LSD post hoc test. Differences were considered statistically significant at p < 0.05. In terms of the significance of differences for the data, letters and asterisks were used for representation. Specifically, letters were used for the significant differences in the same extract liquid but at different pyrolysis temperatures, while asterisk was used for the significant differences between different extract liquids at the same temperature.

3. Results and Discussion

3.1. Release and Characteristic Indices of DOM in Sludge Biochar at Different Pyrolysis Temperatures

Figure 1 presents the amounts of DOM released from sludge biochar produced at different pyrolysis temperatures under two simulated environments: soil pore water and the plant root zone. The results indicate that the dissolved organic carbon (DOC) content extracted from the sludge-derived biochar did not differ significantly (p < 0.05) when using artificial root exudates (ARE) compared to ultrapure water (UP) as the extraction solution. This result diverges from the findings of Li et al. [11], which reported a significant difference (p < 0.05) in DOC released from rice straw biochar extracted with 0.1 mol/L HCl versus UP water. In the present study, however, a difference in DOC content was observed between extractions with HCl and ARE at the same pH, a result potentially attributable to the organic constituents in the ARE solution. This phenomenon may be explained by the complexation of metal cations with functional groups on organic molecules. These cation bridges can then form bridges between biochar and DOM [16,17,18]. In contrast, low-molecular-weight organic acids (e.g., citric acid) present in root exudates can bind to these metal cations, dissolving them and thereby disrupting the bridges between biochar particles and surface-bound DOM [19]. This process promotes the release of DOM into solution [20,21]. Consistently, Sun et al. also observed that such organic acids significantly increase the concentration of metal cations in soil solution [22]. Furthermore, Figure 1 illustrates a pronounced effect of pyrolysis temperature on the DOC released from sludge biochar. Biochar produced at 300 °C exhibited the highest DOC release in both extraction media, while DOC from the 700 °C biochar decreased significantly. This indicates that pyrolysis temperature substantially influences the DOM content of sludge-derived biochar. Specifically, biochar produced at lower pyrolysis temperatures releases a higher amount of DOC, whereas biochar generated at higher temperatures releases less. This trend can be attributed to the progressive reduction in volatile matter and the corresponding increase in fixed carbon content as pyrolysis temperature rises. These changes consequently lead to a decline in DOC release [23,24]. This is mainly due to the key role of pyrolysis temperature. Pyrolysis temperature controls two important processes of thermal cracking and condensation reactions. These processes then determine the physicochemical structure of the biochar. At lower temperatures (e.g., 300 °C), pyrolysis is incomplete. The biochar matrix keeps many aliphatic structures and labile organic matter. Therefore, the DOM released from this biochar usually has certain characteristics. It has a larger molecular weight. It also has lower aromaticity and a higher degree of humification. In contrast, high-temperature pyrolysis (e.g., 700 °C) works differently. It promotes deep condensation of aromatic clusters. It also causes volatilization of aliphatic components. This results in a biochar that is more aromatized and stable. However, the DOM released from such high-temperature biochar is different. It is mainly made of small-molecular-weight organics with low aromaticity. There are two reasons for this. First, potential larger humic substances may be decomposed during the high-temperature pyrolysis. Second, they may become tightly adsorbed within the biochar’s well-developed pore structure. This prevents their release into the solution. Additionally, with increasing pyrolysis temperature, DOM may undergo secondary reactions, such as decomposition, condensation, cyclization, and polymerization [25,26].

