Hyperthermophilic composting of sewage sludge accelerates humic acid formation: Elemental and spectroscopic evidence

Waste Management 103 (2020) 342–351

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Hyperthermophilic composting of sewage sludge accelerates humic acid formation: Elemental and spectroscopic evidence Xiaoming Liu a,b, Yi Hou a, Zhen Li c, Zen Yu b,⇑, Jia Tang b, Yueqiang Wang b, Shungui Zhou b,d a

State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510641, China Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China c College of Packaging and Materials Engineering, Hunan University of Technology, Zhuzhou 412007, China d Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China b

a r t i c l e

i n f o

Article history: Received 21 April 2019 Revised 31 December 2019 Accepted 31 December 2019

Keywords: Hyperthermophilic composting Humic acids Elemental analysis Excitation-emission matrix-parallel factor (EEM-PARAFAC) analysis Two-dimensional FTIR correlation spectroscopy (2D-FTIR-COS) analysis

a b s t r a c t Application of thermophilic composting (TC) is limited due to poor efficiency and long composting period. Hyperthermophilic composting (HTC) could effectively overcome this defect. Here, the transformation of humic acid (HA) in both HTC and TC was characterized and compared to investigate the roles of HTC toward accelerating the formation of HA. In HTC, the highest temperature was 96.6 °C, and the hyperthermophilic and thermophilic phases exceed 18 days. The degree of polymerization (DP) in HTC increased to 1.27 on day 27, while it only increased to 1.15 at the end of TC. The elemental composition of the HA in HTC showed higher O atomic content (36.3%) and lower C/N atomic ratio (6.5) compared with TC. These changes indicated that HTC could significantly accelerate oxidized and polycondensed reactions for HA formation, which resulted in the shortening of composting period to 27 days. The maximum fluorescence intensity (Fmax) of humic-like components were achieved faster in HTC (Fmax = 1649.9) than in TC (Fmax = 1316.9), implying that HTC promoted the polycondensation of small molecular components to form HA with larger molecular weight and higher degree of aromatization. Two-dimensional FTIR correlation spectroscopy (2D-FTIR-COS) analysis demonstrated that HTC prevented the HA precursor from condensing before it was deeply oxidized, and increased the content of small molecules rich in carboxyl moieties. Based on the evolution of the molecular structure of HA, the level of oxidation of HA precursors was a key factor to determine the degree of polymerization and the degree of HA humification. Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction Due to the rapid development of urbanization and industrialization in China, the treatment and disposal of sewage sludge has

Abbreviations: C1, Component I; C2, Component II; C3, Component III; DP, Degree of Polymerization; EEM, Excitation-Emission Matrix; EEM-PARAFAC, Fluorescence EEM-Parallel Factor; Em, Emission; Ex, Excitation; FA, Fulvic Acid; FAC, Fulvic Acid Carbon; Fmax, Maximum Fluorescence Intensity; FTIR, Fourier Transform Infrared; HA, Humic Acid; HAC, Humic Acid Carbon; HTC, Hyperthermophilic Composting; OM, Organic Matter; PARAFAC, Parallel Factor; RH, Rice Husks; SS, Sewage Sludge; SFI, Specific Fluorescence Intensity; TC, Thermophilic Composting; TEC, Total Extractable Carbon; 2D-FTIR-COS, Two-dimensional FTIR Correlation Spectroscopy. ⇑ Corresponding author at: Guangdong Institute of Eco-environmental Science & Technology, Guangzhou 510650, China. E-mail address: [email protected] (Z. Yu). https://doi.org/10.1016/j.wasman.2019.12.053 0956-053X/Ó 2020 Elsevier Ltd. All rights reserved.

become one of the most critical environmental issues (Gao et al., 2015; Meng et al., 2018; Zhang et al., 2018). Composting is an environmentally-friendly and economically feasible technique for the conversion of sewage sludge into safe and stable products (Zhang et al., 2014). However, the application of conventional thermophilic composting (TC) is limited due to poor efficiency, long composting period, and unsatisfying compost quality (Xiao et al., 2009; Meng et al., 2017). Temperature is the key factor that determines the efficiency of composting. Several studies have shown that increasing the temperature of the composting can accelerate the humification process and shorten the composting cycle (Oshima and Moriya, 2008; Tashiro et al., 2016). Studies in TC have shown that the compost temperature cannot exceed 70 °C; otherwise, the effect is detrimental for composting (Bernal et al., 2009). Recently, hyperthermophilic composting (HTC) has been developed, which increases the aerobic fermentation temperature with-

