Influence of biochar amendment on the binding characteristics of dissolved organic matter from chicken manure compost with Cu(II): an integrated analysis using EEM-PARAFAC, 2D-COS, and HPSEC

Influence of biochar amendment on the binding characteristics of dissolved organic matter from chicken manure compost with Cu(II): an integrated analysis using EEM-PARAFAC, 2D-COS, and HPSEC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of biochar amendment on the binding characteristics of dissolved organic matter from chicken manure compost with Cu(II): an integrated analysis using EEM-PARAFAC, 2D-COS, and HPSEC Xiaolong Liu, Ao Cheng, Xufang Yu, Nan Zhang, Ting Li, Dan Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4900729/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Dec, 2024 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract The addition of biochar (BC) during composting significantly influences the concentration and chemical structure of dissolved organic matters (DOM), thereby affecting its binding properties with heavy metals (HMs). This study examines the co-composting of chicken manure with varying BC dosages, specifically 0 wt% (CK), 1 wt% (T1), and 2 wt% (T2), and comparatively analyzes the binding characteristics between compost-derived DOM and copper ions (Cu(II)). EEM-PARAFAC analysis identified three primary components in the DOM: fulvic acid-like (C1), humic acid-like (C2), and protein-like (C3) components, each exhibiting distinct Cu(II) binding properties. The complexation constants (logK) for these components with Cu(II) increased with the BC addition, whereas the fraction of bound sites (f values) decreased, indicating that BC-amended compost DOM forms more stable DOM-Cu(II) complexes but with fewer available binding sites. 2D-COS analysis of synchronous fluorescence spectra revealed that the fulvic-like peaks were more sensitive to Cu(II) concentration variations across all DOM types, while humic-like substances exhibited a stronger preference for Cu(II) binding. HPSEC results showed broad molecular weight (MW) distributions (100–300,000 Da) in BC-amended compost DOM, with a significant decrease in medium MW regions (1000-30,000 Da) as Cu(II) concentration increased, suggesting that medium MW DOM fractions are particularly sensitive to Cu(II) binding, leading to higher weight-averaged MW values with increased Cu(II) concentrations. This study provides new insights into how BC addition to compost can influence the binding behavior of DOM with HMs, highlighting its potential for enhancing HMs immobilization in compost-amended soils. biochar-amended compost dissolved organic matter copper binding EEM-PARAFAC 2DCOS HPSEC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Composting is widely regarded as an efficient and straightforward technology for treating livestock and poultry manure, which can transform organic residues into nutrient-rich fertilizer (He et al., 2019 ; Yin et al., 2023 ; Zhao et al., 2020 ). Dissolved organic matter (DOM) is a crucial chemical component in compost products, readily released into soil ecosystems upon compost application (Beiyuan et al., 2018 ; Sun et al., 2017 ). Compost-derived DOM primarily consists of proteins, fulvic-like and humic-like substances, rich in numerous active functional groups (i.e., phenolic and carboxylic groups) (Sun et al., 2022 ; Xiao et al., 2019 ). These components are essential in modulating the speciation, bioavailability and fate of heavy metals (HMs) within soil environments (Guo et al., 2020 ; Zhu et al., 2021 ). The molecular structure and composition of DOM are pivotal in determining its binding properties toward HMs (Cui et al., 2020 ; Cui et al., 2024 ; Wang et al., 2024 ). For instance, Guo et al. ( 2019 ) investigated the complexation properties of DOM in municipal solid waste compost with Cu(II), and found that the protein-like fluorescence components are more likely to bind Cu(II) than humic-like components (Guo et al., 2019 ). Zhu et al. ( 2021 ) studied the binding behavior of DOM from various livestock manure (i.e., chicken, cow, and pig manure) composts with Cu(II), Pb(II), and Cd(II), demonstrating that chicken manure compost-derived DOM, predominantly due to its higher aromaticity, molecular weight and fulvic-like component, exhibited the greatest affinity for HMs (Zhu et al., 2021 ). Moreover, biochar (BC), as a compost amendment, has been documented to accelerate the composting process, enhance the degree of compost humification, and alter the chemical composition and structure of compost DOM (Song et al., 2023 ; Xie et al., 2023 ). For example, Yang et al. ( 2020 ) found that adding BC to compost can further promote the formation of aromatic structures and the conversion of organic carbon to carboxylic C (Yang et al., 2020 ). Xie et al. ( 2023 ) reported a reduction in aromatic proteins in compost DOM caused by BC amendment (Xie et al., 2023 ). Notably, these functional groups and structural components have been identified as important active sites for DOM interactions with HMs (Huang et al., 2018 ). Consequently, BC incorporation may further influence the binding behavior of compost-derived DOM with HMs (Wei et al., 2020 ; Xie et al., 2023 ). For example, Lee et al. ( 2020 ) demonstrated that BC amendments enhance the conjugational structures and humification degree of compost-derived humic substances (HS), thereby increasing the stability constants for Cu(II) binding in BC-amended composts relative to those without (Lee et al., 2020 ). Furthermore, many previous studies have shown that co-composting of organic waste with varying levels of BC results in distinct changes in the chemical composition and structural characteristics of compost-derived DOM (Song et al., 2023 ; Yang et al., 2020 ). For example, Manu et al. ( 2021 ) compared co-composting of food waste digestate with tobacco BC at levels of 0%, 2.5%, 5%, and 10%, finding that increasing BC levels led to higher aromatic species and humic substances in DOM (Manu et al., 2021 ). However, the subsequent binding properties of DOM derived from compost amended with varying BC levels towards HMs remain poorly understood. The coupling application of modern spectroscopy and multivariate chemometric methods provides a promising approach to deeply interpret the binding behavior of DOM with HMs (Cui et al., 2024 ; Xu et al., 2018 ). Fluorescence excitation-emission matrix (EEM) spectroscopy combined with quenching analysis has been proven to be a reliable method to explore the interaction characteristics between DOM and HMs (Huang et al., 2018 ; Lee et al., 2020 ; Liu et al., 2022a ). Specifically, EEM coupled with parallel factor analysis (EEM-PARAFAC) could further display the interactions of individual fluorescent components, including protein-like, fulvic-like and humic-like substance, with metal ions (Huang et al., 2018 ; Zhu et al., 2021 ). Two-dimensional correlation spectroscopy (2DCOS) analysis, owing to its high sensitivity and selectivity, can identify the heterogeneity of binding sites of DOM with metal ions in terms of subtle variations in optical spectra (i.e., UV-vis, synchronous fluorescence (SF), FTIR), and reveal the sequential order and interaction degree of active components in DOM with metal ions (Cui et al., 2022 ; Liu et al., 2022a ; Xu et al., 2018 ). Recently, high-performance size exclusion chromatography (HPSEC) has been employed to unravel the heterogeneous structures of DOM in terms of molecular size and can further explore the interactions of different molecular size fractions in DOM with metal ions (Li et al., 2022 ; Yang et al., 2024 ). In this study, a representative metal ion (i.e., Cu(II)) and three types of DOM from compost (without (CK) and with 1% (T1) and 2% (T2) BC amendment) were selected to explore the effect of BC addition on the binding behavior of compost-derived DOM with HMs. The EEM-PARAFAC, 2DCOS on SF spectra, and HPSEC were employed to evaluate the binding characteristics of CK, T1, and T2 compost-derived DOM with Cu(II). This study could provide deep insight into the binding characteristics between BC-amended compost-derived DOM and HMs, offering a critical scientific basis for evaluating the contamination control potential of BC-amended compost applied to HM-contaminated soils. 2. Materials and Methods 2.1. Compost sample preparation and DOM extraction Three compost samples with and without BC amendment were collected from a full-scale composting plant in Fuyang, Anhui province, China. The composting process involved the thoroughly mixing of chicken manure and rice husk, which were the predominant raw materials obtained from local farms. The rice husk BC was purchased from a full-scale environmental technology company in Henan province, China. This BC material was prepared at 800 o C with a pyrolysis duration of 4.0 h. The physical and chemical properties of raw materials and BC are listed in Table S1 in the supporting information. For the experiments, the chicken manure and rice husk were initially combined on a dry weight basis in a 3:1 ratio. Approximately 5 tons of pre-composting materials were mechanically mixed with different amounts of rice husk BC to establish three composting treatments: a control group without BC addition (CK), a group with 1% BC addition (T1), and a group with 2% BC addition (T2). The percentage values represent the amount of BC added based on the dry weight of the composting materials. The resulting mixture was then loaded into a designated composting compartment, and adjustments made to attain an initial water content of around 60% and a C/N ratio of approximately 25. The experiments were conducted over 85 days, during which the compost was regularly turned to facilitate the process. Upon completion, at least five sub-samples were collected from different regions within the heap, specifically at a depth of 40–60 cm. These sub-samples were combined to create a composite sample for each compost treatment. The seed germination index of all three composts exceeded 90%, indicating their good maturation for further usage (Ji et al., 2023 ; Kong et al., 2022 ; Yang et al., 2021b ). The compost samples underwent freeze-drying, mechanical crushing, and sieving through a 100-mesh sieve. The extraction of DOM from compost was carried out using a horizontal shaker with ultrapure water at a solid/liquid ratio of 1:10 (w/v) for 24 hours at 25 o C and 150 r/min. The suspensions were subsequently centrifuged at 8,000 rpm for 20 minutes and filtered through a 0.22 µm filter membrane. The concentration of dissolved organic carbon (DOC) was determined utilizing a TOC-VCPN analyzer (Shimadzu, Japan). All DOM samples were stored at 4 o C until further analysis and treatment. 