Figure 2 presents three characteristic indices of DOM: the humification index (HIX), biological index (BIX), and fluorescence index (FI). HIX reflects the degree of DOM humification; a higher HIX value indicates greater humification and a more complex DOM structure [27,28,29]. In both extraction solutions, DOM from the 700 °C biochar exhibited relatively low HIX values (0.71–0.81). In contrast, DOM extracted from the 300 °C biochar showed higher HIX values (2.22–2.29). This suggests that low-temperature pyrolysis released a greater proportion of humic-like substances [30]. Furthermore, HIX values were higher in the artificial root exudate (ARE) than in ultrapure water (UP), indicating a greater degree of humification in ARE-extracted DOM, a finding consistent with the SUVA₂₅₄ results. The FI values of DOM extracted with ARE and UP ranged from 2.12–3.00 and 2.09–2.92, respectively. These values increased gradually with pyrolysis temperature, implying enhanced degradation of DOM at higher temperatures [31]. Additionally, in both extraction solutions, BIX values for DOM derived from the raw (unpyrolyzed) sludge were below 1, indicating insignificant autochthonous characteristics [32]. However, at pyrolysis temperatures of 300 °C and 700 °C, the BIX values exceeded 1, reflecting higher bioavailability compared to the raw sludge. This suggests that high-temperature pyrolysis likely decomposed larger-molecular-weight DOM components in the sludge biochar into smaller, more bioavailable fractions, a trend consistent with the observed changes in the spectral slope ratio (SR) [33].

3.2. Fluorescence Characteristics of DOM in Sludge Biochar at Different Pyrolysis Temperatures

3.2.1. Released DOM Components Analysis of Biochar

Fluorescence excitation-emission matrix (EEM) combined with parallel factor analysis (PARAFAC) was applied to characterize the DOM components released from biochar under different pyrolysis temperatures and in two extraction environments. The identified PARAFAC components are consistent with those identified in previous DOM fluorescence studies, which were matched to reported components based on spectral features. PARAFAC analysis of all sample EEMs revealed four fluorescent components (C1–C4). The probable sources and properties of these components are summarized in

Table 1. Three-dimensional fluorescence spectral positions, contour distribution and spectral load information of the five components identified through fluorescence-PARAFAC analysis.

Component	EEM Wavelength (nm)	EEM Equipotential Surface	Spectrum Characteristic
C1	Ex: 235/275 Em: 416
C2	Ex: 225/320 Em: 390
C3	Ex: 225/365 Em: 438
C4	Ex: 225/280 Em: 304

. The resolved components consist of three humic-like substances (C1, C2, C3) and one protein-like (tyrosine) substance (C4). Component C1, which has emission peaks at 235/275 nm and excitation at 416 nm, is identified as a humic acid-like material. It shows spectral features typical of terrestrial humic substances and resembles humic-like components extracted from marine sediments [34]. Component C2, with peaks at 225/320 nm and emission at 390 nm, is characteristic of a humic-like substance representing stable, aliphatic-rich biogenic organic matter. Component C3, with excitation at 225/365 nm and a distinct emission maximum at 438 nm, corresponds to a UVA humic substance—a widely distributed fluorescent group of yellow humic acid. Component C4, exhibiting excitation maxima at 225/280 nm and emission at 304 nm, is assigned to a tyrosine-like compound [35]. The observed variations in DOM composition and release across pyrolysis temperatures can be explained at the molecular level. DOM released from 300 °C biochar is characterized by larger molecular weight and greater hydrophobicity, which leads to weaker adsorption onto the biochar surface and, consequently, higher release into solution. In contrast, DOM derived from 700 °C biochar consists of smaller molecules. These smaller molecules can be more strongly retained by the highly aromatic biochar matrix, likely through mechanisms such as pore-filling, resulting in reduced release.

