Spectroscopic and molecular insights into humic substances in compost amended with varying biochar levels

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The addition of BC significantly increased the HA content by 13-16% while reducing the FA content by 12-32%, with the most pronounced effects observed at a 5% BC addition. Spectroscopic analyses, including UV-Vis and fluorescence, revealed enhanced aromaticity and molecular weight (MW) of HA, driven by microbial activity and polymerization facilitated by BC amendments, whereas FA aromaticity remained largely unchanged. The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of HA increased significantly, indicating improved humification and stabilization. Fourier-transform infrared (FTIR) spectroscopy showed that BC addition had minimal impact on the functional group composition of HA and FA but altered the proportions of carbon types. Electron paramagnetic resonance (EPR) analysis demonstrated higher persistent free radical (PFR) levels in HA, correlating with increased molecular size and a higher degree of condensation. These findings underscore the potential of BC amendments to enhance compost quality by promoting the formation of stable and complex humic substances with improved reactivity. compost biochar amendments humic substances spectroscopic properties molecular structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The rapid development of the livestock and poultry industry has resulted in an excessive accumulation of manure that surpasses current disposal capacities, becoming a pressing environmental challenge (Guo et al., 2018; He et al., 2019; Huang et al., 2021). Composting stands out as a sustainable and cost-effective strategy for managing this waste, offering a non-polluting alternative to incineration and landfilling (Chen et al., 2017a; Kujawa et al., 2020). This process is predominantly driven by microorganisms, which not only eliminate pathogens through the thermophilic phase but also convert solid organic waste into organic fertilizers, allowing for biological, economical, and sustainable reuse (Altieri et al., 2012; Li et al., 2022). Composting yields a significant by-product in the form of humic substances (HS), which are the result of the humification process. During humification, unstable organic matter is transformed and stabilized into humic acids (HA) and fulvic acids (FA), the primary components of humic substances (Gong et al., 2023). These substances vary in molecular weight, solubility, and degree of polymerization (Ming et al., 2023; Shan et al., 2018). HS is known to enhance soil health through nutrient retention, promoting plant growth, and mitigating heavy metal pollution (Fang et al., 2016; Lee et al., 2020). Previous studies have suggested that the addition of specific additives, such as sawdust, corn stalks, and biochar (BC), can enhance HS formation by stimulating microbial activity and functionality during composting (Chen et al., 2023b; Guo et al., 2019a). Among these additives, BC has gained significant attention as an effective amendment in composting due to its unique properties, such as large surface area, high absorption capacity, cation exchange capacity, and substantial pore volume (Li et al., 2023; Lv et al., 2023). These properties allow BC to improve composting performance by enhancing aeration and promoting microbial proliferation (Li et al., 2023; Ming et al., 2023). The incorporation of BC has been shown to substantially increase the HS content in compost (Altieri et al., 2012; Chen et al., 2023b). For example, Liu et al. (2023) reported that adding 6% maize straw BC to pig manure compost enhanced of HA and FA formation efficiencies by 39.4% and 17.4%, respectively, compared to compost without BC (Liu et al., 2023). Jia et al. reported a 16.5% increase in HA content with 10% corn straw BC additions, although FA content decreased by 18.8% compared to compost without BC (Jia et al., 2023). Similarly, Zhou et al. demonstrated that the compost amended with 5-20% BC exhibited a 1.57-2.29 times increase in HA content, while the FA content was 27.2%-37.4% lower in mature compost with BC compared to without BC (Zhou et al., 2023). While current studies highlight changes in the quantity of HS in compost with varying BC concentrations, there is limited understanding of how BC affects the chemical characteristics of HS. HS are chemically complex and highly heterogeneous, consisting mainly of aromatic, phenolic, and quinone components (Chen et al., 2023b; Zhou et al., 2022). To address this knowledge gap, various spectroscopic techniques such as ultraviolet/visible absorption (UV-vis), fluorescence, and Fourier transform infrared (FTIR) spectroscopy, are invaluable for characterizing the structural and compositional properties of HS (Che et al., 2021; Cui et al., 2020). Additionally, fluorescence excitation-emission matrix (EEM) spectra, combined with fluorescence regional integration (FRI) analysis, allow for detailed semi-quantification of fluorophores within HS (Huang et al., 2021; Lee et al., 2020). Furthermore, high-performance size exclusion chromatography (HPSEC) has emerged as a powerful tool for characterizing the molecular weight (MW) distributions of compost-derived dissolved organic matter (DOM) (Guo et al., 2018; He et al., 2019). Recently, persistent free radicals (PFRs) produced by DOM and HS, which play crucial roles in electron transfer and redox reactions in soils and natural environments, have garnered increasing attention (He et al., 2019; Li et al., 2024; Zhang et al., 2020). These radicals can be directly detected by electron paramagnetic resonance (EPR) spectroscopy (Wu et al., 2022; Zhang et al., 2024). In this study, HA and FA derived from chicken manure compost with rice husk BC amendments at concentrations of 0%, 1%, 2%, and 5% were comparatively analyzed using dissolved organic carbon (DOC), UV-vis, EEM-FRI, FTIR, HPSEC, and EPR protocols. The main objective was to investigate the impact of BC concentration on the structure and properties of HS within compost. By understanding the compositional and structural evolution of HS in response to varying BC levels, this study aims to address a critical knowledge gap in optimizing composting processes. The insights gained from this study could contribute to enhancing the functionality and quality of the final compost product, potentially leading to improved soil health and sustainability in agricultural practices. 2. Materials and Methods 2.1. Compost sampling The aerobic composting was conducted in a designated facility, located in an artificial greenhouse at an organic fertilizer production facility in Fuyang, Anhui Province, China. The primary raw materials used in the composting process were chicken manure and rice husks, sourced from a local breeding company. The rice husk BC was purchased from Henan Lize Environmental Protection Technology Co.Ltd. (Henan, China). The initial C/N ratio of the compost mixture was adjusted to 25:1, utilizing a mixture of 20 tons of chicken manure and rice husks in an 8:1 ratio. The moisture content of the compost was maintained at approximately 60%. The initial compost mixture was divided into four separate piles, containing approximately 5 tons of compost material. The experimental treatments involved the addition of different dosages of BC: 0% BC for the control (CK), 1% BC (T1), 2% BC (T2), and 5% BC (T5). Each pile was thoroughly mixed to ensure an even distribution of the BC throughout the compost. The composting piles were turned every seven days to maintain proper aeration and to facilitate microbial activity. After 80 days of composting, approximately 2 kg of mature compost was sampled from each treatment pile. These samples were freeze-dried, finely ground, and passed through a 100-mesh sieve. The four types of samples (CK, T1, T2, and T5) were then stored for further analysis. The physicochemical properties of each type of compost are listed in Table S1 in the supporting information (SI). 2.2. Extraction and purification of HA and FA Extraction and purification of HA and FA were adapted from the methodologies recommended by the International Humic Substances Society (IHSS) with minor modifications (Fang et al., 2016; He et al., 2019; Mealio et al., 2024). HA and FA were extracted using a 1:1 mixture of aqueous 0.1 M NaOH and 0.1 M Na 2 P 4 O 7 . The extraction solvent was added to the solid compost at a liquid-to-sample ratio of 10:1 (v/w, dry weight basis). The mixture was mechanically shaken under a nitrogen environment for 24 h at 25°C, followed by centrifugation at 8000 rpm to separate the supernatant. The supernatant was then acidified to a pH 1 using 6 M HCl and centrifuged at 8,000 rpm to separate dissolved FA from the precipitated HA. The raw HA was acidified to pH of 1 with 6 M HCl, centrifuged, and washed with ultrapure water to yield pure HA. The raw FA was passed through an XAD-8 resin column, and the adsorbed fraction was eluted with 0.1 M NaOH. It was then purified using a hydrogen-type cation exchange resin column to eliminate cations and obtain pure FA (Huang et al., 2021). Finally, both HA and FA were lyophilized to obtain solid samples, which were then stored at -20 o C until further analysis. 2.3. Dissolved organic carbon analysis For analysis, a portion of solid HA and FA were re-dissolved in ultrapure water. The concentration of dissolved organic carbon (DOC) in HA and FA was measured using a Shimadzu TOC analyzer (TOC-VCPH, Japan) following the non-purgeable organic carbon protocol. The HA and FA solutions were then diluted to 10 mg/L for further spectroscopic analysis. 2.4. Ultraviolet-Visible spectra The UV-vis spectra of HA and FA were recorded using a UV-2600 spectrophotometer (Shimadzu, Japan) with wavelengths ranging from 200 to 700 nm (Castan et al., 2020; Chen et al., 2017b). To characterize the HA and FA, three optical parameters were evaluated: specific UV absorbance at 254 nm (SUVA 254 ), the absorbance ratio between 254 and 365 nm (E 2 /E 3 ), the spectra slope ratios (S R ). Detailed calculation for these parameter are described in Text S1. 