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Spectroscopic evidence for biochar amendment promoting humic acid synthesis and intensifying humification during composting
Journal of Hazardous Materials 280 (2014) 409–416
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Spectroscopic evidence for biochar amendment promoting humic acid synthesis and intensifying humification during composting Cheng Wang a,1 , Qiaoping Tu a,1 , Da Dong a , P.J. Strong b , Hailong Wang c , Bin Sun a , Weixiang Wu a,∗ a
Institute of Environmental Science and Technology, Zhejiang University, Yuhangtang Road 866#, Hangzhou 310058, China Centre for Solid Waste Bioprocessing, School of Civil Engineering, School of Chemical Engineering, University of Queensland, St Lucia, Brisbane 4072, Australia c School of Environmental and Resource Sciences, Zhejiang A & F University, Lin’an, Hangzhou 311300, China b
h i g h l i g h t s • Biochar amendment promoted the neo-synthesis of humic acids during composting. • Higher O-alkyl C/alkyl C ratio and aromaticity of humic acids lead to more intense humification. • Increases in carboxylic groups of biochar may result from oxidation reactions and sorption of humic substances.
a r t i c l e
i n f o
Article history: Received 17 April 2014 Received in revised form 26 July 2014 Accepted 8 August 2014 Available online 27 August 2014 Keywords: EEM FT-IR 13 C-NMR composting biochar
a b s t r a c t Despite the many benefits of biochar amendment in composting, little information is available about its effects on organic matter humification during the process. In this study the analytical results for two in-vessel composting piles were compared, one amended with biochar (VPSB, pig manure + sawdust + biochar) and the other serving as a control (VPS, pig manure + sawdust). During the 74 days of humification, the increased content of humic acid carbon in VPSB is 16.9% more than that of the control. Spectroscopic analyses show a higher O-alkyl C/alkyl C ratio and aromaticity in VPSB at the thermophilic phase, and peak intensities of fulvic-like and humic-like substances were achieved faster in VPSB than VPS. These data inferred that biochar amendment promoted the neo-synthesis of humic acids and intensified the humification of pig manure. Increase in carboxylic groups of biochar as a result of oxidation reactions and sorption of humic substances may correspond to the faster formation of aromatic polymers in biochar-supplemented composting pile. The results suggest that biochar amendment might be a potential method to enhance humification during pig manure composting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the livestock industry in China has expanded rapidly, bringing with it a surge in the volume of animal waste. According to the National Bureau of Statistics (2010), approximately 3.26 billion tons of animal manure was produced in 2009. If the fecal matter is directly applied to soil, it could pose a hazard in the form of antibiotics or pathogenic microorganisms [1]. As such, utilizing and disposing of this animal waste has become a
∗ Corresponding author. Tel.: +86 571 88982020; fax: +86 571 88982020. E-mail address: [email protected] (W. Wu). 1 These authors contributed equally to this work and should be considered co-first authors. http://dx.doi.org/10.1016/j.jhazmat.2014.08.030 0304-3894/© 2014 Elsevier B.V. All rights reserved.
major issue regarding solid waste management [2]. Composting is a treatment method that is a simple and cost-effective way to stabilize and humify this organic waste under aerobic conditions. It is of considerable economic importance as the resultant compost from livestock wastes can be directly recycled as a marketable organic fertilizer. A better understanding of the transformation and humification of organic matter throughout the composting process is essential for assessing compost maturity, achieving maturity more rapidly and improving quality. Humification is generally defined as the process of transforming biologically degradable organic matter into a fully stabilized, microbially recalcitrant humic substance. Successful humification of organic matter via composting is frequently a function of the type and quality of the bulking agent [3]. Carbon-rich bulking agents enhance the humification of animal manure and improve the
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quality of the final composts [4]. This is potentially due to modifying the physicochemical properties of initial composting mixtures, thereby providing a suitable environment for microbial activity. The most commonly used materials are chaff, straw and woody by-products, which typically have high C/N ratios. Recently, attention focused on biochar, a carbon-rich material that has been used successfully as bulking agent in poultry manure compost [5]. Previous studies have indicated that bamboo-derived biochar not only improved nitrogen and cation retention, but also lowered the total N2 O emissions from pig manure composting [6–8]. Furthermore, Steiner et al. [9] found that combining compost with biochar effectively remediated contaminated soils and improved soil nutrient retention. Although biochar amendment benefits composting as bulking agents, it is not known whether it enhances the decomposition rate and humification dynamics. Additionally, the mechanism by which biochar stabilizes and humifies pig manure is poorly defined. Humic substances are usually described as chemically complex and highly heterogenous organic substances comprising large macromolecules with oxidized functional groups [10]. They primarily consist of humic acids (HA), fulvic acids (FA) and humin. Throughout the composting period, microbial degradation lowers the FA and soluble organic carbon content of compost. In parallel, the formation of HA with increasing molecular weight and aromatic characteristics is indicative of a maturing composting [11]. Numerous structural studies have tried to achieve a better understanding of the formation of humic substances. These have been performed using advanced techniques such as 13 C nuclear magnetic resonance (13 C-NMR) and Fourier transform infrared (FT-IR) spectroscopy [12]. The aromatic content and degree of polycondensation increases, while aliphatic groups and peptide and carbohydrate components decreases with increasing composting maturity. Biochar is a carbonaceous residue of biomass pyrolysis and is highly aromatic. It has a high content of oxidized functional groups, such as C O and C O [13], which could be reactive with soluble organic carbon. Additionally, biochar may offer a suitable habitat for microorganisms to attach as it has a strong sorptive capacity due to its high microporosity and large surface area. Jindo et al. [14] suggested that biochar addition could exert an effect on the microbial community, thereby influencing the composting process and the quality of the end product. In this study, two pilot-scale in-vessel aerobic composting experiments were performed with, or without, biochar. We applied 3D excitation-emission matrix (EEM) fluorescence coupled with 13 C-NMR and FT-IR to quantify the changes of chemical composition and structure of the total extractable C (EXC) from samples during the pig manure composting. The primary aim of this study was to investigate the effects of biochar amendment on the dynamics of humification and molecular behavior of HA during composting. We hypothesized that supplementing compost with pyrolytic biochar would affect organic matter decomposition as well as HA structure via chemical interaction with organic matter. Data from this study would provide spectroscopic evidence for the mechanism whereby biochar amendment affected humification during pig manure composting.
