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Removal of antibiotics during the anaerobic digestion of pig manure
Science of the Total Environment 603–604 (2017) 219–225
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Removal of antibiotics during the anaerobic digestion of pig manure Lu Feng a,1, Mònica Escolà Casas b,1, Lars Ditlev Mørck Ottosen a, Henrik Bjarne Møller a, Kai Bester b,⁎ a b
Department of Engineering, Aarhus University, Blichers Allé 20, DK 8830 Tjele, Denmark Department of Environmental Science, Aarhus University, Frederiksborgvej 399, DK 4000 Roskilde, Denmark
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Digestion is able to remove some antibiotics while others are persistent. • Sulfadiazine and sulfamethizole are persistent during manure digestion. • Sulfamethoxazole, erythromycin and trimethoprim are degraded rapidly. • Some transformation products are characterized.
a r t i c l e
i n f o
Article history: Received 20 March 2017 Received in revised form 31 May 2017 Accepted 31 May 2017 Available online xxxx Editor: Simon Pollard Keywords: Macrolide Sulfonamide Erythromycin Thermophilic Psychrophilic
a b s t r a c t Antibiotics are frequently used in animals to treat sickness and prevent infection especially in industrial meat production. Some of the antibiotics cannot be completely metabolized and, as an unavoidable result, are excreted and thus end up in manure which is then spread in the environment. Currently increasing amounts of manure is used in biogas production before spreading the residuals on agricultural fields. In this study, the removal patterns of sulfonamides (sulfadiazine, sulfamethizole, sulfamethoxazole) and macrolides (clarithromycin, erythromycin), as well as trimethoprim, were investigated during the anaerobic digestion of pig manure. Batch kinetic tests were conducted both at thermophilic and psychrophilic condition for 40 days. Some of the antibiotics (clarithromycin, sulfadiazine, sulfamethizole) were persistent in all experiments. Thus, no biodegradation was found for sulfadiazine and sulfamethizole in this study. From the studied compounds, only erythromycin was clearly removed and probably degraded during anaerobic digestion with 99% and 20% removal under thermophilic and psychrophilic condition. The removal of erythromycin was fitted to a single first-order kinetic reaction function, giving reaction rate constant of 0.29 day−1 and 0.005 day−1, respectively. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics are frequently used in humans and animals to treat and prevent infections (Kümmerer, 2008). The conventional livestock ⁎ Corresponding author. E-mail address: [email protected] (K. Bester). 1 Joint first author.
http://dx.doi.org/10.1016/j.scitotenv.2017.05.280 0048-9697/© 2017 Elsevier B.V. All rights reserved.
husbandry has been changed into intensive livestock production due to population growth and economic progress (Hjorth et al., 2010; Udo et al., 2011). The raise of intensive livestock production is inevitably linked with higher demands on animal health thus more antibiotics are used to treat and prevent infections (Kemper, 2008; Widyasari-Mehta et al., 2016). Additionally, another major use of antibiotics is to enhance growth and feed efficiency in animals (Levy, 2013). However this practice is legally outphased in the EU in 2005
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2.2. Instruments
(European Parliament and Council, 2003)), while in the United States, the Food and Drug Administration (FDA) has initiated a voluntary withdrawal of antibiotics for growth promotion by the pharmaceutical companies (Van Boeckel et al., 2015), which have been argued by U.S. food animal and pharmaceutical industries that such restriction have been detrimental to their food animal production. Canada, China, Australia, Brazil and Ukraine do not have any formal national restrictions on antimicrobial use for the purposes of growth promotion (Maron et al., 2013). Depending on the compound used, 50% to 90% of the antibiotics will be absorbed quickly but are excreted via urine and feces after several days (Spielmeyer et al., 2017) resulting in high concentrations in manure (Hamscher et al., 2005) and thus high exposure to antibiotics via dust (Hamscher et al., 2003). Additionally, high concentrations of antibiotics in other compartments of the environment, such as soil and water have been described (Kemper, 2008) as well as their fate in soil (Thiele-Bruhn, 2003) or in treatment systems, such as a biogas plants (Widyasari-Mehta et al., 2016; Wolters et al., 2016). In