2.5. Fluorescence spectroscopy The EEM fluorescence spectra of compost HA and FA were acquired using a fluorescence spectrophotometer (F-4600, Hitachi Japan). The excitation (Ex) wavelength was scanned from 200 to 400 nm, and the corresponding emission (Em) wavelength range extended from 290 to 520 nm. Increments of 5 nm were applied for both Ex and Em wavelengths, and the scanning speed was set at 12,000 nm·min -1 . Background correction was performed using ultrapure water as a blank for all EEM spectra of compost HS samples. for all EEM spectra of compost HS samples. To evaluate compost characteristics, the humification index (HIX), fluorescence index (FI), and biological index (BIX) were calculated, with the specific formulas provided in Text S1. The fluorescence regional integration (FRI) technique was employed on the EEM spectra to characterize the fluorescence components of compost HS. According to previous studies (Mealio et al., 2024; Ren et al., 2020), the EEM spectra were divided into five distinct regions (I-V). These regions could be assigned to simple aromatic proteins (I and II), fulvic acid-like substances (III), soluble microbial byproduct-like materials (IV), and humic acid-like organics (V), respectively(Wei et al., 2014; Zhang et al., 2016). The specific Ex/Em wavelength ranges for each region and their corresponding fluorophore assignments are detailed in Table S2. 2.6. High-performance size exclusion chromatogram analysis For HPSEC analysis, an aqueous gel filtration column (Polysep-GFC-P 3000, Phenomenex) and a guard column (Polysep-GFC-P, Phenomenex) were used. A water/methanol (9:1,v/v) With 25 mM ammonium acetate was used as the mobile phase; the sample injection volume was 100 μL with a flow rate of 1mL min -1. and the detection wavelength was 254 nm. The column was calibrated using a series of polyethylene glycol (PEG) standards (Sigma Aldrich, USA) with molecular weights of 238, 601, 1020, 3450, 4080, 11,100, 17,900, and 41,300 Da (Chen et al., 2023a). The void volumes and exclusion limit were verified using polystyrene sulfonate (MW 210 kDa) and acetone (MW 58 Da), respectively. The calibration curves, depicting the logarithmic molecular weight against retention time, were illustrated in Fig. S1. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (ρ) of HA and FA were calculated using the following formulas (Abid et al., 2020; He et al., 2019): where hi and MWi represent the chromatogram height and the apparent MW of HA and FA samples corresponding to the i th retention time, respectively. It is noted that the Mw and Mn of compost-derived HA and FA were estimated using HPSEC, based on PEG calibration standards applied in this study. 2.7. Fourier-transform infrared (FTIR) spectroscopy The FTIR spectra measurement followed the methods introduced in previous studies (Wang et al., 2021; Xiao et al., 2019). A finely ground 1 mg sample was mixed with 200 mg KBr and compressed under vacuum for 10 min. The resultant solid mixture was thoroughly ground before being compressed into pellets. The FTIR spectrum of the KBr pellet was recorded using an FTIR spectrophotometer (Thermo, USA). The spectra were scanned over a wavenumber range of 4000-400 cm -1 with 4 cm -1 resolution. Each spectrum was generated from a total of 64 scans. A pure KBr pellet was referenced to avoid instrument background signals through subtraction (Dong-Hai et al., 2018; Yu et al., 2019b). 2.8. Electron paramagnetic resonance (EPR) spectroscopy PFRs of HA and FA were determined using EPR spectroscopy (Magnettech ESR5000, Bruker, Ettlingen, Germany). Approximately 5 mg of HA or FA powder was packed into a quartz tube, which was then placed in the EPR instrument for analysis. The EPR detection parameters were set as follows: microwave power at 5 mW, microwave frequency at 9.46 GHz, modulation frequency at 100 kHz, and modulation amplitude at 1 G. The scan range was from 3320 G to 3420 G, with a center field of 3370 G. The scan time was set to 30 seconds, and the resolution along the X-axis was 1024 points. 2.9. Statistical analysis Each analysis was conducted in triplicate, and the resulting mean value was adopted, accompanied by the calculation of respective standard deviations. Data were statistically analyzed using Microsoft Excel to determine significant differences among sample data, followed by further analysis using IBM SPSS Statistics 22.0 software. And the figures were plotted using Origin 2021 (Origin-Lab, USA). 3. Results and Discussion 3.1. Effects of BC addition on HA and FA content The DOC content of HA and FA derived from compost amended with different amounts of BC is shown in Fig. 1. The DOC content of HA in compost with BC additions (T1-T5) ranged from 1.2 to 1.68 mg/g (Fig. 1), representing an increase of 13%-16% compared to CK treatment. This suggests that BC additions promote HA formation, a phenomenon evidenced by many previous studies (He et al., 2019; Jia et al., 2023; Zhang et al., 2022). Moreover, HA content was generally increased with increase of BC addition, with the highest increase observed in T5 treatment (Fig. 1). This suggests that the BC levels might be positively correlated with HA formation. In contrast, the DOC content in FA decreased with increasing BC additions, following the trend of CK (0.92 mg/g) > T1 (0.81 mg/g) > T2 (0.72 mg/g) > T5 (0.63 mg/g). The reduction in TOC of FA for T1-T5 treatment ranged from 12% to 32% compared to CK treatment. This finding indicates that BC additions promote the degradation of FA during composting. The simultaneous HA formation and FA degradation suggest that BC amendment facilitates the conversion of FA to HA. This may be due to enhanced microbial activity, which is stimulated by the presence of BC (Liu et al., 2023; Zhou et al., 2023). These findings are consistent with results reported in previous studies (Liu et al., 2023), which also observed that BC encourages the formation and stabilization of HS (Abbott et al., 2018; Yu et al., 2020). The T5 treatment, with the highest BC addition of 5%, showed the most significant increase in HA content and the greatest decrease in FA content, indicating that the compost with 5% BC became more accessible to microorganisms, leading to the production of more stable humic substances. 3.2. UV-vis spectra of HA and FA UV-vis spectra analysis provides valuable insights into the molecular structure of organic substances. The normalized UV-vis spectra of the HA and FA based on their DOC content are shown in Fig. S2. In general, the absorbance of HA and FA decreased with increasing wavelength, which is a typical characteristic feature of dissolved organic matter (DOM) in compost (Fang et al., 2016; Ren et al., 2020). An absorption platform was observed between 270 and 280 nm, typically associated with lignin and aromatic compounds (Bhat et al., 2017; Guo et al., 2019b). The absorption of HA was significantly higher than that of FA, especially between 200 to 300 nm, indicating the higher abundance of aromatic species within HA. This result indicated that BC improved the degree of humification of compost and promoted the conversion of highly polymeric compounds (Zhou et al., 2023). To gain more insight into the compositional and structural characteristics of HA and FA, spectra parameters such as SUVA 254 , E 2 /E 3 , and S R were investigated (Table 1). SUVA 254 is known to be positively correlated with the aromaticity of OM (Ben Mahmoud et al., 2023). As shown in Table 1, the SUVA 254 for CK HA was 8.4, which was lower than the range of 9.6-10.3 observed for HA from T1-T5 treatments. This suggests a greater degree of aromaticity in HA from T1-T5 compared to CK, implying that BC additions can enhance the aromatization of HA. These results are consistent with previous findings that demonstrated BC amendments facilitate the formation of phenolic, aromatic carboxylic, and polycyclic aromatic groups in compost OM (Guo et al., 2018; Zhou et al., 2024). This implies that composting with BC amendment can improve the stability and humification degree of HA (Ben Mahmoud et al., 2023; Long et al., 2023). Additionally, E 2 /E 3 and S R are generally negatively related to the molecular weight of OM (Guo et al., 2019a). As listed in Table 1, the E 2 /E 3 values for HA from CK were 5.2, similar to those from T1-T5 (5.0-5.4). However, the SR value for CK was 0.71, which was higher than those for T1-T5 (0.51-0.53). This finding indicates that BC addition increases the molecular weight of HA, likely due to enhanced polymerization and the formation of more complex aromatic structures (Yin et al., 2023). On the other hand, it is likely that the abundant carbon functional groups in BC enhance intermolecular interactions, thereby facilitating the formation of more stable HS (Zhou et al., 2023). Table 1. Spectral parameters of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments SUVA 254 E 2 /E 3 S R HA CK 8.4±0.02d 5.2±0.01a 0.71±0.01a T1 10.2±0.01b 5.0±0.08a 0.51±0.02b T2 9.6±0.02c 5.1±0.05a 0.53±0.01b T5 10.3±0.02a 5.0±0.07a 0.52±0.01b FA CK T1 T2 T5 3.3±0.05a 3.2±0.03a 3.3±0.09a 3.0±0.08a 5.4±0.05a 4.9±0.04b 4.9±0.06b 4.8±0.04c 0.52±0.01ab 0.53±0.02ab 0.52±0.01b 0.51±0.01ab *Data in a column with the same letter mean that there were no significant differences at p<0.05 For FA, the SUVA 254 of all treatments did not show significant differences and were within the range of 3.0-3.3. This finding suggests that BC additions did not significantly impact the aromaticity of FA. The E 2 /E 3 values of CK (5.4) were higher than those of the T1-T5 treatment (4.8-4.9). This result indicates that FA from compost amended with BC contains compounds with larger molecular weight and a higher degree of polymerization compared to FA in compost without BC. In general, the spectral parameters confirm that BC addition promotes the formation of more complex and stable HA molecules with higher aromaticity and molecular weight (Lanno et al., 2022; Tan et al., 2022). Meanwhile, BC has less impact on the aromaticity of FA but leads to an increase in its molecular weight, suggesting enhanced polymerization. These findings highlight the potential of BC to modify the structural characteristics of compost HS, thereby improving compost quality and stability. 