16,500 kg flushed Pig manure + 1540 kg Sawdust), which was effectively the control sample, or VPSB (in-vessel, 16,500 kg flushed Pig manure + 1000 kg Sawdust + 540 kg Biochar), the biochar-amended sample. Flushed pig manure was collected from local piggery and its main characteristics were: pH (H2 O) = 6.46; water content = 68.2%; EC = 4.52 mS cm−1 ; and total nitrogen = 33.7 g kg−1 dry matter. Sawdust was used as the bulking agent (obtained locally)and its main characteristics were: pH (H2 O) = 6.73; water content = 11.7%; EC = 0.21 mS cm−1 ; and total nitrogen = 4.3 g kg−1 . The bulking agent was milled to a 5 cm particle size, and then homogeneously mixed with pig manure. Biochar, an abundant residue from bamboo processing was purchased from the Yaoshi Charcoal Production Company (Hangzhou, China). The biochar was pyrolyzed at a temperature of 600 ◦ C for 2 h and its main characteristics were: pH (H2 O) = 10.36; water content = 6.1%; C/N = 118; bulk density = 0.40 g cm−3 ; and specific surface area = 359 m2 g−1 . The compost piles were aerated using natural ventilation and mechanical turning using a tractor-pull windrow turner. Turning frequency varied from 2 to 7 days and was dictated by temperature of the composting windrows. Moisture content of the stock material was initially adjusted to 65 ± 2%, after which there were no further adjustments. The temperature at 30 cm depth below the surface of the compost piles and ambient air was recorded daily with a thermometer. On day 2, 7, 21, 36, 60 and 81 of composting, six subsamples were removed from six sites of the entire profile spanning the whole profile (from top to bottom) and combined to yield one composite sample. This was then divided into two parts that were both tested in triplicate. One part was immediately stored at 4 ◦ C until analyses, while the other part was air-dried, passed through a 0.25 mm sieve and stored in desiccators until further analyses. Germination index (GI) was measured according to the methods described by Chen et al. [6]. 2.2. Extraction of humic compounds The total extractable C (EXC) was obtained via four 0.1 M sodium hydroxide (NaOH) extractions at a ratio of 1:20 (w/v). The fulvic acid carbon (FAC) was obtained by precipitation at pH 1.0–2.0. The humic acid carbon (HAC) was calculated as the difference between the FAC and the EXC [15]. EXC and FAC fractions were quantified using TOC/TN Analyzer (multi N/C 2100/2100S, Analytik Jena AG). 2.3. Extraction and purification of HA Humic acids were extracted and purified according to SánchezMonedero et al. [16]. Briefly, 10 g of each compost sample, air-dried and crushed, were extracted using 0.1 M NaOH 4 times. Suspended solids were removed via settling and centrifugation (6000 g, 15 min). After centrifuging, the supernatants were carefully aspirated and acidified to pH 1.0 using 6 M HCl and left overnight at 4 ◦ C. The HA precipitates were separated via centrifugation (6000 g, 15 min). The precipitates were purified by dissolving in a minimal volume of 0.1 M NaOH and dialyzed with Spectra Por (1000 Dalton MWCO) to eliminate excess salts and finally freeze-dried (72 h).
2. Materials and methods 2.4. 3D-EEM fluorescence spectroscopy 2.1. Composting experiments and sampling Two pilot-scale in-vessel aerobic composting treatments were set up in a suburb of Hangzhou in China and monitored for approximately 12 weeks. Each compost heap consisted of pig manure and bulking agent (sawdust), with one pile receiving the biochar amendment. The two treatments were referred as VPS (in-vessel,
Samples were filtered (0.45 m), and diluted to an appropriate concentration. 3D-EEM fluorescence spectra of compost EXC samples and biochar were then obtained by a fluoromax-4 spectrofluorometer (HORIBA, Japan), using a scanning emission fluorescence from 200 to 500 nm at 3 nm increments and the excitation wavelength from 220 to 600 nm at 4 nm increments. The
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Fig. 1. Changes in total extractable carbon (EXC), humic acid carbon (HAC) and fulvic acid carbon (FAC) during composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
spectra were recorded at a scan rate of 1200 nm min−1 . Samples were blanked against deionized water. 2.5.
13 C-NMR
spectroscopy
The freeze-dried HA was examined using cross-polarization magic angle spinning (CPMAS) 13 C-NMR spectra with a 4-mmwide bore MAS probe, operating at a 13 C resonating frequency of 100.62 MHz on Bruker Avance Z 400WB spectrometer (Bruker BioSpin AG, Switzerland). Experimental parameters consisted of a spectral width of 100 kHz with contact time of 1 ms, spinning speed of 14 kHz, a pulse delay of 0.5 s and acquisition time of 10.2 ms. Spectral distributions (the distribution of total signal intensity among various chemical shift ranges) were calculated by integrating the signal intensity in four chemical shift regions: 0–45 ppm (alkyl C), 45–108 ppm (O-alkyl C), 108–165 ppm (aromatic C) and 165–220 ppm (carbonyls). 2.6. FT-IR spectroscopy FT-IR absorbance spectra were obtained using a Shimadzu IRPrestige-21 (Shimadzu Corp., Japan) at wave numbers from 400 to 4000 cm−1 . Freeze-dried HA (2 mg), Biochar (0.2 mg) was milled to a diameter smaller than 0.25 mm and finely ground using an agate mortar and pestle. To this, 200 mg of dry potassium bromide (KBr) was added, ground to a fine power that was used to prepare a KBr pellet. One hundred scans were averaged with a resolution of 4 cm−1 by subtracting values obtained from pure KBr pellets. 2.7. Statistical analyses All data were expressed as means and standard deviations were compared statistically by Turkey’s t-test at the 5% level. Any differences with p > 0.05 were not considered to be statistically significant. 3. Results and discussion 3.1. Variation of temperature and germination index during the composting Temperature is the main performance indicator of composting and was measured daily. Both treatments exhibited a typical composting temperature profile, which included initial heating, thermophilic and cooling phases (Supplementary Information, Fig. S1). However, biochar addition affected the temperatures in
different composting phases. Adding biochar contributed to more rapid heating and a higher temperature in the thermophilic phase compared to the control. As expected for a batch system with finite resources, this leads to a more rapid transition into the cooling phase. Consistent with our previous study [8], the temperature profiles in this study indicated that biochar incorporation into the raw material enhanced the kinetics of pig manure composting and thereby shortened the composting time required. Germination index is an integrated biological parameter suitable to evaluate the degree of compost maturity [17]. Values of GI for both treatments were relatively low on day 2 (Fig. S2), indicating that phytoinhibitory or phytotoxic compounds were initially present. Of interest was the higher initial GI value for the VPSB sample at the onset of the composting experiment, suggesting that the biochar may have removed some of the inhibitory compounds via its sorptive capacity, although these values were nominal. The GI values increased significantly as composting progressed, reaching 100% at around the 60th day. A GI of 50% is a generally accepted indicator of non-phytotoxic compost [17]. Data indicated that the VPS samples were non-phytotoxic by day 20 and the VPSB samples were non-phytotoxic by day 40.
3.2. Variation of humic substances content during the composting Changes in the concentrations of humic substances of both VPS and VPSB over the composting period are displayed in Fig. 1. HAC and FAC concentrations decreased immediately once composting started, which is largely related to the degradation of partially humified components in the raw material. Thereafter, HAC and FAC concentrations displayed opposite trends. There was a subtle increase in HAC and a notable decrease in FAC between the 7th and 81st day of composting. The FAC decrease corresponded to EXC decrease, and the degradation of FAC derived from easily bio-degradable organic compounds. The increase in the HAC, which is the more complex polymerized component, is linked to the humification of the original organic matter. As expected, biochar amendment led to variation in humic content. During the humification period on days 7–81, the increase in the HAC for VPSB (7.07 g kg−1 ) was notably higher than that in VPS (1.28 g kg−1 ). The HAC/FAC ratio, which represents the degree of polymerization (DP), is one of the most sensitive indicators of the humification process and has been proposed by several investigators as an index for maturity [17]. In this study, the ratio initially declined and then progressively increased (Fig. 2). Initially, less biologically recalcitrant HA and the simple fulvic acids were degraded and consumed by the microbes, leading to a sharp initial drop in these values. As
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Fig. 2. Changes in the degree of polymerisation during composting for biocharsupplemented pig manure (VPSB) and the control (VPS). Significantly different rates (p < 0.05) are indicated by an asterisk.