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out the requirement for external heating by adding extremely thermophilic microorganisms (Tashiro et al., 2016; Yu et al., 2018b). The highest temperature of the HTC process could exceed 90 or even 100 °C, which is remarkably higher than the temperature of TC, and thus increases the bioconversion efficiency of organic matter (OM) (Wu et al., 2018; Yu et al., 2018a, 2018b). Several previous studies reported that HTC could be successfully applied for the disposal of livestock manure and sewage sludge, while achieving significant improvements in composting efficiency (Oshima and Moriya, 2008; Tashiro et al., 2016; Yu et al., 2018b). These studies mainly focused on the influence of HTC on the variation of physicochemical properties and microbial community structure (Liao et al., 2018; Yu et al., 2018b). However, little information is available about the effect of HTC on the transformation of OM. The humification process of OM is the most important process to transform biologically degradable OM into fully stabilized and recalcitrant matter (Yuan et al., 2011; Zhang et al., 2015). Both the contents and structures of humic acid (HA) have been frequently related to the humification degree of OM. Furthermore, the molecular weight and aromatic characteristic of HA in composts are considered as important indicators for the estimation of the biological maturity and chemical stability of the resulting composts (Wang et al., 2014; Li et al., 2017). Thus, understanding HA formation is very important for the OM transformation during the humification process. Over the last decades, most of the studies regarding the formation processes of HA focused on TC and vermicomposting and used either fluorescence excitation-emission matrix (EEM) or Fourier transform infrared (FTIR) analyses (Lee et al., 2015; Shi et al., 2018; Gao et al., 2019). Moreover, previous work provided evidence for the molecular composition and formation mechanism of HA during composting (Amir et al., 2006; Wang et al., 2014). However, as a new composting technology, few studies have explained the mechanism of HA formation for HTC. In addition, HTC has a high fermentation temperature and a unique microbial community (Yu et al., 2018b), which may lead to a different molecular composition and formation mechanism of HA than that of TC during composting. Concurrently, HTC can promote the rapid maturity of compost, which may be attributed to the rapid formation of HA. Therefore, such a formation mechanism of HA has the expected importance to fill the knowledge gaps of HTC accelerated compost maturity. The EEM spectroscopy technique has been used to characterize the structures of HA due to its advantages of rapid speed, selectivity, and sensitivity (Wang et al., 2014). However, the use of EEM spectra alone typically prevents to obtain information for the complete chemical characterization of HA due to the overlap of many fluorescence signals (Li et al., 2014b). Fortunately, the introduction of parallel factor (PARAFAC) analysis to EEM spectra can decrease the interference from overlapping fluorophores among various compounds, which provides quantitative information on the concentration of each individual component (Lv et al., 2014). Although EEM-PARAFAC is capable to study the fluorescence components of HA, it cannot characterize the functional group changes and sequences of HA. FTIR combined with two-dimensional correlation analysis (2DCOS) can provide particular molecular information about the binding process, and is therefore a good solution for this problem (Chen et al., 2015; Liu et al., 2017; Tang et al., 2018). FTIR spectroscopy, which is a commonly used technique applied in the analytical molecular structure, can provide comprehensive insight into the functional groups of the principle organics in HA (Amir et al., 2004; Amir et al., 2010; Provenzano et al., 2014; Jindo et al., 2016). Moreover, 2D-FTIR-COS can confirm information about the relative directions and sequential orders of molecular structure variations by extending spectra along the second dimension (Gao et al., 2019). Due to these appealing features, a combina-

343

tion of EEM-PARAFAC and 2D-FTIR-COS has been utilized to obtain an in-depth understanding of the formation of HA. This study used elemental analysis to investigate variations in the elemental composition of HA. Furthermore, EEM-PARAFAC was applied coupled with 2D-FTIR-COS to assess the molecular composition and formation mechanisms of HA during the composting process. We hypothesized that HTC could promote the oxidation of small molecule components and increase the degree of HA polycondensation. The data obtained in this study provides evidence for the mechanism with which HTC affects HA formation. 2. Materials and methods 2.1. Composting process For compost preparations, the raw materials of sewage sludge (SS) and rice husks (RH) were collected from a municipal wastewater treatment plant (Zhengzhou, China) and a local rice processing plant (Zhengzhou, China), respectively. Composting end products from previous composting round were applied as composting microbial agent in HTC. The moisture content of the raw materials was determined after drying the samples at 105 °C for 24 h. The total organic carbon (TOC) and total nitrogen (TN) contents were determined according to Yu et al. (2018b). The physico-chemical properties of the raw materials are presented in Table S1. Approximately 300 t of composting raw materials were used for HTC and TC in this study. Each pile contained a mixture of 120 t SS (73.6 wt% moisture), 5 t RH (9.0 wt% moisture), and 25 t composting end products (36.8 wt% and 38.9 wt% moisture in HTC and TC, respectively). Both piles were loaded into fermentation compartments (of 7 m length, 5 m width, and 3.5 m height) at about 2.3 m bulk height. Forced ventilation and mechanical turning were used to aerate the materials at a typical turning frequency of 3–5 days, which was chosen according to the composting temperature. The initial moisture content of the mixture for each pile was adjusted to 60 ± 2% without further adjustments of the composting process. The temperature of each pile was monitored daily at 40–50 cm depth with a digital thermometer (Zhong Yi Instrument WSS301, China). The whole period of the experiment was conducted for 45 days and the samples were collected on days 0, 7, 18, 27, and 45. During the sample collection process, each pile was roughly divided into three regions according to its length. Multiple subsamples were removed from different sites of each region (at 50–100 cm depth) and combined to yield one composite sample. Samples were air-dried, passed through a 0.25 mm sieve, and then stored in desiccators for further analyses. 2.2. Extraction of humic compounds Total extractable carbon (TEC) and fulvic acid carbon (FAC) were extracted according to the methods described by Wang et al. (2014). To extract the TEC, the samples was mixed with 0.1 M NaOH (1:20 w/v ratio) and constantly shaken for 4 h. FAC was obtained by precipitation at pH 1.0–2.0. TEC and FAC contents were determined using TOC analyzer (Shimadzu Corporation TOC-L, Japan). Humic acid carbon (HAC) was calculated via the difference between FAC and TEC (Wang et al., 2014). 2.3. Isolation and purification of HA HA fractions were isolated according to procedures as suggested by the International Humic Substances Society (IHSS). Briefly, 30 g of each compost sample were air-dried and crushed, and then added to 100 mL of 0.1 M HCl to remove water-soluble and acid-soluble substances. The residues were extracted with

