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Insights into compositional changes of dissolved organic matter during a full-scale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques
Journal Pre-proofs Insights into compositional changes of dissolved organic matter during a fullscale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques Jiangang Che, Weifen Lin, Jie Ye, Hanpeng Liao, Zhen Yu, Hao Lin, Shungui Zhou PII: DOI: Reference:
S0960-8524(20)30026-2 https://doi.org/10.1016/j.biortech.2020.122757 BITE 122757
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Bioresource Technology
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Please cite this article as: Che, J., Lin, W., Ye, J., Liao, H., Yu, Z., Lin, H., Zhou, S., Insights into compositional changes of dissolved organic matter during a full-scale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech. 2020.122757
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Insights into compositional changes of dissolved organic matter during a full-scale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques Jiangang Chea, Weifen Lina, Jie Yea, Hanpeng Liaoa, Zhen Yub,*, Hao Linc, Shungui Zhoua a
Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College
of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China b
Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and
Management, Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China c
Wuyi University, Nanping 354300, China
*Correspongding author: Zhen Yu, Ph.D Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China Phone number: +86 020 87025872 Fax number: +86 020 87025872 E-mail address: [email protected] (Z. Yu)
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Abstract Various spectroscopic and electrochemical techniques combined was used to investigate the compositional changes of dissolved organic matter (DOM) and the difference in humification degree during full-scale cow dung vermicomposting. This study also investigated that whether the two techniques could be used as humification indices. The physicochemical characteristics of vermicompost were superior to those of the control, indicating that vermicomposting significantly accelerated the humification process, which was confirmed by spectroscopic and electrochemical analyses. Meanwhile, the changes of three components identified and electron transfer capacities in vermicomposting further revealed that vermicomposting resulted in significant compositional changes of DOM and higher humification degree. Partial least squares path modeling and redundancy analysis revealed that the two techniques could be used as humification indices for vermicomposting. These results of this study demonstrated that the combination of spectroscopy and electrochemistry was applicable to characterize the compositional changes of DOM and the humification degree of vermicomposting. Keywords: vermicomposting process; dissolved organic matter; excitation-emission matrix-parallel factor analysis; electron accepting capacity; cyclic voltammograms
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1. Introduction In recent years, the development and prosperity of the livestock industry in China has resulted in an increase in production of biodegradable organic wastes, especially cattle breeding (Lv et al., 2013). Due to environmental impacts, decomposition and efficient recycling of cow dung are needed (Bernal et al., 2009). Vermicomposting refers to the combined action of earthworms and microorganisms, which could accelerate the stabilization of organic matter (OM) and greatly modify the physical, chemical and biochemical properties of the composting products (Sangwan et al., 2008). This method has the advantages of sample operation, as well as being-odorless, cost-effective, pathogen-free and environmentally friendly (Lazcano et al., 2008). Biochemical transformation of OM in composting is a result of the metabolic activity of microorganisms occurring in the water-soluble phase, so dissolved organic matter (DOM) which was easy to change, could directly reflect the most fraction of the OM and the transformation processes (Caricasole et al., 2010). Therefore, the composition of DOM has been suggested as a better indicator of stability for the OM than that of the solid phase. DOM is a heterogeneous mixture consisting of different molecular sizes and complexities. Kinds of spectral analytical methods, including specific ultra-violet absorbance (SUVA), Fourier transform infrared (FTIR), fluorescence excitation-emission matrix (EEM), and nuclear magnetic resonance (NMR) have been used to investigate the properties and compositions of DOM (He et al., 2014). However, using single analytical method to investigate the changes of DOM is insufficient due to the heterogeneous 3
composition of DOM extracted from compost samples (Yu et al., 2019). Thus, more and more researchers are prone to integrate various spectra and other advanced analytical methods to investigate the changes of DOM during composting (Yu et al., 2019). Furthermore, due to the redox active and mediate redox reactions of DOM, previous studies extracted DOM based on its solubility, and characterized its redox properties (Tan et al., 2017). Zhao et al. (2017) reported that the electron transfer capacities (ETC) of humic acids were significantly associated with the functional groups, aromaticity and molecular weight of DOM. In addition, the ETC of DOM could be influenced by the humic-like substances (Tang et al., 2018). Previous study has reported that the electron accepting capacity (EAC) of DOM was highly correlated with the germination index (GI), demonstrating that the ETC could be used to characterize the maturity of composting products (Yuan et al., 2012). However, there was little focus on the redox properties of DOM extracted from vermicomposting products. Herein, the objectives of this study are to (1) characterize the compositional changes of DOM between vermicomposting and the control composting of cow dung using FTIR, 3D-EEM, and EEM-parallel factor analysis (EEM-PARAFAC) methods; (2) investigate the changes of redox properties of functional groups in DOM during the composting processes using electrochemical techniques; (3) evaluate the difference in the humification degree of the two composting processes; and (4) elucidate the relationship among physicochemical properties, EEM properties, electrochemical properties, and the maturity degree of compost using partial least squares path modeling (PLS-PM) and redundancy analysis (RDA). Moreover, the electrochemical techniques were rarely used 4
to measure the redox activity of DOM extracted from vermicomposting samples, and the combination of the two techniques was also seldom used to characterize the compositional changes of DOM. Meanwhile, this study investigated that whether the two techniques could be used as humification indices during vermicomposting. The results of this study will provide an improved understanding of the mechanism of the accelerated humification process during vermicomposting and expanding the application of the two techniques. 2. Materials and Methods 2.1 Preparation of composting materials The fresh cow dung was obtained from a cattle farm in the Hongya Country, Meishan City, Sichuan Province of China. The pH and electrical conductivity (EC) of the cow dung were 8.19 and 2.06 mS cm-1, respectively; the composition of organic substances was 80.56% OM, 35.12% total organic carbon (TOC), and 1.82% total nitrogen (TN); and the C/N ratio was 18.88. In order to avoid the damage of the high water content and anaerobic digestion to earthworm, the cow dung was naturally air-dried for one week with periodic turning over before used. Eisenia fetida were maintained in the laboratory with cow dung as the main culturing substrate. 2.2 The full-scale vermicomposting setup and sampling The full-scale vermicomposting experiment was performed in a plastic greenhouse. Approximately 8 tons air-dried cow dung was arranged in a long ridge measuring 20 × 1.5 × 0.3 m (length × width × height). For vermicomposting, the earthworms randomly picked were inoculated into the long ridges with cow dung. In parallel, the long ridges 5
with the same size containing the same cow dung but without earthworms were used as the control composting. All experiments were carried out under natural conditions. The two composting processes were conducted in triplicate. Three longitudinal sections were randomly selected from different parts of the ridges to ensure that typical samples were collected. Samples were collected on day 0, 10, 20, 30, and 40 of each experiment. Three parallel samples (about 1 kg for each) were collected. The obtained samples were then divided into two parts, one was stored at 4 oC for primary physicochemical analysis, and the other was stored at -80 oC for spectroscopic and electrochemical analyses. 2.3 Determination of physicochemical properties during composting The EC were detected by a conductivity meter. The OM content was measured by dry combustion at 550 oC for 8 h. The TOC and TN were measured with an Elementar instrument (Vario MAX cube, Hanau, Germany), and then the C/N ratio was calculated by TOC and TN (Cui et al., 2019). The GI was tested using the method described by Tiquia and Tam (1998). In order to measure the DOM in the samples, 10 g of dried (40 o