more complex polymeric humic structures (HA) were formed from the polymerization of simple molecules (FA), the ratio increased again [18]. Interestingly, on day 36 of the composting, VPSB had a significantly higher DP value than VPS, suggesting a greater HAC fraction in the biochar-amended compost. Consistent with our observations, Dias et al. [5] also observed a significantly higher DP in poultry manure compost containing biochar compared to mixtures containing coffee husks and sawdust, suggesting that the use of biochar as bulking agents promoted HAC production. Taking this into consideration, it is evident that biochar amendment could contribute to a higher increasing content of more condensed molecules (HA) during the thermophilic stage of composting. Previous research has suggested that biochar strongly adsorbed HA due to its high nano-porosity and large surface area [19], while ligand exchange (carboxyl and hydroxyl groups of HA versus surface hydroxyl groups of biochar) and hydrophobic adsorption may be considered as two potential mechanisms for HA to bind to a biochar surface [20]. Further, Keiluweit and Kleber [21] proposed that the aromatic-systems in biochar were receptive to electron donor–acceptor interactions, and may be crucial to the high adsorption capacity of biochar, as well as instrumental to the persistence of natural organic matter. Additionally, the previous study noted that higher temperature could promote the chemical reactivity and increase the sorption capacity of activated carbon (Filtrasorb 100 from Calgon) [22]. Based on these interpretations, we speculate that the strong sorption of HA to biochar could be one of the mechanisms responsible for the faster formation of humic acids during the thermophilic phase in the composts with biochar. 3.3. Effect of biochar amendment on the evolution of humic-like substances In order to visualize the evolution of humic substances during our in-vessel composting process, 3D-EEM fluorescence spectra were used. As depicted in Fig. 3, the EEM contours of EXC from the composting samples of VPS and VPSB exhibited two maxima at Ex/Em of 250/400–450 nm (peak A) and 350/400–450 nm (peak C). According to the protocol of Chen et al. [23], peak A is attributable to fulvic-like substances, while peak C is attributable to humic-like substances. Dynamics of the two peak intensities directly reflected the evolution of humic substances during composting. An early decrease in peak intensity occurred from the beginning of composting to day 21. This was primarily due to the aerobic microbial degradation of biologically oxidisable and incompletely humified components present in the raw material. The data presented here concurs with the notable decrease in HA and FA content in the early phase (Fig. 1). However, both peaks began to re-emerge on
day 36, and their contour intensities increased strongly thereafter. This indicates the synthesis of new humic substances that are chemically and biologically more stable than their parent compounds. Moreover, the increase in the intensity of peak A and C could be linked to a high degree of humification and maturation of the two composts. As such, our data provides evidence that EEM spectroscopy can be successfully used to assess and monitor the maturity and stability of composts. Although sharing a similar contour pattern during the entire composting process, the notable difference between EEM plots of VPS and VPSB was their relative emission intensities. The intensities of fluorescence peaks in VPSB on day 2 and 7 were clearly higher than those in VPS, due to the humic compounds from the biochar (Fig. S3). However, the humic substances in raw materials were completely degraded by day 21. Thereafter, the peaks A and C reoccurred and rapidly increased in intensity for both treatments. It is apparent that the intensities of fulvic- and humic-like substances in biochar-containing samples from day 21 to day 36 increased much faster than those in the control, suggesting that biochar amendment may accelerate the humification process. These results are reflected in the temperature data and partially supported by the fact that biochar amendment enhanced humification [24]. The data also raise the possibility that compost reaches maturity more rapidly when supplemented with biochar, although identical contour patterns and peaks intensities were observed for both treatments by the end of composting. 3.4. Effect of biochar amendment on the evolution of HA chemical structure The data in sections 3.1 to 3.3 confirm that the evolution of the humic substances serves as a reliable indicator of humification. Yet little is known about the molecular structure evolution of the humic acids, which are postulated to control decomposition rates. In order to better understand the evolution of these chemical structures during composting, 13 C-NMR coupled with FT-IR spectra were used to monitor the humification process. The 13 C-NMR spectra of HA extracted from the control and the biochar-amended compost samples are presented in Fig. 4. The alkyl C region at 30 and 34 ppm dominated in the initial phase of composting, whereas Oalkyl region at 56 and 72 ppm dominated during the thermophilic phase. In the mature phase, there was a significant increase in peak intensity at 148 and 153 ppm, which represent methoxy/hydroxy-substituted phenyl C and oxygen-substituted aromatic C, respectively [25]. Concurrently, the peak intensity of unsubstituted aromatic C at 130 ppm decreased significantly. This data demonstrated that the main types of aromatic C exhibit opposite trends as a measure of peak intensity during the composting. These trends may be sufficient to explain the degradation of the humic substance present in raw materials, which differed chemically and physically from the final product of composting, as illustrated in the EEM fluorescence plots (Fig. 3). A more detailed insight into the structural changes of HA extracted at different stages of composting were provided by the FT-IR spectra (Fig. 5). Table S1 denotes the chemical groups responsible for the main absorbance bands [18]. The spectra of HA from raw compost materials had a high proportion of the O H bonds of alcohols and phenols (3423 cm−1 ) and the aliphatic structures C H (2920 cm−1 and 2855 cm−1 ), which steadily decreased during composting. This may be attributed to the microbes using aliphatic structures and carbohydrates such as polysaccharides, cellulose and hemicellulose to cater for their energy needs [18]. In parallel, an increase in the intensity of aromatic C C and C O bonds of quinones (1609 cm−1 ), as well as C O C ether bonds and amides (1264 cm−1 ) occurred by the end of composting. This suggests that humification process was advanced and HA became highly aromatic with a greater stability. As the FT-IR data correlated with the
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Fig. 3. 3D-excitation emission matrix fluorescence spectra of extractable C from samples at the days 2, 7, 21, 36, 60 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
Fig. 4.
13
C-NMR spectra of humic acids at the days 2, 36 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
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Fig. 5. Fourier transformed infra-red spectroscopy transmittance analysis of humic acids at the days 2, 7, 21, 36, 60 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
13 C-NMR
data, it may be asserted that 13 C-NMR complemented by FT-IR and EEM spectroscopy serve as effective tools to determine the evolution and characteristics of HA during composting. The 13 C-NMR spectra and FT-IR spectra of HA for VPS and VPSB generally exhibited similar resonances, but there was a significant difference in the intensities (Table 1). This suggests that presence and evolution of HA were markedly enhanced by adding biochar. This was particularly evident on day 36, where the biochar-amended compost displayed larger proportions of O-alkylic compounds (56 and 72 ppm, 42.8%) and aromatic compounds (148 and 153 ppm, 24.2%), as well as a lower proportions of alkyl compounds (30 and 34 ppm, 24.0%), compared to the control treatment. However, by the end of composting there were no significant differences in the proportions of functional groups between both trials. The O-alkyl C/alkyl C ratio is typically a useful index to assess the extent of decomposition [26]. In this study, the O-alkyl C/alkyl C ratio increased for both treatments, with the VPSB ratio increasing faster than the VPS ratio over the entire composting period (Table 1). Additionally, the extent of humification during composting can also be indicated by the aromatic value, expressed as a ratio of aromatic C: aliphatic C + aromatic C [27]. These ratios also increased for both treatments, and the VPSB ratio increased faster than the VPS ratio on day 36 (Table 1), corresponding with observations from EEM data. Overall, the higher O-alkyl C/alkyl C ratio and aromatic content in VPSB indicated that biochar amendment contributed to more intensive humification during the thermophilic phase of pig manure composting. Interestingly, changes to the chemical structure of HA were accompanied by surface changes and degradation of biochar in the compost, which was highlighted by the FT-IR spectra of biochar samples. As depicted in Fig. 6, the FT-IR spectra of all biochar samples had intense bands near 3342 cm−1 , 1577 cm−1 and 1069 cm−1 , generally attributed to O H stretching, aromatic C C
Fig. 6. Fourier transformed infra-red spectra of biochars during different phases of composting.
