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Composting of swine manure spiked with sulfadiazine, chlortetracycline and ciprofloxacin
Bioresource Technology 126 (2012) 412–417
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Composting of swine manure spiked with sulfadiazine, chlortetracycline and ciprofloxacin Ammaiyappan Selvam, Zhenyong Zhao, Jonathan W.C. Wong ⇑ Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region
a r t i c l e
i n f o
Article history: Received 10 October 2011 Received in revised form 12 December 2011 Accepted 13 December 2011 Available online 22 December 2011 Keywords: Composting Antibiotics Sulfadiazine Chlortetracycline Ciprofloxacin
a b s t r a c t The fate of chlortetracycline (CTC), sulfadiazine (SDZ) and ciprofloxacin (CIP) during composting of swine manure and their effect on composting process were investigated. Swine manure was spiked with antibiotics, mixed with saw dust (1:1 on DW basis) and composted for 56 d. Antibiotics were spiked to a final concentration of 50 mg/kg CTC + 10 mg/kg SDZ + 10 mg/kg CIP (High-level) or 5 mg/kg CTC + 1 mg/kg SDZ + 1 mg/kg CIP (Low-level), and a control without antibiotics. Antibiotics at high concentrations delayed the initial decomposition that also affected the nitrogen mineralization. CTC and SDZ were completely removed from the composting mass within 21 and 3 d, respectively; whereas, 17–31% of the spiked CIP remained in the composting mass. Therefore, composting could effectively remove the CTC and SDZ spiked even at high concentrations, but the removal of ciprofloxacin (belonging to fluoroquinolone) needs to be improved, indicating this antibiotic may get into the ecosystem through land application of livestock compost. Ó 2011 Published by Elsevier Ltd.
1. Introduction Replenishment with organic matter such as livestock excretions is a major approach to restore the degraded soils due to excessive application of inorganic fertilizer. Use of livestock excretions has been promoted by the Ministry of Agriculture of China since 1992 through the ‘‘Rich Soil Project’’. Besides, it is a common practice for organic farmers to utilize livestock wastes after composting for restoring soil fertility. However, the heavy use of antibiotics for disease prevention and curing in livestock animals has raised the concern it may serve as an important pathway for the spreading of antibiotics into soil because unlike human manure, waste from farms does not undergo tertiary wastewater treatment (Kim et al., 2011). Therefore, removal of antibiotics from livestock waste before their application to soil is becoming an emerging environmental issue. Livestock waste represents a huge issue in many countries due to their discharge into the water bodies without any treatments causing eutrophication. With the implementation of livestock ordinance, all livestock waste needs to be collected and disposed of by landfilling or composting. Composting provides an economical and environment-friendly approach for stabilizing livestock waste and convert it into a good organic fertilizer or soil conditioner, which will be easier to handle than raw wastes. Besides, the application of raw waste will easily lead to leaching of nutrients into the soil aquifer ⇑ Corresponding author. Tel.: +852 34117056; fax: +852 34112355. E-mail address: [email protected] (J.W.C. Wong). 0960-8524/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.biortech.2011.12.073
and contaminate ground water. During composting process, the presence of a wide variety of complex organic compounds will encourage the development of a wide diversity of microorganisms of large population (Díaz et al., 1993). This creates an appropriate composting condition for the removal of antibiotics presence in the livestock waste. Recent studies reported the effectiveness of composting on the removal of several kinds of antibiotics, i.e. Penicillins, tetracyclines (chlortetracycline [CTC] and oxytetracycline [OTC]) and polyether antibiotics (salinomycin) (Arikan et al., 2007, 2009a,b; Kakimoto et al., 2007; Ramaswamy et al., 2010; Zhang et al., 2006). The removal efficiencies of different kinds of antibiotics in livestock and poultry manure varied significantly ranging from 40.2% for oxytetracycline to more than 99% for penicillin, salinomycin and CTC, as well as 100% removal for amoxicillin; while the possible reason for such varying removal efficiencies is largely unknown since there is an extreme lack of information on the mechanisms responsible for the antibiotics removal during composting. Tetracyclines, sulfonamides and fluoroquinolones are frequently used in livestock farming and consequently, livestock excretions were found to contain considerable levels of these antibiotics. A maximum concentration of 200 mg/L of tetracyclines in the swine slurry (Kumar et al., 2005) was reported. Sulfonamides in manures have been found at concentrations from 8.7 to 12.4 mg/kg (Haller et al., 2002). Occasionally, very high concentrations have been reported such as almost 500 mg/kg slurry for sulfadiazine (SDZ) (Grote et al., 2004). In case of CIP, up to 33.98 and 29.59 mg/kg in manure samples from swine and cow, respectively were reported (Winckler et al., 2003). Therefore, the aim of this