Denmark, for instance, there are approximately 80 agricultural biogas plants digesting 2.5 million tons of manure (6–7% of all manure produced) (Luostarinen, 2013). Insam et al. (2015) reported advantages of utilizing digestates from fermenters as organic fertilizer. However, the application of fertilizer containing antibiotics has a risk of altering soil microbial constitution and function, and increased occurrence and abundance of antibiotic resistant genes in various soil bacteria (Knapp et al., 2009). Sengeløv et al. (2003) observed higher occurrence of tetracycline resistance after spreading of pig manure containing 48–698 μg/L of tetracycline. Zhu et al. (2013) also found that the genes potentially conferring resistance to sulfonamide, florfenicol and quaternary ammonium compounds were also found to be enriched broadly in farm samples. The occurrence of antibiotic resistant genes are not only diverse but also offers a higher statistical probability of transfer to the environment (Zhu et al., 2013). Therefore, it is essential to determine reaction rates and residual concentrations of antibiotics in anaerobic digestion to assess the risk connected to the use of this material as fertilizer. In the present study, batch kinetic tests were conducted to determine the bio-degradability of antibiotics at anaerobic digestion of pig manure as a model scenario. Both, thermophilic and psychrophilic (as manure storage) anaerobic digestion were investigated. As a mechanistic and not a merely monitoring study was intended, we focusing on several groups of antibiotics with little isomers to gain clear data: Six commonly used antibiotics (clarithromycin, erythromycin, sulfadiazine, sulfamethizole, sulfamethoxazole, and trimethoprim) were spiked into pig manure which was then digested while sampling during the incubation for quantification.
10 mL of manure were centrifuged for 15 min with 6000 G (6000 rpm) for the quantification of antibiotics during the anaerobic digestion. Subsequently 100 μL of the aqueous phase were taken and placed into an HPLC vial, together with 1000 μL of distilled water. 20 μL of labelled internal standard of erythromycin 13C D3, sulfadiazine 13 C6 and trimethoprim D3 were added to reach a concentration of 1 ng/mL in the samples. 10 μL of each prepared sample were injected into the HPLC-MS/MS in MRM mode. For the identification of transformation products, separate incubations for the individual compounds were conducted: 5–10 mL of manure (containing 1 mg of single antibiotics) were centrifuged for 20 min with 6000 G (6000 rpm). Then, 1 mL of the aqueous phase was transferred to an HPLC vial. First, 10 μL were injected into the HPLC-MS/MS in full-scan mode. After identifying possible transformation products, 5 μL were injected in the HPLC-MS/MS in product ion scan and MRM mode in order to verify and quantify them.
2. Materials and methods
2.4. Incubations
2.1. Substrate and chemicals
Three batch assays were conducted to 1) determine the influence of antibiotics on biogas production from pig manure; 2) investigate the reaction kinetics; and to 3) identify the transformation product. Table 1 shows the concentration (μg/L) of antibiotics in the original unspiked pig manure used for the experiments. It can easily be seen that the conventional operation from which the manure originated, used not only one but several antibiotics. However, since our spike level was at mg/L and not at μg/L as the background, the quantitation and kinetic results should be uninfluenced by the background values. However, the microbial communities will to some extent be adapted to these antibiotics.
40 L of fresh pig manure was collected from a pig farm (Bjørnkærvej 1, Øster Velling, Denmark) on October 2015. The operations at this pig farm mostly produce 7–30 kg pigs. Total solids (TS) and volatile solids (VS) for the manure were 3.14 ± 0.04% and 2.16 ± 0.04%, respectively. It had a pH of 7.05 ± 0.1. After sampling, the coarse particles contained in pig manure were screened out by a 2 mm mesh (Spielmeyer et al., 2015). Half of the pig manure was pre-incubated at 52 °C for 2 weeks serving as inoculum in both biogas yield test and biodegradation kinetic test. The rest was stored at −18 °C prior to using. Clarithromycin, erythromycin, sulfamethoxazole and trimethoprim were obtained from Sigma-Aldrich while sulfadiazine and sulfamethizole were obtained from Dr. Ehrenstorfer, (Wesel, Germany). HPLCgrade methanol and formic acid were obtained from Merck (Darmstadt, Germany) and HPLC-MS-grade water was obtained from Sigma-Aldrich (Brøndby, Denmark).
