Insight into the Effects of Biochar on Manure Composting: Evidence

Article pubs.acs.org/est

Insight into the Effects of Biochar on Manure Composting: Evidence Supporting the Relationship between N2O Emission and Denitrifying Community Cheng Wang,† Haohao Lu,† Da Dong,† Hui Deng,† P. J. Strong,‡ Hailong Wang,§ and Weixiang Wu*,† †

Institute of Environmental Science and Technology, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China Callaghan Innovation Research Ltd, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand § School of Environmental and Resource Sciences, Zhejiang A & F University, Lin’an, Hangzhou 311300, China ‡

S Supporting Information *

ABSTRACT: Although nitrous oxide (N2O) emissions from composting contribute to the accelerated greenhouse effect, it is difficult to implement practical methods to mitigate these emissions. In this study, the effects of biochar amendment during pig manure composting were investigated to evaluate the inter-relationships between N2O emission and the abundance of denitrifying bacteria. Analytical results from two pilot composting treatments with (PWSB, pig manure + wood chips + sawdust + biochar) or without (PWS, pig manure + wood chips + sawdust) biochar (3% w/w) demonstrated that biochar amendment not only lowered NO2−-N concentrations but also lowered the total N2O emissions from pig manure composting, especially during the later stages. Quantification of functional genes involved in denitrification and Spearman rank correlations matrix revealed that the N2O emission rates correlated with the abundance of nosZ, nirK, and nirS genes. Biochar-amended pig manure had a higher pH and a lower moisture content. Biochar amendment altered the abundance of denitrifying bacteria significantly; less N2Oproducing and more N2O-consuming bacteria were present in the PWSB, and this significantly lowered N2O emissions in the maturation phase. Together, the results demonstrate that biochar amendment could be a novel greenhouse gas mitigation strategy during pig manure composting.

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aeration rate, turning frequency and bulk density.8 However, large variations in the technology employed as well as the production scales have rendered parameter optimization for composting impractical or unfeasible. A low-technology solution is required that either lowers N2O production or adsorbs the N2O produced. Biochar amendment is potentially one such solution. Biochar is a carbon-rich residue generated from biomass pyrolysis and has attracted much attention due to its proven benefits in environmental management. In recent times, validating biochar’s potential to mitigate climate change has been focused on and studied extensively. Kammann et al.9 found that biochar-amended soils exhibited significant reductions in N2O emissions, while Yanai et al.10 reported that biochar-amended soils reduced N2O emission by 89%. These beneficial effects may have been caused by the decrease in N available for denitrification, as biochar can efficiently adsorb and

INTRODUCTION Composting is a biological process where naturally occurring microorganisms convert large amounts of biodegradable organic waste into humified end-products. Historically, it has been suggested as a sustainable agricultural method for recycling livestock wastes as the residue may be directly used as a marketable organic fertilizer.1,2 However, the composting of livestock wastes has been universally regarded as a source of anthropogenic greenhouse gas, yielding annual global nitrous oxide (N2O) emissions of 1.2 × 106 metric ton (approximately 3.6 × 108 metric ton of CO2e).3 At this scale it poses a serious environmental pollution risk as the emissions may contribute to global warming and ozone depletion.3−5 As a potent greenhouse gas and a dominant ozone-depleting substance emitted in the 21st century,6 N2O has been calculated to contribute 6% of the global warming effect. More worrying is the rapidly increasing rate of N2O production (currently at an annual rate of 0.25%) and its high global warming potential (298 times greater than the equivalent mass of CO2).7 This provides an impetus to take immediate action to lower anthropogenic N2O emissions. Some success at lowering N2O emissions during manure composting has been achieved by modifying the © XXXX American Chemical Society