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study was to investigate the degradation of antibiotics CTC, SDZ and CIP during composting and assess the effect of antibiotics on the composting process. 2. Methods 2.1. Composting of swine manure spiked with antibiotics Antibiotics sulfadiazine (4-Amino-N-(2-pyrimidinyl) benzenesulfonamide sodium salt, P98%), chlortetracycline (7-Chlorotetracycline hydrochloride, P97%) and ciprofloxacin (1-cyclopropyl-6fluoro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic acid) were purchased from Sigma–Aldrich (St. Louis, MO). The swine manure was collected in a single day, mixed well using the collection equipment, placed in plastic box lined with polythene paper, immediately transported to the lab and stored at 4 °C for 3 d until the samples were analyzed for physicochemical properties including antibiotics. The swine manure was analyzed and selected physicochemical properties were as follows: pH 7.13, moisture content 74%, bulk density 1.04 g/cc, total organic carbon (TOC) 30.7%, total Kjeldahl nitrogen (TKN) 4.03%, organic matter 72.1% and carbon/nitrogen (C/N) ratio 7.6. The swine manure was spiked with SDZ, CTC and CIP at two levels: high (100 mg/kg CTC + 20 mg/kg SDZ + 20 mg/kg CIP) or low (10 mg/kg CTC + 2 mg/kg SDZ + 2 mg/kg CIP). A control treatment was also prepared without the addition of antibiotics. Saw dust was mixed with swine manure (1:1 DW) to adjust the C/N ratio to 29 and moisture content to 55%; and the mixing with saw dust resulted in a reduction in the concentration of the antibiotics spiked
in the composting mass by half. The bench-scale composting of swine manure was conducted in computer controlled 20-L reactors for 56 d with an aeration rate of 0.5 L/kg DW/min. All treatments received 7 kg composting mix consisted of swine manure and saw dust. During the composting period, emission from the reactor was connected to a carbon dioxide (CO2) analyzer for assessing the decomposition process continuously. Periodically, composting mass was removed from the reactors, moistened with water to restore the moisture content, and then thoroughly mixed before 150 g of sample was collected for the analysis of TOC and TKN as per the TMECC (2002) methods and antibiotics as described below. 2.2. Extraction and analysis of antibiotics Antibiotics in the swine manure or compost samples were extracted using the USEPA (2007) method 1694 with modifications. Briefly, to 1 g of the sample, 15 mL phosphate buffer (pH adjusted to 4.0 after addition) and 20 mL acetonitrile was added, sonicated for 30 min, centrifuged at 10,000g, 4 °C for 10 min, and filtered through 1.6 lm glass-fiber filter (Whatman, Banbury, UK). This extraction was repeated once and then for the third time extracted with 20 mL acetonitrile and the extracts were pooled together. The extracts were rotary evaporated to remove the acetonitrile, diluted to 200 mL with MilliQ water, 500 mg of Na4 EDTA was added, pH was adjusted to 4.0 and concentrated using 6 cc/500 mg OasisÒ HLB cartridge (Waters). The concentrated extracts after N2 evaporation was reconstituted in acetonitrile: water (1:9). Antibiotic extracts, 15 lL, were separated using Phenomenex C18 column
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Day Fig. 1. CO2 emission (a) and temperature profiles (b) of the composting mass during swine manure composting.
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Total organic carbon (%)
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Day Fig. 2. Changes in total organic carbon (a), total nitrogen (b) and C/N ratio (c) of the composting mass during the swine manure composting.
(3 lm, 2.0 mm 150 mm) with a Alltima HP C18 (3 lm, 2.1 mm 7.5 mm) guard column in Agilent 1200 Series HPLC and analyzed using Applied Biosystems 3200 Q Trap MS. The mobile phases were (A) 0.5% formic acid and 0.1% ammonium formate and (B) acetonitrile:methanol (1:1). The gradient program was, 5% solvent B for 2 min, solvent B increased to 88% in 12.5 min and 100% in 13 min. Simatone was used as an internal standard in recovery as well as during sample analysis. The recoveries of CTC, SDZ and CIP were 82%, 62% and 73% respectively, and were consistent. 2.3. Statistical analyses Data analyses were performed for triplicate samples and the mean values with standard error were presented. The data were
subjected to one way analysis of variance (ANOVA) and Duncan’s multiple range test using SPSS ver.11.5 software. 3. Results and discussion 3.1. CO2 evolution and temperature profile Concentration of CO2 in the outlet emission is a direct indication of the microbial activity of the composting mass. As shown in Fig. 1a, CO2 emission increased immediately following the commencement of the composting process for all treatments indicating the active decomposition process. For treatments with low level (L-level) of antibiotics and the control, the CO2 emission peaked at day 1 and gradually decreased afterwards due to the depletion of readily soluble nutrients for microorganisms; whereas in
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Residual chlortetracycline (%)
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Residual ciprofloxacin (%)
(c) 100 80 60 40 20 0 0
10
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Days Fig. 3. Changes in the concentrations of chlortetracycline (a), sulfadiazine (b) and ciprofloxacin (c) during swine manure composting. 100% refers to the initial antibiotic concentration of (a) 50 and 5 mg/kg DW CTC for H- and L-level, respectively, (b and c) 10 and 1 mg/kg DW SDZ of CIP for H- and L-level, respectively.