ring stretching and aliphatic ether C O and alcohol C O stretching, respectively. These properties account for the highly recalcitrant nature of biochar. However, as shown below, biochar is not inert. Our study revealed a decline in the peak intensities of aromatic C C
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Table 1 Distribution of the major C-types in the 13 C NMR spectra and calculation of the O-alkyl C/alkyl C ratio and aromatic content during composting for biochar-supplemented pig manure (VPSB) and the control (VPS). Composts
Elapsed time (days)
Alkyl Ca (%)
O-alkyl Ca (%)
Aromatic Ca (%)
Carbonyl Ca (%)
O-alkyl C/alkyl C
Aromaticity
VPS
2 36 81 2 36 81
45.3 31.8 22.6 40.8 24.0 21.3
27.7 37.7 39.4 30.2 42.8 41.5
15.7 21.5 27.1 17.3 24.2 26.6
11.3 9.0 10.9 11.7 9.0 10.6
0.61 1.18 1.74 0.74 1.79 1.95
0.18 0.24 0.30 0.20 0.27 0.30
VPSB
a
Expressed as percentages obtained from ratio of integrated areas of the spectrum to the whole spectrum area.
ring stretching (1577 cm−1 ) and aliphatic CH stretching vibration (2924 cm−1 ), whereas aliphatic CH3 deformation (1377 cm−1 ) and carboxylic bonds (1700 cm−1 ) increased in peak intensity over the composting period. The increase in carboxylic (COOH and COO− ) groups suggests a high degree of oxidation on the biochar surface. The acidic surface of biochar with catalytic abilities appears to be an important contributor to the higher carboxylated and condensed aromatic structures of HA in the composts with biochar amendment. This is strongly supported by Kramer et al. [28], who claimed that natural weathering or oxidative depolymerization of biochar could result in the formation of soil humic substances. A recent study suggests that the enhanced functionalization of biochar surfaces during composting is to some extent caused by the oxidation of the biochar surface or the sorption of humic material [29]. Taken in conjunction, the sorption of humic substances and the chemical oxidation of biochar can, at least in part, explain the faster formation of aromatic polymers, which corresponds to the advanced humification in the biochar-amended compost. The microbial characteristics have a direct and dominant influence on organic matter cycling, not only via decomposition, but also because microbial biomass is an essential component of organic matter. There are significant correlations between humification parameters and microbial properties of compost, including microbial cell count, microbial biomass, oxygen uptake rate, ATP content and microbial activity [30]. Recently, convincing evidence has supported the notion that biochar addition substantially influences microbial activity, biomass and density. Rutigliano et al. [31] found that the soil pH increased after biochar addition, which favored microbial activity (as reflected by greater substrate-induced respiration). Similarly, biochar addition not only promoted changes in compost microbial diversity [32], but also increased the relative abundance of Actinobacteria [33], which are generally able to degrade more organic material. Possible causes of the compositional and functional shifts in microbial communities due to biochar amendment may be the provision of a habitat [34] and carbon source (residual bio-oils and volatile compounds) [35], as well as changing physico-chemical conditions such as pH, gas diffusion, water content and nutrient availability [36]. Thus, it may be speculated that environmental variations caused by biochar amendment may promote microbial activity or communities that are capable of mediating the organic matter humification. This study provides evidence of the effect of biochar amendment on humification, but further research is necessary to determine the interactions between biochar and abiotic environment or microbial processes, as well as their role in enhanced humification. 4. Conclusions Our spectroscopy data not only confirm the usefulness of EEM, FT-IR and 13 C-NMR as measures of compost maturity, but also support the hypothesis that biochar amendment influences the humification process during pig manure composting. EEM spectroscopy revealed that the major effect was enhanced kinetics
in the biochar-amended compost. Analyses of the humic acids’ molecular structure evolution by 13 C-NMR spectroscopy revealed a higher O-alkyl C/alkyl C ratio and greater aromatic content of humic acid, indicating more intensive humification in biocharamended compost. Based on FT-IR spectroscopy of biochars, it appears that the sorption of humic substances and the chemical oxidation of biochar may have resulted in faster formation of aromatic polymers. Together, the results suggest that biochar amendment enhanced humification and promoted humic acid synthesis, but whether this was due to the abiotic environment enhancing microbial growth and metabolism, or the amendment of biochar providing the chemical precursors to enhance aromatic polymer formation, will require further research. Acknowledgments The authors would like to thank Yanghong Shen for research assistance. This work was financially supported by China National Critical Project for Science and Technology on Water Pollution Prevention and Control (2014ZX0710-012), and the National Natural Science Foundation of China (41271247, 41271337). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.08.030. References [1] C.H. Burton, C. Turner, Manure Management: Treatment Strategies for Sustainable Agriculture, second ed., Silsoe Research Institute, Wrest Park, 2003. [2] F. Schuchardt, T. Jiang, G. Li, R.M. Huaitalla, Pig manure systems in Germany and China and the impact on nutrient flow, J. Agr. Sci. Technol. A. 1 (2011) 858–865. [3] S. Goyal, S. Dhull, K. Kapoor, Chemical and biological changes during composting of different organic wastes and assessment of compost maturity, Bioresour. Technol. 96 (2005) 1584–1591. [4] S. Mahimairaja, N. Bolan, M. Hedley, A. Macgregor, Losses and transformation of nitrogen during composting of poultry manure with different amendments: an incubation experiment, Bioresour. Technol. 47 (1994) 265–273. [5] B.O. Dias, C.A. Silva, F.S. Higashikawa, A. Roig, M.A. Sánchez-Monedero, Use of biochar as bulking agent for the composting of poultry manure: effect on organic matter degradation and humification, Bioresour. Technol. 101 (2010) 1239–1246. [6] Y.X. Chen, X.D. Huang, Z.Y. Han, X. Huang, B. Hu, D.Z. Shi, W.X. Wu, Effects of bamboo charcoal and bamboo vinegar on nitrogen conservation and heavy metals immobility during pig manure composting, Chemosphere 78 (2010) 1177–1181. [7] C. Steiner, K. Das, N. Melear, D. Lakly, Reducing nitrogen loss during poultry litter composting using biochar, J. Environ. Qual. 39 (2010) 1236–1242. [8] C. Wang, H.H. Lu, D. Dong, H. Deng, P. Strong, H.L. Wang, W.X. Wu, Insight into the effects of biochar on manure composting: evidence supporting the relationship between N2 O emission and denitrifying community, Environ. Sci. Technol. 47 (2013) 7341–7349. [9] C. Steiner, B. Glaser, W. Geraldes Teixeira, J. Lehmann, W.E. Blum, W. Zech, Nitrogen retention and plant uptake on a highly weathered central Amazonian Ferralsol amended with compost and charcoal, J. Plant Nutr. Soil Sci. 171 (2008) 893–899. [10] H.R. Schulten, B. Plage, M. Schnitzer, A chemical structure for humic substances, Naturwissenschaften. 78 (1991) 311–312.