Received: December 26, 2012 Revised: April 3, 2013 Accepted: June 7, 2013

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dx.doi.org/10.1021/es305293h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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retain ammonia gas and ammonium as well as nitrate ions.11,12 Two other recent studies demonstrated that bulking poultry manure with biochar lessened N loss while simultaneously enhancing humification, thereby producing mature composts with a high fertilizer value.13,14 Furthermore, our previous research has shown that bamboo-derived biochar improved nitrogen and cation retention during composting.15,16 Although the benefits regarding the use of biochar as bulking agent for composting have been specifically addressed, it is an open question whether biochar amendment significantly affects N2O emission patterns. The mechanism by which biochar impacts N2O fluxes over the entire composting period is also poorly defined. It is almost universally accepted that N2O is produced mainly during the microbially mediated process of denitrification.17 Bacterial denitrification was identified as the most crucial source of nitrous oxide production in composting systems by Maeda et al.,18 by way of isotope analysis of N2O emitted from composting piles using the dynamic chamber method.19 Denitrification is a respiratory microbial process where oxidized forms of nitrogen (nitrate, nitrite, nitric oxide, and nitrous oxide) are reduced. Four enzymes with distinctly different functions participate in denitrification: nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos). Therefore, N2O emitted during the denitrification process is a balance between mechanisms that produce or consume N2O. In a recent study by Maeda et al.20 the nosZ (encoding nitrous oxide reductase) diversity in the denitrifying bacterial community positively correlated to N2O emission during composting. However, Hallin et al.21 and Guo et al.22 had previously noted that denitrification activity correlated to total bacterial community size or nosZ gene abundance, rather than just diversity in the genetic composition of denitrifying bacteria. Recent studies reporting a lower abundance of soil bacteria having the nosZ gene relative to other genes, suggested that N2O emissions were the result of a high proportion of the denitrifying bacterial community lacking the genetic capacity to reduce N2O.23,24 Although the majority of cumulative evidence of the relationship between N2O emission and denitrification has been presented from the viewpoint of gene abundance, Yanai et al.10 observed that biochar characteristics affected the enzymes responsible for denitrification. The authors suggested biochar amendment increased the activity of enzymes responsible for the reduction of N2O to N2, thereby lowering soil N2O emissions. Although biochar amended compost has recently received greater attention, the extent to which the approach influences the abundance of the denitrifying community is largely unknown and untested. In this study, two pilot composting experiments were conducted at a practical scale, and the abundance of the denitrifying bacterial community was assessed using real-time quantitative polymerase chain reaction (PCR). The primary aim of our study was to investigate the extent to which biochar addition influences N2O emissions during composting. Changes in emission were related to biochemical parameters and the abundance of the denitrifying bacterial community. We hypothesized that the abundance of these microorganisms between the control (no biochar) and the treatment with biochar would differ considerably throughout the composting period. Additionally, the correlations of N2O emissions, the denitrifying population abundance, and the environmental factors were statistically analyzed. Ultimately, these results

would feed into a development strategy toward mitigating N2O emissions.