2.2. Cu(II) titration experiment The background concentrations of Cu within composts were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES; Optima 7000 DV, Perkin Elmer, Waltham MA, USA), yielding values of 52.4, 48.5 and 47.7 mg/kg for CK, T1 and T2, respectively. These concentrations were lower than 39.7–69.3 mg/kg range reported for manure composts in previous studies (Liu et al., 2022b ; Luo et al., 2024 ). Before the titration experiment, the DOM samples were diluted to a concentration of 10 mg/L DOC using ultrapure water to minimize the influence of inner-filter effects. The pH of sample solution was adjusted at 6.0 by addition 0.1 M NaOH and 0.1 M HCl as necessary. Then, 20 mL of each sample was transferred into a series of brown sealed vials. To initiate the titration, 20 µL of a stock solution of CuCl 2 with concentrations ranging from 0 to 100 mmol/L was added to each vial, resulting in final Cu(II) concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 µmol/L. To ensure coordination equilibrium, all solutions were continuously shaken on an oscillator at 250 rpm and 25°C for 24 hours. 2.3. Fluorescence spectroscopy Synchronous fluorescence (SF) and excitation-emission matrix fluorescence (EEM) spectra were acquired using a Hitachi F-4600 fluorescence spectrometer. For SF measurements, both excitation and emission slits were adjusted to 5 nm, with excitation wavelengths ranging from 250 to 550 nm in 1 nm increments. The scanning speed was set to 1,200 nm/min, with a constant offset (Δλ) of 60 nm. For EEM determination, excitation wavelengths (Ex) ranged from 200 to 400 nm, and emission wavelengths (Em) were set from 290 to 520 nm, with both Ex and Em measured at 5 nm intervals. The scanning speed was maintained at 2400 nm/min. 2.4. High-performance size exclusion chromatography (HPSEC) The molecular size distributions of DOM-Cu(II) complex were analyzed using a Shimadzu high-performance liquid chromatography system, equipped with a refractive index detector (RID, Shimadzu) and a diode array detector (DAD, SPD-6A, Shimadzu). The SEC column used was a Polysep-GFC-P3000 column (Phenomenex, USA) with a Polysep-GFC-P guard column (Phenomenex, USA). The DAD detector wavelength was fixed at 254 nm. The mobile phase consisted of a methanol: ultrapure water mixture (1:9) containing 25 mmol/L ammonium acetate. The flow rate was set at 1 mL/min, and the sample volume injected was 100 µL. Eight polyethylene glycol salt standards (PEG) standards with molecular weights (MW) of 238, 601, 1020, 3450, 4080, 11100, 17900 and 41300 Da were used for molecular size analysis. A good linear correlation between the retention times and the logMW was observed for the calibration curve (R 2 = 0.9937, Fig. S1). The apparent molecular size of DOM-Cu(II) samples was calculated from the elution time using the calibration curve. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (ρ) of DOM-Cu complex were calculated using the following equations (Ignatev and Tuhkanen, 2019 ): \(\:{M}_{n}=\sum\:_{i=1}^{n}{h}_{i}/\sum\:_{i=1}^{n}({h}_{i}/{M}_{i})\) (1) \(\:{M}_{w}=\sum\:_{i=1}^{n}\left({h}_{i}{M}_{i}\right)/\sum\:_{i=1}^{n}{h}_{i}\) (2) \(\:\rho\:={M}_{w}/{M}_{n}\) (3) where, n represents the number of detector responses, hi corresponds to the response value of the detection curve at elution time i , and Mi denotes the relative molecular weight of the molecule eluted at time i . 2.5. 2D-COS Analysis 2D-COS analysis of SF spectra for DOM-Cu(II) complexes was conducted using the 2D Correlation Spectroscopy Analysis (v1.22) plug-in within Origin software (2021), with varying concentrations of Cu(II) serving as external perturbations. The 2D-COS generates two types of the maps: synchronous 2D and asynchronous 2D spectra. These maps were comprehensively interpreted according to Noda’s rule (Noda, 2016 ), which has been widely applied in many previous studies (Aftab and Hur, 2019 ; Guo et al., 2019 ). In brief, the sign of the synchronous cross-peaks indicates the direction of spectral changes. Asynchronous spectra provide information on the sequence of events associated with the external perturbation. If the signs of synchronous and asynchronous cross-peaks are consistent within given wavelength ranges, the spectral change along the x-axis precedes the change along the y-axis. Conversely, if they are dissimilar, the reaction proceeds in the opposite direction. 2.6. EEM-PARAFAC analysis The EEM fluorescence data was modeled using the PARAFAC, implemented through drEEM toolbox ( http://www.models.life.ku.dk/drEEM ) within Matlab software (Cuss et al., 2019 ; Hu et al., 2019 ). The PARAFAC components were identified and validated based on the random analysis, split-half validation and residual error. The resulting maximum fluorescence (Fmax) scores of each component do not represent actual concentrations but can be regarded as proportional to the true concentrations of the various components (Qian et al., 2019 ). 2.7. Determination of copper binding parameters The parameters related to the binding affinity and the relative abundance of the binding sites were calculated using the following modified Stern-Volmer equation (Lee and Hur, 2017 ): $$\:\frac{{F}_{0}}{{F}_{0}-F}=\frac{1}{f·K·\left[Cu\right]}+\frac{1}{f}$$ 4 where F 0 and F denote fluorescence intensity before and after Cu(II) titration, respectively. K represents the conditional stability constant, f indicates the percentage of fluorophores involved in Cu(II) complexation, and [Cu] denotes the Cu(II) concentration. 3. Results and Discussion 3.1. Binding behavior of DOM with Cu(II) using EEM-PARAFAC 3.1.1. EEM spectra with PARAFAC modeling The EEM spectra of DOM derived from three compost (CK, T1, and T2), with and without the addition of Cu(II) (100 µmol/L as an example), are displayed in Fig. S2. Two prominent peaks, Peak A and Peak B, were observed in all EEM spectra at Ex/Em wavelength pairs of 245/425 nm and 325/415 nm, respectively. These peaks could be attributed to fulvic acid-like and humic acid-like substances, respectively (Hu et al., 2019 ; Liu et al., 2018 ). This indicates that all DOM predominantly comprised humic-like substances, suggesting a high degree of compost humification and aromatization (He et al., 2019 ; Huang et al., 2021 ). Furthermore, BC addition did not alter the fluorescent composition of compost DOM, consistent with previous studies (Liu et al., 2023b ; Xie et al., 2023 ; Yang et al., 2020 ). The addition of Cu(II) resulted in significant decreases in fluorescence intensity, with reductions of 59% and 58% for Peak A and Peak B, respectively, indicating complexation-induced fluorescence quenching mainly caused by humic-like substances (Huang et al., 2018 ). PARAFAC analysis decomposed the EEM spectra of the DOM-Cu(II) complexes, identifying three fluorescent components. Their EEM contours and corresponding Ex/Em loadings are presented in Fig. S3. According to previous studies, component C1 (Ex/Em = 235(325)/410 nm) and component C2 (Ex/Em = 265(355)/460 nm) could be attributed to fulvic-like and humic-like substances, respectively (Liu et al., 2024 ; Wu et al., 2012 ; Xie et al., 2023 ). Component C3, with peaks at Ex/Em = 230(280)/355 nm, were likely attributable to protein-like fluorophores (Wang et al., 2024 ; Yang et al., 2021a ). These components were frequently identified as the basic constituents of the compost DOM reported in previous studies (Guo et al., 2019 ; Zhang et al., 2023 ). BC addition led to an increase in the relative abundance of fulvic-like substances (C1) from 40% (CK) to 42% (T1 and T2), while the relative abundance of protein-like substances (C3) decreased from 24% (CK) to 22% (T1 and T2) in compost DOM (Fig. S4). Similar changes were observed in DOM from swine manure compost with the addition of wheat straw BC (Yang et al., 2020 ). These findings suggested that BC addition facilitates the degradation of protein-like substances, potentially through microbial mediation, and promotes the formation or accumulation of humic matters during composting (Song et al., 2023 ). These compositional variations may contribute to distinct binding behaviors of DOM with Cu(II). 