Enhanced antibiotic removal by the addition of bamboo charcoal during pig manure composting

Abstract

While composting is generally an effective way to minimize the adverse environmental impacts of manure prior to land application, some issues may also be introduced during compositing, such as the increased presence of recalcitrant antibiotic residues from feed additives. This study suggested that the addition of bamboo charcoal (BC) during pig manure composting was beneficial for the removal of three antibiotics (ciprofloxacin, chlorotetracycline, and norfloxacin). Addition of 9% (w/w) BC decreased the concentration of ciprofloxacin residues by 98.9% (from 1.85 to 0.02 mg kg⁻¹ dry weight) in 45 days, and decreased the content of norfloxacin and chlorotetracycline below detection limits in less than 5 and 10 days, respectively. In comparison, without added BC, ciprofloxacin levels were decreased by only 82.7% (from 1.85 to 0.32 mg kg⁻¹ dry weight) after 45 days of composting. This indicated that BC could enhance the removal of antibiotics during manure composting, thus reducing the risk of antibiotic residue runoff when composts are used in agriculture.

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1. Introduction

Large amounts of pig manure are produced annually both in China and many other countries worldwide, where it is a common practice to recycle this waste via land application as an organic fertilizer.^1,2 However, some leachable components of the manure, such as organic nitrogen, limit their direct land application. Moreover, heavy metal and antibiotic residues in pig manure can also be released into the terrestrial environment through manure application to agricultural fields.³ Antibiotics are marketed and used worldwide in intensive animal husbandry for the treatment of infectious diseases and as growth promoters.⁴ However, only a small proportion of the supplied antibiotics are metabolized or absorbed by the animal, and most of the antibiotic dose is released in excreta,⁵ entering into the soil and water environments.⁶ Thus, there are growing concerns about the potential risks of antibiotic residues to the terrestrial environment because they may stimulate the development of antibiotic resistance genes or antibiotic-resistant bacteria.⁷

Ciprofloxacin is a popular synthetic antibiotic used worldwide in feed animals. In China ciprofloxacin concentrations in fresh manure were as much as 1.34 mg kg⁻¹.^4,8 In Switzerland, ciprofloxacin concentrations in sewage sludge treatment plants ranged from 1.40 to 2.42 mg g⁻¹.⁹ Ciprofloxacin has also been detected in water from Wisconsin, USA at several hundred ng g⁻¹.¹⁰ Morales-Muñoz et al. (2004) reported a ciprofloxacin concentration of 5.8 mg kg⁻¹ in a farm soil following pig manure application in Córdoba, Spain.¹¹ Consequently, there may be potential health risks for humans when consuming crops and vegetables grown in soil treated with pig manures due to inadvertent dietary exposure to antibiotics.¹² Therefore to avoid unnecessary dietary antibiotic exposures, it is of paramount importance to fully treat pig manure prior to land application in order to decrease the efflux of antibiotics residues into the water and soil environments.

Composting is one remedial treatment that has been generally shown to be effective in organic matter degradation and is applicable to the degradation of antibiotic residues in manure.^7,13 Arikan et al. (2009) observed that chlorotetracycline in beef manure was decreased from 113 to 0.7 μg g⁻¹ following composting at 55 °C when using a mixture of straw and hardwood woodchips.⁷ Kim et al. (2012) also found that concentrations of three common antibiotics (chlorotetracycline, sulfamethazine, and tylosin) were all reduced by composting.¹³ They found that composting pig manure with sawdust; at least on a laboratory scale; decreased the antibiotic concentrations from 20 mg kg⁻¹ to well below the Korea guideline values of (tetracyclines, 0.8 mg kg⁻¹; sulfonamides, 0.2 mg kg⁻¹; and macrolides, 1.0 mg kg⁻¹). Likewise, Ding et al. (2014) found that the extractable kitasamycin concentrations were undetectable in kitasamycin-contaminated composts after 15 days.¹⁴ These studies all indicated that compositing involving a thermophilic phase was potentially suitable for efficiently eliminating antibiotics.