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MATERIALS AND METHODS Composting Experiments. On April 29, 2011, two aerobic windrow compost piles of pig manure (2 tons each) were set up in a suburb of Hangzhou city in China and monitored for 82 days. The original compost material consisted of three parts: pig manure, wood chips, and sawdust. The pig manure was collected from Daguangshan piggery. Sawdust and wood chips were purchased from a local farm. These were used as a bulking agent in pig manure composting due to their higher bulk density and lower moisture content. Biochar (BC), an abundant residue from bamboo processing, was purchased from the Yaoshi Charcoal-Production Company (Hangzhou, China). The biochar resulted from pyrolysis at 600 °C, and its main characteristics were: pH (H2O) = 10.36; water content = 6.1; C/N = 118; bulk density = 0.40 g cm−3; specific surface area = 359 m2 g−1. As per previous research,16 we conducted two different pilotscale treatments. The stock materials for each compost heap consisted of 1200 kg of pig manure mixed with 800 kg of bulking agent (wood chips and sawdust), with one pile receiving an additional 60 kg of biochar amendment. The treatments were referred as PWS (control: pig manure, wood chips, and sawdust) or PWSB (pig manure, wood chips, sawdust, and biochar). The moisture content of the stock material was initially adjusted to approximately 65%. There were no further adjustments to the moisture content throughout the composting period. Aeration of these compost piles occurred via natural ventilation and turning. Both compost piles were periodically remixed and turned over with a wheel loader. Composting Sampling and Chemical Analyses. On days 3, 13, 22, 37, 48, 61, 70, and 82 of composting, subsamples were removed from three random points of the entire profile and combined to yield one composite sample. The composite sample was divided into two parts. One part was immediately stored at 4 °C until analysis, while the other part was air-dried, passed through a 0.25 mm sieve, and stored in a desiccator until further analysis. The temperatures at 20 cm depth below the surface of the compost piles and ambient air were recorded daily with a thermometer. Electrical conductivity (EC) and pH were measured by aqueous compost extracts obtained from fresh samples. The aqueous compost extracts were obtained by mechanically shaking the samples with double-distilled water at a solids−water ratio of 1:10 (w/v, dry weight basis) for 1 h. Water content was determined by weight loss of compost samples after drying at 105 °C for longer than 24 h. C/N ratios were measured with a CNS analyzer (Vario Max CNS, elementar Analysesysteme, Germany). Inorganic nitrogen compounds were determined after a 30 min extraction while shaking in a 10-fold dilution of a 2.0 M KCl solution. Concentrations of NO3−−N, NO2−−N, and NH4+−N were determined using ion chromatography (DX-120, Dionex, USA). N2O Emission Measurement. N2O fluxes were measured in duplicate approximately every 3 days using the closed chamber technique. Gas samples were collected with a syringe at 0, 30, and 60 min after closing the chamber and immediately injected after sampling into a pre-evacuated 18 mL vials fitted with butyl rubber septa. The N2O concentration in gas samples was determined using gas chromatography−mass spectrometry B



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(GC-MS), which was performed with an Agilent 6890 GC instrument coupled to an Agilent 5973 MS using Agilent ChemStation software (Agilent Technologies, Palo Alto, CA, USA). Fluxes in N2O were calculated by linear regression as described by Hutchinson and Mosier.25 Mean N2O flux rates were calculated for each compost pile and subsequently used in statistical analyses. DNA Extraction. Three independent DNA extractions were performed from 0.15 g samples from each compost pile using the MoBio UltraClean Soil DNA isolation kit (MoBio Laboratories, Solana Beach, CA, USA). The extraction was performed according to the manufacturer’s instructions. The concentrations and quality of DNA samples were measured using a NanoDrop (Thermo Scientific, Wilmington, DE, USA). Quantification of Functional Communities. Quantitative PCR (qPCR) of genes encoding the key enzymes involved in nitrate reduction (narG encoding the membrane-bound nitrate reductase) and denitrification (nirS, nirK, and nosZ encoding the cd1 and copper nitrite reductases and the nitrous oxide reductase, respectively) was used to estimate the abundance of functional microbial communities. The quantification was based on the intensity of SYBR Green dye fluorescence, which bound to double-stranded DNA. For the denitrification genes, the PCR reaction mixture consisted of 0.1 μmol L−1 of each primer, 1× SYBR Premix EX Taq (Perfect Real Time) premix reagent (TaKaRa, Dalian, China), and 2 μL of template DNA to a final volume of 20 μL. Thermal cycling conditions and primers used for each reaction are listed in Table S1 (Supporting Information). Reactions were performed in a Bio-Rad CFX1000 Thermal Cycler. All real-time PCR assays were performed using three replicates per sample, and all PCR runs included control reactions without the template. The gene copy numbers were calculated by comparing threshold cycles obtained in each PCR run with those of known standard DNA concentrations. Standard curves were obtained using serial dilutions of linearized plasmids containing cloned narG, nirS, nirK, and nosZ genes. Statistical Analyses. All data were expressed as means and standard deviations were compared statistically by Tukey’s t test at the 5% level. Any differences where p > 0.05 were not considered to be statistically significant. For each compost pile, Spearman rank correlations coefficients (rho) were used to assess the association between N2O emission and microbial parameters, together with several biochemical parameters. A square (9 × 9) correlation matrix was created by MATLAB, resulting in 81 matrices. All statistical calculations were performed with the SPSS11.5 statistical software package.