3.1.2. Fluorescence quenching curves of PARAFAC-derived fluorophores Figure 1 illustrates the fluorescence quenching curves of the PARAFAC-derived fluorophores in the three types of compost-derived DOM. It is evident that C1-C3 fractions exhibit consistent quenching patterns in response to Cu(II) addition. However, it is noteworthy that each fluorophore exhibited distinct quenching degree throughout the entire Cu(II) titration. In comparison, humic-like substances (C2) exhibit a more pronounced decrease (63–66%) in fluorescence intensity compared to the fulvic-like (C1) (51–55%) and protein-like (C3) (49–57%) substances within the compost DOM. The findings suggest that humic-like fluorophores possess a strong affinity for binding with Cu(II), likely due to their abundance of oxygen-containing functional groups (i.e., phenolic and carboxylic groups) (Lee et al., 2020 ; Xu et al., 2021 ). Moreover, it is observed that the quenching effect of fluorescent components, especially for C2 and C3, is alleviated with increased BC addition during the composting process. This phenomenon can be attributed to the reduction in the presence of active fluorophores within the compost-derived DOM that are responsible for binding to HMs, as a result of BC amendment. Previous studies have consistently demonstrated that the BC amendment stimulates the conversion of precursors of humic substances, such as polyphenols and amino acids, into stable and insoluble forms of humic matter forms in compost (Lee et al., 2020 ; Lee et al., 2018 ; Lu et al., 2018 ). These precursors, widely recognized as the primary active components responsible for HMs binding, undergo a transformation process that ultimately leads to a decrease in the quenching effect observed for compost-derived DOM. The logarithmic stability constants (logK) and the relative fraction of the fluorophores acting as the binding sites ( f ) for PARAFAC-derived fluorophores within the three types of compost DOM were determined using the modified Stern-Volmer model (Fig. S5). The corresponding parameters are listed in Table 1 . The logK values for all fluorescent components ranged from 4.29 to 4.61, which fall within the reasonable ranges (3.87–6.08) for compost DOM reported in previous studies (Huang et al., 2018 ; Li et al., 2021 ; Zhu et al., 2021 ). As shown in Table 1 , humic-like substances (C2) generally exhibited larger logK values (4.49–4.61) than fulvic-like (C1) and protein-like (C3) substances (4.30–4.54 and 4.29–4.55, respectively), across the three compost DOM. This suggested that humic-like substances are capable of forming more stable complexes with Cu(II), likely attributable to their abundance of acidic functional groups (e.g., carboxylic groups and phenols) (Yuan et al., 2015 ). Notably, among the three types of compost DOM, the logK values for each fluorescent component follow a decreasing tendency of T2 > T1 > CK. Contrary to the trend of binding constants, the f values of compost DOM gradually decreased after the BC addition, with each fluorescence component showing a decreasing trend of CK > T1 > T2. This suggests that the BC addition leads to a decrease in the fraction of the binding sites within compost DOM available for Cu(II), which might be attributed to the intensified humification of DOM induced by BC addition, consequently limiting the number of metal binding sites. These findings are consistent with a previous study by Lee et al. ( 2020 ), which documented an increase in logK and a decrease in the f value of compost-derived HS fluorescence quenching due to Cu(II) binding after the BC addition (Lee et al., 2020 ). These findings imply that the BC addition results in a more stable compost with an increased ability to bind Cu(II) but with reduced binding sites. Table 1 Binding parameters of fluorescence components within compost DOM with Cu(II). Component log K f R² CK C1 4.30 0.86 0.990 C2 4.49 0.91 0.992 C3 4.29 0.98 0.989 T1 C1 4.44 0.80 0.988 C2 4.60 0.85 0.991 C3 4.47 0.79 0.992 T2 C1 4.54 0.70 0.988 C2 4.61 0.83 0.982 C3 4.55 0.66 0.990 3.2. Binding characteristics of DOM with Cu(II) using 2D-SF-COS The changes in SF spectra of DOM derived from three compost products upon the addition of Cu(II) are shown in Fig. 2 . Three distinct fluorescence regions, within the wavelength ranges of 250–300, 300–380, and 380–520 nm, correspond to protein-like, fulvic-like, and humic-like fluorescence fractions, respectively (Chen et al., 2015 ; Fan et al., 2019 ; Wang et al., 2018 ). Notably, all spectra exhibit pronounced fulvic-like peaks (~ 335 nm) alongside weak protein-like peaks (~ 280 nm), indicative of the prevalent presence of fulvic-like fractions in three compost DOM. With the increase in Cu(II) concentration, a heightened degree of fluorescence quenching across all fractions was observed. Specially, the extents of fluorescence quenching were measured at 59.6%, 58.2% and 51.2% in the protein-like region for CK, T1 and T2 compost-derived DOM, respectively (Fig. 2 ). Similar quenching effects were noted at 54.9%, 55.3% and 53.3% in the fulvic-like region, respectively (Fig. 2 ). These results underscore the occurrence of electronic structural alterations within the fluorescence fractions due to complexes formation with Cu(II). To further elucidate the quenching behavior of compost DOM upon interaction with Cu(II), 2D-COS applied to SF spectra was employed. Figure 3 shows the corresponding synchronous and asynchronous maps for various compost DOM-Cu(II) complexes. The synchronous maps for compost DOM with and without BC addition generally exhibited a similar feature, with one predominant auto-peak centered at 350 nm, accompanied by a small peak at 275 nm. The greater intensity of the peaks at 350 nm compared to those at 275 suggest that the fluorescence of fulvic-like fractions was more susceptible to Cu(II) addition than that of protein-like fractions. Similarly, Cui et al. ( 2020 ) also found that the fluorescence of fulvic-like fractions in compost-derived DOM was more sensitive to Hg(II) than that of protein-like fractions (Cui et al., 2020 ). Moreover, all the cross-peaks exhibited positive values, indicating that the spectral changes proceeded in the same direction as the variation of the Cu(II) concentration. The asynchronous maps provided additional insightful details regarding the sequential relationship among different potential active fractions during Cu(II) binding. As shown in Fig. 3 d-f, a negative cross-peak was discernible at 350/310 nm, while two positive cross peaks emerge at 400/350 and 400/310 nm, situated at the lower right of the diagonal line for compost DOM with and without BC addition. According to Noda's rule (Huang et al., 2020 ; Noda, 2016 ), the DOM binding with Cu(II) followed the sequence of 400 nm→310 nm→350 nm, suggesting that Cu(II) binds to DOM fractions in the order of humic-like fraction → small fulvic-like fraction → large fulvic-like fraction. Lee et al. ( 2020 ) also showed that the efficacy of Cu binding in compost-derived HS may be greater for fulvic-like fluorophores as compared to humic-like fluorophores irrespective (Lee et al., 2020 ). In contrast, Huang et al ( 2020 ) found that fulvic-like substances within compost-derived DOM exhibited a faster response to Cu(II) complexation than humic-like substances (Huang et al., 2020 ). These findings suggest that the reactive sites within compost-derived DOM may vary depending on feedstocks and composting processes. 3.3. Changes in molecular size distributions of DOM binding with Cu(II) The variations in HPSEC chromatograms of compost DOM upon the addition of Cu(II) are shown in Fig. 4 . It is evident that all HPSEC chromatograms displayed MW ranges from 100 to 300,000 Da, characterized by three distinct MW regions: 100–1000, 1000-30,000, and 30,000-200,000 Da. This indicates that compost DOM encompasses a continuous and broad spectrum of MWs, primarily comprising three clusters of MW species (i.e., low, medium, and large MW species). This pattern is consistent with findings from previous studies on DOM derived from various compost (i.e., chicken manure, garden, municipal solid waste) (Yu et al., 2022 ; Zhu et al., 2020 ), which show that compost DOM is a complex mixture of substances with varying molecular weights (Yuan et al., 2019 ). With increasing concentrations of Cu(II), there is a significant reduction in intensity of chromatograms, particularly within the medium MW region (1000-30,000 Da). This suggests the complexation of Cu (II) with compost DOM, especially with medium MW species, highlighting their significant role in complexation with Cu(II). Notably, substantial changes in chromatograms for the three types of DOM occur between 20 and 40 µmol/L of Cu(II), indicative of significant structural transformations in compost DOM due to intensified complexation. These results also suggest that different MW species play distinct roles in HMs complexation. Liu et al. ( 2023a ) revealed that Cu(II) binding with DOM from various sources (i.e., sea, river, and wetland waters) is MW-dependent, with stronger binding ability for low MW fractions in DOM (Liu et al., 2023a ). Given the differences in chemical composition and properties between aqueous and compost DOM, the distribution and chemical properties of MW species differ, affecting their binding properties. Further studies on the MW-dependent binding behavior of compost DOM are warranted. The variations in Mw of compost DOM-Cu(II) complexes were also examined. As shown in Fig. 5 , the Mw of all samples displayed an overall increasing trend with rising Cu(II) concentration, suggesting that Cu(II) binding can enlarger the molecular size of DOM. A significant increase in Mw was observed at low concentrations (0–10 µmol/L), followed by a slower increase. This trend suggests that Cu(II) binding to compost DOM enhances their molecular size. Specially, the Mw of CK DOM-Cu(II) complexes generally surpasses that of T1, followed by T2. These findings suggest that BC addition induces changes in the MW of compost DOM, but does not affect the complexation of Cu(II). 