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Spectroscopic evidence for biochar amendment promoting humic acid synthesis and intensifying humification during composting Cheng Wang a,1 , Qiaoping Tu a,1 , Da Dong a , P.J. Strong b , Hailong Wang c , Bin Sun a , Weixiang Wu a,∗ a
Institute of Environmental Science and Technology, Zhejiang University, Yuhangtang Road 866#, Hangzhou 310058, China Centre for Solid Waste Bioprocessing, School of Civil Engineering, School of Chemical Engineering, University of Queensland, St Lucia, Brisbane 4072, Australia c School of Environmental and Resource Sciences, Zhejiang A & F University, Lin’an, Hangzhou 311300, China b
h i g h l i g h t s • Biochar amendment promoted the neo-synthesis of humic acids during composting. • Higher O-alkyl C/alkyl C ratio and aromaticity of humic acids lead to more intense humification. • Increases in carboxylic groups of biochar may result from oxidation reactions and sorption of humic substances.
a r t i c l e
i n f o
Article history: Received 17 April 2014 Received in revised form 26 July 2014 Accepted 8 August 2014 Available online 27 August 2014 Keywords: EEM FT-IR 13 C-NMR composting biochar
a b s t r a c t Despite the many benefits of biochar amendment in composting, little information is available about its effects on organic matter humification during the process. In this study the analytical results for two in-vessel composting piles were compared, one amended with biochar (VPSB, pig manure + sawdust + biochar) and the other serving as a control (VPS, pig manure + sawdust). During the 74 days of humification, the increased content of humic acid carbon in VPSB is 16.9% more than that of the control. Spectroscopic analyses show a higher O-alkyl C/alkyl C ratio and aromaticity in VPSB at the thermophilic phase, and peak intensities of fulvic-like and humic-like substances were achieved faster in VPSB than VPS. These data inferred that biochar amendment promoted the neo-synthesis of humic acids and intensified the humification of pig manure. Increase in carboxylic groups of biochar as a result of oxidation reactions and sorption of humic substances may correspond to the faster formation of aromatic polymers in biochar-supplemented composting pile. The results suggest that biochar amendment might be a potential method to enhance humification during pig manure composting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the livestock industry in China has expanded rapidly, bringing with it a surge in the volume of animal waste. According to the National Bureau of Statistics (2010), approximately 3.26 billion tons of animal manure was produced in 2009. If the fecal matter is directly applied to soil, it could pose a hazard in the form of antibiotics or pathogenic microorganisms [1]. As such, utilizing and disposing of this animal waste has become a
∗ Corresponding author. Tel.: +86 571 88982020; fax: +86 571 88982020. E-mail address: [email protected] (W. Wu). 1 These authors contributed equally to this work and should be considered co-first authors. http://dx.doi.org/10.1016/j.jhazmat.2014.08.030 0304-3894/© 2014 Elsevier B.V. All rights reserved.
major issue regarding solid waste management [2]. Composting is a treatment method that is a simple and cost-effective way to stabilize and humify this organic waste under aerobic conditions. It is of considerable economic importance as the resultant compost from livestock wastes can be directly recycled as a marketable organic fertilizer. A better understanding of the transformation and humification of organic matter throughout the composting process is essential for assessing compost maturity, achieving maturity more rapidly and improving quality. Humification is generally defined as the process of transforming biologically degradable organic matter into a fully stabilized, microbially recalcitrant humic substance. Successful humification of organic matter via composting is frequently a function of the type and quality of the bulking agent [3]. Carbon-rich bulking agents enhance the humification of animal manure and improve the
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quality of the final composts [4]. This is potentially due to modifying the physicochemical properties of initial composting mixtures, thereby providing a suitable environment for microbial activity. The most commonly used materials are chaff, straw and woody by-products, which typically have high C/N ratios. Recently, attention focused on biochar, a carbon-rich material that has been used successfully as bulking agent in poultry manure compost [5]. Previous studies have indicated that bamboo-derived biochar not only improved nitrogen and cation retention, but also lowered the total N2 O emissions from pig manure composting [6–8]. Furthermore, Steiner et al. [9] found that combining compost with biochar effectively remediated contaminated soils and improved soil nutrient retention. Although biochar amendment benefits composting as bulking agents, it is not known whether it enhances the decomposition rate and humification dynamics. Additionally, the mechanism by which biochar stabilizes and humifies pig manure is poorly defined. Humic substances are usually described as chemically complex and highly heterogenous organic substances comprising large macromolecules with oxidized functional groups [10]. They primarily consist of humic acids (HA), fulvic acids (FA) and humin. Throughout the composting period, microbial degradation lowers the FA and soluble organic carbon content of compost. In parallel, the formation of HA with increasing molecular weight and aromatic characteristics is indicative of a maturing composting [11]. Numerous structural studies have tried to achieve a better understanding of the formation of humic substances. These have been performed using advanced techniques such as 13 C nuclear magnetic resonance (13 C-NMR) and Fourier transform infrared (FT-IR) spectroscopy [12]. The aromatic content and degree of polycondensation increases, while aliphatic groups and peptide and carbohydrate components decreases with increasing composting maturity. Biochar is a carbonaceous residue of biomass pyrolysis and is highly aromatic. It has a high content of oxidized functional groups, such as C O and C O [13], which could be reactive with soluble organic carbon. Additionally, biochar may offer a suitable habitat for microorganisms to attach as it has a strong sorptive capacity due to its high microporosity and large surface area. Jindo et al. [14] suggested that biochar addition could exert an effect on the microbial community, thereby influencing the composting process and the quality of the end product. In this study, two pilot-scale in-vessel aerobic composting experiments were performed with, or without, biochar. We applied 3D excitation-emission matrix (EEM) fluorescence coupled with 13 C-NMR and FT-IR to quantify the changes of chemical composition and structure of the total extractable C (EXC) from samples during the pig manure composting. The primary aim of this study was to investigate the effects of biochar amendment on the dynamics of humification and molecular behavior of HA during composting. We hypothesized that supplementing compost with pyrolytic biochar would affect organic matter decomposition as well as HA structure via chemical interaction with organic matter. Data from this study would provide spectroscopic evidence for the mechanism whereby biochar amendment affected humification during pig manure composting.