Figure 1. Changes in temperature of composting material during the pig manure composting experiment.

Chemical Changes of the Composting Material. Changes in the chemical properties of both composted materials are displayed in Table 1. Significant decreases in moisture content and C/N value and an increase in pH were observed for both treatment processes. Biochar amendment resulted in a higher pH and a lower moisture compared to the PWS treatment. The EC also increased significantly after the active phase for both treatments, and the increase was faster for the PWS control than for the PWSB treatment. Ammonium accounted for most of the inorganic nitrogen in the original composting materials and increased significantly during the active phase of composting. This was followed by the greatest decrease in NH4+ and lowest levels of NO3− during the subsequent maturation stage. Interestingly, a NO2− spike was observed during the sixth week for both treatments, which corresponded to sequential nitrite accumulation. The peak concentration of NO2− in PWS was considerably higher than that in PWSB. In general, the inorganic nitrogen concentrations (NH4+, NO3− plus NO2−) were lower in the treatment containing biochar than the control. N2O Emission Pattern. Lower N2O emission rates were observed for PWSB compared with PWS throughout the composting period (Figure 2). During the thermophilic phase, the N2O emission rate increased slightly and steadily in both PWS and PWSB treatments. When the temperature of the composting material began to decline, the N2O emission rates increased significantly for both treatments and attained its highest value during the 52nd to 55th day. In comparison, the peak N2O emission rate from PWS (184.45 mg m−2 h−1) was significantly higher than that from PWSB (128.13 mg m−2 h−1). Biochar amendment lowered the maximum N2O emission rate by 31% compared to the control treatment. The N2O emission rate in both treatments decreased dramatically after peaking but still remained significantly higher for PWS than PWSB, even during the late composting phase. Consequently, biochar amendment significantly reduced total N2O emission over the total composting period. The PWSB emitted a total of 25.9% less N2O than the control treatment. Quantification of Denitrification Genes. The copy numbers of nitrate reduction and denitrification genes targeting narG, nirK, nirS, and nosZ genes were determined in the composting material of both treatments during the experimental period (Figure 3). The calculated PCR efficiencies for narG, nirK, nirS, and nosZ assays were 103.3%, 84.8%, 93.0%, and 103.8%, respectively. The copy numbers of nitrate reduction and denitrification genes changed significantly over

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RESULTS Compost Temperatures. Temperature was the main parameter used to indicate the performance of the process. Both treatments reflected the typical temperature pattern of many composting processes (Figure 1). The temperature rose immediately once composting started and reached approximately 50 °C. Thermophilic temperatures above 60 °C were maintained for approximately four weeks and were followed by a gradual decrease until the eighth week, after which temperature decreased close to ambient levels. The peak composting temperature reached after initiation was higher for the PWSB treatment (69.9 °C) than the PWS control (66.9 °C). The PWSB treatment maintained its higher temperature for a slightly shorter period and entered the cooling phase more rapidly than PWS. C


a

pig manure (1200 kg) wood chips (400 kg) sawdust (400 kg)

pig manure (1200 kg) wood chips (400 kg) sawdust (400 kg) biochar (60 kg)

PWS

PWSB

EC, electrical conductivity. bDM, dry matter.

materials

composts 1.99 2.97 3.92 4.02 4.01 3.96 3.95 4.03 2.17 2.87 3.77 3.46 3.64 3.74 3.51 3.54