3.4. Environmental implications Currently, the incorporation of BC into composting is vigorously promoted as an optimal method for managing livestock manure within sustainable agriculture systems (Hagemann et al., 2018 ; Yang et al., 2020 ; Yin et al., 2023 ). The findings of this study indicate that the amendment of compost with BC alters the chemical composition and structure of compost DOM, subsequently influencing its Cu(II) binding capacity and interaction dynamics. Typically, the BC addition fosters the maturation of compost, accelerating the accumulation of humic substances within the compost DOM. This study reveals that the humic-like substances exhibit a greater affinity for binding Cu(II) compared to protein-like substances. Moreover, the capacity of BC-amended compost DOM to bind Cu(II) exceeds that of compost DOM without BC addition. These results support the hypothesis that DOM derived from compost with higher levels of BC amendment possesses a more stable structure and an enhanced capacity to immobilize HMs (Li et al., 2021 ; Yin et al., 2023 ). This suggests that the appropriate addition of BC could serve as a beneficial additive in manure compost for the remediation of HM-contaminated soils. By improving the stability and binding capacity of compost DOM, BC amendments can play a crucial role in reducing the mobility and bioavailability of HMs in agricultural soils, thereby mitigating environmental contamination and enhancing soil health. 4. Conclusion In this study, the characteristics of DOM from chicken manure composts amended with varying levels of BC and their interactions with Cu(II) were investigated. The results showed that both the aromaticity and humification of DOM increased with higher BC additions, leading to distinct Cu(II) binding behaviors. Complexation modeling showed that all PARAFAC-derived fluorescence components (i.e., protein-like, fulvic acid-like, and humic acid-like component) were capable of binding Cu(II), with logK values rising and f values decreasing as BC levels increased. 2D-COS analysis of SF indicated a binding sequence of humic-like > fulvic-like substances. BC additions enhanced compost maturation and Cu(II) binding ability, although higher BC levels led to a more stable but less reactive DOM configuration. HPSEC results further showed structural changes with increased Cu(II) concentrations, marked by reduced medium MW intensity and larger molecular size. Overall, BC amendments improve DOM humification and Cu(II) binding, highlighting BC’s potential for enhancing HM stabilization in agricultural soils. Declarations Funding This study was supported by the Science Fund for Distinguished Young Scholars of Anhui Province (2022AH030145, gxyqZD2021126), Provincial-level University Student Innovation and Entrepreneurship Program (S202310879283). Author Contributions Xiaolong Liu: Writing-original draft, Formal analysis, Methodology. Ao Cheng: Investigation, Software, Data curation. Xufang Yu: Investigation, Data curation. Nan Zhang: Investigation, Data curation. Ting Li: Investigation. Dan Chen: Data curation. Wenchao Ji: Software, Formal analysis. Xingjun Fan: Methodology, Supervision, Funding acquisition, Writing-review & editing. Availability of data and materials All data generated or analyzed during this study in this article are available from the corresponding author on reasonable request. Ethics approval and Consent to participate Not applicable Consent to publication Not applicable Competing interests The authors declare that they have no competing interests. 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Xiao, X., Xi, B.-D., He, X.-S., Zhang, H., Li, D., Zhao, X.-Y., Zhang, X.-H., 2019. Hydrophobicity-dependent electron transfer capacities of dissolved organic matter derived from chicken manure compost. Chemosphere 222, 757-765. Xie, J., Xia, H., Guan, M., Huang, K., Chen, J., 2023. Accelerating the humification mechanism of dissolved organic matter using biochar during vermicomposting of dewatered sludge. Waste Management 159, 102-113. Xu, H., Yan, M., Li, W., Jiang, H., Guo, L., 2018. Dissolved organic matter binding with Pb(II) as characterized by differential spectra and 2D UV-FTIR heterospectral correlation analysis. Water Research 144, 435-443. Xu, X., Kang, J., Shen, J., Zhao, S., Wang, B., Zhang, X., Chen, Z., 2021. EEM-PARAFAC characterization of dissolved organic matter and its relationship with disinfection by-products formation potential in drinking water sources of northeastern China. Sci Total Environ 774, 145297. Yang, K., Zhang, Y., Dong, Y., Li, D., Li, W., 2021a. Metal binding by dissolved organic matter in hypersaline water: A size fractionation study using different isolation methods. Limnologica 87, 125849. Yang, K., Zhang, Y., Peng, J., Xu, H., Liu, X., Liu, H., Li, N., Guo, L., Li, W., 2024. Molecular weight-dependent differences in spectral properties and metal-binding behaviors of dissolved organic matter from different lakes. Science of The Total Environment 946, 174245. Yang, Y., Du, W., Cui, Z., Zhao, T., Wang, X., Lv, J., 2020. Spectroscopic characteristics of dissolved organic matter during pig manure composting with bean dregs and biochar amendments. Microchemical Journal 158, 105226. Yang, Y., Wang, G., Li, G., Ma, R., Kong, Y., Yuan, J., 2021b. Selection of sensitive seeds for evaluation of compost maturity with the seed germination index. Waste Management 136, 238-243. Yin, Y., Li, M., Tao, X., Yang, C., Zhang, W., Li, H., Zheng, Y., Wang, X., Chen, R., 2023. Biochar enhanced organic matter transformation during pig manure composting: Roles of the cellulase activity and fungal community. Journal of Environmental Management 333, 117464. Yu, X., Cheng, A., Chen, D., Li, T., Fan, X., Wang, X., Ji, W., Wang, J., Ren, L., 2022. Insight into the evolution characteristics on molecular weight of compost dissolved organic matters using high-performance size exclusion chromatography combined with a two-dimensional correlation analysis. Environmental Science and Pollution Research 30, 37197-37207. Yuan, D.-h., Guo, X.-j., Wen, L., He, L.-s., Wang, J.-g., Li, J.-q., 2015. Detection of Copper (II) and Cadmium (II) binding to dissolved organic matter from macrophyte decomposition by fluorescence excitation-emission matrix spectra combined with parallel factor analysis. Environmental Pollution 204, 152-160. Yuan, Y., Xi, B., He, X.-S., Tan, W., Zhang, H., Li, D., Yang, C., Zhao, X., 2019. Polarity and Molecular Weight of Compost-Derived Humic Acids Impact Bio-dechlorination of Pentachlorophenol. Journal of Agricultural and Food Chemistry 67, 4726-4733. Zhang, X., Li, Y., Cui, K., Zhang, X., Sun, Y., Wei, Z., 2023. Fate of dissolved organic matter affected by oxytetracycline tolerant microorganisms during chicken manure composting. Bioresour Technol 387, 129563. Zhao, S., Schmidt, S., Qin, W., Li, J., Li, G., Zhang, W., 2020. Towards the circular nitrogen economy - A global meta-analysis of composting technologies reveals much potential for mitigating nitrogen losses. Sci Total Environ 704, 135401. Zhu, L., Wei, Z., Yang, T., Zhao, X., Dang, Q., Chen, X., Wu, J., Zhao, Y., 2020. Core microorganisms promote the transformation of DOM fractions with different molecular weights to improve the stability during composting. Bioresource Technology 299, 122575. Zhu, Y., Jin, Y., Liu, X., Miao, T., Guan, Q., Yang, R., Qu, J., 2021. Insight into interactions of heavy metals with livestock manure compost-derived dissolved organic matter using EEM-PARAFAC and 2D-FTIR-COS analyses. J Hazard Mater 420, 126532. Supplementary Materials Supplementary Materials are not available with this version Cite Share Download PDF Status: Published Journal Publication published 14 Dec, 2024 Read the published version in Waste and Biomass Valorization → Version 1 posted Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 12 Nov, 2024 Editor invited by journal 12 Nov, 2024 Editor assigned by journal 12 Nov, 2024 First submitted to journal 11 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Units.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/4926d27eb4bda6159918d0f4.png"},{"id":68942780,"identity":"e4d0beda-b924-4ddb-a40d-552d2009b79f","added_by":"auto","created_at":"2024-11-13 18:27:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":216743,"visible":true,"origin":"","legend":"\u003cp\u003eVariations in synchronous fluorescence spectra of CK-DOM (a), T1-DOM (b) and T2-DOM (c) with varying concentrations of Cu(II).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/dd886a2a7543205e7736d5ed.png"},{"id":68942322,"identity":"444afa6f-d58c-4c49-b8f6-7167b10450fb","added_by":"auto","created_at":"2024-11-13 18:19:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":436236,"visible":true,"origin":"","legend":"\u003cp\u003eSynchronous (a,b,c) and asynchronous (d,e,f) 2DCOS maps generated from SF spectra of CK-DOM (a,d), T1-DOM (b,e) and T2-DOM (c,f) with increasing concentration of Cu(II).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/a7ecd0f80a37cd776596d88a.png"},{"id":68942321,"identity":"d8fccb0c-46b4-4231-872c-a57610bffc97","added_by":"auto","created_at":"2024-11-13 18:19:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210668,"visible":true,"origin":"","legend":"\u003cp\u003eVariations of the HPSEC chromatograms of DOM detected at UV254 with the increasing concentration of Cu(Ⅱ).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/3885b61f04f4d3c633cce36a.png"},{"id":68942320,"identity":"8d43e242-a482-430a-9d0d-c2b299f4a98b","added_by":"auto","created_at":"2024-11-13 18:19:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50161,"visible":true,"origin":"","legend":"\u003cp\u003eVariations of the weight-average molecular weight (Mw) of CK-DOM, T1-DOM and T2-DOM with increasing concentration of Cu(II).