treatment receiving high level of antibiotics (H-level), the CO2 emission peaked at a later stage, i.e. day 7, indicating the initial inhibition of high level of antibiotics on the microbial activity. Similarly, Kakimoto et al. (2007) reported a delay on the CO2 emission during the respiratory test of feces due to the addition of 10– 500 mg kg 1 amoxicillin. In a couple of previous studies, the CTC did not inhibit the composting process (Arikan et al., 2009a,b; Hu et al., 2011) indicating that the CTC spiked in the composting mass of this study might not be the cause responsible for the delay in the CO2 peak emission. Similarly, the effect of the SDZ could also be insignificant as evidenced from the rapid degradation during composting. Therefore, the CIP was expected to affect the composting process significantly. The delayed CO2 emission correlated well with the thermophilic temperature regimes of the composting process. The treatment
with high antibiotics exhibited a comparatively lower temperature than the control and low level antibiotics demonstrating a clear initial inhibition, but notable difference was only observed for the initial 2 weeks and no significant difference was noted among the various treatments thereafter (Fig. 1b). 3.2. Changes in carbon and nitrogen contents The TOC of all treatments declined dramatically during the first 2 weeks, followed by a continuous but more gradual decrease till the end of the composting period (Fig. 2a). As revealed from the CO2 emission, there was a reduced organic decomposition at H-level of antibiotics initially; however, the difference was significant (P < 0.05%) only on day 3. After that there was no significant difference among different treatments indicating the antibiotic based
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inhibition are short lived during the composting process. However, the nitrogen profile was different between L-level and H-level antibiotic treatments. During the first week, TKN decreased on day 1 and increased thereafter for the control and L-level; while, in H-level treatment, an increase was observed that make the N levels significantly different (P < 0.05%) from both control and L-level (Fig. 2b). The initial decrease of TKN may be attributed to the nitrogen loss as ammonia while the increase in H-level might be due to the inhibition of N-mineralization by the antibiotics as also indicated by the less decomposition in TOC (Wong et al., 1997). From day 7 until the end of the experiment, the differences between the H-level and other treatments (control and L-level) were statistically significant (P < 0.05%) indicating the initial inhibition of N-mineralization, probably the ammonification, influenced the overall nitrogen content of the composting mass. One plausible reason is that the microorganism involved in nitrogen transformation may be highly vulnerable to high concentrations of antibiotics. Reports also showed that sulfonamides had effects on soil microbial biomass, structural composition and enzyme activities (Ding and He, 2010; Kotzerke et al., 2008; Sukul and Spiteller, 2006). Mainly, antibiotics caused a shift from bacteria to fungi as the dominant population, especially in the presence of significant quantity of organic matter (Gutiérrez et al., 2010; Zielezny et al., 2006). Hence, antibiotics indeed have a negative influence on the microbial diversity, which correlates with the perturbation on the microbial dynamics during the early stages of composting. It should be noted that after composting TKN for H-level was significantly (P < 0.05%) higher than the other treatments indicating the inhibition of antibiotics on the nitrogen loss during composting. Changes in the carbon and nitrogen contents influenced the profile of the C/N ratio. As shown in Fig. 2c, the solid C/N ratio declined with a high reduction during the first 2 weeks; thereafter gradually declined to 20.1–23. Compared to the control and L-level, H-level shows slightly lower C/N ratio due to the higher TKN.
3.3. Fates of antibiotics during composting The fates of antibiotics during composting are shown in Fig. 3. The CTC levels of both treatments decreased rapidly initially with treatments receiving L-level of CTC reached almost 95% removal within 7 d, while that with higher level of CTC with almost 70% removal during the thermophilic period. No CTC was detected for both treatments after 21 d of composting that agrees with previous reports (Arikan et al., 2009a,b). However, Wu et al. (2011) reported a degradation of about 74% of the 3.1 mg/kg of CTC; and Hu et al. (2011) reported a 97% of CTC degradation with an initial concentration of 60 mg/kg. The complete removal of CTC in the present study indicates that composting conditions were favorable for the biodegradation of CTC. Referring to these previous composting experiments, the inhibition of CTC on the initial composting process as observed in our present study had not been reported. Therefore, the initial reduced organic decomposition of this study may not be due to the presence of CTC. Similar to CTC, the SDZ was also completely removed, but within 3 d of the composting process indicating the biodegradation of SDZ was easier than that of CTC (Fig. 3b). When pig manure containing SDZ was applied to the soil, only a transient perturbation in the microbial dynamics was reported previously (Heuer et al., 2008) and within a day almost 90% of the SDZ was non-extractable in another soil application experiment (Hammesfahr et al., 2008). Major transformation products of SDZ are the non-bioactive conjugate acetyl-SDZ and the metabolite hydroxyl-SDZ that exhibits strongly reduced antibiotic potential. Further, SDZ reduced the rates of nitrification but increased the rate of ammonification in a soil-manure system (Hammesfahr et al., 2011). Therefore, SDZ,