0.03 0.05 0.08 0.06 0.07 0.07 0.05 0.06 0.03 0.14 0.06 0.02 0.03 0.07 0.09 0.05

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.08 0.09 0.07 0.04 0.02 0.05 0.08 0.03 0.09 0.09 0.03 0.06 0.02 0.05 0.09

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 13 22 37 48 61 70 82 3 13 22 37 48 61 70 82

6.49 8.01 8.37 8.22 8.21 8.05 7.93 7.91 6.75 8.01 8.34 8.51 8.20 8.15 8.08 8.13

EC (Ms/cm)

pH

a

elapsed time (days)

Table 1. Chemical Analyses of Materials during the Composting Trial

63.5 55.0 46.6 31.1 22.7 17.2 16.7 12.8 60.0 52.6 42.9 26.1 20.7 16.6 15.9 12.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.2 1.2 2.2 1.2 0.8 0.4 0.8 0.2 2.9 1.9 1.5 0.9 0.6 0.4 0.2 0.3

moisture (%) 6.64 4.55 3.37 2.84 1.57 1.27 1.56 1.61 7.05 5.44 4.01 1.53 1.31 1.25 1.22 1.32

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.16 0.68 0.72 0.37 0.22 0.13 0.16 0.17 1.51 1.06 1.19 0.32 0.11 0.25 0.08 0.24

NH4+ (g/kg DMb) 0.85 0.51 0.37 0.27 0.27 0.32 0.32 0.29 0.65 0.53 0.46 0.21 0.26 0.18 0.26 0.20

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.11 0.09 0.10 0.08 0.03 0.08 0.06 0.06 0.13 0.06 0.08 0.09 0.03 0.07 0.03 0.02

NO3− (g/kg DMb)

chemical component profiles 1.37 3.33 3.74 5.08 13.77 1.81 1.80 1.72 1.25 2.11 2.63 3.38 1.92 1.80 2.38 1.71

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.53 1.17 0.46 0.72 3.23 0.79 0.50 1.08 0.65 0.89 0.57 1.52 1.08 0.40 0.62 0.79

NO2− (mg/kg DMb)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.2 0.3 0.3 0.1 0.2 0.1 0.2 0.2 0.4 0.3 0.2 0.5 0.1 0.4 0.2

C/N ratio 15.4 15.4 15.3 13.5 13.8 13.7 13.1 13.8 17.5 17.3 18.4 16.0 16.5 16.4 15.6 16.3

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differences in denitrifying bacteria numbers when composting with or without biochar amendment (Figure 3). The abundance of narG gene in PWSB and PWS varied significantly for all but one sample point (day 70) and ranged from 1.2 × 106 to 1.6 × 106 copies per gram of dry matter (Figure 3a). The average abundance of denitrifying bacteria bearing the nirS gene was significantly higher in PWSB than PWS and ranged from 2.3 × 107 to 4.4 × 107 copies per gram of dry matter (Figure 3b). The average abundance for nirK ranged from 6.8 × 108 to 9.2 × 108 copies per gram of dry matter in PWSB and PWS. There was significantly more nirK in the PWS compared to the PWSB for all samples, other than days 61 and 70 (Figure 3c). In PWS and PWSB, the abundance of nosZ gene ranged from 7.6 × 105 to 8.8 × 105 copies per gram of dry matter and was significantly lower in the PWS for three sample points: days 3, 37, and 48 (Figure 3d). More importantly, quantification of the nirK and nosZ genes revealed a significantly higher nosZ/nirK ratio in PWSB, where the ratio was approximately 1.57 times that of the control. Linking the N2O Emission Rates to Environmental Properties. Average correlation coefficients were determined for the individual treatments and used to assess a number of parameter relationships. Although PWSB had slightly stronger correlation coefficients between each parameter than PWS, the correlation properties shared by two treatments were very similar (Figure 4). The N2O emission rates measured in both treatments displayed significant positive correlations with the abundance of nosZ (correlation coefficient rho = 0.666, P < 0.01), nirK (rho = 0.793, P < 0.01), and nirS gene copy numbers (rho = 0.582, P < 0.05), together with EC (rho = 0.771, P < 0.01) and moisture (rho = −0.741, P < 0.01). Interestingly, the correlations were mutual and high between the abundance of all three denitrifying genes (nosZ, nirK, and nirS). Moisture had an important influence on N2O emission