C) sample was extracted with 10 mL of deionized water (1:10 w/v ratio) by shaking 24
h at room temperature. The suspensions were centrifuged at 3000 r min-1 for 10 min and then filtered through 0.45 μm filter membranes. Unless otherwise specified, the concentrations of all DOM samples were diluted to 10 mg L-1 with deionized water. 2.4 Spectral analysis of DOM 2.4.1 FTIR spectra In this work, about 1 mg freeze-dried DOM sample was mixed thoroughly with 120 6
mg of dried spectrometry grade KBr and pressed to a pellet under reduced pressure. The pellet was immediately measured after preparation using a Nicolet iS50 FT-IR spectrophotometer (Thermo Nicolet Co., USA). The spectra were recorded in the range of 4000-400 cm-1 with a 4 cm resolution. 2.4.2 Fluorescence spectra analysis Fluorescence EEM spectra were measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies Inc., USA). Spectra were recorded at a scan rate of 2400 nm min-1 using excitation (Ex) and emission (Em) slit bandwidths of 5 nm. Wavelengths were set from 200 to 500 nm for Ex, and from 250 to 550 nm for Em. The voltage of the photomultiplier tube was set at 700 mV for low level light detection. 2.4.3 PARAFAC modeling The PARAFAC analysis was performed on the 3D data array of EEM spectra by MATLAB 2014a (Mathworks Inc. USA) using the DOMFluor toolbox. To minimize the influence of scatter lines and other attributes of the EEM landscape, the EEM spectra data were preprocessed prior to analysis using the following procedure (Yu et al., 2010). The EEM data of a control Milli-Q water sample was first subtracted from each EEM of the DOM samples. The first- and second-order Rayleigh and Roman scatters were then removed using the protocol of Bahram et al. (2006). Finally, a non-negativity constraint was applied to allow only chemically relevant results. The PARAFAC models with two to seven components were computed for the 3D data array, and the correct number of components was determined using residual analysis, split half analysis, and visual inspection. PARAFAC analysis can decompose the EEM 7
spectra into three matrices, including the score, Ex loading and Em loading matrices, through an alternating least squares procedure (Wu et al., 2011). The scores of components could represent the relative concentrations of fluorophores. 2.5 Electrochemical measurements Electrochemical measurements were conducted using the electrochemistry workstation CHI1660 (Chenhua Co. Ltd., Shanghai, China) with a conventional threeelectrode cell at ambient temperature. A graphite plate was used as the working electrode, and Pt net and Ag/AgCl electrodes were employed as the counter and reference electrodes, respectively. The electron-donating capacity (EDC) and EAC of DOM were tested using the method described by Yuan et al. (2011). The cyclic voltammetry (CV) measurements were characterized with a scan rate of 5 mV s-1 in the potential range of -1.5 to 0.5 V and carried out in dimethyl sulfoxide solution amended with 1.0 mM NaClO4 as the electrolyte. To quantitatively describe the capacitive performance of DOM, the areal capacitances (Ca, mF cm-2) of samples were calculated as follows (Ren et al., 2019; Sarkar et al., 2013): I
Ca = fA
Eq. (1)
where I (A) is the average current of the CV loop, f (V/s) is the scan rate, and A (cm2) is the area of the working electrode. 2.6 Statistical analyses PLS-PM was applied to deeply investigate the relationships between various factors and the maturity degree of compost using the R package plspm (v 0.4.7). The model contained the following factors: physicochemical properties, EEM properties, and 8
electrochemical properties, which was proceeded as described by Liao et al. (2018). Moreover, RDA was performed using Canoco 4.5 (Microcomputer Power, USA) to understand the correlations among the maturity (GI), physicochemical properties, EEM properties, and electrochemical properties. All analytical experiments were conducted in triplicate. After confirmation of the normality and homoscedasticity for the data, a Student’s t-test was used for statistical analysis, and a P value < 0.05 was considered statistically significant. Figures were plotted with Origin 8.0 (OriginLab, USA). 3. Results and discussion 3.1 Changes of physicochemical characteristics during vermicomposting The main physicochemical characteristics of the samples from composting processes are shown in Fig. 1. Compared to the vermicomposting, the EC of the control showed an opposite trend as shown in Fig. 1a. Lazcano et al. (2008) reported that the increase of EC in control composting closely related to the release of soluble salts such as ammonium, nitrate, and phosphate during the decomposition of organic compounds. Interestingly, the EC declined significantly from day 0 to day 10 during vermicomposting (Student’s t-test, P < 0.05), which could be contributed to the uptake of the minerals by earthworms and precipitation of the dissolved salts (Zhu et al., 2016). Moreover, the decrease of EC could lower the risk of phytotoxicity (Tiquia and Tam, 1998). The OM contents of the composting samples are displayed in Fig. 1b. The loss of OM during vermicomposting and the control was 27.3% and 10.1%, respectively (Student’s t-test, P < 0.01, Fig. 1b). One reason for the enhancement loss of OM during 9
vermicomposting was the degradation and mineralization of organics, and another reason was that the microbes in the gut of earthworms and earthworms could utilize the OM as the energy source for building their cell structures (Singh et al., 2019). Hence, the OM loss during vermicomposting was attributed to the synergistic effect of the earthworms and microorganism. The change trends of TOC, TN and C/N of the composting samples are shown in Fig. 1c, Fig. 1d and Fig. 1e, respectively. TOC contents dropped during all experiments, and the percent decreases in control and vermicomposting were 1.48% and 42.21%, respectively (Student’s t-test, P < 0.01, Fig. 1c). Relatively high decreases of TOC during vermicomposting indicated stronger mineralization by the microbial activities with released CO2 (Singh et al., 2019). This result of the TOC loss was similar to the result of the organic matter. The final TN content in vermicomposting was higher than that in control (Student’s t-test, P < 0.01, Fig. 1d), which was attributed to the strong degradation of organic carbon substances (Xing et al., 2012). This result was in accordance with Gupta and Garg (2009), who have reported that the increase of TN might be attributed to the action of N-fixing bacteria, which led to the mineralization of C-rich substances. As shown in Fig. 1e, it was obviously observed that C/N ratio decreased in final products, and the C/N ratio in vermicomposting was lower than that in control (Student’s t-test, P < 0.05). The decrease in the C/N ratio could be attributed to the loss of carbon resulting from the respiratory process by earthworms and microorganism (Khatua et al., 2018). This result was in agreement with previous studies, which indicated that the C/N ratio could be reduced significantly through vermicomposting (Zhang et al., 2015; Karmegam 10
et al., 2019) . A C/N ratio less than 15 was achieved in final vermicomposting products, indicating that the degree of humification in vermicomposting was higher than that in the control (Rékási et al., 2019). The result of C/N ratio was in line with EC and OM analyses. The GI values greater than 80% indicate that the final composting products were phytotoxin-free and completely mature (Yu et al., 2018). It was observed (Fig. 1f) that the GI values of vermicomposting showed an increasing trend, and the GI value reached 86% on day 20, which indicated the maturity of vermicomposting was higher and the products of vermicomposting were signified lower present of phytotoxic substances (Student’s t-test, P < 0.05). The GI value of the control on day 40 exceeded 80%, which could illustrate that vermicomposting could obviously accelerate the humification process. The GI value of the control suddenly decreased on day 10, demonstrating that the products of organic decomposition such as organic acids, and NH3 caused phytotoxicity and triggered a decline in GI (Yu et al., 2018). 3.2 FTIR spectra represents the structural changes of dissolved organic matter during vermicomposting The FTIR spectra of DOM after different composting exhibited similar peaks at relative locations, but varied in the relative intensity. The changes of main absorption bands in the final stage were as follows: (1) a decrease of the relative intensity at approximately 2925 cm-1 could be due to the aliphatic C—H stretching, which could be attributed to the biodegradation of fatty acids, carbohydrates and other aliphatics (Gupta and Garg, 2009); (2) a significant increase of the relative intensity at 1660-1600 cm-1 was characteristic of aromatic C=C and C=O vibrations, and C=O of quinone, conjugated 11