similar to CTC, is very unlikely the reason for the initial inhibition on the composting process. Fig. 3c gives the biodegradation of CIP during the composting process, with also an initial sharp decrease in the first 21 d but in contrast to CTC and SDZ, CIP was not completely degraded within the 56 d of composting, indicating fluoroquinolones can be persistent during composting. About 0.31 mg/kg and 1.71 mg/kg of CIP, representing 31% and 17.1% of the spiked concentrations, were persistent in the composting masses of L-level and for H-level treatments, respectively. To our knowledge, the persistence of CIP during composting is being reported for the first time. The residual CIP in the compost may cause potential ecological and health risks by release of antibiotics to environment through application of the composting product to soil. As mentioned above, both CTC and SDZ did not cause any serious inhibition in the composting process, which leads us to suspect that the CIP could be the potential antibiotics that might inhibit the composting process initially. This study also reveals that the effectiveness of composting on the removal of antibiotics is likely to be antibiotics-specific. However, the possible reason is largely unknown since there is an extreme lack of information related to the mechanisms responsible for the removal of antibiotics during composting, which warrants further study. 4. Conclusions The present study revealed that composting could completely degrade the chlortetracycline and sulfadiazine spiked in swine manure; whereas, 17–31% of the ciprofloxacin could be persistent even after the major composting processes. Further, antibiotics at high concentrations, especially, the CIP, inhibited the decomposition of organic matter initially; however, the difference among the treatment disappeared during the later stages. In contrast, antibiotics affected the nitrogen transformation, especially during the initial stages composting that influenced the nitrogen content of the composts. Therefore, the persistence of CIP in the swine manure compost implies their potential impact on the soil ecosystem upon their application which should not be overlooked. Acknowledgements The authors thank the Research Grant Council of the Hong Kong Special Administrative Region, People’s Republic of China for the financial support for this project (Grant HKBU261808). References Arikan, O.A., Sikora, L.J., Mulbry, W.W., Khan, S.U., Foster, G.D., 2007. Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves. Bioresour. Technol. 98, 169–176. Arikan, O.A., Mulbry, W., Ingram, D., Millner, P., 2009a. Minimally managed composting of beef manure at the pilot scale: effect of manure pile construction on pile temperature profiles and on the fate of oxytetracycline and chlortetracycline. Bioresour. Technol. 100, 4447–4453. Arikan, O.A., Mulbry, W., Rice, C., 2009b. Management of antibiotic residues from agricultural sources: use of composting to reduce chlortetracycline residues in beef manure from treated animals. J. Hazard. Mater. 164, 483–489. Díaz, L., Savage, G., Eggerth, L., Golueke, C., 1993. Composting and Recycling Municipal Solid Waste. Lewis publishers, California, USA. Ding, C., He, J., 2010. Effect of antibiotics in the environment on microbial populations. Appl. Microbiol. Biotechnol. 87, 925–941. Grote, M., Vockel, A., Schwarze, D., Mehlich, A., Freitag, M., 2004. Fate of antibiotics in food chain and environment originating from pig fattening (part 1). Fres. Environ. Bull. 13, 1216–1224. Gutiérrez, I.R., Watanabe, N., Harter, T., Glaser, B., Radke, M., 2010. Effect of sulfonamide antibiotics on microbial diversity and activity in a Californian Mollic Haploxeralf. J. Soils Sediments 10, 537–544. Haller, M.Y., Müller, S.R., McArdell, C.S., Alder, A.C., Suter, M.J.F., 2002. Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatography–mass spectrometry. J. Chromatogr. A 952, 111–120.
A. Selvam et al. / Bioresource Technology 126 (2012) 412–417 Hammesfahr, U., Heuer, H., Manzke, B., Small, K., Thiele-Bruhn, S., 2008. Impact of the antibiotic sulfadiazine and pig manure on the microbial community structure in agricultural soils. Soil Biol. Biochem. 40, 1583–1591. Hammesfahr, U., Kotzerke, A., Lamshöft, M., Wilke, B.-M., Kandeler, E., Thiele-Bruhn, S., 2011. Effects of sulfadiazine-contaminated fresh and stored manure on a soil microbial community. Eur. J. Soil Biol. 47, 61–68. Heuer, H., Focks, A., Lamshöft, M., Smalla, K., Matthies, M., Spiteller, M., 2008. Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biol. Biochem. 40, 1892–1900. Hu, Z., Liu, Y., Chen, G., Gui, X., Chen, T., Zhan, X., 2011. Characterization of organic matter degradation during composting of manure-straw mixtures spiked with tetracyclines. Bioresour. Technol. 102, 7329–7334. Kakimoto, T., Osawa, T., Funamizu, N., 2007. Antibiotic effect of amoxicillin on the feces composting process and reactivation of bacteria by intermittent feeding of feces. Bioresour. Technol. 98, 3555–3560. Kim, K.R., Owens, G., Kwon, S.I., So, K.H., Lee, D.B., Ok, Y., 2011. Occurrence and environmental fate of veterinary antibiotics in the terrestrial environment. Water Air Soil Pollut. 214, 163–174. Kotzerke, A., Sharma, S., Schauss, K., Heuer, H., Thiele-Bruhn, S., Smalla, K., Wilke, B., Schloter, M., 2008. Alterations in soil microbial activity and N-transformation processes due to sulfadiazine loads in pig-manure. Environ. Pollut. 153, 315– 322.