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100 mL of 0.3 M NaOH. The mixtures were shaken mechanically for 4 h at 25 °C. The supernatant solutions were separated from the residues via centrifugation at 12000  g for 15 min. This procedure was repeated until a clear supernatant was obtained. The combined alkaline supernatants were acidified using 6 M HCl at 4 °C to permit coagulation of the HA fractions, and then centrifuged at 12000  g for 15 min. Afterwards, the precipitates were repeatedly purified using KOH. The thus obtained HA was repeatedly rinsed with water until Cl- was undetected, and then freezedried. Ash contents was measured by heating the Has for 2 h at 550 °C. 2.4. Elemental analysis Elemental analysis for C, H, N, and S of freeze-dried HA samples was performed using an elementary analyzer (Elementar Vario EL, Germany). The oxygen content was calculated by the difference: O% = 100 - (C + H + N + S)%. All HA samples were extracted and analyzed in triplicate. 2.5. EEM spectra analysis All HA samples were diluted with ultra-pure water to 10 mg/L organic carbon for EEM spectra analysis. The fluorescence EEM spectra were obtained with a FP-7000 fluorescence spectrophotometer (Hitachi, Japan) in a clear quartz cuvette. EEM spectra were scanned using an emission (Em) wavelength from 250 to 600 nm and an excitation (Ex) wavelength from 200 to 500 nm. The emission and excitation slits were fixed at a band width of 5 nm and the scanning speed was set to 2400 min 1. The voltage of the photomultiplier tube was set to 750 mV for low level light detection (Yu et al., 2018a). A total of 30 EEMs (10 samples  3 repetitions) were used for the analysis. The first- and second-order Rayleigh and Raman scatters were removed and adjusted via interpolation. PARAFAC analysis was performed in MATLAB 2013a (Math works, USA) using the DOMFluor toolbox (http://www.models.kvl.dk/) following the process described by Bahram et al. (2006). The PARAFAC model with 3 components was validated. The fluorescence maximum intensity for each PARAFAC component was used as index for the evaluation of HA synthesis efficiencies. 2.6. FTIR spectra FTIR spectra of all HA samples were recorded using a Nicolet Nexus FTIR spectrophotometer equipped with Nicolet Omnic 6.0 software (Nicolet 560E.S.P, America). Spectra were recorded in the range of 4000–400 cm 1 with 2 cm 1 resolution, and 64 scans were performed on each acquisition.

2.7. 2D-FTIR-COS 2D-FTIR-COS was performed using FTIR spectral data according to the method by Noda and Ozaki (2005). In brief, the FTIR spectra data were normalized according to the method of the reference (Li et al., 2014). Subsequently, normalized FTIR spectra were analyzed using 2Dshige software (Kwansei-Gakuin University, Japan). The composting time was employed as external perturbation for 2DFTIR-COS, and a set of time-dependent FTIR spectra data was obtained. The plotting function of Origin 2017b was used to visualize data. Spectral coordinates, intensities, and signs of correlation peaks appearing on the 2D spectra were interpreted by a set of well-established principles (Liu et al., 2017; Tang et al., 2018; Yu et al., 2018a). In the synchronous spectra map, auto-peaks (always positive) suggest spectral intensities, while cross-peaks represent the direction of the intensity change at corresponding spectral coordinates. An asynchronous spectra map exclusively showed cross-peaks. Their signs indicate the sequential order of the dynamics of spectral intensity variations induced by the composting time. The same sign of the cross-peaks (x1, x2) in both synchronous and asynchronous spectra maps indicates that the signal change in x1 is quicker than in that in x2. This order is reversed when the signs of cross-peaks are opposite.