Figure 2. Changes in N2O emission rate during pig manure composting.

the entire composting period. The highest copy numbers of all denitrification genes were observed during the cooling and maturing phase; significantly lower copy numbers were measured during the initial mesophilic and thermophilic phases. Among these four functional genes, the average abundance of nirK was persistently the highest over the composting period. The remaining three genes in order of abundance were: nirS, narG, and nosZ (Figure S1, Supporting Information). Although the denitrifying bacteria can reduce nitrite via either a copper nitrite reductase (NirK) or a cytochrome cd1 nitrite reductase (NirS), our data revealed that the abundance of nirK gene was approximately 24 times higher than that of nirS gene. This dominance of nirK gene over nirS gene in the denitrifying bacteria was consistent throughout the composting period. Statistically significant differences in copy numbers were generally obtained between PWS and PWSB for all genes tested. This supported the hypothesis predicting significant

Figure 3. Changes in gene copy numbers per gram of compost (dry matter) for narG, nirS, nirK, and nosZ at the days 3, 13, 22, 37, 48, 61, and 70 of composting for PWS and PWSB. Error bars indicate standard error of the mean (s.e.) of triplicate qPCR reactions. Significantly different rates (p < 0.05) are indicated by an asterisk. E



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Figure 4. Correlation matrices between N2O emission rate and the likely controlling factors (nitrite, EC, moisture, pH, nirK, nosZ, nirS, and nosZ/ nirK) for PWS and PWSB. Darker boxes represent low-correlation coefficients, whereas lighter boxes represent stronger correlations.

concentration, thereby lowering the amount of nitrite available for transformation and mitigating N2O emission during the maturation phase of pig manure composting. With respect to microbial function during composting, the effects on pH and moisture content were the two most notable changes resulting from biochar amendment. We observed a pH increase in the composting materials with biochar amendment. This impacted N2O emissions and was supported by the results of Dannenmann et al.28 and Simek et al.,29 who reported a decreasing N2O/N2 ratio corresponded with increasing soil pH. This specifically affects the activity of microbes performing N2O reduction.30,31 Similarly, Yanai et al.10 noted that increases in soil pH due to the application of alkaline biochar potentially improved the activity of N2O-reducing organisms but inhibited reductases involved in converting nitrite and nitrate to nitrous oxide.32 Alternately, they proposed that increased reactive sites on biochar surfaces could chemically adsorb and then electrochemically reduce NO2−−N, N2O, and NO formed in soil during denitrification processes. In addition, strong correlations between the compost water content and N2O emission rate, as well as the gene abundance of nirK, nirS, and nosZ indicated the dominant role of moisture in controlling nitrogen cycling and N2O loss. This dominant role of moisture effectively driving N2O fluxes is corroborated by a comprehensive study of 10 steppe grasslands in Inner Mongolia.33 The decrease in moisture content resulting from biochar amendment (potentially due to enhanced compost aeration13) may singularly affect N2O production by altering redox conditions, denitrifying communities, or even gas diffusion. This study demonstrated that biochar incorporation into composts caused significant changes in pH and moisture, thereby affecting functional microbial communities participating in the denitrification process and lowering the N2O emission. Effect of Biochar Amendment on Denitrification Genes Corresponding to N2O Emission. Previous studies had indicated that biochar application to soils lowered denitrification and enhanced N 2O-reductase activity of denitrifiers.10,34 From this, we hypothesized that biochar

and displayed significant negative correlations with the copy numbers of nosZ, nirK, and nirS genes (rho values of −0.859, −0.877, and −0.899, respectively: P < 0.01). Another key regulating factor of N2O emission was pH, which displayed significant correlations to the nitrite concentration and EC. The nosZ/nirK ratio displayed similar matrix correlations to the pH.