ketones (Lv et al., 2013); (3) an increase absorbance peak at 1440-1380 cm-1 represented the C—O of the carboxylic group, and asymmetric stretching of the COO- groups (Jouraiphy et al., 2005); (4) the enhancement of absorbance peaks at 1100-1030 cm-1 demonstrated the characteristics of C—O stretching of secondary alcohols, ethers and polysaccharides (Abouelwafa et al., 2008). The ratios between different peaks can provide some useful information. The peak ratio of 1640/2925 (aromatic C/aliphatic C) increased during vermicomposting, and the finial value of the ratio was higher than that in the control, revealing that vermicomposting could rapidly degrade aliphatic substances (Khan et al., 2019). The peak ratio of 1640/1380 (aromatic C/carboxyl C) in vermicomposting decreased during first 10 days and then increased to the end of the process, whereas it showed no obvious change in the control. It has been reported that organics existed in composting materials could be decomposed and transformed into carboxyl C under aerobic condition (Guo et al., 2018). In this study, the activity of earthworms impacted more oxygen into the vermicomposting process, which would resulted in higher aromatic C/carboxyl C ratio in vermicomposting. The peak ratio of 1640/1094 (aromatic C/alcohol C) displayed a decreasing trend during vermicomposting, suggesting that the alcohol C content increased. Guo et al. (2018) also reported that the final concentration of methanol and ethanol increased at the end of composting. In summary, these results in this study indicated that the aliphatic compounds, carboxyl organics and carbohydrates could be rapidly degraded by earthworms and microorganisms, which could enrich the oxidized and aromatic components of DOM during vermicomposting. Moreover, the FTIR spectra results further confirmed the 12
previous results that the vermicomposting process could cause the decomposition of easily biodegradable organics, increase the aromatic components and accelerate humification. 3.3 The difference in composition of dissolved organic matter during vermicomposting characterized by EEM spectra and PARAFAC analysis 3.3.1 EEM spectra 3D-EEM fluorescence spectra of DOM extracted from composting samples are presented in Fig. 2. According to Marhuenda-Egea et al. (2007) and Xing et al. (2012), the EEM spectra could be divided into five regions: Regions I and II (Ex<250 nm, Em<350 nm) were mainly related to simple aromatic proteins, such as tyrosine; Region III (Ex<250 nm, Em>350 nm) was related to fulvic acid-like matters; Region IV (Ex 250280 nm, Em<380 nm) was related to soluble microbial byproduct-like materials, and Region V (Ex>280 nm, Em>380 nm) was related to humic acid-like substances. The EEM spectra of DOM extracted from initial cow dung showed that the raw materials consisted of aromatic proteins, soluble microbial byproduct-like materials and minor amounts of humic substances. Two distinct peaks were detected in Regions III and V on day 10, whereas the peaks located in Regions I and II weakened over time, indicating that labile organics were rapidly degraded by earthworms and microbial activities during vermicomposting. In contrast, the degradation of organic substances lasted for a longer time in control, because the peaks related to aromatic proteins and soluble microbial byproduct-like materials existed persistently until day 20 (Yu et al., 2018). This finding was in line with Marhuenda-Egea et al. (2007), who found that the peaks located in 13
Regions I, II, and IV disappeared after 43 days of co-composting with exhausted grape marc and cow manure. Two peaks located in Regions III and V appeared from day 10, and the intensities of the peaks increased steadily to the end of vermicomposting, while the appearance and increasing intensities of the peaks happened from day 20 in control (Fig. 2). Yu et al. (2010) reported that the peaks at Ex/Em of 230/420 and 330/420 relating to fulvic acidlike and humic acid-like substances (in Regions III and V) were observed in the EEM spectra of mature composts. Thus, this phenomenon suggested that the formation of fulvic acid-like and humic acid-like substances in control were remarkably later than those in vermicomposting. It is well known that humification occurs predominantly in the maturation stage of composting (Yuan et al., 2012; Yu et al., 2019) . In this study, vermicomposting resulted in rapid degradation of the labile organics in first 10 days, and accelerated the formation of humic substances to the end of vermicomposting, suggesting a more rapid humification process. 3.3.2 EEM-PARAFAC analysis To obtain more information about the characterization of DOM extracted from composting samples, three fluorescent components characterized by Ex/Em (270, 320)/430 nm (Component 1, C1), (220, 350)/450 nm (Component 2, C2) and 220/340 nm (Component 3, C3) were identified by EEM-PARAFAC analysis. C1 and C2 were related to humic-like and fulvic-like substances, respectively, while C3 was attributed to proteinlike substances (Zhu et al., 2016). The changes in the maximum fluorescence intensity (Fmax) of the three DOM 14
components are shown in Fig. 3. The initial Fmax of the three components were similar in vermicomposting and the control. However, the Fmax exhibited a different change trend as the composting process proceeded. The Fmax of C3 showed a continuous decline to the end of vermicomposting. This could be attributed to intense bio-oxidation of protein-like substances by microbial activities (He et al., 2011). By comparison, the Fmax of C3 in the control on day 40 was higher than that in the vermicomposting on day 10, which indicated that the degree of mineralization in vermicomposting was obviously higher than that in control. This result was in line with the previous results that the vermicomposting can accelerate the decomposition of organics. The Fmax of C2 reached its maximum value on day 10 (Fmax = 59.2), and then decreased rapidly to the end of vermicomposting (Fmax = 19.4). The shape increase of the fulvic-like substances in the first 10 days of vermicomposting was related to the remarkable decrease of protein-like substances. Fulvic acid-like substances were characterized by prevalent aliphatic character, large contents of C, S-containing groups, proteinaceous materials and polysaccharide components (Plaza et al., 2007). Thus, the protein-like substances were likely a prime precursor for the synthesis of fulvic acid-like substances, and this was confirmed by the rapid shift of fluorescence peaks from Regions I and II to Region III in the 3D-EEM spectra. Moreover, one reason for the decrease of the fulvic acid-like substances was that the humic acid-like substances were formed by the polymerization of sample fulvic acid-like substances, another reason was that the fulvic acid-like substances were biodegraded due to its non-humic or easily decomposable properties (Jouraiphy et al., 2005). The Fmax of C1 in vermicomposting 15
showed an increasing trend, which suggested the enrichment of the humic acid-like substances and the enhancement of humification degree. Meantime, opposite trends of Fmax between the increased C1 and decreased C3 was obviously observed, indicating that protein-like substances may act as the organic precursors for humification. This phenomenon was in accordance with the previous study (He et al., 2014), finding that the rapid decrease of protein-like substances can accelerate the formation of humus substances during composting. The Fmax of C1 and C2 increased (Fmax = 38.2 and Fmax = 61.8, respectively) as the control process proceeded, which indicated an increase of aromatic polycondensation and humification degree with composting time increasing (Yu et al., 2010). By comparison, the Fmax of C1 was much lower at the end of control than that in vermicomposting, which clearly revealed that vermicomposting resulted in a higher humification degree. This could be attributed to the intense bio-oxidation of protein-like substances, which could produce rather high concentrations of N-containing precursors, as well as the activities of microbial and earthworms could use the precursors to form stable humus through polymerization (Wu et al., 2017). 3.4 The changes of electron transfer capacity of dissolved organic matter during vermicomposting It has been widely accepted that redox-active functional groups existed in DOM act as an electron shuttle in environmental redox processes (He et al., 2019). Therefore, the redox properties of the extracted DOM during composting were systematically measured by quantifying the EAC and EDC. With the addition of the DOM extracted from 16