Gas chromatography (Agilent technologies 7890A, Santa Clara, CA 95051, USA) equipped with a thermal conductivity detector (TCD), an Alltech® CTR 1 double column (Grace, MD, USA), and helium as the carrier gas was used to determine the biogas composition. The temperature of oven, injector port, and detector was 120, 150, and 150 °C, respectively. Biodegradation experiments were conducted by using an incubation unit (Bioreactor simulator, Bioprocess Control, Lund, Sweden) consisting of 2 L reactors with rubber plug, mechanical agitation (100 rpm) and a temperature control system. HPLC-MS/MS was used to quantify the antibiotics in the manure. The HPLC was equipped with dual low-pressure mixing ternary-gradient system Ultimate 3000 (Dionex, Germering, Germany). The system had a pump of the 3000 series (DGP-3600 M), a 3000 TSL autosampler (WPS 3000 TSL) and a column oven and degasser also from the Dionex 3000 series. The HPLC operated with two ten-port Valco valves. The mass spectrometer was an API 4000 (ABSciex, Framingham, MA, USA). The API 4000 was operated with an ESI source in positive mode which was set at 400 °C with a capillary voltage of 5500 V. The method used methanol and water, both containing 0.2% of formic acid, as mobile phases. The column used was a Synergy 4 μm Polar-RP (150 × 2 mm) (Phenomenex, Torrance, CA, USA). Further details of the HPLC-MS/MS conditions are described in (Escolà-Casas et al., 2015). The details of the mass spectrometrical operation including the conditions for the multi reaction monitoring (MRMs) are shown in the supplementary material Table S1. The pH value was measured using portable pH meter (Portamess 911, Knick, Germany). 2.3. Sampling and sample preparation
2.4.1. Determining the influence of antibiotics on biogas production Anaerobic incubations were performed to determine the influence of antibiotic on biogas production (bio-methane yield). The experiments were carried out following the procedures suggested by Moset et al. (2015). Incubation bottles (1 L) containing a mixture of pig manure and pre-incubated inoculum at a mixing ratio of approximately 1:1 based on volume, were dosed with all targeted antibiotics to reach
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Table 1 Concentrations of antibiotics in fresh unspiked pig manure. Antibiotics
Sulfadiazine (SDZ)
Sulfamethizole (SMZ)
Sulfamethoxazole (SMX)
Clarithromycin (CLA)
Erythromycin (Eryc)
Trimethoprim (TMP)
3.1
3.2
1.0
b0.0016
0.2
0.2
Concentration [μg/L]
a concentration of 1 mg/Lmanure. As a control, manure and inoculum were incubated without adding antibiotics. A blank control with only inoculum was also included. All bottles were tightly sealed with rubber stoppers and screw caps and then purged with nitrogen gas for two minutes to create anaerobic conditions. All bottles were incubated at 52 °C for 90 d in triplicates. Biogas yield and composition was periodically measured at day 4, 7, 10, 20, 30, 60 and 90. Biomethane production from each sample was calculated by subtracting the volume of methane produced from the blank control. The specific methane yield was adjusted to standard conditions (0 °C and 1.013 bar). 2.4.2. Antibiotics kinetic degradation experiment Anaerobic incubations were performed to measure the biodegradation kinetics of the antibiotics, which were conducted in triplicates at 52 °C. The bottle contained a mixture of pig manure and inoculum (pre-incubated pig manure) at a mixing ratio of approximately 1:1 based on volume, and all aimed antibiotics at concentration of 1 mg/Lmanure. The working volume for each unit was 1.5 L. The headspace was filled with nitrogen gas for 15 min prior to securing the rubber plug and septum cap as well as after sampling. During the batch assay, 10 mL samples were taken from each anaerobic unit at predefined time from 24 h to 40 days. Additional bottles were prepared in parallel with the same mixture of pig manure, inoculum and aimed antibiotics at the same concentration but incubated at 15 °C (psychrophilic condition) without agitation. The samples obtained from these sets were used to mimic the bio-degradation potential during storage process. All bottles were re-flushed with nitrogen gas after sampling. 2.4.3. Characterization of transformation products Antibiotics for which significant bio-degradation was determined in batch assay 2 (clarithromycin, erythromycin, sulfamethoxazole and trimethoprim) were used in a second spiked batch test to identify the transformation products during anaerobic digestion. The batch assay was conducted in triplicates using 500 mL glass bottles fitted with rubber plug and septum cap and controlled at the same condition with batch assay 2. The bottles contained mixture of pig manure and inoculum at a ratio of approximately 1:1 based on volume and single antibiotics at a concentration of 1 mg/Lmanure spiked into the incubations. During the batch assay, 10 mL samples were taken from each replica at predefined time from 1 h to 20 days. All samples were preserved in a freezer at −18 °C and transported under cool condition when the experiment was finished. All bottles were re-flushed with nitrogen gas for 2 min after sampling.