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Kumar, K., Gupta, S.C., Chander, Y., Singh, A.K., 2005. Antibiotic use in agriculture and its impact on the terrestrial environment. Adv. Agron. 87, 1–54. Ramaswamy, J., Prasher, S.O., Patel, R.M., Hussain, S.A., Barrington, S.F., 2010. The effect of composting on the degradation of a veterinary pharmaceutical. Bioresour. Technol. 101, 2294–2299. Sukul, P., Spiteller, M., 2006. Sulfonamides in the environment as veterinary drugs: present status and future scopes – a review. Rev. Environ. Contam. Toxicol. 187, 67–102. USEPA, 2007. Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment and Biosolids by HPLC/MS/MS. Winckler, C., Engels, H., Hund-Rinke, K., Luckow, T., Simon, M., Steffens, G., 2003. Verhalten von Tetracyclinen und anderen Veterinärantibiotika in Wirtschaftsdünger und Boden, UFOPLAN 20073 248, Berlin. Wong, J.W.C., Fang, M., Li, G.X., Wong, M.H., 1997. Feasibility of co-composting coal ash residues with sewage sludge. Environ. Technol. 18, 563–567. Wu, X., Wei, Y., Zheng, J., Zhao, X., Zhong, W., 2011. The behavior of tetracyclines and their degradation products during swine manure composting. Bioresour. Technol. 102, 5924–5931. Zhang, S.Q., Zhang, F.D., Liu, X.M., Wang, Y.J., Zhang, J.F., 2006. Degradation of antibiotics and passivation of heavy metals during thermophilic composting process. Sci. Agr. Sin. 39, 337–343. Zielezny, Y., Groeneweg, J., Vereecken, H., Tappe, W., 2006. Impact of sulfadiazine and chlorotetracycline on soil bacterial community structure and respiratory activity. Soil Biol. Biochem. 38, 2372–2380.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Composting of swine manure spiked with sulfadiazine, chlortetracycline and ciprofloxacin Ammaiyappan Selvam, Zhenyong Zhao, Jonathan W.C. Wong ⇑ Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region
a r t i c l e
i n f o
Article history: Received 10 October 2011 Received in revised form 12 December 2011 Accepted 13 December 2011 Available online 22 December 2011 Keywords: Composting Antibiotics Sulfadiazine Chlortetracycline Ciprofloxacin
a b s t r a c t The fate of chlortetracycline (CTC), sulfadiazine (SDZ) and ciprofloxacin (CIP) during composting of swine manure and their effect on composting process were investigated. Swine manure was spiked with antibiotics, mixed with saw dust (1:1 on DW basis) and composted for 56 d. Antibiotics were spiked to a final concentration of 50 mg/kg CTC + 10 mg/kg SDZ + 10 mg/kg CIP (High-level) or 5 mg/kg CTC + 1 mg/kg SDZ + 1 mg/kg CIP (Low-level), and a control without antibiotics. Antibiotics at high concentrations delayed the initial decomposition that also affected the nitrogen mineralization. CTC and SDZ were completely removed from the composting mass within 21 and 3 d, respectively; whereas, 17–31% of the spiked CIP remained in the composting mass. Therefore, composting could effectively remove the CTC and SDZ spiked even at high concentrations, but the removal of ciprofloxacin (belonging to fluoroquinolone) needs to be improved, indicating this antibiotic may get into the ecosystem through land application of livestock compost. Ó 2011 Published by Elsevier Ltd.
1. Introduction Replenishment with organic matter such as livestock excretions is a major approach to restore the degraded soils due to excessive application of inorganic fertilizer. Use of livestock excretions has been promoted by the Ministry of Agriculture of China since 1992 through the ‘‘Rich Soil Project’’. Besides, it is a common practice for organic farmers to utilize livestock wastes after composting for restoring soil fertility. However, the heavy use of antibiotics for disease prevention and curing in livestock animals has raised the concern it may serve as an important pathway for the spreading of antibiotics into soil because unlike human manure, waste from farms does not undergo tertiary wastewater treatment (Kim et al., 2011). Therefore, removal of antibiotics from livestock waste before their application to soil is becoming an emerging environmental issue. Livestock waste represents a huge issue in many countries due to their discharge into the water bodies without any treatments causing eutrophication. With the implementation of livestock ordinance, all livestock waste needs to be collected and disposed of by landfilling or composting. Composting provides an economical and environment-friendly approach for stabilizing livestock waste and convert it into a good organic fertilizer or soil conditioner, which will be easier to handle than raw wastes. Besides, the application of raw waste will easily lead to leaching of nutrients into the soil aquifer ⇑ Corresponding author. Tel.: +852 34117056; fax: +852 34112355. E-mail address: [email protected] (J.W.C. Wong). 0960-8524/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.biortech.2011.12.073