3. Results and discussion 3.1. Variation of temperature and humic substance content during composting The temperature evolutions at different composting stages are shown in Table 1. The three distinct phases of HTC, namely hyperthermophilic (80 °C), thermophilic (50–80 °C), and mature phases (50 °C) can be clearly distinguished. The temperature in HTC reached a maximum value of 96.6 °C on day 7, and its change trend indicated that HTC ran at the hyperthermophilic and thermophilic phases for more than 18 days under the lower initial C/N condition, which was consistent with the existing researches (Oshima and Moriya, 2008; Liao et al., 2018; Yu et al., 2018a). The temperature profile has also been applied to monitor the HTC stabilization and microbial activity (Yu et al., 2018b). The temperature in TC maintained a decreasing trend during the whole composting period; however, the temperature in HTC decreased to 35.1 °C on the 27th day, where it remained until the end of the experiment, indicating that OM can be quickly transferred into increasingly stable states. These results suggested that increasing the temperature during the composting process accelerated the humification process and thereby contributes to the fast stabiliza-

Table 1 Temperature evolution of piles and elemental composition of humic acids isolated from hyperthermophilic compost (HTC) and thermophilic compost (TC) at different stages of composting. Composts

HTC

TC

Elapsed time (d)

0 7 18 27 45 0 7 18 27 45

Temperature (°C)

35.2 96.6 61.3 35.1 32.2 31.7 55.9 50.4 52.3 45.8

C (%)

53.93 53.03 48.17 48.13 47.34 53.97 53.89 51.11 49.65 48.04

N (%)

± ± ± ± ± ± ± ± ± ±

0.27 0.17 0.06 0.07 0.09 0.16 0.12 0.23 0.29 0.07

7.22 7.20 8.28 8.38 8.49 7.22 6.95 6.97 8.02 8.25

± ± ± ± ± ± ± ± ± ±

H (%)

0.04 0.04 0.04 0.04 0.03 0.04 0.07 0.06 0.06 0.03

6.33 6.29 5.43 5.21 5.10 6.37 6.53 6.15 5.44 5.33

± ± ± ± ± ± ± ± ± ±

O (%)

0.03 0.03 0.04 0.04 0.05 0.05 0.09 0.06 0.04 0.03

29.84 30.86 35.50 35.66 36.34 29.81 29.93 33.16 33.84 35.51

Atomic ratios

± ± ± ± ± ± ± ± ± ±

0.22 0.17 0.11 0.08 0.11 0.17 0.29 0.12 0.32 0.08

C/N

C/H

C /O

8.72 8.60 6.79 6.70 6.51 8.73 9.05 8.55 7.23 6.77

0.71 0.70 0.74 0.77 0.77 0.71 0.69 0.69 0.75 0.75

2.41 2.29 1.81 1.81 1.75 2.41 2.39 2.06 1.95 1.81

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tion of OM, which was similar to previous reports (Tashiro et al., 2016; Yu et al., 2018a). Changes in the contents of humic substance during HTC and TC are shown in Fig. 1. The TEC decrease corresponded to the FAC decrease (Fig. 1a), which is possibly due to the dramatic decrease of fulvic acid (FA). This noticeable reduction in FA indicates that the FA contained easily bio-degradable organic compounds (Li et al., 2017). Moreover, the decrease of FA content in the composts implied that easily available carbon was reduced and increased the stability of composts (Li et al., 2017). The contents of FAC in HTC decreased significantly from 45.3 g/kg to 24.2 g/kg between the start of the experiment and the 27st day of composting. Thereafter, these contents remained almost constant until the end of experiment, indicating a high degree of stabilization. The increase of the HAC content was related to the humification degree of the organic matter during composting (Wang et al., 2014). HAC contents (Fig. 1b) increased in TC and HTC, which indicated that composting led to the humification of organic matter. HAC content in HTC increased from 18.83 g/kg to 32.75 g/kg during the composting process, while that in TC increased from 20.1 g/kg to 23.2 g/kg. The HAC content in HTC increased faster than that in TC throughout the entire composting process. This was particularly evident between the 7th and the 18th day of composting, which indicated that the humification process was closely related to the composting temperature. The HAC/FAC ratio represents the degree of polymerization (DP), which is one of the most sensitive indicators for humification and has been proposed as an index of maturity by several investigators (Wang et al., 2014; Zhang et al., 2018). Compost with DP  1 has been identified as mature compost (Roletto et al., 1985). The value of DP in HTC increased to 1.28 on day 27, while it only increased to 0.72 in TC, suggesting that HTC could significantly shorten the composting period compared to TC. In addition, since more complex polymeric humic structures were formed from the polymerization of simple molecules, the DP value increased. HTC achieved a significantly higher DP value than TC during the composting process. This may be because HTC promotes the polycondensation of small molecules such as HA precursors to rapidly synthesize HA. 3.2. Elemental analysis The elemental composition of HA extracted from both composting treatments at different stages of composting are listed in Table 1. The variations of C, H, O, and N contents suggest that oxidation, dehydrogenation, and the incorporation of N-containing compounds into HA structures occurred over the whole compost-