The removal (%) of antibiotics was calculated by comparing concentrations according to Eq. (3): Removal ð%Þ ¼
Concentration ðt Þ 100 1− Concentration0
ð3Þ
where Concentration (t) represents the concentration of the respective antibiotic at time t and Concentration0 is the initial concentration. Considering experimental uncertainties, degradation was considered significant when the numerical value exceeded 20% (Escolà-Casas et al., 2015). JMP 13.0 (SAS Insititute Inc., Cary, NC, USA) was used for graphing and modelling. 3. Results and discussion 3.1. Influence of antibiotics on bio-methane production As shown in Fig. 1, the final bio-methane yield from pig manure and pig manure with antibiotics were determined to be 443.67 ± 11.01 and 452 ± 1.82 L/kgVS. The bio-methane yield of pig manure observed in this study was similar to those estimated by the IPCC as ultimate biomethane yield in developed countries (Eggleston et al., 2006). There is no significant difference on bio-methane yield from pig manure with/ without antibiotics at the given concentrations. The result indicates that there is no significant toxic or inhibition effect with mixture of antibiotics at concentration of 1 mg/Lmanure on the biogas producing communities. 3.2. Removal of antibiotics The removal of six antibiotics during anaerobic digestion is shown in Table.2. In Fig. 2 the concentrations of antibiotics in the spiked incubations are shown over time. All incubations start at the spiked level (considering an uncertainty of 10%) thus demonstrating the capability of the used methods to determine the concentrations correctly. Different
2.5. Calculations and statistics The concentrations of all the compounds during anaerobic digestion of pig manure were plotted for each unit and compound. A first-order reaction equation was fitted with no weighting Eq. (1): C ¼ ðC 0 Þ e−kt
ð1Þ
where C0 and C represents the initial and concentration at a given point in time, respectively; k is the removal rate constant, and t stands for time. The half-life of antibiotics was calculated based on Eq. (2)
t1 ¼ 2
ln 2 k
ð2Þ
Fig. 1. Bio-methane yield from pig manure with (1 mg/L) and without antibiotics.
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Table 2 Removal of antibiotics during anaerobic digestion of pig manure (40 days). Removal [%]
Current research Thermophilic Psychrophilic Literature (Mohring et al., 2009; Spielmeyer et al., 2015)
Sulfadiazine (SDZ)
Sulfamethizole (SMZ)
Sulfamethoxazole (SMX)
Clarithromycin (CLA)
Erythromycin (Eryc)
Trimethoprim (TMP)
ns ns 33%–99.9%
ns 26 na
98.5 ± 0.5 99.88 98.7–99.9%
36 ± 12 33 na
99.0 ± 0.2 20 40%
99.97 ± 0.05 99.94 Rapidly removed
ns: not significant; na – not available.
removal patterns were observed as some antibiotics were removed rapidly, while others reacted slowly or were persistent. Concerning thermophilic and psychrophilic anaerobic digestion processes, temperature generally influenced the reaction rate constants. However, there was no general trend towards whether higher or lower temperatures resulted in higher reaction rates. Sulfamethoxazole and trimethoprim were quantitatively removed (degraded) in one day under both conditions. 3.2.1. Fast removing compounds: sulfamethoxazole (SMX) and trimethoprim (TMP) During the kinetic experiment, a rapid decrease leading to removal close to 100% was determined from sulfamethoxazole and trimethoprim (Table 2). This might be connected to the fact that the operation from which the manure was taken is using some of the compounds as demonstrated by the concentrations found in unspiked manure (Table 1). Thus the microbial community in the manure is adapted to some of the antibiotics in question. The concentration of sulfamethoxazole and trimethoprim from first sample was found to be 1.21 ± 0.20 and 0.16 ± 0.03 μg/L, corresponding to close to 100% removal. The results from this study agree to the findings of Mohring et al. (2009) who also detected a rapid removal of sulfamethoxazole and
Fig. 2. Measured concentrations of antibiotics during batch test 2 (anaerobic thermophilic and psychrophilic digestion). The lines are just a smooth connection between dots to guide the eye. (Erythromycin (Eryc), Clarithromycin (CLA), Sulfadiazine (SDZ), Trimethoprim (TMP), Sulfamethoxazole (SMX), Sulfamethizole (SMZ)).