composting samples, cathodic currents were clearly observed, which suggested that the redox-active moieties existed in DOM were directly reduced at graphite electrode (Yuan et al., 2013). As shown in Fig. 4a, the EAC value of DOM increased during composting. DOM extracted from final vermicomposting products had EAC of 2347.3 μmol e- g·C-1, which was twice the value of the EAC from final control products (Student’s t-test, P < 0.001). DOM contains a mass of quinones/aromatic substances due to the oxidation of lignin during composting process. The quinone and/or aromatic structures were considered as the main redox-active functional groups (He et al., 2019). In this study, the EAC results suggested that vermicomposting could accelerate the oxidation of lignin, resulting in higher concentrations of quinones/aromatic substances. As shown in Fig. 4b, the EDC values were significantly higher than the EAC values, which indicated that the number of electron-donating groups of DOM was rather higher than the number of electron-accepting groups (Xiao et al., 2019). The EDC of DOM extracted from vermicomposting exhibited an increasing trend in general, whereas that of the control was increased initially and then decreased. Previous studies have reported that the fulvic acids were composed of polymer proteins and low molecular weight aromatic acids. Due to the degradation of polymer proteins and the polymerization of aromatic substances, the EDC value decreased during the control composting (He et al., 2019). This result was accordant with the analysis of 3D-EEM and EEM-PARAFAC. DOM could be reversibly oxidized and reduced, by which the electron transfer is sustained (Huang et al., 2010). CV experiments were conducted to further characterize the redox properties of DOM. As shown in Fig. 5, similar redox characteristics were 17
observed for DOM extracted from composting samples. All DOM samples had redoxactive, showing a couple of redox peaks in the range of 0 to -1.5 V (Ye et al., 2018a; Ye et al., 2018b). The DOM had weak reduction peaks at -0.75 V and weak oxidation peaks at 0.20 V. The peak currents increased gradually during vermicomposting process, which indicated more redox active moieties at the electrode surface (Yuan et al., 2013). Previous studies have reported that the capacitive behavior had a positive correlation with the CV rectangular shape (Tang et al., 2015). To quantitatively describe the capacitive performance of the DOM, the Ca of all DOM were calculated, and the calculated Ca values of the vermicomposting and control were range from 0.7 to 1.3 and 0.7 to 0.9 mF cm-2, respectively. The results indicated that the capacitances of DOM extracted from vermicomposting were significantly enhanced, which could be the result of higher concentrations of quinones/aromatic substances. These results were also accordant with the analysis of 3D-EEM and EEM-PARAFAC results. 3.5 Relative contributions of various factors to the maturity degree during vermicomposting GI combines the measure of seed germination and root growth of seeds, which is one of effective indices used to evaluate the toxicity and the degree of maturity of compost (Yu et al, 2018). Meanwhile, the properties of DOM could reflect the maturity degree of compost (Tang et al., 2019). Thus, in this study, the PLS-PM was used to deeply explore the relationships among physicochemical properties, EEM properties, electrochemical properties, and the maturity degree of compost. Interestingly, the different composting processes represented different mechanisms by which the factors affected the maturity 18
degree. For vermicomposting, electrochemical properties were the dominant factor affecting the maturity degree, and EEM properties were the secondary factor. For the control, EEM properties were the main factor, and the physicochemical and electrochemical properties were the minor factors (Fig. 6a). In particular, all the factors had a positive effect on the maturity degree in vermicomposting, while the physicochemical properties had an inhibitory effect in the control. For example, the higher EC in the control result in higher risk of phytotoxicity, which was in accordance with the GI results, revealing that the maturity degree of the control was lower than that in vermicomposting. Moreover, the PLS-PM model found that the EEM and electrochemical properties showed positive correlation with the maturity. Thus, the two factors could be used to present the maturity degree of vermicomposting. Standardized direct and indirect mean further revealed that the factors affecting the maturity in vermicomposting were different from those in control. The results were further supported by the RDA results. 4. Conclusions This study has evaluated the compositional changes of DOM during vermicomposting with spectroscopic and electrochemical methods. For vermicomposting, GI value on day 20 reached 86%, C1 was 63.3, EAC was 2347.3 μmol e- g·C-1, and Ca was 1.3 mF cm-2, respectively. These results revealed that vermicomposting resulted in the compositional changes of DOM, and significantly accelerated the humification process. Moreover, the PLS-PM and RDA enabled that the two techniques could be used as humification indices. These findings revealed that the combination of the two 19
techniques was able to characterize the compositional changes of DOM and the humification degree. CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition. Acknowledgements This research was supported by the Key Research & Development Plan of Fujian Province (2017NZ0001-1), the National Natural Science Foundation of China (51608121, 41977281), Natural Science Foundation of Fujian (2018J01709), the University-Industry Cooperation Project of Fujian (2018Y4011), and the Project of the Fuzhou Municipal Department of Science and Technology of China (No. 2019-G-32). Supplementary data E-supplementary data of this work can be found in online version of the paper. Conflict to interest The authors declare no conflicts of interest.
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Figure captions Fig. 1 Dynamic changes of physicochemical properties during control composting and the vermicomposting: (a) Electrical conductivity (EC); (b) Organic matter (OM); (c) Total organic carbon (TOC); (d) Total nitrogen (TN); (e) TOC/TN (C/N); (f) Germination index (GI). Significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). Fig. 2 Fluorescence excitation-emission matrix spectra of DOM extracted from control and vermicomposting samples at different days. Em (nm): emission wavelength (nm); Ex (nm): excitation wavelength (nm). Regions I and II: simple aromatic proteins, such as tyrosine; Region III: fulvic acid-like matters; Region IV: soluble microbial byproductlike materials, Region V: humic acid-like substances. Fig. 3 Distribution of three PARAFAC-derived components in DOM extracted from control and vermicomposting samples at different days: (a) Control; (b) Vermicomposting. Fmax (a.u.): the maximum fluorescence intensity (arbitrary units). Fig. 4 Changes in the electron-accepting capacity (EAC) and electron-donating capacity (EDC) of the DOM extracted from control and vermicomposting samples at different days: (a) EAC; (b) EDC. Significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). Fig. 5 Cyclic voltammograms of the DOM extracted from control and vermicomposting samples at different days: (a) Control; (b) Vermicomposting. Fig. 6 PLS-PM revealing the direct and indirect effects of different factors on maturity in different composting processes. (a): PLS-PM showing the relationships among the maturity, physicochemical properties, EEM properties, and electrochemical properties in 28
different composting processes. Large path coefficients were described as wider arrows, and blue and red colors represent positive and negative effects, respectively. Path coefficients and coefficients of determination (R2) were calculated after 999 bootstraps, and significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). The GoF values for vermicomposting and control were 0.72 and 0.67, respectively. (b): Standardized direct and indirect mean effects derived from PLS-PMs.
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Fig. 6 CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Graphical abstract
Highlights: Vermicomposting can accelerate the humification process. Vermicomposting led to higher changes in DOM composition and humification degree.
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The two techniques were used as humification indices during vermicomposting. The compositional changes of DOM were represented by the two techniques.
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CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition.