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/02f3ff81f355d6b0ca06af59.png"},{"id":71552552,"identity":"367d581d-d944-4640-812b-b065d79cd4dd","added_by":"auto","created_at":"2024-12-16 16:07:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1430332,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4900729/v1/f93d1351-988d-4580-9528-ca858f85fd70.pdf"}],"financialInterests":"","formattedTitle":"Influence of biochar amendment on the binding characteristics of dissolved organic matter from chicken manure compost with Cu(II): an integrated analysis using EEM-PARAFAC, 2D-COS, and HPSEC","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eComposting is widely regarded as an efficient and straightforward technology for treating livestock and poultry manure, which can transform organic residues into nutrient-rich fertilizer (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Dissolved organic matter (DOM) is a crucial chemical component in compost products, readily released into soil ecosystems upon compost application (Beiyuan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Compost-derived DOM primarily consists of proteins, fulvic-like and humic-like substances, rich in numerous active functional groups (i.e., phenolic and carboxylic groups) (Sun et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These components are essential in modulating the speciation, bioavailability and fate of heavy metals (HMs) within soil environments (Guo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe molecular structure and composition of DOM are pivotal in determining its binding properties toward HMs (Cui et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cui et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For instance, Guo et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) investigated the complexation properties of DOM in municipal solid waste compost with Cu(II), and found that the protein-like fluorescence components are more likely to bind Cu(II) than humic-like components (Guo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Zhu et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) studied the binding behavior of DOM from various livestock manure (i.e., chicken, cow, and pig manure) composts with Cu(II), Pb(II), and Cd(II), demonstrating that chicken manure compost-derived DOM, predominantly due to its higher aromaticity, molecular weight and fulvic-like component, exhibited the greatest affinity for HMs (Zhu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, biochar (BC), as a compost amendment, has been documented to accelerate the composting process, enhance the degree of compost humification, and alter the chemical composition and structure of compost DOM (Song et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, Yang et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that adding BC to compost can further promote the formation of aromatic structures and the conversion of organic carbon to carboxylic C (Yang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Xie et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported a reduction in aromatic proteins in compost DOM caused by BC amendment (Xie et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, these functional groups and structural components have been identified as important active sites for DOM interactions with HMs (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, BC incorporation may further influence the binding behavior of compost-derived DOM with HMs (Wei et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, Lee et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated that BC amendments enhance the conjugational structures and humification degree of compost-derived humic substances (HS), thereby increasing the stability constants for Cu(II) binding in BC-amended composts relative to those without (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, many previous studies have shown that co-composting of organic waste with varying levels of BC results in distinct changes in the chemical composition and structural characteristics of compost-derived DOM (Song et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, Manu et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) compared co-composting of food waste digestate with tobacco BC at levels of 0%, 2.5%, 5%, and 10%, finding that increasing BC levels led to higher aromatic species and humic substances in DOM (Manu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the subsequent binding properties of DOM derived from compost amended with varying BC levels towards HMs remain poorly understood.\u003c/p\u003e \u003cp\u003eThe coupling application of modern spectroscopy and multivariate chemometric methods provides a promising approach to deeply interpret the binding behavior of DOM with HMs (Cui et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Fluorescence excitation-emission matrix (EEM) spectroscopy combined with quenching analysis has been proven to be a reliable method to explore the interaction characteristics between DOM and HMs (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Specifically, EEM coupled with parallel factor analysis (EEM-PARAFAC) could further display the interactions of individual fluorescent components, including protein-like, fulvic-like and humic-like substance, with metal ions (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Two-dimensional correlation spectroscopy (2DCOS) analysis, owing to its high sensitivity and selectivity, can identify the heterogeneity of binding sites of DOM with metal ions in terms of subtle variations in optical spectra (i.e., UV-vis, synchronous fluorescence (SF), FTIR), and reveal the sequential order and interaction degree of active components in DOM with metal ions (Cui et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Recently, high-performance size exclusion chromatography (HPSEC) has been employed to unravel the heterogeneous structures of DOM in terms of molecular size and can further explore the interactions of different molecular size fractions in DOM with metal ions (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, a representative metal ion (i.e., Cu(II)) and three types of DOM from compost (without (CK) and with 1% (T1) and 2% (T2) BC amendment) were selected to explore the effect of BC addition on the binding behavior of compost-derived DOM with HMs. The EEM-PARAFAC, 2DCOS on SF spectra, and HPSEC were employed to evaluate the binding characteristics of CK, T1, and T2 compost-derived DOM with Cu(II). This study could provide deep insight into the binding characteristics between BC-amended compost-derived DOM and HMs, offering a critical scientific basis for evaluating the contamination control potential of BC-amended compost applied to HM-contaminated soils.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Compost sample preparation and DOM extraction\u003c/h2\u003e \u003cp\u003eThree compost samples with and without BC amendment were collected from a full-scale composting plant in Fuyang, Anhui province, China. The composting process involved the thoroughly mixing of chicken manure and rice husk, which were the predominant raw materials obtained from local farms. The rice husk BC was purchased from a full-scale environmental technology company in Henan province, China. This BC material was prepared at 800 \u003csup\u003eo\u003c/sup\u003eC with a pyrolysis duration of 4.0 h. The physical and chemical properties of raw materials and BC are listed in Table S1 in the supporting information. For the experiments, the chicken manure and rice husk were initially combined on a dry weight basis in a 3:1 ratio. Approximately 5 tons of pre-composting materials were mechanically mixed with different amounts of rice husk BC to establish three composting treatments: a control group without BC addition (CK), a group with 1% BC addition (T1), and a group with 2% BC addition (T2). The percentage values represent the amount of BC added based on the dry weight of the composting materials. The resulting mixture was then loaded into a designated composting compartment, and adjustments made to attain an initial water content of around 60% and a C/N ratio of approximately 25. The experiments were conducted over 85 days, during which the compost was regularly turned to facilitate the process. Upon completion, at least five sub-samples were collected from different regions within the heap, specifically at a depth of 40\u0026ndash;60 cm. These sub-samples were combined to create a composite sample for each compost treatment. The seed germination index of all three composts exceeded 90%, indicating their good maturation for further usage (Ji et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kong et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe compost samples underwent freeze-drying, mechanical crushing, and sieving through a 100-mesh sieve. The extraction of DOM from compost was carried out using a horizontal shaker with ultrapure water at a solid/liquid ratio of 1:10 (w/v) for 24 hours at 25 \u003csup\u003eo\u003c/sup\u003eC and 150 r/min. The suspensions were subsequently centrifuged at 8,000 rpm for 20 minutes and filtered through a 0.22 \u0026micro;m filter membrane. The concentration of dissolved organic carbon (DOC) was determined utilizing a TOC-VCPN analyzer (Shimadzu, Japan). All DOM samples were stored at 4 \u003csup\u003eo\u003c/sup\u003eC until further analysis and treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cu(II) titration experiment\u003c/h2\u003e \u003cp\u003eThe background concentrations of Cu within composts