Composting also has the advantages of simultaneously increasing the fulvic- and humic-like substance content,¹⁵ and transforming manures into a safer and environmentally friendly source of nutrients beneficial for plant growth.^16–18 However, one common issue is that the nitrogen content of manure derived composts decreases rapidly with increasing temperature during the composting process. According to Raviv et al. (2002), the loss of total nitrogen was as much as 76% during the composting of organic waste, which lowered the quality of the composted products and also caused new pollution issues due to ammonia (NH₃) emissions.¹⁹ Thus, to date, while several articles have been published related to the conservation of nitrogen during composting,^2,20 low efficiency, potential negative impacts on soil pH, non-renewability, and cost factors have generally retarded the uptake of composting as an effective remediation and value adding technology. Hence, it is vital to develop efficient amendments which simultaneously conserve nutrients and eliminate harmful residues present in the parent manure during composting.

BC is a biochar derived from bamboo stem, which has both a large microporous physical structure and a large surface area.²¹ Thus, because of its high adsorption capacity, BC may be an ideal amendment for simultaneous nutrient stabilization and antibiotic degradation or adsorption allows subsequent degradation. The addition of BC during manure compositing has already been shown to significantly decrease the mobility of heavy metals and conserve nutrients.¹ Furthermore, biochar could also act as a soil fertilizer or conditioner to increase the yield of crops and vegetables by beneficially supplying nutrients and/or adjusting soil pH.^22,23 Therefore, BC has great potential as a composting amendment. However, there is currently little knowledge on the potential impact of BC on antibiotic depletion during manure compositing. The objective of the current study was thus to explore the effects of BC on antibiotic degradation during pig manure composting.

2. Materials and methods

2.1. Chemicals and material

Ciprofloxacin dehydrate (C₁₇H₁₈FN₃O₃), chlorotetracycline (C₂₂H₂₃ClN₂O₈), and norfloxacin (C₁₆H₁₈FN₃O₃) (>99.0% purity, Table 1) were supplied by the National Institute for the Control of Pharmaceutical and Biological Products, China. All organic solvents (methanol, acetonitrile, formic acid and ethyl acetate) were purchased from Fisher and were of HPLC-grade. Citric acid monohydrate (C₆H₈O₇·H₂O), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O), and ethylenediaminetetra acetic acid disodiumsalt (Na₂EDTA), were all of analytical purity. Ultra-pure water (Millipore Milli-Q system) with a resistivity of 18 MΩ cm was used in all antibiotic extractions.

Table 1 Molecular structure and characteristics of ciprofloxacin, chlorotetracycline, and norfloxacin

Compound	Ciprofloxacin	Chlorotetracycline	Norfloxacin
Molecular weight (g mol^−1)	331.3	515.3	319.2
Formula	C17H18FN3O3	C22H23ClN2O8	C16H18FN3O3
Structure
pKa,1	5.90	3.30	6.34
pKa,2	8.89	7.44	8.75
pKa,3		9.27
log Kow (25 °C)	0.40	−1.43	−0.88
Water solubility (mg L^−1)	30.00	92.75	300.00

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Fresh and matured pig manure compost was collected from a farm near Hangzhou city, Zhejiang province, China. Sawdust of China fir (Cunninghamia lanceolata (Lamb.) Hook) was purchased from Fuyang wood-working factory, China and used to adjust the ratio of C/N in the composting mixture. BC was purchased from Yaoshi Charcoal Production Company, China. The major characteristics of these materials are summarized in Table 2. Of all the materials used pig manure contained the highest nitrogen content, with a total Kjeldahl nitrogen concentration of 24.1 ± 0.8 g kg⁻¹. BC had the highest organic carbon content (317 ± 3.0 g kg⁻¹) and C/N ratio (310 ± 10). Sawdust was used as a regulator during manure composting because of its higher organic carbon content and low moisture content.¹ The specific surface area of BC was 359 ± 22 m² g⁻¹, which was similar to Chen et al.¹

Table 2 Physicochemical characteristics of composting materials used in this study