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DISCUSSION In the present study, we investigated composting as an agricultural source of nitrous oxide and also a potential means to lower these emissions. We aimed to better understand the process by addressing the effect of biochar amendment on N2O emission, as well as improve the mechanistic understanding of the role of microbial denitrification contributing to these emissions. Effect of Biochar Amendment on Biochemical Parameters and N2O Fluxes. In the current study, PWSB displayed higher initial temperatures, a higher peak temperature, quicker entry to the cooling phase, and lower moisture and EC values. The temperature differences indicated that biochar amendment increased the kinetics of composting and thereby shortened the required time period. These results may be attributed to the positive effect of biochar on natural aeration within the composting pile due to its high nanoporosity and large surface area.26 A greater concentration of NO2−−N occurred in the PWS control around the 48th day, suggesting that the use of biochar also significantly affected NO2−−N in this study. The N2O emission rate continuously increased during the most intense composting phase and then peaked after the thermophilic phase for both treatments. As hypothesized, the N2O emission rates were lower for the PWSB than the control treatment over the entire composting period, most notably after day 48 and especially between the 52nd to 55th days. According to He et al.,27 NO2−−N was transformed into N2O during the later period of composting, and a clear linear correlation between nitrite and N2O concentration was observed in composting trials. By coupling the N2O emission and NO2−−N concentration data in our study, we deduced that biochar amendment significantly reduced the NO2 −-N F



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of greenhouse gas emission in soil systems. Also, Philippot et al.40 stated that N2O production correlated with the nosZ/16S rRNA ratio and nosZ/narG ratio. This suggested that the relative abundance of bacteria possessing the nosZ gene in the total bacterial community was a strong predictor of the N2O/ (N2O + N2) ratio. Contrary to the research regarding soil ecology, denitrifying bacteria possessing the nirK gene were approximately 24 times more prevalent than those with the nirS gene. This occurred in both PWSB and PWS and was consistent over the composting trial. No correlation was observed between N2O emission and nirS−nosZ gene abundance or the nosZ/16S rRNA ratio. Although not universally compatible with all types of environmental conditions, the relationship between N2O emission and the abundance of denitrifying genes were generally observed. The data in the current study particularly suggests that the abundances of nirK and nosZ genes were likely to play a dominant role in controlling N2O emissions. Although targeted denitrifying gene abundances were strongly linked to measured N2O emission rates in this study, copy numbers of key genes represent the standing community and its potential activity rather than the actual gene expression. Thus, adopting innovative technologies (such as metatranscriptomics or metaproteomics) to accurately assess the key environmental functions and activities is highly desirable for future work. Environmental Implications. To our knowledge, the present study is the first research quantifying the effect of biochar addition on N2O emission patterns and the abundance of denitrifying bacteria during the pig manure composting process. It is also the first study that provides robust evidence supporting the relationship between the abundance of denitrifying bacteria and their functional genes in controlling N2O emission from a pig manure composting system. Our data confirm that biochar addition affects N2O emission and the abundance of the nitrate reducing species and denitrifying bacterial community, illustrating the importance of the relative abundance of N2O-producing and N2O-consuming denitrifying bacteria for regulating and lowering N2O emissions. On the basis of the results of this study, biochar amendment to composting has the potential to lessen GHG emissions in the form of lowering the total N2O emitted, thereby offering a potential strategy to mitigate greenhouse gas release and deliver environmental benefits. Although Zeman et al.41 demonstrated composting is not regarded as a net source of CO2 for agricultural wastes, and there is evidence to suggest that biochar amendment does not affect compost CO2 emissions,13 it would be necessary to devise mitigation programs that consider the other key greenhouse gas, CO2, in order to predict and reduce global warming.