were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES; Optima 7000 DV, Perkin Elmer, Waltham MA, USA), yielding values of 52.4, 48.5 and 47.7 mg/kg for CK, T1 and T2, respectively. These concentrations were lower than 39.7\u0026ndash;69.3 mg/kg range reported for manure composts in previous studies (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBefore the titration experiment, the DOM samples were diluted to a concentration of 10 mg/L DOC using ultrapure water to minimize the influence of inner-filter effects. The pH of sample solution was adjusted at 6.0 by addition 0.1 M NaOH and 0.1 M HCl as necessary. Then, 20 mL of each sample was transferred into a series of brown sealed vials. To initiate the titration, 20 \u0026micro;L of a stock solution of CuCl\u003csub\u003e2\u003c/sub\u003e with concentrations ranging from 0 to 100 mmol/L was added to each vial, resulting in final Cu(II) concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 \u0026micro;mol/L. To ensure coordination equilibrium, all solutions were continuously shaken on an oscillator at 250 rpm and 25\u0026deg;C for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eSynchronous fluorescence (SF) and excitation-emission matrix fluorescence (EEM) spectra were acquired using a Hitachi F-4600 fluorescence spectrometer. For SF measurements, both excitation and emission slits were adjusted to 5 nm, with excitation wavelengths ranging from 250 to 550 nm in 1 nm increments. The scanning speed was set to 1,200 nm/min, with a constant offset (Δλ) of 60 nm. For EEM determination, excitation wavelengths (Ex) ranged from 200 to 400 nm, and emission wavelengths (Em) were set from 290 to 520 nm, with both Ex and Em measured at 5 nm intervals. The scanning speed was maintained at 2400 nm/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. High-performance size exclusion chromatography (HPSEC)\u003c/h2\u003e \u003cp\u003eThe molecular size distributions of DOM-Cu(II) complex were analyzed using a Shimadzu high-performance liquid chromatography system, equipped with a refractive index detector (RID, Shimadzu) and a diode array detector (DAD, SPD-6A, Shimadzu). The SEC column used was a Polysep-GFC-P3000 column (Phenomenex, USA) with a Polysep-GFC-P guard column (Phenomenex, USA). The DAD detector wavelength was fixed at 254 nm. The mobile phase consisted of a methanol: ultrapure water mixture (1:9) containing 25 mmol/L ammonium acetate. The flow rate was set at 1 mL/min, and the sample volume injected was 100 \u0026micro;L. Eight polyethylene glycol salt standards (PEG) standards with molecular weights (MW) of 238, 601, 1020, 3450, 4080, 11100, 17900 and 41300 Da were used for molecular size analysis. A good linear correlation between the retention times and the logMW was observed for the calibration curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9937, Fig. S1). The apparent molecular size of DOM-Cu(II) samples was calculated from the elution time using the calibration curve.\u003c/p\u003e \u003cp\u003eThe weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (ρ) of DOM-Cu complex were calculated using the following equations (Ignatev and Tuhkanen, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{M}_{n}=\\sum\\:_{i=1}^{n}{h}_{i}/\\sum\\:_{i=1}^{n}({h}_{i}/{M}_{i})\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{M}_{w}=\\sum\\:_{i=1}^{n}\\left({h}_{i}{M}_{i}\\right)/\\sum\\:_{i=1}^{n}{h}_{i}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\rho\\:={M}_{w}/{M}_{n}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere, \u003cem\u003en\u003c/em\u003e represents the number of detector responses, \u003cem\u003ehi\u003c/em\u003e corresponds to the response value of the detection curve at elution time \u003cem\u003ei\u003c/em\u003e, and \u003cem\u003eMi\u003c/em\u003e denotes the relative molecular weight of the molecule eluted at time \u003cem\u003ei\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. 2D-COS Analysis\u003c/h2\u003e \u003cp\u003e2D-COS analysis of SF spectra for DOM-Cu(II) complexes was conducted using the 2D Correlation Spectroscopy Analysis (v1.22) plug-in within Origin software (2021), with varying concentrations of Cu(II) serving as external perturbations. The 2D-COS generates two types of the maps: synchronous 2D and asynchronous 2D spectra. These maps were comprehensively interpreted according to Noda\u0026rsquo;s rule (Noda, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which has been widely applied in many previous studies (Aftab and Hur, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In brief, the sign of the synchronous cross-peaks indicates the direction of spectral changes. Asynchronous spectra provide information on the sequence of events associated with the external perturbation. If the signs of synchronous and asynchronous cross-peaks are consistent within given wavelength ranges, the spectral change along the x-axis precedes the change along the y-axis. Conversely, if they are dissimilar, the reaction proceeds in the opposite direction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. EEM-PARAFAC analysis\u003c/h2\u003e \u003cp\u003eThe EEM fluorescence data was modeled using the PARAFAC, implemented through drEEM toolbox (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.models.life.ku.dk/drEEM\u003c/span\u003e\u003cspan address=\"http://www.models.life.ku.dk/drEEM\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) within Matlab software (Cuss et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The PARAFAC components were identified and validated based on the random analysis, split-half validation and residual error. The resulting maximum fluorescence (Fmax) scores of each component do not represent actual concentrations but can be regarded as proportional to the true concentrations of the various components (Qian et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of copper binding parameters\u003c/h2\u003e \u003cp\u003eThe parameters related to the binding affinity and the relative abundance of the binding sites were calculated using the following modified Stern-Volmer equation (Lee and Hur, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\frac{{F}_{0}}{{F}_{0}-F}=\\frac{1}{f\u0026middot;K\u0026middot;\\left[Cu\\right]}+\\frac{1}{f}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eF\u003c/em\u003e denote fluorescence intensity before and after Cu(II) titration, respectively. \u003cem\u003eK\u003c/em\u003e represents the conditional stability constant, \u003cem\u003ef\u003c/em\u003e indicates the percentage of fluorophores involved in Cu(II) complexation, and [Cu] denotes the Cu(II) concentration.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Binding behavior of DOM with Cu(II) using EEM-PARAFAC\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. EEM spectra with PARAFAC modeling\u003c/h2\u003e \u003cp\u003eThe EEM spectra of DOM derived from three compost (CK, T1, and T2), with and without the addition of Cu(II) (100 \u0026micro;mol/L as an example), are displayed in Fig. S2. Two prominent peaks, Peak A and Peak B, were observed in all EEM spectra at Ex/Em wavelength pairs of 245/425 nm and 325/415 nm, respectively. These peaks could be attributed to fulvic acid-like and humic acid-like substances, respectively (Hu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This indicates that all DOM predominantly comprised humic-like substances, suggesting a high degree of compost humification and aromatization (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, BC addition did not alter the fluorescent composition of compost DOM, consistent with previous studies (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The addition of Cu(II) resulted in significant decreases in fluorescence intensity, with reductions of 59% and 58% for Peak A and Peak B, respectively, indicating complexation-induced fluorescence quenching mainly caused by humic-like substances (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePARAFAC analysis decomposed the EEM spectra of the DOM-Cu(II) complexes, identifying three fluorescent components. Their EEM contours and corresponding Ex/Em loadings are presented in Fig. S3. According to previous studies, component C1 (Ex/Em\u0026thinsp;=\u0026thinsp;235(325)/410 nm) and component C2 (Ex/Em\u0026thinsp;=\u0026thinsp;265(355)/460 nm) could be attributed to fulvic-like and humic-like substances, respectively (Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Component C3, with peaks at Ex/Em\u0026thinsp;=\u0026thinsp;230(280)/355 nm, were likely attributable to protein-like fluorophores (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). These components were frequently identified as the basic constituents of the compost DOM reported in previous studies (Guo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). BC addition led to an increase in the relative abundance of fulvic-like substances (C1) from 40% (CK) to 42% (T1 and T2), while the relative abundance of protein-like substances (C3) decreased from 24% (CK) to 22% (T1 and T2) in compost DOM (Fig. S4). Similar changes were observed in DOM from swine manure compost with the addition of wheat straw BC (Yang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings suggested that BC addition facilitates the degradation of protein-like substances, potentially through microbial mediation, and promotes the formation or accumulation of humic matters during composting (Song et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These compositional variations may contribute to distinct binding behaviors of DOM with Cu(II).