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S0960-8524(20)30026-2 https://doi.org/10.1016/j.biortech.2020.122757 BITE 122757
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
12 November 2019 31 December 2019 3 January 2020
Please cite this article as: Che, J., Lin, W., Ye, J., Liao, H., Yu, Z., Lin, H., Zhou, S., Insights into compositional changes of dissolved organic matter during a full-scale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech. 2020.122757
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© 2020 Published by Elsevier Ltd.
Insights into compositional changes of dissolved organic matter during a full-scale vermicomposting of cow dung by combined spectroscopic and electrochemical techniques Jiangang Chea, Weifen Lina, Jie Yea, Hanpeng Liaoa, Zhen Yub,*, Hao Linc, Shungui Zhoua a
Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College
of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China b
Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and
Management, Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China c
Wuyi University, Nanping 354300, China
*Correspongding author: Zhen Yu, Ph.D Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China Phone number: +86 020 87025872 Fax number: +86 020 87025872 E-mail address: [email protected] (Z. Yu)
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Abstract Various spectroscopic and electrochemical techniques combined was used to investigate the compositional changes of dissolved organic matter (DOM) and the difference in humification degree during full-scale cow dung vermicomposting. This study also investigated that whether the two techniques could be used as humification indices. The physicochemical characteristics of vermicompost were superior to those of the control, indicating that vermicomposting significantly accelerated the humification process, which was confirmed by spectroscopic and electrochemical analyses. Meanwhile, the changes of three components identified and electron transfer capacities in vermicomposting further revealed that vermicomposting resulted in significant compositional changes of DOM and higher humification degree. Partial least squares path modeling and redundancy analysis revealed that the two techniques could be used as humification indices for vermicomposting. These results of this study demonstrated that the combination of spectroscopy and electrochemistry was applicable to characterize the compositional changes of DOM and the humification degree of vermicomposting. Keywords: vermicomposting process; dissolved organic matter; excitation-emission matrix-parallel factor analysis; electron accepting capacity; cyclic voltammograms
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1. Introduction In recent years, the development and prosperity of the livestock industry in China has resulted in an increase in production of biodegradable organic wastes, especially cattle breeding (Lv et al., 2013). Due to environmental impacts, decomposition and efficient recycling of cow dung are needed (Bernal et al., 2009). Vermicomposting refers to the combined action of earthworms and microorganisms, which could accelerate the stabilization of organic matter (OM) and greatly modify the physical, chemical and biochemical properties of the composting products (Sangwan et al., 2008). This method has the advantages of sample operation, as well as being-odorless, cost-effective, pathogen-free and environmentally friendly (Lazcano et al., 2008). Biochemical transformation of OM in composting is a result of the metabolic activity of microorganisms occurring in the water-soluble phase, so dissolved organic matter (DOM) which was easy to change, could directly reflect the most fraction of the OM and the transformation processes (Caricasole et al., 2010). Therefore, the composition of DOM has been suggested as a better indicator of stability for the OM than that of the solid phase. DOM is a heterogeneous mixture consisting of different molecular sizes and complexities. Kinds of spectral analytical methods, including specific ultra-violet absorbance (SUVA), Fourier transform infrared (FTIR), fluorescence excitation-emission matrix (EEM), and nuclear magnetic resonance (NMR) have been used to investigate the properties and compositions of DOM (He et al., 2014). However, using single analytical method to investigate the changes of DOM is insufficient due to the heterogeneous 3
composition of DOM extracted from compost samples (Yu et al., 2019). Thus, more and more researchers are prone to integrate various spectra and other advanced analytical methods to investigate the changes of DOM during composting (Yu et al., 2019). Furthermore, due to the redox active and mediate redox reactions of DOM, previous studies extracted DOM based on its solubility, and characterized its redox properties (Tan et al., 2017). Zhao et al. (2017) reported that the electron transfer capacities (ETC) of humic acids were significantly associated with the functional groups, aromaticity and molecular weight of DOM. In addition, the ETC of DOM could be influenced by the humic-like substances (Tang et al., 2018). Previous study has reported that the electron accepting capacity (EAC) of DOM was highly correlated with the germination index (GI), demonstrating that the ETC could be used to characterize the maturity of composting products (Yuan et al., 2012). However, there was little focus on the redox properties of DOM extracted from vermicomposting products. Herein, the objectives of this study are to (1) characterize the compositional changes of DOM between vermicomposting and the control composting of cow dung using FTIR, 3D-EEM, and EEM-parallel factor analysis (EEM-PARAFAC) methods; (2) investigate the changes of redox properties of functional groups in DOM during the composting processes using electrochemical techniques; (3) evaluate the difference in the humification degree of the two composting processes; and (4) elucidate the relationship among physicochemical properties, EEM properties, electrochemical properties, and the maturity degree of compost using partial least squares path modeling (PLS-PM) and redundancy analysis (RDA). Moreover, the electrochemical techniques were rarely used 4
to measure the redox activity of DOM extracted from vermicomposting samples, and the combination of the two techniques was also seldom used to characterize the compositional changes of DOM. Meanwhile, this study investigated that whether the two techniques could be used as humification indices during vermicomposting. The results of this study will provide an improved understanding of the mechanism of the accelerated humification process during vermicomposting and expanding the application of the two techniques. 2. Materials and Methods 2.1 Preparation of composting materials The fresh cow dung was obtained from a cattle farm in the Hongya Country, Meishan City, Sichuan Province of China. The pH and electrical conductivity (EC) of the cow dung were 8.19 and 2.06 mS cm-1, respectively; the composition of organic substances was 80.56% OM, 35.12% total organic carbon (TOC), and 1.82% total nitrogen (TN); and the C/N ratio was 18.88. In order to avoid the damage of the high water content and anaerobic digestion to earthworm, the cow dung was naturally air-dried for one week with periodic turning over before used. Eisenia fetida were maintained in the laboratory with cow dung as the main culturing substrate. 2.2 The full-scale vermicomposting setup and sampling The full-scale vermicomposting experiment was performed in a plastic greenhouse. Approximately 8 tons air-dried cow dung was arranged in a long ridge measuring 20 × 1.5 × 0.3 m (length × width × height). For vermicomposting, the earthworms randomly picked were inoculated into the long ridges with cow dung. In parallel, the long ridges 5
with the same size containing the same cow dung but without earthworms were used as the control composting. All experiments were carried out under natural conditions. The two composting processes were conducted in triplicate. Three longitudinal sections were randomly selected from different parts of the ridges to ensure that typical samples were collected. Samples were collected on day 0, 10, 20, 30, and 40 of each experiment. Three parallel samples (about 1 kg for each) were collected. The obtained samples were then divided into two parts, one was stored at 4 oC for primary physicochemical analysis, and the other was stored at -80 oC for spectroscopic and electrochemical analyses. 2.3 Determination of physicochemical properties during composting The EC were detected by a conductivity meter. The OM content was measured by dry combustion at 550 oC for 8 h. The TOC and TN were measured with an Elementar instrument (Vario MAX cube, Hanau, Germany), and then the C/N ratio was calculated by TOC and TN (Cui et al., 2019). The GI was tested using the method described by Tiquia and Tam (1998). In order to measure the DOM in the samples, 10 g of dried (40 o