Characteristic	Material
	Pig manure	Matured pig manure	Bamboo charcoal	Sawdust
a ND, not detected.
pH	7.89–8.32	7.24–7.45	7.35–7.64	7.21–7.39
Moisture content (%)	70.1 ± 0.1	33.2 ± 0.07	17.30 ± 0.09	17.40 ± 0.03
Electrical conductivity (mS cm^−1)	5.32 ± 0.04	5.12 ± 0.02	0.09 ± 0.01	0.35 ± 0.01
Organic carbon (g kg^−1)	151 ± 2.0	142 ± 4.0	317 ± 3.0	211 ± 2.0
Total Kjeldahl nitrogen (g kg^−1)	24.1 ± 0.8	2.8 ± 0.9	2.8 ± 0.1	2.0 ± 0.3
C/N	6.0 ± 1.0	17.0 ± 3.0	310 ± 10.0	104 ± 6.0
Density (g cm^−3)	ND^a	ND	0.40 ± 0.05	ND
Pyrolysis temperature (°C)	ND	ND	800	ND
Specific surface (m^2 g^−1)	ND	ND	359 ± 22.0	ND

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2.2. Composting

All composting was conducted under controlled laboratory conditions. The composters consisted of 180 L rectangular plastic containers (80 (L) × 50 (W) × 45 (H) cm³). Four different treatments each having a different ratio of BC to pig manure (control, BC1, BC2 and BC3) on a dry weight basis were used. Each treatment was triplicated and prepared as indicated below.

Control: 90 kg pig manure (fresh) + 7 kg sawdust + 6 kg matured pig manure;

BC1: 90 kg pig manure (fresh) + 7 kg sawdust + 6 kg matured pig manure + 2.7 kg BC;

BC2: 90 kg pig manure (fresh) + 7 kg sawdust + 6 kg matured pig manure + 5.4 kg BC;

BC3: 90 kg pig manure (fresh) + 7 kg sawdust + 6 kg matured pig manure + 8.1 kg BC.

The initial moisture content was adjusted to approximately 65% (w/w) using tap water. Sufficient aeration was ensured by manually turning the pile every 5 d during the incubation. The daily temperature of the composting pile was recorded using a thermometer. Composite samples (20 g) from three discrete points within the pile were periodically collected (1, 3, 7, 10, 20, 30 and 45 d) during compositing and stored in a refrigerator at −4 °C prior to extraction and analysis.

2.3. Analytical procedures

Moisture content, organic matter, total phosphorus, total potassium, pH and electrical conductivity were all analyzed using standard methods.²⁴ Total nitrogen was determined via the Kjeldahl method by digesting the material with sulfuric acid at 400 °C.²⁵ Ammonia nitrogen and nitrate nitrogen were extracted from the material (10 g) with 2 mol L⁻¹ KCl (50 mL) at a ratio of 1 : 5 (m/V) with shaking at 160 rpm for 1 h and filtered through a 0.45 μm filter paper prior to analytical determination of concentrations. NH₄⁺–N was colorimetrically determined via the indophenol blue method,²⁶ and NO₃⁻–N was determined via the nitron gravimetric method.²⁷ Ash content was determined following combustion in a muffle furnace at 550 °C for 8 h and thereafter cooled in the furnace before transferring to a desiccator, until no change in dry weight was observed.²⁸ Previous studies have shown that theses experimental conditions are sufficient to accurately determine ash content.^1,28

Ciprofloxacin, chlorotetracycline, and norfloxacin were extracted in triplicate from composting subsamples according to the method of Wang et al. (2013).²⁹ Briefly, compost subsamples (1 g) were extracted with a 20 mL mixed buffer of methanol, acetic acid and water (6 : 3 : 1 v/v). The mixture was initially briefly vortexed for 30 s, prior to being ultrasonically extracted for 15 min using a 100 W ultrasonic bath. Subsequently the sample was centrifuged at 10 000 rpm for 10 min and the supernatant collected. The extraction step was repeated thrice and pooled supernatants were passed through a 60 mg hydrophilic–lipophilic balance solid phase extraction cartridge for enrichment and purification. The resulting eluent was concentrated to approximately 0.5 mL under a continuous flow of N₂ before being made up to 1 mL via addition of pure water. Samples were filtered through a 0.22 μm syringe filter prior to measurement by high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS, Waters Corp., USA).