3.3. EEM-FRI analysis of HA and FA The EEM spectra of HA and FA in compost with and without BC additions are shown in Fig. 2. It is evident that four types of HA exhibited comparable fluorescence patterns, characterized by a dominant peak at Ex/Em = 240-270/430-470 nm and a weaker band at 330-380/430-470 nm. These regions are indicative of humic acids, representing their complex aromatic structures and high molecular weight (Cui et al., 2020; Musadji and Geffroy-Rodier, 2019). Additionally, all FA samples displayed similar spectral characteristics, with a pronounced peak at Ex/Em = 220-235/410-430 nm, which is typical for fulvic acids-like substances (Fang et al., 2016; Zhou et al., 2023). FA are known for their lower molecular weight and reduced aromaticity compared to HA (Masmoudi et al., 2024; Song et al., 2017). These findings suggest that the BC additions did not significantly affect the fluorescence characteristics of HS in the compost. The spectral profiles of HA and FA remain distinct, with HA showing fluorescence at higher Ex/Em wavelengths than FA. This difference reflects the inherent structural differences between HA and FA, with HA containing larger, more aromatic molecular structures compared to FA (Masmoudi et al., 2024; Yu et al., 2019a). The relative distribution of the five distinct fluorescent components within HA and FA is shown in Fig. 3. It is observable that HA was dominated by fulvic acid-like (Region Ⅲ) and humic acid-like (Region V) substances, with proportions within the range of 53-61% and 34-36%, respectively (Fig. 3a). As a comparison, region V for HA from T1-T5 were 4%-8% higher than that from CK, suggesting that BC additions increase humic acid-like substances within HA. A possible reason for this result was that BC enhanced biological oxidation reactions during composting and consequently promoted the formation of stable structures such as HA (Lu et al., 2024). For FA, the highest content was found in Region III, accounting for 30%-33% of total fluorescence, followed by Region V (23%-28%), and Region II (23-26%). These results suggest that FA was dominated by fulvic acid-like substance. Additionally, the relative content of tryptophan-like substances in Region II was 23%-26%, indicating that tryptophan-like and humic acid-like substances are important components of FA. Compared to the control group, the fluorescence intensity of humic acid-like substances in HA increased significantly for T1 (59%), T2 (57%), and T5 (61%), as shown in Figure 4a, with increases of 6%, 4%, and 8%, respectively, compared to CK (53%). These results indicate that BC enhanced the biodegradation reactions in the compost, promoting the formation of stable structures like HA, and making humic-like substances the main component of HA (D' Silva et al., 2023). As illustrated in Figures 4(a) and 4(b), there are significant differences between the EEM spectra of HA and FA. The contents of Regions III and V in HA compost are higher than those in FA, with values of 34%-36% and 53%-61% for HA, and 30%-33% and 23%-28% for FA, respectively. This indicates that HA possesses higher humification intensity and greater stability. 3.4. MW distributions of HA and FA Molecular weight (MW) is a critical attribute of HS, offering insights into their structure and reactivity (Chen et al., 2020). The corresponding HPSEC chromatograms of compost HA and FA, both with and without BC amendments, are shown in Fig. 4. The MW distribution of HA generally ranges from 1×10 2 to 2×10 5 Da, while FA ranges from 1×10 2 and 2×10 4 Da (Fig. 4). The broader MW range observed for HA compared to FA suggests that HA contains a larger proportion of high MW fractions, consistent with the previously observed higher SUVA 254 and lower E 2 /E 3 values. Interestingly, the chromatogram patterns of HA and FA across different compost samples, whether BC-amended or not, are quite similar. This suggests that BC addition does not significantly alter the MW distributions of HA and FA within compost. However, some variations are observed, particularly for HA (Fig. 4a). For HA, four prominent peaks were identified at MW of 0.56, 2.1, 12.4, and 93 kDa, suggesting the presence of four distinct clusters of MW fractions (Fig. 4a). With the addition of BC, the relative intensity of the smaller MW peaks (0.57 and 2.14 kDa) decreased, while the largest MW peak at 93 kDa increased in intensity. This trend suggests that BC facilitates the formation of larger MW fractions while reducing the presence of smaller MW fractions within HA. This is consistent with the previous studies that reported the transformation of MW fractions in compost OM into larger ones with BC amendment. Additionally, the levels of BC added during composting also influenced the MW distribution of HA. For example, the relative intensity of peak at 93 kDa followed the trend of T2 > T5 > T1 > CK, indicating that the 2% BC addition significantly promotes the formation of larger MW fractions within HA. Furthermore, 5% BC addition appeared to promote the degradation of smaller MW fractions (0.57 and 2.14 kDa) or their transformation into larger MW fractions. In contrast, the FA chromatograms revealed two dominant peaks at MW of 0.56 and 2.1 kDa, along with a weak band at 4.1 kDa. The smaller MW fractions in FA were less significant compared to the larger ones in HA, reinforcing that HA contains more large MW moieties than FA. Notably, the similarity of peaks at MW of 0.56 and 2.1 kDa in both HA and FA suggests that these MW fractions are fundamental components of HS. Additionally, FA in compost amended with BC generally exhibits higher intensities at MW of 0.56 and 2.1 kDa, indicating a higher proportion of smaller molecular clusters in FA with BC additions. Moreover, Mw and Mn of HA and FA were investigated to quantify their molecular size in compost with and without BC amendments. As shown in Table 2, the Mw of HA in compost amended with BC (T1, T2, and T5) ranged from 27422 to 28754 Da, significantly higher than the 24594 Da observed for HA in compost without BC. Similarly, the corresponding Mn values for BC-amended HA ranged from 2334 to 2742 Da, which was higher than 2283 Da for HA without BC amendment. These results suggest that BC addition effectively increases the molecular size of HA. For FA, the Mw values ranged from 5772 to 6635 Da for T1-T5 treatments, slightly higher than the 5306 Da observed for CK ones. The Mn values for FA ranged from 1163 to 1318 Da, with no significant differences. These findings suggest that while BC additions promote the molecular size of HA, and effect on FA is minimal, likely due to the relatively small range of BC levels (1-5%) used in this study. Further study using higher levels of BC (>5%) would be necessary to explore deeper effects. Comparative analysis shows that the Mw and Mn of HA are 4.3-4.8 and 1.7-2.3 times higher than those of FA, confirming that HA generally has a significantly larger molecular size than FA. These findings are consistent with the SUVA 254 results, supporting the relationship between molecular weight and the presence of aromatic structures and unsaturated double bonds in conjugated systems (Chen et al., 2020). Table 2. The Mw, Mn, and ρ of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments Mw(Da) Mn(Da) ρ HA CK 24594 ± 780b 2283 ± 19c 10.9 ± 0.4ab T1 27422 ± 972ab 2334 ± 118bc 11.7 ± 0.4ab T2 28469 ± 1811a 2442 ± 102ab 11.7 ± 0.8a T5 28754 ± 146a 2742 ± 80a 11.6 ± 0.6ab FA CK 5306 ± 387ab 1318 ± 63a 4.0 ± 0.1c T1 5772 ± 676ab 1276 ± 94a 4.5 ± 0.3bc T2 5819 ± 456ab 1191 ± 118a 3.2 ± 0.3c T5 6635 ± 650a 1163 ± 99a 6.3 ± 0.7a *Data in a column with the same letter mean that there were no significant differences at ρ <0.05 3.5. FTIR spectra of HA and FA FTIR spectra of HA and FA in different types of compost are shown in Fig.5. The HA spectra display consistent peaks at 3345, 2930, and 1655 cm -1 , with additional absorptions observed in the 1035-1511 cm -1 range. The FA spectra feature dominant peaks at 3354, 2939, 1711, 1385, and 1230 cm -1 . The broad peak around 3300 cm -1 indicates O-H stretching of carboxylic acids, alcohols, and phenols (Abid et al., 2020). The bands at 2930 and 2939 cm -1 suggest the presence of aliphatic C-H stretching vibrations (Abid et al., 2020). The peak at 1711 cm -1 indicates C=O bonds, likely from amides, while the peak at 1655 cm -1 is associated with aromatic and carboxylate C=O vibrations (Dong et al., 2021; Ming et al., 2023). The band at 1514 cm -1 corresponds to lignin aromatic C-C stretching, and the region around 1420-1460 cm -1 is attributed to carboxylic O-H deformation (Monda et al., 2017; Savy and Piccolo, 2014). Peaks at 1224 and 1230 cm -1 are indicative of lignin diary ether bonds. The fingerprint region below 1200 cm -1 represents C-C, C-O, and C-N vibrations (Niu et al., 2021; Zhu et al., 2021). Overall, both HA and FA exhibit complex functional group structures, primarily involving C-O, C=O, C-H, C=C, and C-N bonds. The spectral characteristics of HA and FA with BC amendments in composting are quite similar, suggesting that BC addition does not significantly alter the functional group structures of compost-derived HS. The FTIR spectra exhibit similar profiles for HA and FA across all samples, yet their intensities vary with BC addition, indicating alterations in the chemical composition. The intensity ratios of the major peaks at 2930 and 1655 cm -1 for HA, and at 2939 and 1711 cm -1 for FA, were investigated. The ratio of 2930/1655 (aliphatic C/aromatic C) remained relatively consistent for HA across all treatments, ranging from 0.73 to 0.77, suggesting that BC amendments did not significantly affect the formation of key functional groups. In contrast, the ratio of 2939/1711 (aliphatic C/carboxyl C) for FA derived from CK was 0.77, which was notably higher than that for FA from the BC treatments (T1-T5), which ranged from 0.55 to 0.69. This suggests that the BC amendment may promote the formation of carboxyl C groups or facilitate the degradation of aliphatic C. Therefore, the results indicate that BC amendment influences the transformation of organic matter, affecting the solubility and reactivity of FA, while also altering the relative proportions of carbon types in both HA and FA. 3.6. PFRs generation The EPR spectra of HA and FA derived from compost amending with different levels of BC are shown in Fig. 6. It is evident that both HA and FA exhibited a single, well-defined resonance with good symmetry, indicating the presence of free radicals. In comparison, the resonance intensity of HA was stronger than that of FA, suggesting a higher production of PFRs in HA samples. Zhang et al. (2020) highlighted that the MW and the degree of condensation of HS positively influence the generation of PFRs (Zhang et al., 2020). As revealed by the aforementioned UV-vis and EEM spectra and HPSEC analysis, the HA was indeed more condensed and had a larger MW than FA. Moreover, the g factors of HA ranged from 2.00411 to 2.00415, which were slightly lower than those of FA (2.00468-2.00541). Previous studies have shown that g factors greater than 2.004 correspond to oxygen-centered organic radicals (Dellinger et al., 2007; Wu et al., 2022), which are predominantly attributed to semiquinone free radicals (g = 2.0010-2.0051) (Lodygin et al., 2024; Pan et al., 2019). These findings imply that both HA and FA derived from compost, with and without BC amendment, can generate semiquinone-type PFRs. As shown in Fig. 6, the BC amendment resulted in alterations to the EPR signals of compost-derived HA and FA. Notably, the signals for HA exhibited a decreasing trend in the order of CK > T2 > T5 > T1 (Fig. 6a), while the FA signals followed the trend of T5 > T1 > T2 > CK. These results indicate that the BC amendment weakens the formation of HA PFRs, but promotes the formation of FA PFRs. However, the levels of BC additions did not exhibit a consistent trend across treatments. In addition, g factors for CK HA (2.00415) were slightly higher than those for T1-T5 (2.0041-2.00412), and g factors for CK FA (2.00468) were lower than those for T1-T5 (2.00530-2.00541). This suggests that BC amendment may alter the type of PFRs formed in HS derived from compost. 