C) sample was extracted with 10 mL of deionized water (1:10 w/v ratio) by shaking 24
h at room temperature. The suspensions were centrifuged at 3000 r min-1 for 10 min and then filtered through 0.45 μm filter membranes. Unless otherwise specified, the concentrations of all DOM samples were diluted to 10 mg L-1 with deionized water. 2.4 Spectral analysis of DOM 2.4.1 FTIR spectra In this work, about 1 mg freeze-dried DOM sample was mixed thoroughly with 120 6
mg of dried spectrometry grade KBr and pressed to a pellet under reduced pressure. The pellet was immediately measured after preparation using a Nicolet iS50 FT-IR spectrophotometer (Thermo Nicolet Co., USA). The spectra were recorded in the range of 4000-400 cm-1 with a 4 cm resolution. 2.4.2 Fluorescence spectra analysis Fluorescence EEM spectra were measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies Inc., USA). Spectra were recorded at a scan rate of 2400 nm min-1 using excitation (Ex) and emission (Em) slit bandwidths of 5 nm. Wavelengths were set from 200 to 500 nm for Ex, and from 250 to 550 nm for Em. The voltage of the photomultiplier tube was set at 700 mV for low level light detection. 2.4.3 PARAFAC modeling The PARAFAC analysis was performed on the 3D data array of EEM spectra by MATLAB 2014a (Mathworks Inc. USA) using the DOMFluor toolbox. To minimize the influence of scatter lines and other attributes of the EEM landscape, the EEM spectra data were preprocessed prior to analysis using the following procedure (Yu et al., 2010). The EEM data of a control Milli-Q water sample was first subtracted from each EEM of the DOM samples. The first- and second-order Rayleigh and Roman scatters were then removed using the protocol of Bahram et al. (2006). Finally, a non-negativity constraint was applied to allow only chemically relevant results. The PARAFAC models with two to seven components were computed for the 3D data array, and the correct number of components was determined using residual analysis, split half analysis, and visual inspection. PARAFAC analysis can decompose the EEM 7
spectra into three matrices, including the score, Ex loading and Em loading matrices, through an alternating least squares procedure (Wu et al., 2011). The scores of components could represent the relative concentrations of fluorophores. 2.5 Electrochemical measurements Electrochemical measurements were conducted using the electrochemistry workstation CHI1660 (Chenhua Co. Ltd., Shanghai, China) with a conventional threeelectrode cell at ambient temperature. A graphite plate was used as the working electrode, and Pt net and Ag/AgCl electrodes were employed as the counter and reference electrodes, respectively. The electron-donating capacity (EDC) and EAC of DOM were tested using the method described by Yuan et al. (2011). The cyclic voltammetry (CV) measurements were characterized with a scan rate of 5 mV s-1 in the potential range of -1.5 to 0.5 V and carried out in dimethyl sulfoxide solution amended with 1.0 mM NaClO4 as the electrolyte. To quantitatively describe the capacitive performance of DOM, the areal capacitances (Ca, mF cm-2) of samples were calculated as follows (Ren et al., 2019; Sarkar et al., 2013): I
Ca = fA
Eq. (1)
where I (A) is the average current of the CV loop, f (V/s) is the scan rate, and A (cm2) is the area of the working electrode. 2.6 Statistical analyses PLS-PM was applied to deeply investigate the relationships between various factors and the maturity degree of compost using the R package plspm (v 0.4.7). The model contained the following factors: physicochemical properties, EEM properties, and 8
electrochemical properties, which was proceeded as described by Liao et al. (2018). Moreover, RDA was performed using Canoco 4.5 (Microcomputer Power, USA) to understand the correlations among the maturity (GI), physicochemical properties, EEM properties, and electrochemical properties. All analytical experiments were conducted in triplicate. After confirmation of the normality and homoscedasticity for the data, a Student’s t-test was used for statistical analysis, and a P value < 0.05 was considered statistically significant. Figures were plotted with Origin 8.0 (OriginLab, USA). 3. Results and discussion 3.1 Changes of physicochemical characteristics during vermicomposting The main physicochemical characteristics of the samples from composting processes are shown in Fig. 1. Compared to the vermicomposting, the EC of the control showed an opposite trend as shown in Fig. 1a. Lazcano et al. (2008) reported that the increase of EC in control composting closely related to the release of soluble salts such as ammonium, nitrate, and phosphate during the decomposition of organic compounds. Interestingly, the EC declined significantly from day 0 to day 10 during vermicomposting (Student’s t-test, P < 0.05), which could be contributed to the uptake of the minerals by earthworms and precipitation of the dissolved salts (Zhu et al., 2016). Moreover, the decrease of EC could lower the risk of phytotoxicity (Tiquia and Tam, 1998). The OM contents of the composting samples are displayed in Fig. 1b. The loss of OM during vermicomposting and the control was 27.3% and 10.1%, respectively (Student’s t-test, P < 0.01, Fig. 1b). One reason for the enhancement loss of OM during 9
vermicomposting was the degradation and mineralization of organics, and another reason was that the microbes in the gut of earthworms and earthworms could utilize the OM as the energy source for building their cell structures (Singh et al., 2019). Hence, the OM loss during vermicomposting was attributed to the synergistic effect of the earthworms and microorganism. The change trends of TOC, TN and C/N of the composting samples are shown in Fig. 1c, Fig. 1d and Fig. 1e, respectively. TOC contents dropped during all experiments, and the percent decreases in control and vermicomposting were 1.48% and 42.21%, respectively (Student’s t-test, P < 0.01, Fig. 1c). Relatively high decreases of TOC during vermicomposting indicated stronger mineralization by the microbial activities with released CO2 (Singh et al., 2019). This result of the TOC loss was similar to the result of the organic matter. The final TN content in vermicomposting was higher than that in control (Student’s t-test, P < 0.01, Fig. 1d), which was attributed to the strong degradation of organic carbon substances (Xing et al., 2012). This result was in accordance with Gupta and Garg (2009), who have reported that the increase of TN might be attributed to the action of N-fixing bacteria, which led to the mineralization of C-rich substances. As shown in Fig. 1e, it was obviously observed that C/N ratio decreased in final products, and the C/N ratio in vermicomposting was lower than that in control (Student’s t-test, P < 0.05). The decrease in the C/N ratio could be attributed to the loss of carbon resulting from the respiratory process by earthworms and microorganism (Khatua et al., 2018). This result was in agreement with previous studies, which indicated that the C/N ratio could be reduced significantly through vermicomposting (Zhang et al., 2015; Karmegam 10
et al., 2019) . A C/N ratio less than 15 was achieved in final vermicomposting products, indicating that the degree of humification in vermicomposting was higher than that in the control (Rékási et al., 2019). The result of C/N ratio was in line with EC and OM analyses. The GI values greater than 80% indicate that the final composting products were phytotoxin-free and completely mature (Yu et al., 2018). It was observed (Fig. 1f) that the GI values of vermicomposting showed an increasing trend, and the GI value reached 86% on day 20, which indicated the maturity of vermicomposting was higher and the products of vermicomposting were signified lower present of phytotoxic substances (Student’s t-test, P < 0.05). The GI value of the control on day 40 exceeded 80%, which could illustrate that vermicomposting could obviously accelerate the humification process. The GI value of the control suddenly decreased on day 10, demonstrating that the products of organic decomposition such as organic acids, and NH3 caused phytotoxicity and triggered a decline in GI (Yu et al., 2018). 3.2 FTIR spectra represents the structural changes of dissolved organic matter during vermicomposting The FTIR spectra of DOM after different composting exhibited similar peaks at relative locations, but varied in the relative intensity. The changes of main absorption bands in the final stage were as follows: (1) a decrease of the relative intensity at approximately 2925 cm-1 could be due to the aliphatic C—H stretching, which could be attributed to the biodegradation of fatty acids, carbohydrates and other aliphatics (Gupta and Garg, 2009); (2) a significant increase of the relative intensity at 1660-1600 cm-1 was characteristic of aromatic C=C and C=O vibrations, and C=O of quinone, conjugated 11