trimethoprim in swine manure at 37 °C. The longer degradation time in that study maybe due to the lower digestion temperature compared to our study. Haller et al. (2002) were only able to detect trimethoprim in very selected samples and discussed selective sorption and rapid degradation. The fate of sulfamethoxazole and trimethoprim could vary in terms of the treatment system. No kinetic fit was applied to sulfamethoxazole and trimethoprim due to the high speed of the degradation which generated a lack of data between initial concentrations and the 100% degradation. No transformation products could be observed for sulfamethoxazole or trimethoprim in our study. 3.2.2. Sulfamethizole (SMZ) and sulfadiazine (SDZ) No-removal of sulfadiazine and sulfamethizole was found from the kinetic experiments. Mathematically a non-significant production of sulfadiazine and sulfamethizole is detected, which is probably artefact and easily covered by the stated uncertainty. In detail: an insignificantly increase from the initial measured concentration of 822 μg/L to 938 ± 124 μg/L at day 1 was observed, followed by a period of 40 days of no further change in concentration (Fig. 2). Similar observations were determined for sulfadiazine: A non-significant productions followed by a 40 d period of non-changed concentrations (Fig. 2). García Galán et al. (2012) reported that sulfamethizole was the most recalcitrant sulfonamide among nine investigated sulfonamide antibiotics when using conventional activated sludge and membrane bioreactor to determine the removal of sulfonamide antibiotics. This is an indication that sulfamethizole is recalcitrant at both aerobic and anaerobic condition. Oppositely, Mohring et al. (2009) found similar results for sulfamethizole while these authors reported rapid degradation of sulfadiazine. Mohring et al. (2009) detected the hydroxy metabolite of sulfadiazine. This difference in sulfadiazine degradation may be attributed to the different microorganisms in the two systems which might be connected to different incubation temperatures or different substrates. In our study, all microorganisms were from (anaerobically pre-incubated) pig manure and no inoculum from digesters under long-term operations were used. 3.2.3. Macrolides: clarithromycin (CLA) and erythromycin (Eryc) The removal of the macrolides (clarithromycin and erythromycin) were determined to be 36% and 99% in the kinetic experiment. As shown in Fig. 2, clarithromycin is slowly degraded from initial concentration of 557 μg/L to 356 ± 65 μg/L, with a total degradation rate of around 36% during 40 days. There is no information found about degradation of clarithromycin during anaerobic digestion of animal manure. However, the removal rate of clarithromycin was in agreement with that found in wastewater treatment. Significant adsorption and low biodegradability of clarithromycin to sewage sludge had been reported by Abegglen et al. (2009) using single-house MBR treatment. EstradaArriaga et al. (2016) also found that clarithromycin was one of the most persistent compounds from effluent of different WWTPs, with degradation rates between 0 and 50%. Higher degradation rate could be expected with longer solids retention time in the activated sludge reactor of the wastewater treatment plant. Results reported in the
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to that, there were differences considering erythromycin, which showed an exponential degradation curve at thermophilic conditions (Fig. 3), while a slower degradation rate was determined at psychrophilic conditions. The (first order) removal rate constant of erythromycin at thermophilic and psychrophilic condition was 0.287 day−1 (R2 = 0.9834) and 0.005 day− 1 (R2 = 0.8995), respectively. This is corresponding to a half-life of 3 and 142 days. 3.4. Fate of antibiotic compounds during anaerobic degradation of pig manure (transformation)
Fig. 3. Concentrations of erythromycin under thermophilic and psychrophilic conditions fitted with first-order reaction rates.