345

ing process (Zhang et al., 2015; Li et al., 2017). Through the hyperthermophilic phase in HTC, both the C and H contents decreased from 53.9% (day 0) to 48.2% (day 18) and 6.3% (day 0) to 5.4% (day 18), while the O and N contents increased from 29.8% to 35.5% and 7.2% to 8.3%, respectively. The O and N contents in TC were 35.5% and 8.2% at the end of composting, while the C and H contents decreased to 48.0% and 5.3%, respectively. This suggests that high temperature could promote the formation of stable Nrich structures and recalcitrant oxygenated compounds. Additionally, these results reflected that organic carbon compounds were actively degraded during the hyperthermophilic phase. The atomic ratio changes of C/H, C/N, and C/O are also shown in Table 1. In the present study, HA in HTC and TC showed the low C/N ratio (8.72 and 8.73, respectively) at the beginning of composting. This was mainly due to the high proportion and the characteristics of sewage sludge in initial mixtures (Zhang et al., 2014). The C/N atomic ratio in HTC and TC decreased significantly, suggesting that composting could concentrate N-rich structures with progressing composting (Marine et al., 2005; Amir et al., 2010). This result could be attributed to the decomposition of easily biodegradable matters, such as protein decomposition products, which were formed from the nitrogen-rich compounds by condensation (Adani et al., 2006; Li et al., 2014a). In addition, the C/N ratio in TC was increased on days 0–7, which might be that N-containing components were converted into volatile NH3 by decomposition, thereby reducing the N content of TC. Furthermore, during the hyperthermophilic and thermophilic period on days 7–18, the decrease in the C/N atomic ratio for HTC was notably higher than that in TC. This result indicated that high temperature could accelerate the decline of C/N ratio. The C/N atomic ratio is typically used to indicate structural changes of HA during composting (Zhang et al., 2015; Li et al., 2017). The C/N atomic ratio in HTC decreased to 6.70 on day 27, while it only decreased to 6.77 at the end of TC. These observations are supported by the fact that HTC enhanced humification, which shows that HTC facilitates the aromatic condensation of HA. Amir et al. attributed the increasing C/H to either an increment of aromatic structures or an increment of aliphatic structures (Amir et al., 2010). At the end of composting, the slight increase of C/H values in HTC and TC suggests that the proportion of aromatics in HA increased. Based on these interpretations, we concluded that HTC may accelerate the oxidation, polycondensation, and aromatization reactions during HA formation. 3.3. Fluorescence EEM analysis and EEM-PARAFAC analysis EEM spectra of HA samples at different composting stages are displayed in Fig. 2. The Ex/Em wavelength pairs and specific fluo-

Fig. 1. Change in (a) total extractible carbon (TEC) and fulvic acid carbon (FAC) (b) degree of polymerization (DP) and humic acid carbon (HAC) during hyperthermophilic composting (HTC) and thermophilic composting (TC).

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Fig. 2. Fluorescent excitation emission matrix (EEM) spectra of the humic acids from samples at the days 7, 18, 27, and 45 of composting for hyperthermophilic composting (HTC) and thermophilic composting (TC).

rescence intensity (SFI) of peaks are listed in Table S2. Two peaks (Peak A and Peak B) appeared in HTC and TC. The Ex/Em wavelength pairs of Peak A were recorded at 270/430–470 nm, while Peak B presented Ex/Em wavelength pairs of 350/430–470 nm. According to the protocol of Chen et al., Peak A and Peak B could be attributed to humic-like substances (Chen et al., 2003). The fluorescence intensity could be linked to the degree of compost humification and aromatization (He et al., 2011; Wang et al., 2014). During the initial 27 day of composting, the intensities of Peak A and B in HTC showed an increasing trend, while their intensities from day 27 to day 45 remained nearly identical, indicating that compost had matured. Compared with the EEM plots of HTC, although they share a similar contour pattern, a notable difference between HTC and TC was the relative fluorescence intensities of peaks. Peak A and B increased in intensity for both treatments. It is apparent that the intensities of both peaks were significantly higher in HTC after the 7th day than in TC. PARAFAC analysis was used to decompose the fluorescence EEM spectra of the obtained HA. Fig. 3 shows the EEM contours and the relative distribution of the three components. Fig. S2 shows the excitation and emission wavelength pairs of the main peaks of each component as well as a description of similar components of previous studies. Comparison with the previously identified components indicated that samples contain protein-like and humic-like fluorophores (Bikovens et al., 2012; Zhang et al., 2016b). Component I (C1) was composed of two peaks with excitation/emission (Ex/Em) wavelengths at 270, 350/438 nm. Component II (C2) was composed of two peaks at 270, 400/495 nm excitation/emission wavelengths. C1 and C2 were similar to the components associated with humic-like substances. According to previous reports, C1 and C2 have often been found in a wide range of aquatic and terrestrial environments as well as in mature composts (Wu et al., 2011; Lv et al., 2014; Tang et al., 2018; Yu et al., 2018a). Moreover, both components were often associated with OM composed of high molecular weight and aromatic organic compounds with longer Ex/Em (Henderson et al., 2009). Therefore, the increasing amounts of C1 and C2 in this study suggest increased molecular weight and aromaticity of humic substance during composting. C3 showed two dominant peaks around Ex/E m = 220/365 nm and 280/365 nm, and also two minor bands cen-