ketones (Lv et al., 2013); (3) an increase absorbance peak at 1440-1380 cm-1 represented the C—O of the carboxylic group, and asymmetric stretching of the COO- groups (Jouraiphy et al., 2005); (4) the enhancement of absorbance peaks at 1100-1030 cm-1 demonstrated the characteristics of C—O stretching of secondary alcohols, ethers and polysaccharides (Abouelwafa et al., 2008). The ratios between different peaks can provide some useful information. The peak ratio of 1640/2925 (aromatic C/aliphatic C) increased during vermicomposting, and the finial value of the ratio was higher than that in the control, revealing that vermicomposting could rapidly degrade aliphatic substances (Khan et al., 2019). The peak ratio of 1640/1380 (aromatic C/carboxyl C) in vermicomposting decreased during first 10 days and then increased to the end of the process, whereas it showed no obvious change in the control. It has been reported that organics existed in composting materials could be decomposed and transformed into carboxyl C under aerobic condition (Guo et al., 2018). In this study, the activity of earthworms impacted more oxygen into the vermicomposting process, which would resulted in higher aromatic C/carboxyl C ratio in vermicomposting. The peak ratio of 1640/1094 (aromatic C/alcohol C) displayed a decreasing trend during vermicomposting, suggesting that the alcohol C content increased. Guo et al. (2018) also reported that the final concentration of methanol and ethanol increased at the end of composting. In summary, these results in this study indicated that the aliphatic compounds, carboxyl organics and carbohydrates could be rapidly degraded by earthworms and microorganisms, which could enrich the oxidized and aromatic components of DOM during vermicomposting. Moreover, the FTIR spectra results further confirmed the 12
previous results that the vermicomposting process could cause the decomposition of easily biodegradable organics, increase the aromatic components and accelerate humification. 3.3 The difference in composition of dissolved organic matter during vermicomposting characterized by EEM spectra and PARAFAC analysis 3.3.1 EEM spectra 3D-EEM fluorescence spectra of DOM extracted from composting samples are presented in Fig. 2. According to Marhuenda-Egea et al. (2007) and Xing et al. (2012), the EEM spectra could be divided into five regions: Regions I and II (Ex<250 nm, Em<350 nm) were mainly related to simple aromatic proteins, such as tyrosine; Region III (Ex<250 nm, Em>350 nm) was related to fulvic acid-like matters; Region IV (Ex 250280 nm, Em<380 nm) was related to soluble microbial byproduct-like materials, and Region V (Ex>280 nm, Em>380 nm) was related to humic acid-like substances. The EEM spectra of DOM extracted from initial cow dung showed that the raw materials consisted of aromatic proteins, soluble microbial byproduct-like materials and minor amounts of humic substances. Two distinct peaks were detected in Regions III and V on day 10, whereas the peaks located in Regions I and II weakened over time, indicating that labile organics were rapidly degraded by earthworms and microbial activities during vermicomposting. In contrast, the degradation of organic substances lasted for a longer time in control, because the peaks related to aromatic proteins and soluble microbial byproduct-like materials existed persistently until day 20 (Yu et al., 2018). This finding was in line with Marhuenda-Egea et al. (2007), who found that the peaks located in 13
Regions I, II, and IV disappeared after 43 days of co-composting with exhausted grape marc and cow manure. Two peaks located in Regions III and V appeared from day 10, and the intensities of the peaks increased steadily to the end of vermicomposting, while the appearance and increasing intensities of the peaks happened from day 20 in control (Fig. 2). Yu et al. (2010) reported that the peaks at Ex/Em of 230/420 and 330/420 relating to fulvic acidlike and humic acid-like substances (in Regions III and V) were observed in the EEM spectra of mature composts. Thus, this phenomenon suggested that the formation of fulvic acid-like and humic acid-like substances in control were remarkably later than those in vermicomposting. It is well known that humification occurs predominantly in the maturation stage of composting (Yuan et al., 2012; Yu et al., 2019) . In this study, vermicomposting resulted in rapid degradation of the labile organics in first 10 days, and accelerated the formation of humic substances to the end of vermicomposting, suggesting a more rapid humification process. 3.3.2 EEM-PARAFAC analysis To obtain more information about the characterization of DOM extracted from composting samples, three fluorescent components characterized by Ex/Em (270, 320)/430 nm (Component 1, C1), (220, 350)/450 nm (Component 2, C2) and 220/340 nm (Component 3, C3) were identified by EEM-PARAFAC analysis. C1 and C2 were related to humic-like and fulvic-like substances, respectively, while C3 was attributed to proteinlike substances (Zhu et al., 2016). The changes in the maximum fluorescence intensity (Fmax) of the three DOM 14
components are shown in Fig. 3. The initial Fmax of the three components were similar in vermicomposting and the control. However, the Fmax exhibited a different change trend as the composting process proceeded. The Fmax of C3 showed a continuous decline to the end of vermicomposting. This could be attributed to intense bio-oxidation of protein-like substances by microbial activities (He et al., 2011). By comparison, the Fmax of C3 in the control on day 40 was higher than that in the vermicomposting on day 10, which indicated that the degree of mineralization in vermicomposting was obviously higher than that in control. This result was in line with the previous results that the vermicomposting can accelerate the decomposition of organics. The Fmax of C2 reached its maximum value on day 10 (Fmax = 59.2), and then decreased rapidly to the end of vermicomposting (Fmax = 19.4). The shape increase of the fulvic-like substances in the first 10 days of vermicomposting was related to the remarkable decrease of protein-like substances. Fulvic acid-like substances were characterized by prevalent aliphatic character, large contents of C, S-containing groups, proteinaceous materials and polysaccharide components (Plaza et al., 2007). Thus, the protein-like substances were likely a prime precursor for the synthesis of fulvic acid-like substances, and this was confirmed by the rapid shift of fluorescence peaks from Regions I and II to Region III in the 3D-EEM spectra. Moreover, one reason for the decrease of the fulvic acid-like substances was that the humic acid-like substances were formed by the polymerization of sample fulvic acid-like substances, another reason was that the fulvic acid-like substances were biodegraded due to its non-humic or easily decomposable properties (Jouraiphy et al., 2005). The Fmax of C1 in vermicomposting 15
showed an increasing trend, which suggested the enrichment of the humic acid-like substances and the enhancement of humification degree. Meantime, opposite trends of Fmax between the increased C1 and decreased C3 was obviously observed, indicating that protein-like substances may act as the organic precursors for humification. This phenomenon was in accordance with the previous study (He et al., 2014), finding that the rapid decrease of protein-like substances can accelerate the formation of humus substances during composting. The Fmax of C1 and C2 increased (Fmax = 38.2 and Fmax = 61.8, respectively) as the control process proceeded, which indicated an increase of aromatic polycondensation and humification degree with composting time increasing (Yu et al., 2010). By comparison, the Fmax of C1 was much lower at the end of control than that in vermicomposting, which clearly revealed that vermicomposting resulted in a higher humification degree. This could be attributed to the intense bio-oxidation of protein-like substances, which could produce rather high concentrations of N-containing precursors, as well as the activities of microbial and earthworms could use the precursors to form stable humus through polymerization (Wu et al., 2017). 3.4 The changes of electron transfer capacity of dissolved organic matter during vermicomposting It has been widely accepted that redox-active functional groups existed in DOM act as an electron shuttle in environmental redox processes (He et al., 2019). Therefore, the redox properties of the extracted DOM during composting were systematically measured by quantifying the EAC and EDC. With the addition of the DOM extracted from 16