16,500 kg flushed Pig manure + 1540 kg Sawdust), which was effectively the control sample, or VPSB (in-vessel, 16,500 kg flushed Pig manure + 1000 kg Sawdust + 540 kg Biochar), the biochar-amended sample. Flushed pig manure was collected from local piggery and its main characteristics were: pH (H2 O) = 6.46; water content = 68.2%; EC = 4.52 mS cm−1 ; and total nitrogen = 33.7 g kg−1 dry matter. Sawdust was used as the bulking agent (obtained locally)and its main characteristics were: pH (H2 O) = 6.73; water content = 11.7%; EC = 0.21 mS cm−1 ; and total nitrogen = 4.3 g kg−1 . The bulking agent was milled to a 5 cm particle size, and then homogeneously mixed with pig manure. Biochar, an abundant residue from bamboo processing was purchased from the Yaoshi Charcoal Production Company (Hangzhou, China). The biochar was pyrolyzed at a temperature of 600 ◦ C for 2 h and its main characteristics were: pH (H2 O) = 10.36; water content = 6.1%; C/N = 118; bulk density = 0.40 g cm−3 ; and specific surface area = 359 m2 g−1 . The compost piles were aerated using natural ventilation and mechanical turning using a tractor-pull windrow turner. Turning frequency varied from 2 to 7 days and was dictated by temperature of the composting windrows. Moisture content of the stock material was initially adjusted to 65 ± 2%, after which there were no further adjustments. The temperature at 30 cm depth below the surface of the compost piles and ambient air was recorded daily with a thermometer. On day 2, 7, 21, 36, 60 and 81 of composting, six subsamples were removed from six sites of the entire profile spanning the whole profile (from top to bottom) and combined to yield one composite sample. This was then divided into two parts that were both tested in triplicate. One part was immediately stored at 4 ◦ C until analyses, while the other part was air-dried, passed through a 0.25 mm sieve and stored in desiccators until further analyses. Germination index (GI) was measured according to the methods described by Chen et al. [6]. 2.2. Extraction of humic compounds The total extractable C (EXC) was obtained via four 0.1 M sodium hydroxide (NaOH) extractions at a ratio of 1:20 (w/v). The fulvic acid carbon (FAC) was obtained by precipitation at pH 1.0–2.0. The humic acid carbon (HAC) was calculated as the difference between the FAC and the EXC [15]. EXC and FAC fractions were quantified using TOC/TN Analyzer (multi N/C 2100/2100S, Analytik Jena AG). 2.3. Extraction and purification of HA Humic acids were extracted and purified according to SánchezMonedero et al. [16]. Briefly, 10 g of each compost sample, air-dried and crushed, were extracted using 0.1 M NaOH 4 times. Suspended solids were removed via settling and centrifugation (6000 g, 15 min). After centrifuging, the supernatants were carefully aspirated and acidified to pH 1.0 using 6 M HCl and left overnight at 4 ◦ C. The HA precipitates were separated via centrifugation (6000 g, 15 min). The precipitates were purified by dissolving in a minimal volume of 0.1 M NaOH and dialyzed with Spectra Por (1000 Dalton MWCO) to eliminate excess salts and finally freeze-dried (72 h).
2. Materials and methods 2.4. 3D-EEM fluorescence spectroscopy 2.1. Composting experiments and sampling Two pilot-scale in-vessel aerobic composting treatments were set up in a suburb of Hangzhou in China and monitored for approximately 12 weeks. Each compost heap consisted of pig manure and bulking agent (sawdust), with one pile receiving the biochar amendment. The two treatments were referred as VPS (in-vessel,
Samples were filtered (0.45 m), and diluted to an appropriate concentration. 3D-EEM fluorescence spectra of compost EXC samples and biochar were then obtained by a fluoromax-4 spectrofluorometer (HORIBA, Japan), using a scanning emission fluorescence from 200 to 500 nm at 3 nm increments and the excitation wavelength from 220 to 600 nm at 4 nm increments. The
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Fig. 1. Changes in total extractable carbon (EXC), humic acid carbon (HAC) and fulvic acid carbon (FAC) during composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
spectra were recorded at a scan rate of 1200 nm min−1 . Samples were blanked against deionized water. 2.5.
13 C-NMR
spectroscopy
The freeze-dried HA was examined using cross-polarization magic angle spinning (CPMAS) 13 C-NMR spectra with a 4-mmwide bore MAS probe, operating at a 13 C resonating frequency of 100.62 MHz on Bruker Avance Z 400WB spectrometer (Bruker BioSpin AG, Switzerland). Experimental parameters consisted of a spectral width of 100 kHz with contact time of 1 ms, spinning speed of 14 kHz, a pulse delay of 0.5 s and acquisition time of 10.2 ms. Spectral distributions (the distribution of total signal intensity among various chemical shift ranges) were calculated by integrating the signal intensity in four chemical shift regions: 0–45 ppm (alkyl C), 45–108 ppm (O-alkyl C), 108–165 ppm (aromatic C) and 165–220 ppm (carbonyls). 2.6. FT-IR spectroscopy FT-IR absorbance spectra were obtained using a Shimadzu IRPrestige-21 (Shimadzu Corp., Japan) at wave numbers from 400 to 4000 cm−1 . Freeze-dried HA (2 mg), Biochar (0.2 mg) was milled to a diameter smaller than 0.25 mm and finely ground using an agate mortar and pestle. To this, 200 mg of dry potassium bromide (KBr) was added, ground to a fine power that was used to prepare a KBr pellet. One hundred scans were averaged with a resolution of 4 cm−1 by subtracting values obtained from pure KBr pellets. 2.7. Statistical analyses All data were expressed as means and standard deviations were compared statistically by Turkey’s t-test at the 5% level. Any differences with p > 0.05 were not considered to be statistically significant. 3. Results and discussion 3.1. Variation of temperature and germination index during the composting Temperature is the main performance indicator of composting and was measured daily. Both treatments exhibited a typical composting temperature profile, which included initial heating, thermophilic and cooling phases (Supplementary Information, Fig. S1). However, biochar addition affected the temperatures in
different composting phases. Adding biochar contributed to more rapid heating and a higher temperature in the thermophilic phase compared to the control. As expected for a batch system with finite resources, this leads to a more rapid transition into the cooling phase. Consistent with our previous study [8], the temperature profiles in this study indicated that biochar incorporation into the raw material enhanced the kinetics of pig manure composting and thereby shortened the composting time required. Germination index is an integrated biological parameter suitable to evaluate the degree of compost maturity [17]. Values of GI for both treatments were relatively low on day 2 (Fig. S2), indicating that phytoinhibitory or phytotoxic compounds were initially present. Of interest was the higher initial GI value for the VPSB sample at the onset of the composting experiment, suggesting that the biochar may have removed some of the inhibitory compounds via its sorptive capacity, although these values were nominal. The GI values increased significantly as composting progressed, reaching 100% at around the 60th day. A GI of 50% is a generally accepted indicator of non-phytotoxic compost [17]. Data indicated that the VPS samples were non-phytotoxic by day 20 and the VPSB samples were non-phytotoxic by day 40.
3.2. Variation of humic substances content during the composting Changes in the concentrations of humic substances of both VPS and VPSB over the composting period are displayed in Fig. 1. HAC and FAC concentrations decreased immediately once composting started, which is largely related to the degradation of partially humified components in the raw material. Thereafter, HAC and FAC concentrations displayed opposite trends. There was a subtle increase in HAC and a notable decrease in FAC between the 7th and 81st day of composting. The FAC decrease corresponded to EXC decrease, and the degradation of FAC derived from easily bio-degradable organic compounds. The increase in the HAC, which is the more complex polymerized component, is linked to the humification of the original organic matter. As expected, biochar amendment led to variation in humic content. During the humification period on days 7–81, the increase in the HAC for VPSB (7.07 g kg−1 ) was notably higher than that in VPS (1.28 g kg−1 ). The HAC/FAC ratio, which represents the degree of polymerization (DP), is one of the most sensitive indicators of the humification process and has been proposed by several investigators as an index for maturity [17]. In this study, the ratio initially declined and then progressively increased (Fig. 2). Initially, less biologically recalcitrant HA and the simple fulvic acids were degraded and consumed by the microbes, leading to a sharp initial drop in these values. As
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Fig. 2. Changes in the degree of polymerisation during composting for biocharsupplemented pig manure (VPSB) and the control (VPS). Significantly different rates (p < 0.05) are indicated by an asterisk.