2.4. Analysis of ciprofloxacin, chlorotetracycline and norfloxacin

The three antibiotics of ciprofloxacin, chlorotetracycline and norfloxacin were determined using HPLC-MS/MS on a symmetry C₁₈ column (150 mm × 3.9 mm, 5 μm, Waters, USA) at 35 °C and an injection volume of 10 μL. The mobile phase was acetonitrile (A) and 0.3% (v/v) formic acid (B) at a constant flow rate of 0.50 mL min⁻¹ using a gradient program of: Step 1 – initially 20% A increased linearly to 50% A in 5 min, Step 2 – maintain constant elution at 50% A for 5 min, Step 3 – decrease from 50 to 20% A over 4 min and finally Step 4 – maintain constant elution at 20% A for 1 min to maintain reaction equilibration. Tandem mass spectrometry conditions were set up as follows: source temperature, 80 °C, desolvation temperature, 350 °C, capillary voltage, 3.0 kV, desolvation gas flow, 500 L h⁻¹ and cone gas flow, 50 L h⁻¹. Acquisition was in multiple reactions monitoring mode.

The extraction recoveries of three antibiotics from 1 g samples of sawdust, pig manure and matured pig manure compost, spiked at different concentrations (1, 5, 10 μg g⁻¹ (DW)) were shown in Table 3. Those recoveries appeared to be independent of the initial spiked concentration and were good irrespective of the material spiked. This initial analysis gave confidence that antibiotic residues could be accurately quantified from the materials and at the concentrations expected in this experiment.

Table 3 Recoveries of ciprofloxacin, chlorotetracycline, and norfloxacin in composting materials

Materials	Spiked (μg g^−1)	Recoveries (%)
		Ciprofloxacin	Chlorotetracycline	Norfloxacin
Sawdust	1	85.0 ± 2.6	60.0 ± 8.9	125.4 ± 3.5
	5	92.0 ± 4.1	66.2 ± 2.8	128.0 ± 6.6
	10	95.5 ± 2.3	68.74 ± 5.2	134.6 ± 9.5
Pig manure	1	117.0 ± 7.9	51.50 ± 4.7	127.9 ± 4.6
	5	119.0 ± 5.6	58.0 ± 7.5	131.0 ± 3.1
	10	124.0 ± 5.2	65.5 ± 3.7	134.1 ± 6.8
Matured pig manure	1	95.5 ± 3.6	86.0 ± 6.4	116.2 ± 3.1
	5	86.0 ± 7.9	98.5 ± 3.6	129.8 ± 3.5
	10	102.0 ± 4.8	102.0 ± 6.4	123.7 ± 6.2

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Reproducibility was evaluated by run-to-run recoveries (six successive injections) whereas the method precision was expressed via the relative standard deviation (RSD) of triplicate measurements. The RSDs were good for sawdust (3.9–6.1%), pig manure (5.2–7.9%) and matured pig mature (2.8–9.8%), suggesting high precision.

The limits of detection and quantification of three antibiotics were detected with a signal-to-noise (S/N) ratio of 3 and 10, respectively (Table 4), suggesting a high sensitivity. Linear calibration curves of three antibiotics were constructed with standard concentrations of 50, 100, 200, 400, 600 and 1000 μg L⁻¹, with correlation coefficient (R²) > 0.99 (Table 4).

Table 4 Limits of detection (LOD, S/N = 3), limits of quantification (LOQ, S/N = 10) and regression analysis of calibration curves of ciprofloxacin, chlorotetracycline, and norfloxacin

Antibiotics	LOD (μg L^−1)	LOQ (μg L^−1)	Regression equation	R^2
Ciprofloxacin	0.16	0.53	Y = 22.12X − 471.49	0.994
Chlorotetracycline	7.18	23.94	Y = 12.77X − 224.57	0.995
Norfloxacin	3.16	10.52	Y = 12.17X − 311.57	0.990

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2.5. Data analysis

Antibiotics depletion kinetics was analyzed using the zero-order, first-order and second-order kinetic equations, which have been widely used to describe the depletion of organic pollutants during composting.^30–32where C is the measured antibiotic concentration (mg kg⁻¹) at time t (day), k is the removal rate constant (day⁻¹), n is the order of reaction.

When n = 0 the kinetic equation was zero-order kinetic equation (eqn (2)).

C = −kt + C₀ (2)

where C₀ is the initial antibiotic concentration (mg kg⁻¹).

As n = 1 the kinetic equation was first-order kinetic equation (eqn (3)).

According to the eqn (3), the corresponding antibiotics composting half-life was consequently calculated using eqn (4).

t_1/2 = ln(2)/k (4)

The second-order kinetic equation was given as eqn (5).

The total Kjeldahl nitrogen (TKN) loss was calculated using eqn (5).³³

N_loss = (N₁ − N₂)/(N₁) (6)

where N₁ and N₂ was the initial and the sample' TKN concentrations at any particular sampling time during composting.