and contaminate ground water. During composting process, the presence of a wide variety of complex organic compounds will encourage the development of a wide diversity of microorganisms of large population (Díaz et al., 1993). This creates an appropriate composting condition for the removal of antibiotics presence in the livestock waste. Recent studies reported the effectiveness of composting on the removal of several kinds of antibiotics, i.e. Penicillins, tetracyclines (chlortetracycline [CTC] and oxytetracycline [OTC]) and polyether antibiotics (salinomycin) (Arikan et al., 2007, 2009a,b; Kakimoto et al., 2007; Ramaswamy et al., 2010; Zhang et al., 2006). The removal efficiencies of different kinds of antibiotics in livestock and poultry manure varied significantly ranging from 40.2% for oxytetracycline to more than 99% for penicillin, salinomycin and CTC, as well as 100% removal for amoxicillin; while the possible reason for such varying removal efficiencies is largely unknown since there is an extreme lack of information on the mechanisms responsible for the antibiotics removal during composting. Tetracyclines, sulfonamides and fluoroquinolones are frequently used in livestock farming and consequently, livestock excretions were found to contain considerable levels of these antibiotics. A maximum concentration of 200 mg/L of tetracyclines in the swine slurry (Kumar et al., 2005) was reported. Sulfonamides in manures have been found at concentrations from 8.7 to 12.4 mg/kg (Haller et al., 2002). Occasionally, very high concentrations have been reported such as almost 500 mg/kg slurry for sulfadiazine (SDZ) (Grote et al., 2004). In case of CIP, up to 33.98 and 29.59 mg/kg in manure samples from swine and cow, respectively were reported (Winckler et al., 2003). Therefore, the aim of this
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study was to investigate the degradation of antibiotics CTC, SDZ and CIP during composting and assess the effect of antibiotics on the composting process. 2. Methods 2.1. Composting of swine manure spiked with antibiotics Antibiotics sulfadiazine (4-Amino-N-(2-pyrimidinyl) benzenesulfonamide sodium salt, P98%), chlortetracycline (7-Chlorotetracycline hydrochloride, P97%) and ciprofloxacin (1-cyclopropyl-6fluoro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic acid) were purchased from Sigma–Aldrich (St. Louis, MO). The swine manure was collected in a single day, mixed well using the collection equipment, placed in plastic box lined with polythene paper, immediately transported to the lab and stored at 4 °C for 3 d until the samples were analyzed for physicochemical properties including antibiotics. The swine manure was analyzed and selected physicochemical properties were as follows: pH 7.13, moisture content 74%, bulk density 1.04 g/cc, total organic carbon (TOC) 30.7%, total Kjeldahl nitrogen (TKN) 4.03%, organic matter 72.1% and carbon/nitrogen (C/N) ratio 7.6. The swine manure was spiked with SDZ, CTC and CIP at two levels: high (100 mg/kg CTC + 20 mg/kg SDZ + 20 mg/kg CIP) or low (10 mg/kg CTC + 2 mg/kg SDZ + 2 mg/kg CIP). A control treatment was also prepared without the addition of antibiotics. Saw dust was mixed with swine manure (1:1 DW) to adjust the C/N ratio to 29 and moisture content to 55%; and the mixing with saw dust resulted in a reduction in the concentration of the antibiotics spiked
in the composting mass by half. The bench-scale composting of swine manure was conducted in computer controlled 20-L reactors for 56 d with an aeration rate of 0.5 L/kg DW/min. All treatments received 7 kg composting mix consisted of swine manure and saw dust. During the composting period, emission from the reactor was connected to a carbon dioxide (CO2) analyzer for assessing the decomposition process continuously. Periodically, composting mass was removed from the reactors, moistened with water to restore the moisture content, and then thoroughly mixed before 150 g of sample was collected for the analysis of TOC and TKN as per the TMECC (2002) methods and antibiotics as described below. 2.2. Extraction and analysis of antibiotics Antibiotics in the swine manure or compost samples were extracted using the USEPA (2007) method 1694 with modifications. Briefly, to 1 g of the sample, 15 mL phosphate buffer (pH adjusted to 4.0 after addition) and 20 mL acetonitrile was added, sonicated for 30 min, centrifuged at 10,000g, 4 °C for 10 min, and filtered through 1.6 lm glass-fiber filter (Whatman, Banbury, UK). This extraction was repeated once and then for the third time extracted with 20 mL acetonitrile and the extracts were pooled together. The extracts were rotary evaporated to remove the acetonitrile, diluted to 200 mL with MilliQ water, 500 mg of Na4 EDTA was added, pH was adjusted to 4.0 and concentrated using 6 cc/500 mg OasisÒ HLB cartridge (Waters). The concentrated extracts after N2 evaporation was reconstituted in acetonitrile: water (1:9). Antibiotic extracts, 15 lL, were separated using Phenomenex C18 column
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Day Fig. 1. CO2 emission (a) and temperature profiles (b) of the composting mass during swine manure composting.
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Total organic carbon (%)
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Day Fig. 2. Changes in total organic carbon (a), total nitrogen (b) and C/N ratio (c) of the composting mass during the swine manure composting.