tered at Ex/Em = 220/480 nm and 280/480 nm. These peaks were similar to tryptophan-like components in un-denatured protein structures and could also be contributed by polyphenol materials (Liu et al., 2018). These substances can be broken down into HA precursors (Wu et al., 2017). Thus, rapid decomposition of C3 can increase its concentration and promote HA synthesis. EEM-PARAFAC analysis also provided additional quantitative information for accessing the degree of humification during composting (Zhang et al., 2016b). As shown in Fig. 3b, the maximum fluorescence intensity (Fmax) of C3 in both treatments increased between the start of experiment and day 7 of composting, suggesting that large organic molecules were broken down into smaller, soluble molecules (He et al., 2014; Lv et al., 2014). Thereafter, C3 decreased in Fmax for both treatments. It is apparent that the C3 in HTC achieved stabilization much faster than that in TC, indicating that HTC can promote the formation of HA precursors. Fmax of C1 and C2 in HTC showed an increasing trend between day 0 and day 18 of composting, while these components in TC immediately showed a decreasing trend as soon as composting started and an increasing trend thereafter. This suggests that the molecular weight and aromaticity of HA were markedly enhanced by HTC. According to previous reports, these two components were similar to humic components produced by microbial activity (Lv et al., 2014; Liu et al., 2018; Yu et al., 2018a). The Fmax of C1 and C2 in both treatments rapidly increased between day 7 and day 45. Interestingly, two components in HTC from day 7 to day 18 increased much faster than those in TC, indicating that HTC contributed to a more intensive aromatization. Additionally, Fmax of two components in HTC was remarkably higher than that in TC at the end of composting. These results further ascertained the findings of EEM spectra, indicating that HTC could improve the conversion efficiency of small molecules to macromolecules and increase their aromaticity during HA formation. 3.4. FTIR spectra FTIR spectra of HA samples extracted from both treatments are shown in Fig. S3. The main absorption bands and corresponding assignments are listed in Table 2 according to previous publications (Li et al., 2011; Chen et al., 2014; Wang et al., 2014; Chen

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Fig. 3. Three fluorescent components decomposed by the PARAFAC model according to the EEMs of humic acid samples (a); distribution of three PARAFAC-derived components (b).

Table 2 Infrared indicator bands in the HA-FTIR and the characteristic IR bands present as 2D-FTIR-COS maps. Bands and peaks (cm

1

)

1

2923, 2855 cm 1710–1690 cm 1 1660, 1650, 1644 cm 1575–1577 cm 1 1560 cm 1 1541–1550 cm 1 1490 cm 1 1460 cm 1 1430 cm 1 1365 cm 1 1330 cm 1 1260, 1245 cm-1 1230 cm 1 1200 cm 1 1125–1145 cm 1 1090–1030 cm 1

1

Vibration

Functional group or component

C–H stretching C = O stretching C = O stretching C = C stretching C = O stretching N–H deformation and C = N stretching C = C stretching C–H bending C = O stretching C-N stretching C-N stretching C-O stretching C-O stretching, O-H deformation, C-O stretching C-O stretching C-O stretching C-O stretching

Aliphatic methylene groups carboxylic acid group Quinone, Amide, or ketone Imidazole, aromatic ring modes Carboxylate Amide II Aromatic ring CH2 or CH3 groups Carboxylate Amide II Aromatic primary and secondary amines Aryl ethers Carboxylic acids, aliphatic Aromatic acid, aliphatic acid ester Aliphatic OH Polysaccharides or polysaccharide-like substances

et al., 2015; Liu et al., 2017). FTIR bands and peaks displayed a marked difference during different compost stages. At the final stage of composting, the absorption intensities at 2923, 2855, and 1715 cm 1 (stretching of aliphatic C-H and not conjugated carboxylic C = O) had clearly decreased, indicating a decrease of aliphatic materials. The absorption intensity at 1650 cm 1 (aromatic C = C, C = O stretching, amide I) increased significantly, which indicated the enrichment of carboxylic acids and aromatic structures during the composting process. The FTIR spectra of HA for TC and HTC generally exhibited similar resonances; however, a significant difference of peak ratios was found. The peak ratios have been used to monitor chemical changes during the composting (He et al., 2011). This was particularly evident on day 18, when 1650/2923 (aromatic C/aliphatic C-H groups) in HTC was 1.39,

which exceeded that of TC (1.21). This suggests that HTC promoted the rapid conversion to fatty substances into small molecules of HA precursors and contribute to the formation of HA substances with high aromaticity and polycondensation. 3.5. 2D-FTIR-COS analysis Overlapping IR peaks are major obstacles for band assignment and for the analysis of the conformational changes of HA; therefore, 2D-FTIR-COS was applied to obtain more details on the process of HA formation (Li et al., 2014b; Yu et al., 2018a). To understand the changing sequence of functional groups of HA with composting time, the 1850–800 cm 1 and 3750–2250 cm 1 regions of 2D-FTIR-COS were analyzed, and both the synchronous

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Fig. 4. Synchronous (a, c) and asynchronous (b, d) two-dimensional FTIR correlation spectra (2D-FTIR-COS) maps from the 800–1850 cm infrared spectra of humic acid samples during hyperthermophilic composting (HTC) and thermophilic composting (TC).