All statistical analysis was conducted using least significant difference (LSD) tests at a significance level of 0.05 with SPSS v.13.0 (SPSS, Chicago, IL, USA).

3. Results and discussion

3.1. Composting characteristics

During composting the core temperature of the compositing pile increased well above the ambient temperature, indicating that thermophilic conditions were met for all treatments (Fig. 1A) and this was taken to be indicative of significant self-heating due to elevated microbial activity resulting from utilization of carbon and nitrogen.¹³ For all 3 treatments having BC addition the thermophilic stage (>40 °C) was reached within 3 days and was maintained above 50 °C for around 7 days (Fig. 1A), which was in agreement with Malińska et al. (2014) and indicated rapid establishment of microbial activity.³⁴ In contrast, for the control compost the onset of the thermophilic stage was reached much later (15 days) and the temperature declined much faster than in all 3 treatments (Fig. 1A). During the cooling stage, although the temperature remained higher than the ambient temperature, the temperature of all treatments gradually declined with time and became similar at day 38. The temperature of the composting piles also progressively increased with the addition of BC (BC3 > BC2 > BC1 > control), which suggested that BC increased the heat generated due to organic matter degradation and speed up the composting process by provision of more nutrition to microorganisms. After cooling, the compost entered the maturation stage until composting was finished. The final compost produced was dark in color, had a C/N ratios of 22.8 ± 1.5, 17.1 ± 2.7, 16.6 ± 0.6, and 14.9 ± 0.9 for control, BC1, BC2 and BC3, respectively, indicating that the compost was of good quality.³⁵

Fig. 1 Variation of temperature (A) and pH (B) with composting time (days).

The addition of BC slightly increased the pH of the compositing mixtures (Fig. 1B). During compositing the pH increased from 7.15 to a maximum of 8.25 on day 7, and thereafter gradually declined to 7.36 on day 45 for the control treatment. Increases in pH were expected due to an increase in ammonium production through the degradation of proteins contained in the initial pig manure, and ammonia's reaction with H₂O to form NH₄⁺ and free OH⁻ causing consequential pH increases.² Whereas the subsequent decreases in pH were ascribed to the volatilization of ammonic nitrogen,³⁶ the formation of low molecular weight fatty acids, and CO₂ production during organic matter degradation.³⁷ From day 7, the pH of all 3 treatments were lower than that of the control, which was attributed to the absorption of ammonia onto BC, which therefore inhibited ammonia dissolution into the compost and OH release.³⁰

During composing moisture content decreased from 60.9 ± 0.7% at the beginning of compositing to 48.3 ± 1.3% at the end of composting (Fig. 2A). The change in moisture content reflected a combination of decomposition, evaporation of water and the release of gases.

Fig. 2 Changes in physiochemical compost characteristics before and after composting.

For all treatments electrical conductivity (EC) decreased due to composting (Fig. 2B). Low EC was attributed to the precipitation of organic salts, precipitation of phosphate, and the volatilization of carbon dioxide.³⁸ The addition of BC slightly decreased EC in comparison to the control which was taken to be indicative of more efficient composting (Fig. 2B).

The organic matter decreased by 24.4 ± 1.3% for the control, compared to 33.8 ± 1.7% for treatment BC3 (Fig. 2C). The loss of organic matter was mostly attributed to the degradation of protein, cellulose, and hemicelluloses,³⁰ which can be utilized as C and N sources by microorganisms.³⁹ The addition of BC decreased the organic matter content much more than that of control, which suggested that BC increased the degradation of organic matter.

The ash content significantly increased after compositing for all treatments (Fig. 2D) which was partially ascribed to decomposition of organic matter into inorganic compounds. The slightly higher ash content in all 3 treatments (33.1 to 37.1%) compared to the control (30.3 ± 3.0%) was ascribed to the greater ash content contained in the BC itself (Fig. 2D) which was much higher than that reported by Ramaswamy et al.(2010), where the discrepancy may potentially be ascribed to the different types of feed materials used.³⁰

3.2. Antibiotics depletion and the kinetics during composting

Three types of antibiotics, ciprofloxacin, chlorotetracycline, and norfloxacin, were all initially detected in the pig manure, with initial concentrations of 1.85, 0.39, and 0.11 mg kg⁻¹ DW, respectively.