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Fluorescence quenching curves of PARAFAC-derived fluorophores\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the fluorescence quenching curves of the PARAFAC-derived fluorophores in the three types of compost-derived DOM. It is evident that C1-C3 fractions exhibit consistent quenching patterns in response to Cu(II) addition. However, it is noteworthy that each fluorophore exhibited distinct quenching degree throughout the entire Cu(II) titration. In comparison, humic-like substances (C2) exhibit a more pronounced decrease (63\u0026ndash;66%) in fluorescence intensity compared to the fulvic-like (C1) (51\u0026ndash;55%) and protein-like (C3) (49\u0026ndash;57%) substances within the compost DOM. The findings suggest that humic-like fluorophores possess a strong affinity for binding with Cu(II), likely due to their abundance of oxygen-containing functional groups (i.e., phenolic and carboxylic groups) (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, it is observed that the quenching effect of fluorescent components, especially for C2 and C3, is alleviated with increased BC addition during the composting process. This phenomenon can be attributed to the reduction in the presence of active fluorophores within the compost-derived DOM that are responsible for binding to HMs, as a result of BC amendment. Previous studies have consistently demonstrated that the BC amendment stimulates the conversion of precursors of humic substances, such as polyphenols and amino acids, into stable and insoluble forms of humic matter forms in compost (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These precursors, widely recognized as the primary active components responsible for HMs binding, undergo a transformation process that ultimately leads to a decrease in the quenching effect observed for compost-derived DOM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cp\u003eThe logarithmic stability constants (logK) and the relative fraction of the fluorophores acting as the binding sites (\u003cem\u003ef\u003c/em\u003e) for PARAFAC-derived fluorophores within the three types of compost DOM were determined using the modified Stern-Volmer model (Fig. S5). The corresponding parameters are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The logK values for all fluorescent components ranged from 4.29 to 4.61, which fall within the reasonable ranges (3.87\u0026ndash;6.08) for compost DOM reported in previous studies (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, humic-like substances (C2) generally exhibited larger logK values (4.49\u0026ndash;4.61) than fulvic-like (C1) and protein-like (C3) substances (4.30\u0026ndash;4.54 and 4.29\u0026ndash;4.55, respectively), across the three compost DOM. This suggested that humic-like substances are capable of forming more stable complexes with Cu(II), likely attributable to their abundance of acidic functional groups (e.g., carboxylic groups and phenols) (Yuan et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Notably, among the three types of compost DOM, the logK values for each fluorescent component follow a decreasing tendency of T2\u0026thinsp;\u0026gt;\u0026thinsp;T1\u0026thinsp;\u0026gt;\u0026thinsp;CK.\u003c/p\u003e \u003cp\u003eContrary to the trend of binding constants, the \u003cem\u003ef\u003c/em\u003e values of compost DOM gradually decreased after the BC addition, with each fluorescence component showing a decreasing trend of CK\u0026thinsp;\u0026gt;\u0026thinsp;T1\u0026thinsp;\u0026gt;\u0026thinsp;T2. This suggests that the BC addition leads to a decrease in the fraction of the binding sites within compost DOM available for Cu(II), which might be attributed to the intensified humification of DOM induced by BC addition, consequently limiting the number of metal binding sites. These findings are consistent with a previous study by Lee et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which documented an increase in logK and a decrease in the \u003cem\u003ef\u003c/em\u003e value of compost-derived HS fluorescence quenching due to Cu(II) binding after the BC addition (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings imply that the BC addition results in a more stable compost with an increased ability to bind Cu(II) but with reduced binding sites.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding parameters of fluorescence components within compost DOM with Cu(II).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elog\u003cem\u003eK\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ef\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.992\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.989\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.991\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.992\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.982\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Binding characteristics of DOM with Cu(II) using 2D-SF-COS\u003c/h2\u003e \u003cp\u003eThe changes in SF spectra of DOM derived from three compost products upon the addition of Cu(II) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Three distinct fluorescence regions, within the wavelength ranges of 250\u0026ndash;300, 300\u0026ndash;380, and 380\u0026ndash;520 nm, correspond to protein-like, fulvic-like, and humic-like fluorescence fractions, respectively (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, all spectra exhibit pronounced fulvic-like peaks (~\u0026thinsp;335 nm) alongside weak protein-like peaks (~\u0026thinsp;280 nm), indicative of the prevalent presence of fulvic-like fractions in three compost DOM. With the increase in Cu(II) concentration, a heightened degree of fluorescence quenching across all fractions was observed. Specially, the extents of fluorescence quenching were measured at 59.6%, 58.2% and 51.2% in the protein-like region for CK, T1 and T2 compost-derived DOM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similar quenching effects were noted at 54.9%, 55.3% and 53.3% in the fulvic-like region, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results underscore the occurrence of electronic structural alterations within the fluorescence fractions due to complexes formation with Cu(II).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the quenching behavior of compost DOM upon interaction with Cu(II), 2D-COS applied to SF spectra was employed. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the corresponding synchronous and asynchronous maps for various compost DOM-Cu(II) complexes. The synchronous maps for compost DOM with and without BC addition generally exhibited a similar feature, with one predominant auto-peak centered at 350 nm, accompanied by a small peak at 275 nm. The greater intensity of the peaks at 350 nm compared to those at 275 suggest that the fluorescence of fulvic-like fractions was more susceptible to Cu(II) addition than that of protein-like fractions. Similarly, Cui et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also found that the fluorescence of fulvic-like fractions in compost-derived DOM was more sensitive to Hg(II) than that of protein-like fractions (Cui et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, all the cross-peaks exhibited positive values, indicating that the spectral changes proceeded in the same direction as the variation of the Cu(II) concentration.\u003c/p\u003e \u003cp\u003eThe asynchronous maps provided additional insightful details regarding the sequential relationship among different potential active fractions during Cu(II) binding. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f, a negative cross-peak was discernible at 350/310 nm, while two positive cross peaks emerge at 400/350 and 400/310 nm, situated at the lower right of the diagonal line for compost DOM with and without BC addition. According to Noda's rule (Huang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Noda, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the DOM binding with Cu(II) followed the sequence of 400 nm\u0026rarr;310 nm\u0026rarr;350 nm, suggesting that Cu(II) binds to DOM fractions in the order of humic-like fraction \u0026rarr; small fulvic-like fraction \u0026rarr; large fulvic-like fraction. Lee et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also showed that the efficacy of Cu binding in compost-derived HS may be greater for fulvic-like fluorophores as compared to humic-like fluorophores irrespective (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, Huang et al (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that fulvic-like substances within compost-derived DOM exhibited a faster response to Cu(II) complexation than humic-like substances (Huang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings suggest that the reactive sites within compost-derived DOM may vary depending on feedstocks and composting processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Changes in molecular size distributions of DOM binding with Cu(II)\u003c/h2\u003e \u003cp\u003eThe variations in HPSEC chromatograms of compost DOM upon the addition of Cu(II) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It is evident that all HPSEC chromatograms displayed MW ranges from 100 to 300,000 Da, characterized by three distinct MW regions: 100\u0026ndash;1000, 1000-30,000, and 30,000-200,000 Da. This indicates that compost DOM encompasses a continuous and broad spectrum of MWs, primarily comprising three clusters of MW species (i.e., low, medium, and large MW species). This pattern is consistent with findings from previous studies on DOM derived from various compost (i.e., chicken manure, garden, municipal solid waste) (Yu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which show that compost