amendment to composting material would alter the bacterial gene abundance for key denitrification enzymes, which would correlate to changes in N2O emissions. As was expected, biochar amendment produced significant changes in the copy numbers of all functional genes. This was notable by day 48, where the PWSB contained more nosZ and less nirK than the control treatment. Although N2O generation was highest in both treatments from day 48 to 60, the N2O emission rate of biochar-amended treatment was significantly lower than the control treatment. There are a number of hypotheses surrounding biochar’s influence on N2O emissions, which include improved soil aeration, differences in the N2O/N2 ratio, changes in activities of enzymes for denitrification, shifts in microbial composition, and gross N transformation.32,35 However, we propose that the primary cause in biocharamended pig manure composting was due to variations in the abundance of denitrifying bacteria (especially for producing and consuming N2O). Apart from variations in pH, nitrite, and moisture mentioned above, changes in other factors due to biochar amendment could directly and indirectly affect the abundance of denitrifying bacteria. For example, ethylene produced by biochar could act as a microbial inhibitor and suppress the formation of N2O,36 while metal oxides on or near biochar surfaces such as TiO2 can catalyze the reduction of N2O to N2.37 In addition, improved aeration with biochar addition would also decrease either the duration or the extent of anoxia, thus depressing the activity of denitrifying bacteria and mitigating N2O emission.38 Based on these interpretations, our findings demonstrated that less N2O-producing and more N2O-consuming bacteria in the PWSB significantly lowered N2O emissions during the maturation phase of composting. Consistent with our observations, Regan et al.24 attributed substantially higher N2O emissions to the relative abundance of denitrifying bacteria either generating or degrading N2O (under elevated CO2 levels in a grassland ecosystem). Our data is also partially supported by the result of Philippot et al.,23 who concluded that a specific portion of denitrifying bacteria community with the nosZ gene reduced N2O to N2. Relationships among Microbial Parameters and N2O Emissions during Composting. Numerous studies have focused on elucidating the correlation between denitrifying bacterial populations and N fluxes to determine the nature of the denitrification end product. In the current study, the first to apply a quantitative approach to predict the relationship between denitrification and N2O emission in composting system, the highest denitrification gene copy numbers were observed between the cooling and maturation phase. Significantly lower gene copy numbers occurred during the mesophilic and thermophilic phases. As the temperature gradually decreased, the copy numbers of each denitrification gene increased moderately in both treatments. The increase in denitrification gene copy numbers correlated to the highest NO2− concentrations and N2O emission rates in this trial. All experimental data were used to determine Spearman rank correlations to compare the N2O emission rates with biochemical and microbial gene data. Of interest, we observed a significant correlation between the N2O emission rate and nirK, nirS, and nosZ gene abundance (Figure 4). However, a strong correlation between N2O emission and nirS gene abundance minus nosZ gene abundance has been reported by Morales et al.,39 who found the predominant denitrifying bacteria contained nirS genes rather than nirK genes, and suggested that bacterial gene abundances were good indicators

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ASSOCIATED CONTENT

* Supporting Information S

Additional methods and results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-571-88982020; fax: +86-571-88982020; e-mail: [email protected]. Author Contributions

C.W. and H.L. contributed equally to this work and should be considered cofirst authors. G



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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Yao Zhu and Wei Li for assistance with drawing the figure of matrices by MATLAB. The authors acknowledge the financial support of the Natural Science Foundation of Zhejiang Province (R5100044), the National Natural Science Foundation of China (41271247, 41271337), the Environment Protection Agency of Zhejiang Province, China (No. 2011B12), and the Specialized Research Fund for the Doctoral Program of Higher Education (20110101110083).

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