composting samples, cathodic currents were clearly observed, which suggested that the redox-active moieties existed in DOM were directly reduced at graphite electrode (Yuan et al., 2013). As shown in Fig. 4a, the EAC value of DOM increased during composting. DOM extracted from final vermicomposting products had EAC of 2347.3 μmol e- g·C-1, which was twice the value of the EAC from final control products (Student’s t-test, P < 0.001). DOM contains a mass of quinones/aromatic substances due to the oxidation of lignin during composting process. The quinone and/or aromatic structures were considered as the main redox-active functional groups (He et al., 2019). In this study, the EAC results suggested that vermicomposting could accelerate the oxidation of lignin, resulting in higher concentrations of quinones/aromatic substances. As shown in Fig. 4b, the EDC values were significantly higher than the EAC values, which indicated that the number of electron-donating groups of DOM was rather higher than the number of electron-accepting groups (Xiao et al., 2019). The EDC of DOM extracted from vermicomposting exhibited an increasing trend in general, whereas that of the control was increased initially and then decreased. Previous studies have reported that the fulvic acids were composed of polymer proteins and low molecular weight aromatic acids. Due to the degradation of polymer proteins and the polymerization of aromatic substances, the EDC value decreased during the control composting (He et al., 2019). This result was accordant with the analysis of 3D-EEM and EEM-PARAFAC. DOM could be reversibly oxidized and reduced, by which the electron transfer is sustained (Huang et al., 2010). CV experiments were conducted to further characterize the redox properties of DOM. As shown in Fig. 5, similar redox characteristics were 17
observed for DOM extracted from composting samples. All DOM samples had redoxactive, showing a couple of redox peaks in the range of 0 to -1.5 V (Ye et al., 2018a; Ye et al., 2018b). The DOM had weak reduction peaks at -0.75 V and weak oxidation peaks at 0.20 V. The peak currents increased gradually during vermicomposting process, which indicated more redox active moieties at the electrode surface (Yuan et al., 2013). Previous studies have reported that the capacitive behavior had a positive correlation with the CV rectangular shape (Tang et al., 2015). To quantitatively describe the capacitive performance of the DOM, the Ca of all DOM were calculated, and the calculated Ca values of the vermicomposting and control were range from 0.7 to 1.3 and 0.7 to 0.9 mF cm-2, respectively. The results indicated that the capacitances of DOM extracted from vermicomposting were significantly enhanced, which could be the result of higher concentrations of quinones/aromatic substances. These results were also accordant with the analysis of 3D-EEM and EEM-PARAFAC results. 3.5 Relative contributions of various factors to the maturity degree during vermicomposting GI combines the measure of seed germination and root growth of seeds, which is one of effective indices used to evaluate the toxicity and the degree of maturity of compost (Yu et al, 2018). Meanwhile, the properties of DOM could reflect the maturity degree of compost (Tang et al., 2019). Thus, in this study, the PLS-PM was used to deeply explore the relationships among physicochemical properties, EEM properties, electrochemical properties, and the maturity degree of compost. Interestingly, the different composting processes represented different mechanisms by which the factors affected the maturity 18
degree. For vermicomposting, electrochemical properties were the dominant factor affecting the maturity degree, and EEM properties were the secondary factor. For the control, EEM properties were the main factor, and the physicochemical and electrochemical properties were the minor factors (Fig. 6a). In particular, all the factors had a positive effect on the maturity degree in vermicomposting, while the physicochemical properties had an inhibitory effect in the control. For example, the higher EC in the control result in higher risk of phytotoxicity, which was in accordance with the GI results, revealing that the maturity degree of the control was lower than that in vermicomposting. Moreover, the PLS-PM model found that the EEM and electrochemical properties showed positive correlation with the maturity. Thus, the two factors could be used to present the maturity degree of vermicomposting. Standardized direct and indirect mean further revealed that the factors affecting the maturity in vermicomposting were different from those in control. The results were further supported by the RDA results. 4. Conclusions This study has evaluated the compositional changes of DOM during vermicomposting with spectroscopic and electrochemical methods. For vermicomposting, GI value on day 20 reached 86%, C1 was 63.3, EAC was 2347.3 μmol e- g·C-1, and Ca was 1.3 mF cm-2, respectively. These results revealed that vermicomposting resulted in the compositional changes of DOM, and significantly accelerated the humification process. Moreover, the PLS-PM and RDA enabled that the two techniques could be used as humification indices. These findings revealed that the combination of the two 19
techniques was able to characterize the compositional changes of DOM and the humification degree. CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition. Acknowledgements This research was supported by the Key Research & Development Plan of Fujian Province (2017NZ0001-1), the National Natural Science Foundation of China (51608121, 41977281), Natural Science Foundation of Fujian (2018J01709), the University-Industry Cooperation Project of Fujian (2018Y4011), and the Project of the Fuzhou Municipal Department of Science and Technology of China (No. 2019-G-32). Supplementary data E-supplementary data of this work can be found in online version of the paper. Conflict to interest The authors declare no conflicts of interest.
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Figure captions Fig. 1 Dynamic changes of physicochemical properties during control composting and the vermicomposting: (a) Electrical conductivity (EC); (b) Organic matter (OM); (c) Total organic carbon (TOC); (d) Total nitrogen (TN); (e) TOC/TN (C/N); (f) Germination index (GI). Significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). Fig. 2 Fluorescence excitation-emission matrix spectra of DOM extracted from control and vermicomposting samples at different days. Em (nm): emission wavelength (nm); Ex (nm): excitation wavelength (nm). Regions I and II: simple aromatic proteins, such as tyrosine; Region III: fulvic acid-like matters; Region IV: soluble microbial byproductlike materials, Region V: humic acid-like substances. Fig. 3 Distribution of three PARAFAC-derived components in DOM extracted from control and vermicomposting samples at different days: (a) Control; (b) Vermicomposting. Fmax (a.u.): the maximum fluorescence intensity (arbitrary units). Fig. 4 Changes in the electron-accepting capacity (EAC) and electron-donating capacity (EDC) of the DOM extracted from control and vermicomposting samples at different days: (a) EAC; (b) EDC. Significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). Fig. 5 Cyclic voltammograms of the DOM extracted from control and vermicomposting samples at different days: (a) Control; (b) Vermicomposting. Fig. 6 PLS-PM revealing the direct and indirect effects of different factors on maturity in different composting processes. (a): PLS-PM showing the relationships among the maturity, physicochemical properties, EEM properties, and electrochemical properties in 28
different composting processes. Large path coefficients were described as wider arrows, and blue and red colors represent positive and negative effects, respectively. Path coefficients and coefficients of determination (R2) were calculated after 999 bootstraps, and significance levels are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.001). The GoF values for vermicomposting and control were 0.72 and 0.67, respectively. (b): Standardized direct and indirect mean effects derived from PLS-PMs.
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Fig. 6 CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Graphical abstract
Highlights: Vermicomposting can accelerate the humification process. Vermicomposting led to higher changes in DOM composition and humification degree.
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The two techniques were used as humification indices during vermicomposting. The compositional changes of DOM were represented by the two techniques.
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CRediT authorship contribution statement Jiangang Che: Investigation, Writing - original draft, Formal analysis, Weifen Lin: Investigation, Data curation, Methodology. Jie Ye: Methodology, Funding acquisition. Hanpeng Liao: Data curation, Funding acquisition. Zhen Yu: Supervision, Writing review & editing. Hao Lin: Funding acquisition. Shungui Zhou: Funding acquisition.
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