4. Conclusion This study highlights the critical role of BC amendment in enhancing the composition, structure, and stability of humic substances (HS) during composting. The addition of BC significantly increased the humic acid (HA) content and decreased the fulvic acid (FA) content, promoting the transformation of FA into HA. UV-vis, EEM, and FTIR analyses revealed that BC amendments enhanced the degree of aromaticity, molecular weight, and polymerization of HA, while minimally impacting these properties in FA. This structural modification suggests that BC facilitates the formation of more stable and complex HA molecules. Additionally, the molecular weight distributions and spectral parameters confirm that BC promotes the condensation of high-molecular-weight fractions, further improving the quality and stability of composted organic matter. The findings also indicate that BC amendments selectively alter the chemical functionalities of HS, with significant implications for compost quality. Enhanced PFR production and structural stability of HA in BC-amended composts suggest increased microbial activity and oxidative degradation, facilitated by BC's unique properties. While BC’s impact on FA was less pronounced, the observed increases in molecular weight and polymerization highlight its role in improving the solubility and reactivity of FA. These results demonstrate the potential of BC as an effective amendment for improving compost quality and sustainability by enhancing humification processes. Further research should explore the effects of higher BC levels and diverse feedstocks to optimize composting practices and fully harness BC's benefits for agricultural applications. Declarations Acknowledgment This study was supported by the Science Fund for Distinguished Young Scholars of Anhui Province (2022AH030145, gxyqZD2021126), and the Anhui Provincial University Student Innovation and Entrepreneurship Training Program (2023cxcysj182) Author contributions Nennen Zhu : writing-original draft, formal analysis, methodology. Ao Chen g : data curation, software, formal analysis. Xufang Yu: methodology, supervision, writing-review and editing. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5509938","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":395065750,"identity":"70ce271c-3203-48e7-904d-3b16f6bee638","order_by":0,"name":"Nennen Zhu","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Nennen","middleName":"","lastName":"Zhu","suffix":""},{"id":395065751,"identity":"bdcaec1d-415b-4d1c-8479-2c087d6be426","order_by":1,"name":"Ao Cheng","email":"","orcid":"","institution":"Anhui Science and Technology 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with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/22920fe944b0075ebdb0913a.png"},{"id":72591981,"identity":"b0219574-1bce-45dd-9d36-f8f4360d1be5","added_by":"auto","created_at":"2024-12-30 07:22:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43971,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence EEM spectra of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/075a79b8bd9644da4cb583d6.png"},{"id":72593196,"identity":"14d017ff-5596-4fe2-aae9-bedaafb1362f","added_by":"auto","created_at":"2024-12-30 07:30:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20508,"visible":true,"origin":"","legend":"\u003cp\u003eThe proportions of fluorescence regional integration of (a) HA and (b) FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/b6f9790ea4558421105a43c8.png"},{"id":72593193,"identity":"22467616-4c6f-4f1d-943f-96f63c3ac71e","added_by":"auto","created_at":"2024-12-30 07:30:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26288,"visible":true,"origin":"","legend":"\u003cp\u003eThe HPSEC chromatograms of HA(a) and FA(b) in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/f336b5fbb9572971043d4acb.png"},{"id":72591985,"identity":"242a09fd-e627-4325-862c-332a6c5cd8dc","added_by":"auto","created_at":"2024-12-30 07:22:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24305,"visible":true,"origin":"","legend":"\u003cp\u003eThe FTIR spectra of (a) HA and (b) FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/f0d02201c500a0f17fae015a.png"},{"id":72593509,"identity":"9bd905dc-e122-4ea6-a632-dda952920520","added_by":"auto","created_at":"2024-12-30 07:38:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21332,"visible":true,"origin":"","legend":"\u003cp\u003eEPR spectra of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/771365d8cbef0ff361159c2f.png"},{"id":83386217,"identity":"1d82cd88-b608-4ebf-a6dd-ee7eb5b37148","added_by":"auto","created_at":"2025-05-24 13:19:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":822505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5509938/v1/18cf6370-6385-4c2f-9279-744edf105057.pdf"}],"financialInterests":"","formattedTitle":"Spectroscopic and molecular insights into humic substances in compost amended with varying biochar levels","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rapid development of the livestock and poultry industry has resulted in an excessive accumulation of manure that surpasses current disposal capacities, becoming a pressing environmental challenge (Guo et al., 2018; He et al., 2019; Huang et al., 2021). Composting stands out as a sustainable and cost-effective strategy for managing this waste, offering a non-polluting alternative to incineration and landfilling (Chen et al., 2017a; Kujawa et al., 2020). This process is predominantly driven by microorganisms, which not only eliminate pathogens through the thermophilic phase but also convert solid organic waste into organic fertilizers, allowing for biological, economical, and sustainable reuse (Altieri et al., 2012; Li et al., 2022). Composting yields a significant by-product in the form of humic substances (HS), which are the result of the humification process. During humification, unstable organic matter is transformed and stabilized into humic acids (HA) and fulvic acids (FA), the primary components of humic substances (Gong et al., 2023). These substances vary in molecular weight, solubility, and degree of polymerization (Ming et al., 2023; Shan et al., 2018). HS is known to enhance soil health through nutrient retention, promoting plant growth, and mitigating heavy metal pollution (Fang et al., 2016; Lee et al., 2020).\u003c/p\u003e\n\u003cp\u003ePrevious studies have suggested that the addition of specific additives, such as sawdust, corn stalks, and biochar (BC), can enhance HS formation by stimulating microbial activity and functionality during composting (Chen et al., 2023b; Guo et al., 2019a). Among these additives, BC has gained significant attention as an effective amendment in composting due to its unique properties, such as large surface area, high absorption capacity, cation exchange capacity, and substantial pore volume (Li et al., 2023; Lv et al., 2023). These properties allow BC to improve composting performance by enhancing aeration and promoting microbial proliferation (Li et al., 2023; Ming et al., 2023). The incorporation of BC has been shown to substantially increase the HS content in compost (Altieri et al., 2012; Chen et al., 2023b). For example, Liu et al. (2023) reported that adding 6% maize straw BC to pig manure compost enhanced of HA and FA \u0026nbsp;formation efficiencies by 39.4% and 17.4%, respectively, compared to compost without BC (Liu et al., 2023). Jia et al. reported a 16.5% increase in HA content with 10% corn straw BC additions, although FA content decreased by 18.8% compared to compost without BC (Jia et al., 2023). Similarly, Zhou et al. demonstrated that the compost amended with 5-20% BC exhibited a 1.57-2.29 times increase in HA content, while the FA content was 27.2%-37.4% lower in mature compost with BC compared to without BC (Zhou et al., 2023). While current studies highlight changes in the quantity of HS in compost with varying BC concentrations, there is limited understanding of how BC affects the chemical characteristics of HS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHS are chemically complex and highly heterogeneous, consisting mainly of aromatic, phenolic, and quinone components (Chen et al., 2023b; Zhou et al., 2022). To address this knowledge gap, various spectroscopic techniques such as ultraviolet/visible absorption (UV-vis), fluorescence, and Fourier transform infrared (FTIR) spectroscopy, are invaluable for characterizing the structural and compositional properties of HS (Che et al., 2021; Cui et al., 2020). Additionally, fluorescence excitation-emission matrix (EEM) spectra, combined with fluorescence regional integration (FRI) analysis, allow for detailed semi-quantification of fluorophores within HS (Huang et al., 2021; Lee et al., 2020). Furthermore, high-performance size exclusion chromatography (HPSEC) has emerged as a powerful tool for characterizing the molecular weight (MW) distributions of compost-derived dissolved organic matter (DOM) (Guo et al., 2018; He et al., 2019). Recently, persistent free radicals (PFRs) produced by DOM and HS, which play crucial roles in electron transfer and redox reactions in soils and natural environments, have garnered increasing attention (He et al., 2019; Li et al., 2024; Zhang et al., 2020). These radicals can be directly detected by electron paramagnetic resonance (EPR) spectroscopy (Wu et al., 2022; Zhang et al., 2024).\u003c/p\u003e\n\u003cp\u003eIn this study, HA and FA derived from chicken manure compost with rice husk BC amendments at concentrations of 0%, 1%, 2%, and 5% were comparatively analyzed using dissolved organic carbon (DOC), UV-vis, EEM-FRI, FTIR, HPSEC, and EPR protocols. The main objective was to investigate the impact of BC concentration on the structure and properties of HS within compost. By understanding the compositional and structural evolution of HS in response to varying BC levels, this study aims to address a critical knowledge gap in optimizing composting processes. The insights gained from this study could contribute to enhancing the functionality and quality of the final compost product, potentially leading to improved soil health and sustainability in agricultural practices.