3.2.2. Distribution of DOM Components in Biochar at Different Pyrolysis Temperatures

To evaluate the influence of pyrolysis temperature on the release of DOM from biochar, the distribution of fluorescent components was assessed based on fluorescence intensity. Figure 3 shows the relative abundance of DOM components released from sludge-derived biochar. The biochar was prepared at different pyrolysis temperatures and extracted in two environments (ARE and UP). Overall, the distribution of the four DOM components varied considerably with pyrolysis temperature, while only minor differences were observed between the two-extraction media—a trend consistent with the DOC results presented in Figure 1. In the raw (unpyrolyzed) sludge, two components (C1 and C4) were identified in both ARE and UP extracts. Their relative abundances were C1: 40.79% (ARE) and 47.25% (UP), and C4: 59.21% (ARE) and 52.75% (UP). C4 was the dominant component in ARE, whereas the difference was less pronounced in UP. As the pyrolysis temperature increased to 300 °C and 700 °C, the DOM composition changed markedly. At 300 °C, component C1 was absent in both extracts, component C4 decreased, and new components C2 and C3 emerged as the dominant fractions. At 700 °C, component C3 disappeared, leaving only C2 and C4 in both extracts, with C4 remaining the major constituent. This pattern may be attributed to secondary reactions such as decomposition, condensation, cyclization, and polymerization that DOM can undergo as pyrolysis temperature increases [25,36]. In summary, pyrolysis temperature significantly altered the distribution of DOM components in biochar, with low-temperature pyrolysis (300 °C) promoting the substantial release of humic acid-like DOM.

3.3. FTIR Spectroscopy and 2D-COS Analysis

Figure 4 shows the infrared spectrum of DOM, with notable changes observed mainly in the 3600–2800 cm⁻¹ and 1800–800 cm⁻¹ regions. Compared to the raw sludge, the sludge biochar attenuated -OH vibration under high-temperature pyrolysis. This is likely attributed to the dehydration of cellulose, hemicellulose, and lignin during heating. The aliphatic C-H absorption peaks in the 2850–2950 cm⁻¹ range progressively diminished and eventually disappeared as pyrolysis temperature increased. This is likely due to the decomposition of organic aliphatic hydrocarbons in the biochar at elevated temperatures, or their conversion into condensed aromatic structures. Additionally, the absorption peak for the aromatic C=C skeletal vibration intensified with rising temperature but weakened at 700 °C. This suggests that pyrolysis initially promotes aromatization before high-temperature decomposition of larger molecules reduces aromaticity. This observation aligns with the SUVA₂₅₄ trends. Meanwhile, the C=O vibrations from carbonyl/carboxyl groups gradually weakened, indicating carbonate decomposition. The peak near 1260 cm⁻¹, corresponding to a C-O/C-C stretch, also decreased in intensity at higher temperatures, likely a result of the thermal decomposition of carbon-containing compounds. In summary, the intensities of aliphatic -CH, C-O, and C=O groups in DOM from sludge biochar pyrolyzed at 300 °C and 700 °C decreased with increasing temperature, which can be attributed to the breakdown of macromolecular substances into smaller units under high-temperature conditions. No clear differences in functional groups were evident between DOM extracted with ARE and UP, possibly due to the resolution of the infrared analysis.

To elucidate the evolution of DOM functional groups as a function of pyrolysis temperature, the FTIR spectral data were analyzed using two-dimensional correlation spectroscopy (2D-COS). The synchronous and asynchronous maps in the 1800–800 cm⁻¹ region are presented in Figure 5a. In the ARE extracts, three main autopeaks were observed at approximately 1600, 1400, and 1150 cm⁻¹, ordered by intensity. Based on Noda’s rule, the sequence of spectral changes was determined as follows: C-O of polysaccharides > C-N/C=N > aromatic ring C=C > aromatic C-H > ester C=O > aliphatic C-H > carboxyl C=O. These results indicate that the molecular structure of DOM undergoes distinct changes as pyrolysis temperature increases. The autopeaks of DOM are concentrated in the 3600–3000 cm⁻¹ region, corresponding to O-H groups. In the synchronous map, the positive cross-peaks for both ARE and UP indicate that the O-H groups respond to temperature changes in a similar manner. In the asynchronous map, the cross-peak sign is negative for CB but positive for SB and DB. This pattern reveals that changes in CH₂ precede those of O-H in CB and SB, whereas the sequence is reversed in DB.