(3 lm, 2.0 mm 150 mm) with a Alltima HP C18 (3 lm, 2.1 mm 7.5 mm) guard column in Agilent 1200 Series HPLC and analyzed using Applied Biosystems 3200 Q Trap MS. The mobile phases were (A) 0.5% formic acid and 0.1% ammonium formate and (B) acetonitrile:methanol (1:1). The gradient program was, 5% solvent B for 2 min, solvent B increased to 88% in 12.5 min and 100% in 13 min. Simatone was used as an internal standard in recovery as well as during sample analysis. The recoveries of CTC, SDZ and CIP were 82%, 62% and 73% respectively, and were consistent. 2.3. Statistical analyses Data analyses were performed for triplicate samples and the mean values with standard error were presented. The data were
subjected to one way analysis of variance (ANOVA) and Duncan’s multiple range test using SPSS ver.11.5 software. 3. Results and discussion 3.1. CO2 evolution and temperature profile Concentration of CO2 in the outlet emission is a direct indication of the microbial activity of the composting mass. As shown in Fig. 1a, CO2 emission increased immediately following the commencement of the composting process for all treatments indicating the active decomposition process. For treatments with low level (L-level) of antibiotics and the control, the CO2 emission peaked at day 1 and gradually decreased afterwards due to the depletion of readily soluble nutrients for microorganisms; whereas in
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Residual chlortetracycline (%)
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Days Fig. 3. Changes in the concentrations of chlortetracycline (a), sulfadiazine (b) and ciprofloxacin (c) during swine manure composting. 100% refers to the initial antibiotic concentration of (a) 50 and 5 mg/kg DW CTC for H- and L-level, respectively, (b and c) 10 and 1 mg/kg DW SDZ of CIP for H- and L-level, respectively.
treatment receiving high level of antibiotics (H-level), the CO2 emission peaked at a later stage, i.e. day 7, indicating the initial inhibition of high level of antibiotics on the microbial activity. Similarly, Kakimoto et al. (2007) reported a delay on the CO2 emission during the respiratory test of feces due to the addition of 10– 500 mg kg 1 amoxicillin. In a couple of previous studies, the CTC did not inhibit the composting process (Arikan et al., 2009a,b; Hu et al., 2011) indicating that the CTC spiked in the composting mass of this study might not be the cause responsible for the delay in the CO2 peak emission. Similarly, the effect of the SDZ could also be insignificant as evidenced from the rapid degradation during composting. Therefore, the CIP was expected to affect the composting process significantly. The delayed CO2 emission correlated well with the thermophilic temperature regimes of the composting process. The treatment
with high antibiotics exhibited a comparatively lower temperature than the control and low level antibiotics demonstrating a clear initial inhibition, but notable difference was only observed for the initial 2 weeks and no significant difference was noted among the various treatments thereafter (Fig. 1b). 3.2. Changes in carbon and nitrogen contents The TOC of all treatments declined dramatically during the first 2 weeks, followed by a continuous but more gradual decrease till the end of the composting period (Fig. 2a). As revealed from the CO2 emission, there was a reduced organic decomposition at H-level of antibiotics initially; however, the difference was significant (P < 0.05%) only on day 3. After that there was no significant difference among different treatments indicating the antibiotic based
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inhibition are short lived during the composting process. However, the nitrogen profile was different between L-level and H-level antibiotic treatments. During the first week, TKN decreased on day 1 and increased thereafter for the control and L-level; while, in H-level treatment, an increase was observed that make the N levels significantly different (P < 0.05%) from both control and L-level (Fig. 2b). The initial decrease of TKN may be attributed to the nitrogen loss as ammonia while the increase in H-level might be due to the inhibition of N-mineralization by the antibiotics as also indicated by the less decomposition in TOC (Wong et al., 1997). From day 7 until the end of the experiment, the differences between the H-level and other treatments (control and L-level) were statistically significant (P < 0.05%) indicating the initial inhibition of N-mineralization, probably the ammonification, influenced the overall nitrogen content of the composting mass. One plausible reason is that the microorganism involved in nitrogen transformation may be highly vulnerable to high concentrations of antibiotics. Reports also showed that sulfonamides had effects on soil microbial biomass, structural composition and enzyme activities (Ding and He, 2010; Kotzerke et al., 2008; Sukul and Spiteller, 2006). Mainly, antibiotics caused a shift from bacteria to fungi as the dominant population, especially in the presence of significant quantity of organic matter (Gutiérrez et al., 2010; Zielezny et al., 2006). Hence, antibiotics indeed have a negative influence on the microbial diversity, which correlates with the perturbation on the microbial dynamics during the early stages of composting. It should be noted that after composting TKN for H-level was significantly (P < 0.05%) higher than the other treatments indicating the inhibition of antibiotics on the nitrogen loss during composting. Changes in the carbon and nitrogen contents influenced the profile of the C/N ratio. As shown in Fig. 2c, the solid C/N ratio declined with a high reduction during the first 2 weeks; thereafter gradually declined to 20.1–23. Compared to the control and L-level, H-level shows slightly lower C/N ratio due to the higher TKN.