Ciprofloxacin content in the composted mixture decreased dramatically during composting (Fig. 3A), from an initial concentration of 1.85 mg kg⁻¹ DW, to 1.0 mg kg⁻¹ DW on day 5, and to 0.80 mg kg⁻¹ DW on day 10, reaching 0.32 mg kg⁻¹ DW by the end of composting on day 45. Thus, even for the control, without added BC, simple composting alone was able to remove >82.7% of the initial dose of ciprofloxacin within 45 days. The ciprofloxacin removal efficiency observed here was significantly higher than that reported by Nadia et al.,³⁹ who found residual ciprofloxacin concentrations of 0.31 mg kg⁻¹ in pig manure after 56 d of composting when initially spiked with 1.0 mg kg⁻¹. These recalcitrant residues indicated that fluoroquinolones were generally persistent during composting.

Fig. 3 The evolution of ciprofloxacin (A), chlorotetracycline (B) and norfloxacin (C) during composting (error bar represents the standard deviation of triplicate samples).

Ciprofloxacin depletion followed first-order but not zero-order and second-order kinetics equations during composting, with high linear correlations (R² > 0.917) for all treatments (Fig. 4). The removal rate constants for the control, BC1, BC2, BC3 were 0.048, 0.059, 0.072 and 0.085 day⁻¹, respectively and the half-life of ciprofloxacin depletion varied with treatment. Ciprofloxacin was most rapidly removed, with a half-life of 8.2 days in treatment BC3; which thereafter increased with decreasing amounts of BC; having half-lives of 9.6, 11.7 and 14.4 days for BC2, BC1 and the control, respectively.

Fig. 4 The fitting of antibiotic depletion kinetics: zero-order (A), first-order (B) and second-order (C) kinetic equations.

In comparison, the decrease in the antibiotic concentration of both chlorotetracycline, and norfloxacin during composting was even more dramatic and both decreased to below detection limits within 5–10 days (Fig. 3B and C). For example, chlorotetracycline levels in the composted mixture decreased from 0.39 mg kg⁻¹ DW to 0.26 mg kg⁻¹ DW on day 3, and to 0.13 mg kg⁻¹ DW on day 5, and to below the detection limit on day 10 (Fig. 3B). Thus, more than 65.8% chlorotetracycline removal was achieved with a 5 day composting period for the control, and BC treatments were able to increase the removal percent >71.2% in the same period. Likewise, the norfloxacin concentration in the composted mixture decreased from 0.11 mg kg⁻¹ DW to 0.07 mg kg⁻¹ DW on day 3, and to below the detection limit by day 5 (Fig. 3C). The addition of BC slightly increased both the removal of chlorotetracycline and norfloxacin. Since the content of chlorotetracycline and norfloxacin were lower and to below the detection limit on day 5, we did not have enough data to analyze the depletion and the kinetics during composting.

Generally, temperature dependent abiotic processes; including adsorption and degradation; are commonly proposed as the main mechanisms for antibiotic depletion during composting.^7,12,13 The rapid removal of antibiotic within the first 5 days of composting was thus related to the rapid temperature and pH increase which accelerated ciprofloxacin degradation (Fig. 3A). Hartlieb et al. (2003) also indicated, that in addition to degradation, composting also generates more available sites for antibiotics adsorption.⁴¹ Thus the observed rapid depletion of ciprofloxacin within the first 5 days of composting can also partially be ascribed to the presence of organic matter originating from compost, which potentially initially generates more ciprofloxacin adsorption sites; thus making ciprofloxacin less extractable.⁴² Thereafter biotic processes, such as microbial degradation, may still occur to some degree and continue to degrade the antibiotics during the entire composting period.¹³ Information on the specific microorganisms that may be involved in antibiotic depletion and the exact biotic processes involved is currently lacking and is certainly an area were further research is required.