3.4. Ultraviolet-Visible Spectroscopy Analysis of DOM in Sludge Biochar

Furthermore, the chemical properties of DOM in sludge biochar were examined using the SUVA₂₅₄ index and spectral slope ratio (SR) analysis (Figure 6). In both extraction environments, the SUVA₂₅₄ values of DOM ranged from 0.29 to 2.84 L·mg C⁻¹·m⁻¹, indicating overall low aromaticity. For the raw sludge, the SUVA₂₅₄ of DOM extracted with ARE was significantly lower than that with UP (p < 0.05). As the pyrolysis temperature increased, however, the SUVA₂₅₄ of the ARE extract became significantly higher than that of the UP extract (p < 0.05). This suggests that DOM in ARE attained relatively higher aromaticity under elevated temperatures. The SR value, an indicator of DOM molecular weight (higher SR corresponding to larger molecular size) [1,37], varied as follows: for raw sludge, it was 0.99 (ARE) and 2.08 (UP); for 300 °C biochar, 0.23 (ARE) and 0.73 (UP); and for 700 °C biochar, 1.25 (ARE) and 1.20 (UP). The higher SUVA₂₅₄ combined with lower SR observed for DOM released from 300 °C biochar indicates that DOM at this temperature possesses a higher molecular weight and a greater degree of humification compared to both raw sludge and 700 °C biochar. According to previous studies, smaller, aliphatic DOM molecules are more strongly adsorbed onto biochar surfaces than larger aromatic molecules [25,38]. Therefore, the more aliphatic, higher molecular weight DOM in 300 °C biochar is more readily released. This aligns with the relatively higher DOC content detected at this temperature. As pyrolysis temperature rises, large aromatic components in biochar gradually decompose into smaller fragments. This leads to reduced aromaticity and molecular weight of DOM [31,39]. Thus, pyrolysis temperature influences both the size and humification degree of DOM molecules, thereby modulating DOM release from the biochar matrix.

4. Conclusions

This study systematically investigated sludge biochar under two extraction environments—ultrapure water (UP) and artificial root exudates (ARE)—and at different pyrolysis temperatures (300 °C and 700 °C). The release behavior, chemical composition, and structural characteristics of DOM across different environmental media were examined, leading to the following main conclusions. The DOC content released from the sludge biochar did not differ significantly between the two extraction environments. This suggests that in the root-simulating (acidic) environment, DOM release from biochar is not inhibited by the lower pH. This can be attributed to the presence of organic acids, which enhance DOM solubility. Pyrolysis temperature significantly influences the content and composition of dissolved organic matter (DOM) in sludge-derived biochar. Biochar produced at lower temperatures contains a greater diversity of DOM components and exhibits a higher DOM release potential. This is likely due to the larger molecular size and higher degree of humification of low-temperature DOM, which reduces its adsorption affinity to the biochar matrix and facilitates desorption. In contrast, at higher pyrolysis temperatures, DOM undergoes thermal decomposition, producing smaller molecular fragments that adsorb more strongly to biochar surfaces, thereby limiting its overall release. The DOM released from sludge-derived biochar comprised four main components. At lower pyrolysis temperatures, the composition was dominated by components C2, C3, and C4. As the temperature increased, the composition shifted towards a predominance of C2 and C4. The ecological effects of sludge-derived biochar are strongly influenced by pyrolysis temperature. Low-temperature biochar releases diverse dissolved organic matter (DOM), which can stimulate soil microbial activity and enhance short-term nutrient cycling. Due to its stable structure and strong adsorption capacity, high-temperature biochar releases less DOM but is more effective for long-term carbon sequestration and contaminant immobilization. Simulated root exudates did not inhibit DOM release, suggesting that in real root-soil systems, plant activity may interact with biochar DOM, influencing biochar aging and the fate of nutrients and pollutants. In terms of practical applications, low-temperature biochar can serve as a soil amendment or biostimulant to quickly improve fertility and support microbial degradation of organic pollutants. High-temperature biochar is more suitable for long-term stabilization of heavy metals and organic contaminants, soil carbon storage, or as an adsorbent material. It should be noted that DOM release may also mobilize adsorbed pollutants, so biochar produced from contaminated sludge requires careful risk assessment. Matching biochar type to specific goals and conducting systematic risk evaluations are essential to maximize benefits and control potential risks.