more complex polymeric humic structures (HA) were formed from the polymerization of simple molecules (FA), the ratio increased again [18]. Interestingly, on day 36 of the composting, VPSB had a significantly higher DP value than VPS, suggesting a greater HAC fraction in the biochar-amended compost. Consistent with our observations, Dias et al. [5] also observed a significantly higher DP in poultry manure compost containing biochar compared to mixtures containing coffee husks and sawdust, suggesting that the use of biochar as bulking agents promoted HAC production. Taking this into consideration, it is evident that biochar amendment could contribute to a higher increasing content of more condensed molecules (HA) during the thermophilic stage of composting. Previous research has suggested that biochar strongly adsorbed HA due to its high nano-porosity and large surface area [19], while ligand exchange (carboxyl and hydroxyl groups of HA versus surface hydroxyl groups of biochar) and hydrophobic adsorption may be considered as two potential mechanisms for HA to bind to a biochar surface [20]. Further, Keiluweit and Kleber [21] proposed that the aromatic-systems in biochar were receptive to electron donor–acceptor interactions, and may be crucial to the high adsorption capacity of biochar, as well as instrumental to the persistence of natural organic matter. Additionally, the previous study noted that higher temperature could promote the chemical reactivity and increase the sorption capacity of activated carbon (Filtrasorb 100 from Calgon) [22]. Based on these interpretations, we speculate that the strong sorption of HA to biochar could be one of the mechanisms responsible for the faster formation of humic acids during the thermophilic phase in the composts with biochar. 3.3. Effect of biochar amendment on the evolution of humic-like substances In order to visualize the evolution of humic substances during our in-vessel composting process, 3D-EEM fluorescence spectra were used. As depicted in Fig. 3, the EEM contours of EXC from the composting samples of VPS and VPSB exhibited two maxima at Ex/Em of 250/400–450 nm (peak A) and 350/400–450 nm (peak C). According to the protocol of Chen et al. [23], peak A is attributable to fulvic-like substances, while peak C is attributable to humic-like substances. Dynamics of the two peak intensities directly reflected the evolution of humic substances during composting. An early decrease in peak intensity occurred from the beginning of composting to day 21. This was primarily due to the aerobic microbial degradation of biologically oxidisable and incompletely humified components present in the raw material. The data presented here concurs with the notable decrease in HA and FA content in the early phase (Fig. 1). However, both peaks began to re-emerge on
day 36, and their contour intensities increased strongly thereafter. This indicates the synthesis of new humic substances that are chemically and biologically more stable than their parent compounds. Moreover, the increase in the intensity of peak A and C could be linked to a high degree of humification and maturation of the two composts. As such, our data provides evidence that EEM spectroscopy can be successfully used to assess and monitor the maturity and stability of composts. Although sharing a similar contour pattern during the entire composting process, the notable difference between EEM plots of VPS and VPSB was their relative emission intensities. The intensities of fluorescence peaks in VPSB on day 2 and 7 were clearly higher than those in VPS, due to the humic compounds from the biochar (Fig. S3). However, the humic substances in raw materials were completely degraded by day 21. Thereafter, the peaks A and C reoccurred and rapidly increased in intensity for both treatments. It is apparent that the intensities of fulvic- and humic-like substances in biochar-containing samples from day 21 to day 36 increased much faster than those in the control, suggesting that biochar amendment may accelerate the humification process. These results are reflected in the temperature data and partially supported by the fact that biochar amendment enhanced humification [24]. The data also raise the possibility that compost reaches maturity more rapidly when supplemented with biochar, although identical contour patterns and peaks intensities were observed for both treatments by the end of composting. 3.4. Effect of biochar amendment on the evolution of HA chemical structure The data in sections 3.1 to 3.3 confirm that the evolution of the humic substances serves as a reliable indicator of humification. Yet little is known about the molecular structure evolution of the humic acids, which are postulated to control decomposition rates. In order to better understand the evolution of these chemical structures during composting, 13 C-NMR coupled with FT-IR spectra were used to monitor the humification process. The 13 C-NMR spectra of HA extracted from the control and the biochar-amended compost samples are presented in Fig. 4. The alkyl C region at 30 and 34 ppm dominated in the initial phase of composting, whereas Oalkyl region at 56 and 72 ppm dominated during the thermophilic phase. In the mature phase, there was a significant increase in peak intensity at 148 and 153 ppm, which represent methoxy/hydroxy-substituted phenyl C and oxygen-substituted aromatic C, respectively [25]. Concurrently, the peak intensity of unsubstituted aromatic C at 130 ppm decreased significantly. This data demonstrated that the main types of aromatic C exhibit opposite trends as a measure of peak intensity during the composting. These trends may be sufficient to explain the degradation of the humic substance present in raw materials, which differed chemically and physically from the final product of composting, as illustrated in the EEM fluorescence plots (Fig. 3). A more detailed insight into the structural changes of HA extracted at different stages of composting were provided by the FT-IR spectra (Fig. 5). Table S1 denotes the chemical groups responsible for the main absorbance bands [18]. The spectra of HA from raw compost materials had a high proportion of the O H bonds of alcohols and phenols (3423 cm−1 ) and the aliphatic structures C H (2920 cm−1 and 2855 cm−1 ), which steadily decreased during composting. This may be attributed to the microbes using aliphatic structures and carbohydrates such as polysaccharides, cellulose and hemicellulose to cater for their energy needs [18]. In parallel, an increase in the intensity of aromatic C C and C O bonds of quinones (1609 cm−1 ), as well as C O C ether bonds and amides (1264 cm−1 ) occurred by the end of composting. This suggests that humification process was advanced and HA became highly aromatic with a greater stability. As the FT-IR data correlated with the
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Fig. 3. 3D-excitation emission matrix fluorescence spectra of extractable C from samples at the days 2, 7, 21, 36, 60 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
Fig. 4.
13
C-NMR spectra of humic acids at the days 2, 36 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
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Fig. 5. Fourier transformed infra-red spectroscopy transmittance analysis of humic acids at the days 2, 7, 21, 36, 60 and 81 of composting for biochar-supplemented pig manure (VPSB) and the control (VPS).
13 C-NMR
data, it may be asserted that 13 C-NMR complemented by FT-IR and EEM spectroscopy serve as effective tools to determine the evolution and characteristics of HA during composting. The 13 C-NMR spectra and FT-IR spectra of HA for VPS and VPSB generally exhibited similar resonances, but there was a significant difference in the intensities (Table 1). This suggests that presence and evolution of HA were markedly enhanced by adding biochar. This was particularly evident on day 36, where the biochar-amended compost displayed larger proportions of O-alkylic compounds (56 and 72 ppm, 42.8%) and aromatic compounds (148 and 153 ppm, 24.2%), as well as a lower proportions of alkyl compounds (30 and 34 ppm, 24.0%), compared to the control treatment. However, by the end of composting there were no significant differences in the proportions of functional groups between both trials. The O-alkyl C/alkyl C ratio is typically a useful index to assess the extent of decomposition [26]. In this study, the O-alkyl C/alkyl C ratio increased for both treatments, with the VPSB ratio increasing faster than the VPS ratio over the entire composting period (Table 1). Additionally, the extent of humification during composting can also be indicated by the aromatic value, expressed as a ratio of aromatic C: aliphatic C + aromatic C [27]. These ratios also increased for both treatments, and the VPSB ratio increased faster than the VPS ratio on day 36 (Table 1), corresponding with observations from EEM data. Overall, the higher O-alkyl C/alkyl C ratio and aromatic content in VPSB indicated that biochar amendment contributed to more intensive humification during the thermophilic phase of pig manure composting. Interestingly, changes to the chemical structure of HA were accompanied by surface changes and degradation of biochar in the compost, which was highlighted by the FT-IR spectra of biochar samples. As depicted in Fig. 6, the FT-IR spectra of all biochar samples had intense bands near 3342 cm−1 , 1577 cm−1 and 1069 cm−1 , generally attributed to O H stretching, aromatic C C
Fig. 6. Fourier transformed infra-red spectra of biochars during different phases of composting.