1

region of the Fourier transformed

Table 3 2D-FTIR-COS results on the assignment and sign of each cross-peak and asynchronous maps of humic acids with composting time (a) HTC-2D-FTIR-COS results on the assignment and sign of each cross-peak and asynchronous maps of humic acids with composting time; (b) TC-2D-FTIR-COS results on the assignment and sign of each cross-peak and asynchronous maps of humic acids with composting time. Position (cm

1

)

(a) 1695 1650 1575 1550 1490 1462 1430 1330 1260 1245 1200 1090 Position (cm

(b) 1710 1675 1575 1541 1490 1458 1365 1230 1120 1050

1

)

Sign 1695

1650

1575

1550

1490

1462

1430

1330

1260

1245

1200

1090

+

+ (+) +

+ (-) + (-) +

+ (-) + (-) + (+) +

+ + + + +

+ + + + + +

+ + + + + + +

+ + + + + + + +

+ + + + + + + +

+(+) + (+) + (+) + (+) + (+) + (+) + (+) + (+) + (-) +

+ + + + + + + + + + +

+ + + + + + + + + + + +

(-) (-) (-) (-)

(+) (+) (+) (-) (+)

(+) (+) (+) (+) (+) (-)

(-) (-) (+) (+) (+) (-) (-)

(+) (+) (+) (+) (+) (+) (+) (+)

(+) (-) (+) (+) (+) (+) (+) (+) (-) (-)

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)

Sign 1710

1675

1575

1541

1490

1458

1365

1230

1120

1050

+

+ (-) +

+ (-) + (+) +

+ (-) + (-) + (+) +

+ + + + +

+ + + + + +

+ + + + + + +

+ + + + + + + +

+ + + + + + + + +

+ + + + + + + + + +

(-) (-) (-) (-)

(-) (-) (+) (+) (+)

(-) (-) (+) (-) (+) (-)

(-) (-) (+) (+) (+) (-) (+)

(-) (-) (+) (+) (+) (-) (+) (-)

(-) (-) (+) (+) (+) (-) (+) (-) (+)

X. Liu et al. / Waste Management 103 (2020) 342–351

and asynchronous spectra are shown in Fig. 4 and S4. The 1850–800 cm 1 region includes bands corresponding to amides, carboxylic acids, esters, aliphatic groups, and carbohydrates (Li et al., 2014b). Thus, this region of the FTIR was mainly analyzed and discussed. Fig. 4a, b show the 2D-FTIR-COS of the HA interaction with composting time in the HTC. The sign and assignment of each cross-peak are listed in Tables 2 and 3, respectively. In the synchronous maps, the predominant auto-peaks appeared at 1034, 1060, 1220, 1331, 1440, 1461, 1546, 1607, 1625, and 1720 cm 1 (Fig. 4a). The signs of all cross-peaks in the synchronous map were positive, indicating that OM transformed into new HA in the same direction. In particularly, the most significant changes were found at 1606 and 1625 cm 1, which could be attributed to an enrichment of carboxylic acids and aromatic structures in the process of HA formation. Compared to the synchronous map, the asynchronous map displays distinctive characteristics. In general, 12 main cross-peaks were found above the diagonal line in the asynchronous map (Fig. 5b). According to the rule of Noda and Ozaki (2005), it could be concluded that the structural changing sequence of HA with composting time followed the order: 1490 > 1575 > 1546 > 1331 > 1430 > 1695 > 1200 > 1650 > 1245 > 1 260 > 1462 > 1090 cm 1. This translates to C = C stretching of aromatic ring modes and imidazole ? C-N and C = N stretching in amides ? C = O stretching vibration in the carboxylic acid, quinone, or ketone ? C-O stretching of aryl ethers ? C-H bending of CH2 or CH3 groups ? C-O stretching of polysaccharides. For TC, the synchronous map (Fig. 4c) shows seven major autopeaks centered at 1710, 1656, 1541, 1456, 1400, 1230, and 1051 cm 1. These changes showed a similar trend than the peak of HTC, indicating that the compost could influence the humification of OM. However, the asynchronous map in TC displayed different characteristics compared to HTC. There were 10 main crosspeaks in the asynchronous map (Fig. 4d). The sign of each crosspeak is listed in Table 3. Thus, the final sequence order of the HA formation could be concluded on the basis of the rule of Noda and Ozaki (2005) as: 1490 > 1575 > 1365 > 1541 > 1120 > 1050 > 1230 > 1458 > 1675 > 1710 cm 1. This translates to C = C stretching of aromatic ring modes and imidazole ? C = N stretching in amide II ? C-O stretching of polysaccharide, alcohols and phenols ? C-H bending of CH2 or CH3 groups ? C = O stretching vibration in the carboxylic acid, quinone, or ketone.