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Compost sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aerobic composting was conducted in a designated facility, located in an artificial greenhouse at an organic fertilizer production facility in Fuyang, Anhui Province, China. The primary raw materials used in the composting process were chicken manure and rice husks, sourced from a local breeding company. The rice husk BC was purchased from Henan Lize Environmental Protection Technology Co.Ltd. (Henan, China). The initial C/N ratio of the compost mixture was adjusted to 25:1, utilizing a mixture of 20 tons of chicken manure and rice husks in an 8:1 ratio. The moisture content of the compost was maintained at approximately 60%. The initial compost mixture was divided into four separate piles, containing approximately 5 tons of compost material. The experimental treatments involved the addition of different dosages of BC: 0% BC for the control (CK), 1% BC (T1), 2% BC (T2), and 5% BC (T5). Each pile was thoroughly mixed to ensure an even distribution of the BC throughout the compost. The composting piles were turned every seven days to maintain proper aeration and to facilitate microbial activity. After 80 days of composting, approximately 2 kg of mature compost was sampled from each treatment pile. These samples were freeze-dried, finely ground, and passed through a 100-mesh sieve. The four types of samples (CK, T1, T2, and T5) were then stored for further analysis. The physicochemical properties of each type of compost are listed in Table S1 in the supporting information (SI).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Extraction and purification of HA and FA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtraction and purification of HA and FA were adapted from the methodologies recommended by the International Humic Substances Society (IHSS) with minor modifications (Fang et al., 2016; He et al., 2019; Mealio et al., 2024). HA and FA were extracted using a 1:1 mixture of aqueous 0.1 M NaOH and 0.1 M Na\u003csub\u003e2\u003c/sub\u003eP\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e. The extraction solvent was added to the solid compost at a liquid-to-sample ratio of 10:1 (v/w, dry weight basis). The mixture was mechanically shaken under a nitrogen environment for 24 h at 25\u0026deg;C, followed by centrifugation at 8000 rpm to separate the supernatant. The supernatant was then acidified to a pH 1 using 6 M HCl and centrifuged at 8,000 rpm to separate dissolved FA from the precipitated HA. The raw HA was acidified to pH of 1 with 6 M HCl, centrifuged, and washed with ultrapure water to yield pure HA. The raw FA was passed through an XAD-8 resin column, and the adsorbed fraction was eluted with 0.1 M NaOH. It was then purified using a hydrogen-type cation exchange resin column to eliminate cations and obtain pure FA (Huang et al., 2021). Finally, both HA and FA were lyophilized to obtain solid samples, which were then stored at -20 \u003csup\u003eo\u003c/sup\u003eC until further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Dissolved organic carbon analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor analysis, a portion of solid HA and FA were re-dissolved in ultrapure water. The concentration of dissolved organic carbon (DOC) in HA and FA was measured using a Shimadzu TOC analyzer (TOC-VCPH, Japan) following the non-purgeable organic carbon protocol. The HA and FA solutions were then diluted to 10 mg/L for further spectroscopic analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Ultraviolet-Visible spectra\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UV-vis spectra of HA and FA were recorded using a UV-2600 spectrophotometer (Shimadzu, Japan) with wavelengths ranging from 200 to 700 nm (Castan et al., 2020; Chen et al., 2017b). To characterize the HA and FA, three optical parameters were evaluated: specific UV absorbance at 254 nm (SUVA\u003csub\u003e254\u003c/sub\u003e), the\u0026nbsp;absorbance ratio between 254 and 365 nm (E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e), the spectra slope ratios (S\u003csub\u003eR\u003c/sub\u003e). Detailed calculation for these parameter are described in Text S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Fluorescence spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EEM fluorescence spectra of compost HA and FA were acquired using a fluorescence spectrophotometer (F-4600, Hitachi Japan). The excitation (Ex) wavelength was scanned from 200 to 400 nm, and the corresponding emission (Em) wavelength range extended from 290 to 520 nm. Increments of 5 nm were applied for both Ex and Em wavelengths, and the scanning speed was set at 12,000 nm\u0026middot;min\u003csup\u003e-1\u003c/sup\u003e. Background correction was performed using ultrapure water as a blank for all EEM spectra of compost HS samples. for all EEM spectra of compost HS samples. To evaluate compost characteristics, the humification index (HIX), fluorescence index (FI), and biological index (BIX) were calculated, with the specific formulas provided in Text S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fluorescence regional integration (FRI) technique was employed on the EEM spectra to characterize the fluorescence components of compost HS. According to previous studies (Mealio et al., 2024; Ren et al., 2020), the EEM spectra were divided into five distinct regions (I-V). These regions could be assigned to simple aromatic proteins (I and II), fulvic acid-like substances (III), soluble microbial byproduct-like materials (IV), and humic acid-like organics (V), respectively(Wei et al., 2014; Zhang et al., 2016). The specific Ex/Em wavelength ranges for each region and their corresponding fluorophore assignments are detailed in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. High-performance size exclusion chromatogram analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor HPSEC analysis, an aqueous gel filtration column (Polysep-GFC-P 3000, Phenomenex) and a guard column (Polysep-GFC-P, Phenomenex) were used. A water/methanol (9:1,v/v) With 25 mM ammonium acetate was used as the mobile phase; the sample injection volume was 100 \u0026mu;L with a flow rate of 1mL min\u003csup\u003e-1.\u0026nbsp;\u003c/sup\u003eand the detection wavelength was 254 nm. The column was calibrated using a series of polyethylene glycol (PEG) standards (Sigma Aldrich, USA) with molecular weights of 238, 601, 1020, 3450, 4080, 11,100, 17,900, and 41,300 Da (Chen et al., 2023a). The void volumes and exclusion limit were verified using polystyrene sulfonate (MW 210 kDa) and acetone (MW 58 Da), respectively. The calibration curves, depicting the logarithmic molecular weight against retention time, were illustrated in Fig. S1.\u003c/p\u003e\n\u003cp\u003eThe weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (\u0026rho;) of HA and FA were calculated using the following formulas (Abid et al., 2020; He et al., 2019):\u003c/p\u003e\n\u003cp\u003e\u003cimg 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wYvfMEuAJUCF/3Rw2xs+AGEbeD+vDXXwwXbz3jRBV0SSY98P76Ps0+yffuDgeVg4ADmRgYQv4og1vCK+zC2v19qIj6NftjWuQE2CTIvK0KT4Xr4/bSBwKk0Df5S4WhTrq3R7EBeIWM7/I/wnJYi6/02SNJDmLIFOROIRDCjLw9zBHtiH/aDixB0UR5aQrMRFKFfgUADwRa624goE7nk/y3iNZgm3QUMVBu5z5W7hQikN1wzHeiptyqEwZhv8dKVp5oo7ek+qkjTwGd4f9Ufwm9lc/qfJm3LrZrn73zj8f/ge/gsEiCTI3yx5JiLR3P4bdyDJa+VejyvPbgskFDCuIPBXSGHDDk7zhV8g8JzCicKCSMVz1sF9liBQLeWa6ZIg3dxcukhjmnSRV1HRP420aQgX5j5+Q7aJdPCnKvfeKORD1BEJ20i+8H9x6yO/+NPx/+A7+WwSbh18XkSqx+1bUQeClAUc8LF/856ociuuvLht1BYzqksFp31c2vnN0aNHbY/+lStX7Kk8ent7TVtbm30d7733nj1f1NmzZ+11xXnO5DpGHDEaYKQxF6N0RG9KGRQsSYf0wqWLNE6fPt0uGwojpUo/mFm4cGGwpDJp03DhwoXBIcRhXMue32bDhg329+F68PzOSXHt++effz54dtORI0fMU089ZaZMmWKf85/54osvzMsvv2wmTpxo58XMmTPHvpbUmTNngkciMpzYX9etW2enH3R2dppSwLCjuFiexG2BZN++fWbBggX2MQUnBc3q1avtyjmNR1SBwFAyCszXXnstWHKjAJ4/f37wLJobWlvuxnuyICjOnDnTZlBaBAIK3XfffTdYciNd4fQzbDVqaC/LGDpHPmaRJQ1hbP9bb701uE0EFv/AAIwbjxray/Lm5mYzd+7cYMlNnIV49uzZg+v9888/zbfffmu3mYMTcAoYH8MJ055XTEQqN2HCBHt/6dIle+/s3LnzluG97P8cjFKRCGuJOklsUDMZRFOEq/ZQzeEt3CPcP+JQ3aEd3+HzUe+rNbab7YqqxiVBM5CfLte/kARp5718JotK0sBnaEoKc9vvbxPrjmsPDWOdUf1DbJvfbkoTF9/j1kuTVtT2xHHV76TbJSLJsa+Gm61ck5W/z0XtgzR7RTVt31Iquh3YBQDXnu7QV+C/7rBiXnP4nHtf0nbxanNpCWdYGqTJT5croP10UWj6wcYJ510lKk2D+1OEhQOj/3uTHu5JI8vCAdAFofBvj3DgIs9c/vB+/rjhvGBdft76wv9DEake9lfKrTC377sb+2EY+3LU8ltKG7eCob4k/Dor949U+SIKLAqPrEfklXCFV1xBlRRp8NPlamSk3xVypDXqe6KifhpZ0uBqhOFaDOvy18frpJHvCddSwuKORFyhz43/SBh5FfU6j/289fEa2yQiw4P9KyoglEN5FhUbkKydpsFQWJJRtTiipTAMF4gukGb5/qxp4AePKtiHQkAJBy+2gfSk/eOVw3fEHWRwAFLJtotIchzcJT3YHergLnfn2qJDmI5fziGTtZM7CTqr7777btPT0xMsudFxlaWTvRppYATG5s2b7QCJNI4dO2amTZtmt8H58ccfYzvZK0Wn/5gxY24bFUI+MsBh+fLlwRIRGQ6l8t+WMUOd346y4Pz583Zkbqwb8SQfXFNNLY9mXc3BHV1z9E62VtqkV800cLTB0X2aWg21K77fP1JhHXHNUJXiO/wmQpBnfl6KSGPIxRUSHc6wixMnTtj7RlTtNHDE//HHH9shzMz3qVcMlWYo8JtvvlkX849EJLncBBJOQ05TDvNcGrUgykMaRKR4chFIOOqeN2+e6erqsrPvG1Ee0iAixdTwgYTOZGZMt7a23tJBnBRNKnv27DH79+8PltReHtIgIsXV8KO2Xn31VXv+q08++SRYkg6jrkZaHtIgIsXV0IHEDZPdu3dvRcNkaU7i8yPZH5GHNIhIsTVsIGHss7sgPX0LUSd8HOrG58D1iEdCHtIgItKwgaSjo8NOXGtkeUiDiEiu5pGIiEjt5e4UKSIiUlu5CCR0WEddiKkcPuNfyGUk0VfCuazSoJOdi35xLyIyknIRSAgibv4FBWtUp7R/pUVmkKOvr8/ejzRGXDH814nafm4Oab148WL5k6iJiNRIwwcSggKFrLtkK2eopdsnfPMn63H517Q1mOHiAp9/qd6o7efmsO3c6qVGJSLF1vCBhKDQ1NRU1yckLIfA197ebq93LiLSiBo+kFATaWtrC54la9qqNzRR+YEwavu5iYjUo4YPJOELMSVp2oJrCkt74adqYztooiIAugvMRG0/Nx/bTQC6evVqsEREZGTkorOdU6+nQe3koYceso/vuOOOwc73kXLgwAHzww8/JD7NCdvLdjNYYNGiRXVd2xKR/NOERBERySQXNRIRERk5CiQiIpKJAomIiGSiQCIiIpkokIiISCYKJCIikokCiYiIZKJAIiIimSiQiIhIJgokIiKSiQKJiIhkokAiIiKZKJCIiEgmCiQ14i5OFXXKd65D0tzcbG89PT3BUhGRxqBAUiPd3d32nmuPhK1atcrer1y50jz++OP2sYhIo1AgqZE//vjDLFmyJHh2E1d2HDdunLl8+bKZM2dOsFREpHEokNTIzz//bJ588kn7mMvqgsvlrlu3zrzwwgv2+axZs+y9iEgj0RUSa4CAwaVxyWr6SQ4fPmyvLb906VKzYMECc+HCBdPb22tOnDgRfEJEpHGoRlIDx48fH2zWam1tNUePHrW1kitXrpgVK1bYINLW1mZfFxFpNAokNbBv3z5b88D48ePNwMCAWb16tens7LS1lZMnT6p/REQalgJJDVDjWLhwoX38yCOPmE2bNplnnnnGTJo0ydZWoP4REWlUCiTDjCasvr4+M3bsWPuce5q31qxZY5/v2LHD3jsMBaYfxWHeieucZ7kbKiwiUi/U2T7MXFBob283+/fvt4+drVu3mrfffts+dq+7jnmCD1paWkxXV5ftS1m7dq1Zvny5rcmIiNQLBZI6NHnyZLN9+3azc+dO09TUZGsxixcvNt99991gTUZEpF4okNQhmrOmT59u7rnnHnP//febjRs32lrIhg0bbGe9iEg9UR9JHaJDnqYtmrPGjBljfv31V/Pmm28qiIhIVdH6wbn+wFk2aIrnxnKH5UP1zapGIiJSMAQP+l/d5GiCxXPPPWcnTYPAwWjTs2fP2uf053722WeDz8MUSERECsb1wxJEQHP6iy++aM+24VAz2bVr1+Aygsn58+ft/LewwjVtnT592maMq8IxEqocMti9N+4WdWp4EZF6REAgkLgggqizkjOS9NChQ8EzYwf6UEtx0xF8hQokBBFmmHOjInbq1Ck7OZBqXRyG5PLecrfwsF4RkXpFExW1jzA/aMThVE6MJg0rVCB5//33bUbQiY2pU6fae06aOByiai/VuomIpEXfCAN5Jk6cGCy5gWshffrpp4Md74jqD5k/f76tlYQVJpAw0W/37t2Dp2wHy8Aw2zhZmraiai/VuomIpHXp0iV7P2HCBHvv0O9BUxYd8K5cI+AQOMLcZGlfYQLJmTNn7L1/BUIm+DHhz50HK4qatkSkCPyybsuWLTao+J3v5RQmkHDqdoKGP2b63XfftZFY8zNEpAhcTcTVTKJQRnLqJkZ1RSHAhBUmkHCFQvpHuFFt48qEX375ZeKIKyLS6DhDBoHg4sWLwZJbMaKL1xn264/qcuiQj7p2UmHmkRA83OQbEZGiIlhwYO03ydPPyxBggkjcpEOE5584haiRuHHPIx1EXCdWVOc81cnm5mZ76+npCZaKiFQX80EIFv58ENc/Ui6I0B0Qnn/iFCKQ0D/CiISR1t3dbe+jJv+4c9kwDM8fECAiUm0EjHnz5pWdQ+ejFkN3QNzAokIEEiJwPYys+uOPPwav3e7jxxw3bpy5fPmyLrkrIjVBDYRmKn/uSBTKJ06NUq62onNt1RBNWswo5eRorr+GuSxcZvejjz4yixYtMv39/RpFJiINRYGkRtyVD8luv+OfUWOcsoXZ9cwYPXHiRPAJEZHGUJjhvyPt+PHjg81aXLOdfhs6u65cuWJP2UIQiRpWJyJS7xRIamTfvn225gGargYGBszq1avthEhqKydPnlT/iIg0JAWSGqHG4U7FwhUQOevwM888YycIUVsBfSUiIo1GgaQGaMLiRGdjx461z7mneYvRZNixY4e9dzjdPeO16T9hxARzS2bMmBG8KiJSXxRIaoDx2li2bJm9p0/EdaozPpuzEsO9/vfff9tRXNRiaAL766+/7NBgEZF6pFFbdWrbtm22ZkIfCjPd6WOJusSliMhIU42kTh08eHDwWgBHjhyxjwkuIiL1RoGkTvmd8+AUKrNnzw6eiYjUDzVtiYhIJqqRiIhIJgokIiKSiQKJiIhkYMz/AyF+jeSDvI97AAAAAElFTkSuQmCC\" width=\"402\" height=\"174\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003ehi\u003c/em\u003e