DOM is a complex mixture of substances with varying molecular weights (Yuan et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith increasing concentrations of Cu(II), there is a significant reduction in intensity of chromatograms, particularly within the medium MW region (1000-30,000 Da). This suggests the complexation of Cu (II) with compost DOM, especially with medium MW species, highlighting their significant role in complexation with Cu(II). Notably, substantial changes in chromatograms for the three types of DOM occur between 20 and 40 \u0026micro;mol/L of Cu(II), indicative of significant structural transformations in compost DOM due to intensified complexation. These results also suggest that different MW species play distinct roles in HMs complexation. Liu et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) revealed that Cu(II) binding with DOM from various sources (i.e., sea, river, and wetland waters) is MW-dependent, with stronger binding ability for low MW fractions in DOM (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Given the differences in chemical composition and properties between aqueous and compost DOM, the distribution and chemical properties of MW species differ, affecting their binding properties. Further studies on the MW-dependent binding behavior of compost DOM are warranted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe variations in Mw of compost DOM-Cu(II) complexes were also examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the Mw of all samples displayed an overall increasing trend with rising Cu(II) concentration, suggesting that Cu(II) binding can enlarger the molecular size of DOM. A significant increase in Mw was observed at low concentrations (0\u0026ndash;10 \u0026micro;mol/L), followed by a slower increase. This trend suggests that Cu(II) binding to compost DOM enhances their molecular size. Specially, the Mw of CK DOM-Cu(II) complexes generally surpasses that of T1, followed by T2. These findings suggest that BC addition induces changes in the MW of compost DOM, but does not affect the complexation of Cu(II).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Environmental implications\u003c/h2\u003e \u003cp\u003eCurrently, the incorporation of BC into composting is vigorously promoted as an optimal method for managing livestock manure within sustainable agriculture systems (Hagemann et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The findings of this study indicate that the amendment of compost with BC alters the chemical composition and structure of compost DOM, subsequently influencing its Cu(II) binding capacity and interaction dynamics. Typically, the BC addition fosters the maturation of compost, accelerating the accumulation of humic substances within the compost DOM. This study reveals that the humic-like substances exhibit a greater affinity for binding Cu(II) compared to protein-like substances. Moreover, the capacity of BC-amended compost DOM to bind Cu(II) exceeds that of compost DOM without BC addition. These results support the hypothesis that DOM derived from compost with higher levels of BC amendment possesses a more stable structure and an enhanced capacity to immobilize HMs (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This suggests that the appropriate addition of BC could serve as a beneficial additive in manure compost for the remediation of HM-contaminated soils. By improving the stability and binding capacity of compost DOM, BC amendments can play a crucial role in reducing the mobility and bioavailability of HMs in agricultural soils, thereby mitigating environmental contamination and enhancing soil health.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, the characteristics of DOM from chicken manure composts amended with varying levels of BC and their interactions with Cu(II) were investigated. The results showed that both the aromaticity and humification of DOM increased with higher BC additions, leading to distinct Cu(II) binding behaviors. Complexation modeling showed that all PARAFAC-derived fluorescence components (i.e., protein-like, fulvic acid-like, and humic acid-like component) were capable of binding Cu(II), with logK values rising and f values decreasing as BC levels increased. 2D-COS analysis of SF indicated a binding sequence of humic-like \u0026gt;\u0026thinsp;fulvic-like substances. BC additions enhanced compost maturation and Cu(II) binding ability, although higher BC levels led to a more stable but less reactive DOM configuration. HPSEC results further showed structural changes with increased Cu(II) concentrations, marked by reduced medium MW intensity and larger molecular size. Overall, BC amendments improve DOM humification and Cu(II) binding, highlighting BC\u0026rsquo;s potential for enhancing HM stabilization in agricultural soils.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\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), Provincial-level University Student Innovation and Entrepreneurship Program (S202310879283).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXiaolong Liu:\u0026nbsp;\u003c/strong\u003eWriting-original draft, Formal analysis, Methodology. \u003cstrong\u003eAo Cheng:\u003c/strong\u003e Investigation, Software, Data curation. \u003cstrong\u003eXufang Yu:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eNan Zhang:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eTing Li:\u003c/strong\u003e Investigation. \u003cstrong\u003eDan Chen:\u0026nbsp;\u003c/strong\u003eData curation. \u003cstrong\u003eWenchao Ji:\u0026nbsp;\u003c/strong\u003eSoftware, Formal analysis. \u003cstrong\u003eXingjun Fan:\u003c/strong\u003e Methodology, Supervision, Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study in this article are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics approval and Consent to participate\u003c/em\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent to publication\u003c/em\u003e Not applicable\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\u003eAftab, B., Hur, J., 2019. 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J Hazard Mater 420, 126532.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Materials","content":"\u003cp\u003eSupplementary Materials are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"biochar-amended compost, dissolved organic matter, copper binding, EEM-PARAFAC, 2DCOS, HPSEC","lastPublishedDoi":"10.21203/rs.3.rs-4900729/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4900729/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe addition of biochar (BC) during composting significantly influences the concentration and chemical structure of dissolved organic matters (DOM), thereby affecting its binding properties with heavy metals (HMs). This study examines the co-composting of chicken manure with varying BC dosages, specifically 0 wt% (CK), 1 wt% (T1), and 2 wt% (T2), and comparatively analyzes the binding characteristics between compost-derived DOM and copper ions (Cu(II)). EEM-PARAFAC analysis identified three primary components in the DOM: fulvic acid-like (C1), humic acid-like (C2), and protein-like (C3) components, each exhibiting distinct Cu(II) binding properties. The complexation constants (logK) for these components with Cu(II) increased with the BC addition, whereas the fraction of bound sites (f values) decreased, indicating that BC-amended compost DOM forms more stable DOM-Cu(II) complexes but with fewer available binding sites. 2D-COS analysis of synchronous fluorescence spectra revealed that the fulvic-like peaks were more sensitive to Cu(II) concentration variations across all DOM types, while humic-like substances exhibited a stronger preference for Cu(II) binding. HPSEC results showed broad molecular weight (MW) distributions (100\u0026ndash;300,000 Da) in BC-amended compost DOM, with a significant decrease in medium MW regions (1000-30,000 Da) as Cu(II) concentration increased, suggesting that medium MW DOM fractions are particularly sensitive to Cu(II) binding, leading to higher weight-averaged MW values with increased Cu(II) concentrations. This study provides new insights into how BC addition to compost can influence the binding behavior of DOM with HMs, highlighting its potential for enhancing HMs immobilization in compost-amended soils.\u003c/p\u003e","manuscriptTitle":"Influence of biochar amendment on the binding characteristics of dissolved organic matter from chicken manure compost with Cu(II): an integrated analysis using EEM-PARAFAC, 2D-COS, and HPSEC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 18:19:03","doi":"10.21203/rs.3.rs-4900729/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-16T10:48:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-12T16:59:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2024-11-12T16:53:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-12T12:19:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2024-11-11T23:27:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7fe8ff7a-a73d-4ef6-83f1-ae1bb58faeb1","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:04:45+00:00","versionOfRecord":{"articleIdentity":"rs-4900729","link":"https://doi.org/10.1007/s12649-024-02834-4","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2024-12-14 15:57:35","publishedOnDateReadable":"December 14th, 2024"},"versionCreatedAt":"2024-11-13 18:19:03","video":"","vorDoi":"10.1007/s12649-024-02834-4","vorDoiUrl":"https://doi.org/10.1007/s12649-024-02834-4","workflowStages":[]},"version":"v1","identity":"rs-4900729","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4900729","identity":"rs-4900729","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}