ring stretching and aliphatic ether C O and alcohol C O stretching, respectively. These properties account for the highly recalcitrant nature of biochar. However, as shown below, biochar is not inert. Our study revealed a decline in the peak intensities of aromatic C C
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Table 1 Distribution of the major C-types in the 13 C NMR spectra and calculation of the O-alkyl C/alkyl C ratio and aromatic content during composting for biochar-supplemented pig manure (VPSB) and the control (VPS). Composts
Elapsed time (days)
Alkyl Ca (%)
O-alkyl Ca (%)
Aromatic Ca (%)
Carbonyl Ca (%)
O-alkyl C/alkyl C
Aromaticity
VPS
2 36 81 2 36 81
45.3 31.8 22.6 40.8 24.0 21.3
27.7 37.7 39.4 30.2 42.8 41.5
15.7 21.5 27.1 17.3 24.2 26.6
11.3 9.0 10.9 11.7 9.0 10.6
0.61 1.18 1.74 0.74 1.79 1.95
0.18 0.24 0.30 0.20 0.27 0.30
VPSB
a
Expressed as percentages obtained from ratio of integrated areas of the spectrum to the whole spectrum area.
ring stretching (1577 cm−1 ) and aliphatic CH stretching vibration (2924 cm−1 ), whereas aliphatic CH3 deformation (1377 cm−1 ) and carboxylic bonds (1700 cm−1 ) increased in peak intensity over the composting period. The increase in carboxylic (COOH and COO− ) groups suggests a high degree of oxidation on the biochar surface. The acidic surface of biochar with catalytic abilities appears to be an important contributor to the higher carboxylated and condensed aromatic structures of HA in the composts with biochar amendment. This is strongly supported by Kramer et al. [28], who claimed that natural weathering or oxidative depolymerization of biochar could result in the formation of soil humic substances. A recent study suggests that the enhanced functionalization of biochar surfaces during composting is to some extent caused by the oxidation of the biochar surface or the sorption of humic material [29]. Taken in conjunction, the sorption of humic substances and the chemical oxidation of biochar can, at least in part, explain the faster formation of aromatic polymers, which corresponds to the advanced humification in the biochar-amended compost. The microbial characteristics have a direct and dominant influence on organic matter cycling, not only via decomposition, but also because microbial biomass is an essential component of organic matter. There are significant correlations between humification parameters and microbial properties of compost, including microbial cell count, microbial biomass, oxygen uptake rate, ATP content and microbial activity [30]. Recently, convincing evidence has supported the notion that biochar addition substantially influences microbial activity, biomass and density. Rutigliano et al. [31] found that the soil pH increased after biochar addition, which favored microbial activity (as reflected by greater substrate-induced respiration). Similarly, biochar addition not only promoted changes in compost microbial diversity [32], but also increased the relative abundance of Actinobacteria [33], which are generally able to degrade more organic material. Possible causes of the compositional and functional shifts in microbial communities due to biochar amendment may be the provision of a habitat [34] and carbon source (residual bio-oils and volatile compounds) [35], as well as changing physico-chemical conditions such as pH, gas diffusion, water content and nutrient availability [36]. Thus, it may be speculated that environmental variations caused by biochar amendment may promote microbial activity or communities that are capable of mediating the organic matter humification. This study provides evidence of the effect of biochar amendment on humification, but further research is necessary to determine the interactions between biochar and abiotic environment or microbial processes, as well as their role in enhanced humification. 4. Conclusions Our spectroscopy data not only confirm the usefulness of EEM, FT-IR and 13 C-NMR as measures of compost maturity, but also support the hypothesis that biochar amendment influences the humification process during pig manure composting. EEM spectroscopy revealed that the major effect was enhanced kinetics
in the biochar-amended compost. Analyses of the humic acids’ molecular structure evolution by 13 C-NMR spectroscopy revealed a higher O-alkyl C/alkyl C ratio and greater aromatic content of humic acid, indicating more intensive humification in biocharamended compost. Based on FT-IR spectroscopy of biochars, it appears that the sorption of humic substances and the chemical oxidation of biochar may have resulted in faster formation of aromatic polymers. Together, the results suggest that biochar amendment enhanced humification and promoted humic acid synthesis, but whether this was due to the abiotic environment enhancing microbial growth and metabolism, or the amendment of biochar providing the chemical precursors to enhance aromatic polymer formation, will require further research. Acknowledgments The authors would like to thank Yanghong Shen for research assistance. This work was financially supported by China National Critical Project for Science and Technology on Water Pollution Prevention and Control (2014ZX0710-012), and the National Natural Science Foundation of China (41271247, 41271337). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.08.030. References [1] C.H. Burton, C. Turner, Manure Management: Treatment Strategies for Sustainable Agriculture, second ed., Silsoe Research Institute, Wrest Park, 2003. [2] F. Schuchardt, T. Jiang, G. Li, R.M. Huaitalla, Pig manure systems in Germany and China and the impact on nutrient flow, J. Agr. Sci. Technol. A. 1 (2011) 858–865. [3] S. Goyal, S. Dhull, K. Kapoor, Chemical and biological changes during composting of different organic wastes and assessment of compost maturity, Bioresour. Technol. 96 (2005) 1584–1591. [4] S. Mahimairaja, N. Bolan, M. Hedley, A. Macgregor, Losses and transformation of nitrogen during composting of poultry manure with different amendments: an incubation experiment, Bioresour. Technol. 47 (1994) 265–273. [5] B.O. Dias, C.A. Silva, F.S. Higashikawa, A. Roig, M.A. Sánchez-Monedero, Use of biochar as bulking agent for the composting of poultry manure: effect on organic matter degradation and humification, Bioresour. Technol. 101 (2010) 1239–1246. [6] Y.X. Chen, X.D. Huang, Z.Y. Han, X. Huang, B. Hu, D.Z. Shi, W.X. Wu, Effects of bamboo charcoal and bamboo vinegar on nitrogen conservation and heavy metals immobility during pig manure composting, Chemosphere 78 (2010) 1177–1181. [7] C. Steiner, K. Das, N. Melear, D. Lakly, Reducing nitrogen loss during poultry litter composting using biochar, J. Environ. Qual. 39 (2010) 1236–1242. [8] C. Wang, H.H. Lu, D. Dong, H. Deng, P. Strong, H.L. Wang, W.X. Wu, Insight into the effects of biochar on manure composting: evidence supporting the relationship between N2 O emission and denitrifying community, Environ. Sci. Technol. 47 (2013) 7341–7349. [9] C. Steiner, B. Glaser, W. Geraldes Teixeira, J. Lehmann, W.E. Blum, W. Zech, Nitrogen retention and plant uptake on a highly weathered central Amazonian Ferralsol amended with compost and charcoal, J. Plant Nutr. Soil Sci. 171 (2008) 893–899. [10] H.R. Schulten, B. Plage, M. Schnitzer, A chemical structure for humic substances, Naturwissenschaften. 78 (1991) 311–312.
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