and \u003cem\u003eMWi\u003c/em\u003e represent the chromatogram height and the apparent MW of HA and FA samples corresponding to the \u003cem\u003ei\u003c/em\u003eth retention time, respectively. It is noted that the \u003cem\u003eMw\u003c/em\u003e and \u003cem\u003eMn\u003c/em\u003e of compost-derived HA and FA were estimated using HPSEC, based on PEG calibration standards applied in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Fourier-transform infrared (FTIR) spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra measurement followed the methods introduced in previous studies (Wang et al., 2021; Xiao et al., 2019). A finely ground 1 mg sample was mixed with 200 mg KBr and compressed under vacuum for 10 min. The resultant solid mixture was thoroughly ground before being compressed into pellets. The FTIR spectrum of the KBr pellet was recorded using an FTIR spectrophotometer (Thermo, USA). The spectra were scanned over a wavenumber range of 4000-400 cm\u003csup\u003e-1\u003c/sup\u003e with 4 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eresolution. Each spectrum was generated from a total of 64 scans. A pure KBr pellet was referenced to avoid instrument background signals through subtraction (Dong-Hai et al., 2018; Yu et al., 2019b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Electron paramagnetic resonance (EPR) spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePFRs of HA and FA were determined using EPR spectroscopy (Magnettech ESR5000, Bruker, Ettlingen, Germany). Approximately 5 mg of HA or FA powder was packed into a quartz tube, which was then placed in the EPR instrument for analysis. The EPR detection parameters were set as follows: microwave power at 5 mW, microwave frequency at 9.46 GHz, modulation frequency at 100 kHz, and modulation amplitude at 1 G. The scan range was from 3320 G to 3420 G, with a center field of 3370 G. The scan time was set to 30 seconds, and the resolution along the X-axis was 1024 points.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach analysis was conducted in triplicate, and the resulting mean value was adopted, accompanied by the calculation of respective standard deviations. Data were statistically analyzed using Microsoft Excel to determine significant differences among sample data, followed by further analysis using IBM SPSS Statistics 22.0 software. And the figures were plotted using Origin 2021 (Origin-Lab, USA).\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Effects of BC addition on HA and FA content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DOC content of HA and FA derived from compost amended with different amounts of BC is shown in Fig. 1. The DOC content of HA in compost with BC additions (T1-T5) ranged from 1.2 to 1.68 mg/g (Fig. 1), representing an increase of 13%-16% compared to CK treatment. This suggests that BC additions promote HA formation, a phenomenon evidenced by many previous studies (He et al., 2019; Jia et al., 2023; Zhang et al., 2022). Moreover, HA content was generally increased with increase of BC addition, with the highest increase observed in T5 treatment (Fig. 1). This suggests that the BC levels might be positively correlated with HA formation. In contrast, the DOC content in FA decreased with increasing BC additions, following the trend of CK (0.92 mg/g) \u0026gt; T1 (0.81 mg/g) \u0026gt; T2 (0.72 mg/g) \u0026gt; T5 (0.63 mg/g). The reduction in TOC of FA for T1-T5 treatment ranged from 12% to 32% compared to CK treatment. This finding indicates that BC additions promote the degradation of FA during composting. The simultaneous HA formation and FA degradation suggest that BC amendment facilitates the conversion of FA to HA. This may be due to enhanced microbial activity, which is stimulated by the presence of BC (Liu et al., 2023; Zhou et al., 2023). These findings are consistent with results reported in previous studies (Liu et al., 2023), which also observed that BC encourages the formation and stabilization of HS (Abbott et al., 2018; Yu et al., 2020). The T5 treatment, with the highest BC addition of 5%, showed the most significant increase in HA content and the greatest decrease in FA content, indicating that the compost with 5% BC became more accessible to microorganisms, leading to the production of more stable humic substances.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. UV-vis spectra of HA and FA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV-vis spectra analysis provides valuable insights into the molecular structure of organic substances. The normalized UV-vis spectra of the HA and FA based on their DOC content are shown in Fig. S2. In general, the absorbance of HA and FA decreased with increasing wavelength, which is a typical characteristic feature of dissolved organic matter (DOM) in compost (Fang et al., 2016; Ren et al., 2020). An absorption platform was observed between 270 and 280 nm, typically associated with lignin and aromatic compounds (Bhat et al., 2017; Guo et al., 2019b). The absorption of HA was significantly higher than that of FA, especially between 200 to 300 nm, indicating the higher abundance of aromatic species within HA. This result indicated that BC improved the degree of humification of compost and promoted the conversion of highly polymeric compounds (Zhou et al., 2023). To gain more insight into the compositional and structural characteristics of HA and FA, spectra parameters such as SUVA\u003csub\u003e254\u003c/sub\u003e, E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e, and S\u003csub\u003eR\u003c/sub\u003e were investigated (Table 1). SUVA\u003csub\u003e254\u003c/sub\u003e is known to be positively correlated with the aromaticity of OM (Ben Mahmoud et al., 2023). As shown in Table 1, the SUVA\u003csub\u003e254\u003c/sub\u003e for CK HA was 8.4, which was lower than the range of 9.6-10.3 observed for HA from T1-T5 treatments. This suggests a greater degree of aromaticity in HA from T1-T5 compared to CK, implying that BC additions can enhance the aromatization of HA. These results are consistent with previous findings that demonstrated BC amendments facilitate the formation of phenolic, aromatic carboxylic, and polycyclic aromatic groups in compost OM (Guo et al., 2018; Zhou et al., 2024). This implies that composting with BC amendment can improve the stability and humification degree of HA (Ben Mahmoud et al., 2023; Long et al., 2023). Additionally, E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e and S\u003csub\u003eR\u003c/sub\u003e are generally negatively related to the molecular weight of OM (Guo et al., 2019a). As listed in Table 1, the E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e values for HA from CK were 5.2, similar to those from T1-T5 (5.0-5.4). However, the SR value for CK was 0.71, which was higher than those for T1-T5 (0.51-0.53). This finding indicates that BC addition increases the molecular weight of HA, likely due to enhanced polymerization and the formation of more complex aromatic structures (Yin et al., 2023). On the other hand, it is likely that the abundant carbon functional groups in BC enhance intermolecular interactions, thereby facilitating the formation of more stable HS (Zhou et al., 2023).\u003c/p\u003e\n\u003cp\u003eTable 1. Spectral parameters of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"67%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eSUVA\u003csub\u003e254\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003eS\u003csub\u003eR\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 10px;\"\u003e\n \u003cp\u003eHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e8.4\u0026plusmn;0.02d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5.2\u0026plusmn;0.01a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.71\u0026plusmn;0.01a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e10.2\u0026plusmn;0.01b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5.0\u0026plusmn;0.08a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.51\u0026plusmn;0.02b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e9.6\u0026plusmn;0.02c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5.1\u0026plusmn;0.05a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.53\u0026plusmn;0.01b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e10.3\u0026plusmn;0.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5.0\u0026plusmn;0.07a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.52\u0026plusmn;0.01b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e3.3\u0026plusmn;0.05a\u003c/p\u003e\n \u003cp\u003e3.2\u0026plusmn;0.03a\u003c/p\u003e\n \u003cp\u003e3.3\u0026plusmn;0.09a\u003c/p\u003e\n \u003cp\u003e3.0\u0026plusmn;0.08a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e5.4\u0026plusmn;0.05a\u003c/p\u003e\n \u003cp\u003e4.9\u0026plusmn;0.04b\u003c/p\u003e\n \u003cp\u003e4.9\u0026plusmn;0.06b\u003c/p\u003e\n \u003cp\u003e4.8\u0026plusmn;0.04c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28px;\"\u003e\n \u003cp\u003e0.52\u0026plusmn;0.01ab\u003c/p\u003e\n \u003cp\u003e0.53\u0026plusmn;0.02ab\u003c/p\u003e\n \u003cp\u003e0.52\u0026plusmn;0.01b\u003c/p\u003e\n \u003cp\u003e0.51\u0026plusmn;0.01ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Data in a column with the same letter mean that there were no significant differences at p\u0026lt;0.05\u003c/p\u003e\n\u003cp\u003eFor FA, the SUVA\u003csub\u003e254\u003c/sub\u003e of all treatments did not show significant differences and were within the range of 3.0-3.3. This finding suggests that BC additions did not significantly impact the aromaticity of FA. The E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e values of CK (5.4) were higher than those of the T1-T5 treatment (4.8-4.9). This result indicates that FA from compost amended with BC contains compounds with larger molecular weight and a higher degree of polymerization compared to FA in compost without BC. In general, the spectral parameters confirm that BC addition promotes the formation of more complex and stable HA molecules with higher aromaticity and molecular weight (Lanno et al., 2022; Tan et al., 2022). Meanwhile, BC