3.3. Fates of antibiotics during composting The fates of antibiotics during composting are shown in Fig. 3. The CTC levels of both treatments decreased rapidly initially with treatments receiving L-level of CTC reached almost 95% removal within 7 d, while that with higher level of CTC with almost 70% removal during the thermophilic period. No CTC was detected for both treatments after 21 d of composting that agrees with previous reports (Arikan et al., 2009a,b). However, Wu et al. (2011) reported a degradation of about 74% of the 3.1 mg/kg of CTC; and Hu et al. (2011) reported a 97% of CTC degradation with an initial concentration of 60 mg/kg. The complete removal of CTC in the present study indicates that composting conditions were favorable for the biodegradation of CTC. Referring to these previous composting experiments, the inhibition of CTC on the initial composting process as observed in our present study had not been reported. Therefore, the initial reduced organic decomposition of this study may not be due to the presence of CTC. Similar to CTC, the SDZ was also completely removed, but within 3 d of the composting process indicating the biodegradation of SDZ was easier than that of CTC (Fig. 3b). When pig manure containing SDZ was applied to the soil, only a transient perturbation in the microbial dynamics was reported previously (Heuer et al., 2008) and within a day almost 90% of the SDZ was non-extractable in another soil application experiment (Hammesfahr et al., 2008). Major transformation products of SDZ are the non-bioactive conjugate acetyl-SDZ and the metabolite hydroxyl-SDZ that exhibits strongly reduced antibiotic potential. Further, SDZ reduced the rates of nitrification but increased the rate of ammonification in a soil-manure system (Hammesfahr et al., 2011). Therefore, SDZ,
similar to CTC, is very unlikely the reason for the initial inhibition on the composting process. Fig. 3c gives the biodegradation of CIP during the composting process, with also an initial sharp decrease in the first 21 d but in contrast to CTC and SDZ, CIP was not completely degraded within the 56 d of composting, indicating fluoroquinolones can be persistent during composting. About 0.31 mg/kg and 1.71 mg/kg of CIP, representing 31% and 17.1% of the spiked concentrations, were persistent in the composting masses of L-level and for H-level treatments, respectively. To our knowledge, the persistence of CIP during composting is being reported for the first time. The residual CIP in the compost may cause potential ecological and health risks by release of antibiotics to environment through application of the composting product to soil. As mentioned above, both CTC and SDZ did not cause any serious inhibition in the composting process, which leads us to suspect that the CIP could be the potential antibiotics that might inhibit the composting process initially. This study also reveals that the effectiveness of composting on the removal of antibiotics is likely to be antibiotics-specific. However, the possible reason is largely unknown since there is an extreme lack of information related to the mechanisms responsible for the removal of antibiotics during composting, which warrants further study. 4. Conclusions The present study revealed that composting could completely degrade the chlortetracycline and sulfadiazine spiked in swine manure; whereas, 17–31% of the ciprofloxacin could be persistent even after the major composting processes. Further, antibiotics at high concentrations, especially, the CIP, inhibited the decomposition of organic matter initially; however, the difference among the treatment disappeared during the later stages. In contrast, antibiotics affected the nitrogen transformation, especially during the initial stages composting that influenced the nitrogen content of the composts. Therefore, the persistence of CIP in the swine manure compost implies their potential impact on the soil ecosystem upon their application which should not be overlooked. Acknowledgements The authors thank the Research Grant Council of the Hong Kong Special Administrative Region, People’s Republic of China for the financial support for this project (Grant HKBU261808). References Arikan, O.A., Sikora, L.J., Mulbry, W.W., Khan, S.U., Foster, G.D., 2007. Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves. Bioresour. Technol. 98, 169–176. Arikan, O.A., Mulbry, W., Ingram, D., Millner, P., 2009a. Minimally managed composting of beef manure at the pilot scale: effect of manure pile construction on pile temperature profiles and on the fate of oxytetracycline and chlortetracycline. Bioresour. Technol. 100, 4447–4453. Arikan, O.A., Mulbry, W., Rice, C., 2009b. Management of antibiotic residues from agricultural sources: use of composting to reduce chlortetracycline residues in beef manure from treated animals. J. Hazard. Mater. 164, 483–489. Díaz, L., Savage, G., Eggerth, L., Golueke, C., 1993. Composting and Recycling Municipal Solid Waste. Lewis publishers, California, USA. Ding, C., He, J., 2010. Effect of antibiotics in the environment on microbial populations. Appl. Microbiol. Biotechnol. 87, 925–941. Grote, M., Vockel, A., Schwarze, D., Mehlich, A., Freitag, M., 2004. Fate of antibiotics in food chain and environment originating from pig fattening (part 1). Fres. Environ. Bull. 13, 1216–1224. Gutiérrez, I.R., Watanabe, N., Harter, T., Glaser, B., Radke, M., 2010. Effect of sulfonamide antibiotics on microbial diversity and activity in a Californian Mollic Haploxeralf. J. Soils Sediments 10, 537–544. Haller, M.Y., Müller, S.R., McArdell, C.S., Alder, A.C., Suter, M.J.F., 2002. Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatography–mass spectrometry. J. Chromatogr. A 952, 111–120.
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