349

3.6. Effects of HTC on the HA formation process Composting is a humification process where microorganisms mineralize OM and promote HA formation. Undoubtedly, understanding the HA synthesis process is central for the improvement of the humification degree. To better understand the evolution of HA chemical structures during composting, 2D-FTIR-COS analysis was used to study the HA formation process in the present work (Fig. 5). In HTC, C = C, C-N, and C = N stretching implied that the protein-like material was broken down into HA precursors. The detailed transformation order of the protein decomposition product is listed in the following: tryptophan-like ? tyrosine-like ? histidine-like. These findings agreed with the report of Li et al. (2014). Thereafter, C = O bonds stretched and changed, which could be explain by small molecule organic matter transformation into matters of carboxylic acids, quinones, and ketones by oxidation. This result indicates that the oxidation played an importance role during the humification process. The C-O stretching of aryl ethers indicates that the carboxylic acids, quinones, and ketones lead to the formation of HA following self-polycondensation and/ or combination with other nitrogen-containing compounds (Martin and Haider, 1971; Amir et al., 2006). In addition, the C-H of the aliphatic group and the C-O of polysaccharides changed, which could be attributed to the death of most of the microbial population at the end of composting, typified by a low respiratory rate. In contrast, the sequence order of HA in the TC synthesis exhibited a significant difference for HTC (Fig. 5). The C-O and C-H bonds changed faster in TC than in HTC, indicating that small molecules, such as proteins and polysaccharide decomposition products, attached to the C skeleton of HA in advance via condensation. In addition, the C = O bonds in TC showed a slower change than the C-O bonds. This result indicates that small-molecules aggregated to form macromolecules with uneven structures that were oxidized and formed carboxylic acids, quinones, and ketones. However, macromolecular substances were difficult to be oxidized to form HA precursors that are rich in carboxyl and hydroxyl moieties. Simultaneously, elemental analysis also indicated that the degree of oxidation of organic matter was significantly lower in TC than in HTC. Consequently, this suggests that the premature polycondensation of small molecules could be an important reason hindering the further oxidation of HA precursors.

Fig. 5. The structural changing sequence of HA with composting time during hyperthermophilic composting (HTC) and thermophilic composting (TC).

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Considering the afore-mentioned information, we speculated about the mechanism underlying the HTC acceleration of the formation of HA during composting. During the composting process, deep oxidation of unpolymerized small molecules promoted the formation of HA precursors rich in carboxyl and hydroxyl moieties. This improved the external environment of the condensation reaction and promoted HA synthesis. This explains why the content of HA was significantly higher in HTC compared to TC. Moreover, HA precursors were tightly bound to each other by condensation reactions, reducing the number of available aromatic binding sites. These results testified that HA in HTC has a compact structure and a high degree of humification. The DP value and EEMPARAFAC analysis also showed that HTC facilitated the condensation of small molecules into macromolecules. Gao et al. also showed that increasing carboxyl-rich humic precursors could promote the formation of macromolecules with complex and uneven structure by aggregation (Gao et al., 2018). These results indicate that HTC enhanced the condensation reaction by increasing the oxidation of small molecules, thus accelerating the formation of HA with high molecular weight and aromatization degree. Therefore, the combined application of 2D-FTIR-COS and EEMPARAFAC provided theoretical support and evidence for accelerating HA formation. 4. Conclusion The formation processes of HA in both HTC and TC was investigated and compared by a series of chemical and spectroscopic methods. During the HTC process, the temperature climbed rapidly to the thermophilic phase with a maximum value of 96.6 °C and maintained for>18 days. The DP value in HTC was 1.27 on day 27, while it was only 1.15 at the end of TC, implying that HTC could significantly shortened the composting period from 45 days to 27 days. Elemental analysis showed that HA in HTC had higher degrees of oxidation and polycondensation due to their low C/N atom ratio (6.51) and high O content (36.3%). The synthesis of humic-like components (C1 and C2) was faster in HTC than that of TC. FTIR and 2D-FTIR-COS analyses showed that the change of carboxylic acids and aromatic structures in HTC were faster than those in TC, suggesting that intensifying the oxidation of the HA precursor was key to accelerate the polycondensation reactions and enhance both the quantity and quality of HA. These results provide a more detailed theoretical basis for HTC to accelerate the formation of HA at the molecular level. Acknowledgments This study was supported by the GDAS’ Project of Science and Technology Development (No. 2019GDASYL-0501005), the Science and Technology Planning Project of Guangdong Province, China (No. 2019B110205003), the Science and Technology Planning Project of Guangzhou City, China (No. 201903010071), and the Scientific Research Project of Hunan University of Technology (No. 2013HZX02). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.12.053. References Adani, F., Genevini, P., Tambone, F., Montoneri, E., 2006. Compost effect on soil humic acid: A NMR study. Chemosphere. 65, 1414–1418.

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