has less impact on the aromaticity of FA but leads to an increase in its molecular weight, suggesting enhanced polymerization. These findings highlight the potential of BC to modify the structural characteristics of compost HS, thereby improving compost quality and stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. EEM-FRI analysis of HA and FA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EEM spectra of HA and FA in compost with and without BC additions are shown in Fig. 2. It is evident that four types of HA exhibited comparable fluorescence patterns, characterized by a dominant peak at Ex/Em = 240-270/430-470 nm and a weaker band at 330-380/430-470 nm. These regions are indicative of humic acids, representing their complex aromatic structures and high molecular weight (Cui et al., 2020; Musadji and Geffroy-Rodier, 2019). Additionally, all FA samples displayed similar spectral characteristics, with a pronounced peak at Ex/Em = 220-235/410-430 nm, which is typical for fulvic acids-like substances (Fang et al., 2016; Zhou et al., 2023). FA are known for their lower molecular weight and reduced aromaticity compared to HA (Masmoudi et al., 2024; Song et al., 2017). These findings suggest that the BC additions did not significantly affect the fluorescence characteristics of HS in the compost. The spectral profiles of HA and FA remain distinct, with HA showing fluorescence at higher Ex/Em wavelengths than FA. This difference reflects the inherent structural differences between HA and FA, with HA containing larger, more aromatic molecular structures compared to FA (Masmoudi et al., 2024; Yu et al., 2019a).\u003c/p\u003e\n\u003cp\u003eThe relative distribution of the five distinct fluorescent components within HA and FA is shown in Fig. 3. It is observable that HA was dominated by fulvic acid-like (Region Ⅲ) and humic acid-like (Region V) substances, with proportions within the range of 53-61% and 34-36%, respectively (Fig. 3a). As a comparison, region V for HA from T1-T5 were 4%-8% higher than that from CK, suggesting that BC additions increase humic acid-like substances within HA. A possible reason for this result was that BC enhanced biological oxidation reactions during composting and consequently promoted the formation of stable structures such as HA (Lu et al., 2024). For FA, the highest content was found in Region III, accounting for 30%-33% of total fluorescence, followed by Region V (23%-28%), and Region II (23-26%). These results suggest that FA was dominated by fulvic acid-like substance. Additionally, the relative content of tryptophan-like substances in Region II was 23%-26%, indicating that tryptophan-like and humic acid-like substances are important components of FA. Compared to the control group, the fluorescence intensity of humic acid-like substances in HA increased significantly for T1 (59%), T2 (57%), and T5 (61%), as shown in Figure 4a, with increases of 6%, 4%, and 8%, respectively, compared to CK (53%). These results indicate that BC enhanced the biodegradation reactions in the compost, promoting the formation of stable structures like HA, and making humic-like substances the main component of HA (D\u0026apos; Silva et al., 2023). As illustrated in Figures 4(a) and 4(b), there are significant differences between the EEM spectra of HA and FA. The contents of Regions III and V in HA compost are higher than those in FA, with values of 34%-36% and 53%-61% for HA, and 30%-33% and 23%-28% for FA, respectively. This indicates that HA possesses higher humification intensity and greater stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. MW distributions of HA and FA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular weight (MW) is a critical attribute of HS, offering insights into their structure and reactivity (Chen et al., 2020). The corresponding HPSEC chromatograms of compost HA and FA, both with and without BC amendments, are shown in Fig. 4. The MW distribution of HA generally ranges from 1\u0026times;10\u003csup\u003e2\u003c/sup\u003e to 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e Da, while FA ranges from 1\u0026times;10\u003csup\u003e2\u003c/sup\u003e and 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e Da (Fig. 4). The broader MW range observed for HA compared to FA suggests that HA contains a larger proportion of high MW fractions, consistent with the previously observed higher SUVA\u003csub\u003e254\u003c/sub\u003e and lower E\u003csub\u003e2\u003c/sub\u003e/E\u003csub\u003e3\u003c/sub\u003e values. Interestingly, the chromatogram patterns of HA and FA across different compost samples, whether BC-amended or not, are quite similar. This suggests that BC addition does not significantly alter the MW distributions of HA and FA within compost. However, some variations are observed, particularly for HA (Fig. 4a).\u003c/p\u003e\n\u003cp\u003eFor HA, four prominent peaks were identified at MW of 0.56, 2.1, 12.4, and 93 kDa, suggesting the presence of four distinct clusters of MW fractions (Fig. 4a). With the addition of BC, the relative intensity of the smaller MW peaks (0.57 and 2.14 kDa) decreased, while the largest MW peak at 93 kDa increased in intensity. This trend suggests that BC facilitates the formation of larger MW fractions while reducing the presence of smaller MW fractions within HA. This is consistent with the previous studies that reported the transformation of MW fractions in compost OM into larger ones with BC amendment. Additionally, the levels of BC added during composting also influenced the MW distribution of HA. For example, the relative intensity of peak at 93 kDa followed the trend of T2 \u0026gt; T5 \u0026gt; T1 \u0026gt; CK, indicating that the 2% BC addition significantly promotes the formation of larger MW fractions within HA. Furthermore, 5% BC addition appeared to promote the degradation of smaller MW fractions (0.57 and 2.14 kDa) or their transformation into larger MW fractions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, the FA chromatograms revealed two dominant peaks at MW of 0.56 and 2.1 kDa, along with a weak band at 4.1 kDa. The smaller MW fractions in FA were less significant compared to the larger ones in HA, reinforcing that HA contains more large MW moieties than FA. Notably, the similarity of peaks at MW of 0.56 and 2.1 kDa in both HA and FA suggests that these MW fractions are fundamental components of HS. Additionally, FA in compost amended with BC generally exhibits higher intensities at MW of 0.56 and 2.1 kDa, indicating a higher proportion of smaller molecular clusters in FA with BC additions. Moreover, Mw and Mn of HA and FA were investigated to quantify their molecular size in compost with and without BC amendments. As shown in Table 2, the Mw of HA in compost amended with BC (T1, T2, and T5) ranged from 27422 to 28754 Da, significantly higher than the 24594 Da observed for HA in compost without BC. Similarly, the corresponding Mn values for BC-amended HA ranged from 2334 to 2742 Da, which was higher than 2283 Da for HA without BC amendment. These results suggest that BC addition effectively increases the molecular size of HA. For FA, the Mw values ranged from 5772 to 6635 Da for T1-T5 treatments, slightly higher than the 5306 Da observed for CK ones. The Mn values for FA ranged from 1163 to 1318 Da, with no significant differences. These findings suggest that while BC additions promote the molecular size of HA, and effect on FA is minimal, likely due to the relatively small range of BC levels (1-5%) used in this study. Further study using higher levels of BC (\u0026gt;5%) would be necessary to explore deeper effects. Comparative analysis shows that the Mw and Mn of HA are 4.3-4.8 and 1.7-2.3 times higher than those of FA, confirming that HA generally has a significantly larger molecular size than FA. These findings are consistent with the SUVA\u003csub\u003e254\u003c/sub\u003e results, supporting the relationship between molecular weight and the presence of aromatic structures and unsaturated double bonds in conjugated systems (Chen et al., 2020).\u003c/p\u003e\n\u003cp\u003eTable 2. The Mw, Mn, and \u0026rho; of HA and FA in composts with (T1, T2, and T5) and without (CK) BC amendments\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 43px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003eMw(Da)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eMn(Da)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e\u0026rho;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 43px;\"\u003e\n \u003cp\u003eHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e24594 \u0026plusmn; 780b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e2283 \u0026plusmn; 19c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e10.9 \u0026plusmn; 0.4ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e27422 \u0026plusmn; 972ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e2334 \u0026plusmn; 118bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e11.7 \u0026plusmn; 0.4ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e28469 \u0026plusmn; 1811a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e2442 \u0026plusmn; 102ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e11.7 \u0026plusmn; 0.8a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e28754 \u0026plusmn; 146a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e2742 \u0026plusmn; 80a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e11.6 \u0026plusmn; 0.6ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 43px;\"\u003e\n \u003cp\u003eFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e5306 \u0026plusmn; 387ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e1318 \u0026plusmn; 63a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e4.0 \u0026plusmn; 0.1c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e5772 \u0026plusmn; 676ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e1276 \u0026plusmn; 94a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e4.5 \u0026plusmn; 0.3bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e5819 \u0026plusmn; 456ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e1191 \u0026plusmn; 118a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e3.2 \u0026plusmn; 0.3c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e6635 \u0026plusmn; 650a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e1163 \u0026plusmn; 99a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 163px;\"\u003e\n \u003cp\u003e6.3 \u0026plusmn; 0.7a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Data in a column with the same letter mean that there were no significant differences at \u0026rho; \u0026lt;0.05\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. FTIR spectra of HA and FA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR spectra of HA and FA in different types of compost are shown in Fig.5. The HA spectra display consistent peaks at 3345, 2930, and 1655 cm\u003csup\u003e-1\u003c/sup\u003e, with additional absorptions observed in the 1035-1511 cm\u003csup\u003e-1\u003c/sup\u003e range. The FA spectra feature dominant peaks at 3354, 2939, 1711, 1385, and 1230 cm\u003csup\u003e-1\u003c/sup\u003e. The broad peak around 3300 cm\u003csup\u003e-1\u003c/sup\u003e indicates O-H stretching of carboxylic acids, alcohols, and phenols (Abid et al., 2020). The bands at 2930 and 2939 cm\u003csup\u003e-1\u003c/sup\u003e suggest the presence of aliphatic C-H stretching vibrations (Abid et al., 2020). The peak at 1711 cm\u003csup\u003e-1\u003c/sup\u003e indicates C=O bonds, likely from amides, while the peak at 1655 cm\u003csup\u003e-1\u003c/sup\u003e is associated with aromatic and carboxylate C=O vibrations (Dong et al., 2021; Ming et al., 2023). The band at 1514 cm\u003csup\u003e-1\u003c/sup\u003e corresponds to lignin aromatic C-C stretching, and the region around 1420-1460 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to carboxylic O-H deformation (Monda et al., 2017; Savy and Piccolo, 2014). Peaks at 1224 and 1230 cm\u003csup\u003e-1\u003c/sup\u003e are indicative of lignin diary ether bonds. The fingerprint region below 1200 cm\u003csup\u003e-1\u003c/sup\u003e represents C-C, C-O, and C-N vibrations (Niu et al., 2021; Zhu et al., 2021). Overall, both HA and FA exhibit complex functional group structures, primarily involving C-O, C=O, C-H, C=C, and C-N bonds. The spectral characteristics of HA and FA with BC amendments in composting are quite similar, suggesting that BC addition does not significantly alter the functional group structures of compost-derived HS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra exhibit similar profiles for HA and FA across all samples, yet their intensities vary with BC addition, indicating alterations in the chemical composition. The intensity ratios of the major peaks at 2930 and 1655 cm\u003csup\u003e-1\u003c/sup\u003e for HA, and at 2939 and 1711 cm\u003csup\u003e-1\u003c/sup\u003e for FA, were investigated. The ratio of 2930/1655 (aliphatic C/aromatic C) remained relatively consistent for HA across all treatments, ranging from 0.73 to 0.77, suggesting that BC amendments did not significantly affect the formation of key functional groups. In contrast, the ratio of 2939/1711 (aliphatic C/carboxyl C) for FA derived from CK was 0.77, which was notably higher than that for FA from the BC treatments (T1-T5), which ranged from 0.55 to 0.69. This suggests that the BC amendment may promote the formation of carboxyl C groups or facilitate the degradation of aliphatic C. Therefore, the results indicate that BC amendment influences the transformation of organic matter, affecting the solubility and reactivity of FA, while also altering the relative proportions of carbon types in both HA and FA.\u003c/p\u003e\n\u003cp\u003e3.6. PFRs generation\u003c/p\u003e\n\u003cp\u003eThe EPR spectra of HA and FA derived from compost amending with different levels of BC are shown in Fig. 6. It is evident that both HA and FA exhibited a single, well-defined resonance with good symmetry, indicating the presence of free radicals. In comparison, the resonance intensity of HA was stronger than that of FA, suggesting a higher production of PFRs in HA samples. Zhang et al. (2020) highlighted that the MW and the degree of condensation of HS positively influence the generation of PFRs (Zhang et al., 2020). As revealed by the aforementioned UV-vis and EEM spectra and HPSEC analysis, the HA was indeed more condensed and had a larger MW than FA. Moreover, the g factors of HA ranged from 2.00411 to 2.00415, which were slightly lower than those of FA (2.00468-2.00541). Previous studies have shown that g factors greater than 2.004 correspond to oxygen-centered organic radicals (Dellinger et al., 2007; Wu et al., 2022), which are predominantly attributed to semiquinone free radicals (g = 2.0010-2.0051) (Lodygin et al., 2024; Pan et al., 2019). These findings imply that both HA and FA derived from compost, with and without BC amendment, can generate semiquinone-type PFRs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 6, the BC amendment resulted in alterations to the EPR signals of compost-derived HA and FA. Notably, the signals for HA exhibited a decreasing trend in the order of CK \u0026gt; T2 \u0026gt; T5 \u0026gt; T1 (Fig. 6a), while the FA signals followed the trend of T5 \u0026gt; T1 \u0026gt; T2 \u0026gt; CK. These results indicate that the BC amendment weakens the formation of HA PFRs, but promotes the formation of FA PFRs. However, the levels of BC additions did not exhibit a consistent trend across treatments. In addition, g factors for CK HA (2.00415) were slightly higher than those for T1-T5 (2.0041-2.00412), and g factors for CK FA (2.00468) were lower than those for T1-T5 (2.00530-2.00541). This suggests that BC amendment may alter the type of PFRs formed in HS derived from compost.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study highlights the critical role of BC amendment in enhancing the composition, structure, and stability of humic substances (HS) during composting. The addition of BC significantly increased the humic acid (HA) content and decreased the fulvic acid (FA) content, promoting the transformation of FA into HA. UV-vis, EEM, and FTIR analyses revealed that BC amendments enhanced the degree of aromaticity, molecular weight, and polymerization of HA, while minimally impacting these properties in FA. This structural modification suggests that BC facilitates the formation of more stable and complex HA molecules. Additionally, the molecular weight distributions and spectral parameters confirm that BC promotes the condensation of high-molecular-weight fractions, further improving the quality and stability of composted organic matter.\u003c/p\u003e\n\u003cp\u003eThe findings also indicate that BC amendments selectively alter the chemical functionalities of HS, with significant implications for compost quality. Enhanced PFR production and structural stability of HA in BC-amended composts suggest increased microbial activity and oxidative degradation, facilitated by BC\u0026apos;s unique properties. While BC\u0026rsquo;s impact on FA was less pronounced, the observed increases in molecular weight and polymerization highlight its role in improving the solubility and reactivity of FA.\u003c/p\u003e\n\u003cp\u003eThese results demonstrate the potential of BC as an effective amendment for improving compost quality and sustainability by enhancing humification processes. Further research should explore the effects of higher BC levels and diverse feedstocks to optimize composting practices and fully harness BC\u0026apos;s benefits for agricultural applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Science Fund for Distinguished Young Scholars of Anhui Province (2022AH030145, gxyqZD2021126), and the Anhui Provincial University Student Innovation and Entrepreneurship Training Program (2023cxcysj182)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNennen Zhu\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e writing-original draft, formal analysis, methodology. \u003cstrong\u003eAo Chen\u003c/strong\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e data curation, software, formal analysis. \u003cstrong\u003eXufang Yu:\u003c/strong\u003e methodology, supervision, writing-review and editing. \u003cstrong\u003eXiujuan\u003c/strong\u003e\u003cstrong\u003eZhou\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e investigation. \u003cstrong\u003eXiaolong Liu:\u003c/strong\u003e investigation, software, data curation. \u003cstrong\u003eWenchao Ji:\u003c/strong\u003e data curation. \u003cstrong\u003eTao Cao:\u0026nbsp;\u003c/strong\u003edata curation, software, formal analysis. \u003cstrong\u003eXingjun Fan:\u003c/strong\u003e funding acquisition, writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and Consent to participate\u003c/em\u003e Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent to publication\u003c/em\u003e Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e The authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbott, L.K., Macdonald, L.M., Wong, M.T.F., Webb, M.J., Jenkins, S.N., Farrell, M., 2018. 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Journal of Hazardous Materials 420, 126532.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"compost, biochar amendments, humic substances, spectroscopic properties, molecular structure","lastPublishedDoi":"10.21203/rs.3.rs-5509938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5509938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the effects of biochar (BC) amendments at varying levels (0%, 1%, 2%, and 5%) on the molecular and structural characteristics of humic substances (HS), specifically humic acids (HA) and fulvic acids (FA), in compost. The addition of BC significantly increased the HA content by 13-16% while reducing the FA content by 12-32%, with the most pronounced effects observed at a 5% BC addition. Spectroscopic analyses, including UV-Vis and fluorescence, revealed enhanced aromaticity and molecular weight (MW) of HA, driven by microbial activity and polymerization facilitated by BC amendments, whereas FA aromaticity remained largely unchanged. The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of HA increased significantly, indicating improved humification and stabilization. Fourier-transform infrared (FTIR) spectroscopy showed that BC addition had minimal impact on the functional group composition of HA and FA but altered the proportions of carbon types. Electron paramagnetic resonance (EPR) analysis demonstrated higher persistent free radical (PFR) levels in HA, correlating with increased molecular size and a higher degree of condensation. These findings underscore the potential of BC amendments to enhance compost quality by promoting the formation of stable and complex humic substances with improved reactivity.\u003c/p\u003e","manuscriptTitle":"Spectroscopic and molecular insights into humic substances in compost amended with varying biochar levels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 07:22:10","doi":"10.21203/rs.3.rs-5509938/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46c29340-b6a3-4415-9c41-ac22501e05a8","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-24T13:11:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 07:22:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5509938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5509938","identity":"rs-5509938","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}