While ciprofloxacin was reportedly normally persistent during composting,⁴⁰ this study has shown that the addition of BC to pig manure could increase ciprofloxacin removal. Specifically, the levels of ciprofloxacin in the BC3 treatment decreased from 1.85 to 0.43 mg kg⁻¹ DW on day 5, to 0.29 mg kg⁻¹ DW on day 10 and to 0.02 mg kg⁻¹ DW at the end of composting on day 45. This corresponded to a 98.9% ciprofloxacin removal. The enhanced removal of ciprofloxacin in BC treatments observed here was specifically attributed to the adsorption of ciprofloxacin onto BC due to BC's relatively large surface area (Table 2). Biochar is known to have a high capacity for antibiotic adsorption because of its surface area.⁴² Chen et al. (2010) also reported that the immobilization of Cu and Zn was increased by 35 and 39%, respectively due to the addition of 9% BC relative to the control,¹ and attributed this increase to the high adsorption capacity of BC via its larger surface area and functional groups. However, elucidation of the exact mechanisms responsible for enhanced removal of ciprofloxacin following addition of BC during manure composting is still not clear and warrants further study.

3.3. Evolution of N, P and K

At least one previous study has reported that the addition of BC during manure composting could conserve nutrient levels.¹ In this study, total nitrogen was generally significantly decreased with composting time (Fig. 5A) for all treatments, due mainly to nitrogen loss via volatilization in the form of ammonia at relatively high temperatures. However, addition of BC significantly (P < 0.01) decreased nitrogen loss. The amount of nitrogen loss decreased with the amount of BC added during composting. Thus nitrogen loss was reduced to 38.4 ± 0.9, 32.9 ± 1.5, and 28.1 ± 1.3% with BC1, BC2, and BC3 treatments, respectively, in sharp contrast to a significantly higher nitrogen loss of 51.5 ± 2.1% in the control which contained no added biochar. Ramaswamy et al. (2010) reported that nitrogen loss was reduced to 36% during pig manure composting in sealed and insulated containers compared to a 60% loss when poultry manure was composted in open containers.³⁰ In the current study, the addition of BC significantly reduced nitrogen loss due mainly to the large specific surface area and microporous structure of BC.^1,36

Fig. 5 Variation of total N (A), NH₄⁺–N (B), NO₃⁻–N (C), total P (D) and total K (E) during composting (days) (the different letters mean the significant differences at a significance level of 0.05 among treatments).

The NH₄⁺–N concentrations increased rapidly at the beginning of composting (Fig. 5B) due to ammonization which was favored by an increase in both temperature and pH. Subsequent decreases in of NH₄⁺–N concentrations were probably due to volatilization as NH₃. The addition of BC 6% and 9% significantly decreased NH₄⁺–N loss relative to the control, which was ascribed to immobilization of NH₄⁺ ions and reduction of ammonia volatilization BC.³⁴

Nitrification generally increases during compost cooling resulting in increases in NO₃⁻–N. Consistent with this trend NO₃⁻–N concentrations remained stable for 20 days, then increased relatively rapidly until day 30, and thereafter increased only at a moderate rate after day 30 (Fig. 5C). The addition of BC slightly increased NO₃⁻–N concentration while showing non-significant differences among BC treatments. With the exception of the initial stages of compositing for the control and BC1, total phosphorus and potassium generally increased with composting time and all treatments had increased phosphorus at maturation (Fig. 5D and E), which can be partially ascribed to the innate phosphorus and potassium content of the biochar.²² This observation suggested that organic matter degradation during compositing, not only releases carbon dioxide, but may also result in the reduction of dry mass and a continuous increase in total phosphorus.

4. Conclusion

Simple composting of pig manure efficiently decreases residual antibiotics, ciprofloxacin, chlorotetracycline, and norfloxacin, concentrations and addition of modest BC amendments significantly (P < 0.05) enhances antibiotics removal. Composting decreased the content of chlorotetracycline and norfloxacin below detection limits in less than 5 and 10 days, respectively. Ciprofloxacin was decreased >98.9% within 45 days of composting compared to only 82.7% removal when no BC was added. Addition of BC to a pig manure compost also conserved TKN, thus retaining fertilizer value and increasing the adsorption of ammonia by reducing volatilization. Thus BC is an effective material capable of reducing many of the detrimental effects associated with pig manure compost, making the application of composted pig manure safer for field applications.

Acknowledgements

This work was supported by Hangzhou Science and Technology Committee (20132231E), and the Research Institute of Subtropical Forestry, Chinese Academy of Forestry (RISF 2013001). Dr Gary Owens gratefully acknowledges the support of the Australian Research Council Future Fellowship Scheme (FT120100799) for funding his salary.

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