literature show that the degradation of clarithromycin increased to 87– 90% with a solid retention time of 60–80 days compared to the lower solid retention times (Göbel et al., 2007). No transformation products from clarithromycin could be identified in our study. Erythromycin was the only antibiotic that showed an exponential degradation curve, with a half-life of 3 days. A quantitative removal of 89.9% was determined at day 8 and 99.9% at day 40. The degradation pattern of erythromycin was similar as reported by Schlüsener et al. (2006) which has a typical first-order degradation curve during storage of pig manure in manure tanks. In contrast, the poor removal of erythromycin has been reported for advanced membrane bioreactor (MBR) and conventional activated sludge (CAS) (Göbel et al., 2007; Radjenović et al., 2009). 3.3. Influence of temperature The degradation of sulfonamides (sulfadiazine, sulfamethizole, sulfamethoxazole), clarithromycin, and trimethoprim antibiotics followed similar patterns at thermophilic and psychrophilic condition. Opposite
In the kinetic biodegradation experiment, some antibiotics degraded in anaerobic digestion of pig manure. Thus in the last batch assay, selected antibiotics were incubated in individual assays for eventual identification of transformation products. For these experiments, clarithromycin, erythromycin, sulfamethoxazole and trimethoprim were used as they were degraded in the initial biodegradation experiment. The identification of transformation products of the tested compounds was made by full-scan analysis using Q1 scans on the HPLCMS. Only for the samples of the degradation of erythromycin transformation products could be identified. For the other antibiotics, no peaks could be identified as being originated by potential transformation products in these experiments. There are two possible reasons for that: 1) there was not enough amount of parent compound degraded (e.g. clarithromycin) or 2) the degradation of the parent compound was fast and probably giving small transformation products due to the relatively small molecular size of the parent compounds (e.g. trimethoprim and sulfamethoxazole) or 3) the degradation of the transformation products was occurring with a similar speed as their formation. The metabolite of erythromycin was detected with mass 716 Da (Fig.4). This mass would correspond to a structure of erythromycin minus a water molecule, and the metabolite was denominated erythromycin-H2O. To further elucidate the identity of the metabolite, Q3 fragmentation assessment was performed. This process proved that the loss of water was in the lactone ring (Fig.5). In the literature, erythromycin-H2O has been inconsistently linked to the structures of anhydroerythromycin (CAS 23893-13-2) (McArdell et al., 2003; Yang et al., 2006) and erythromycin enol ether (CAS 33396-29-1) (McArdell et al., 2003; Nakada et al., 2007). However, none of these structures was the one matching with the erythromycinH2O identified in our manure samples. On the one hand, the Q3
Fig. 4. HPLC Chromatogram with electrospray (+) ionization and MS detection in Q1 fullscan mode of the erythromycin- H2O metabolite at RT 23.20 min giving the mass signal of 716.8 Da. As a comparison the peak of Erythromycin (m/z) [M + 1] = 734.8 Da is superimposed as TIC.
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Fig. 5. Comparison erythromycin-H2O product ion spectra (ESI+). a) of the suspect compound in the manure samples and b) of the standard Anhydroerythromycin A obtained from Sigma-Aldrich.
fragmentation pattern of the erythromycin-H2O in our samples did not coincide with the one of anhydroerythromycin standard. On the other hand, erythromycin enol ether had a somehow similar Q3 fragmentation pattern as the erythromycin-H2O in the samples, but the retention times did not coincide. Alternatively, other literature suggests pseudoerythromycin enol ether (also 716 Da) as a possible metabolite (Kim et al., 2004). Also, other structures implying a loss of water in the lactone ring may be possible. Furthermore, other transformation products of erythromycin with different masses than 716 Da have been described (Llorca et al., 2015). Thus the real structure of the erythromycin-H2O found in this study remains unresolved. Some loss of water from erythromycin has been reported by Hirsch et al., 1999 and Roth and Fenner, 1994 as simple hydrolysis of erythromycin at low pH. As the used manure contained relative high concentrations of sulfadiazine, sulfamethizole, sulfamethoxazole, and traces of erythromycin and trimethoprim, but no clarithromycine, the selectivity cannot be linked to the known preconditioning. 4. Conclusion In this study, sulfamethoxazole, erythromycin and trimethoprim were removed rapidly during anaerobic digestion of pig manure, concerning these compounds digestion provides a functional barrier against dissipation into the environment. For sulfadiazine, sulfamethizole, and clarithromycine this barrier was less pronounced (or nonexistent). No adverse effects of the presence of antibiotic compounds on biogas/methane yield during digestion was observed during the batch tests. Erythromycin is hydrolysed during digestion to form an unknown derivative of erythromycine-H2O. Acknowledgement The authors wish to thank the post-doc, Radziah Wahid at Biogas Plant, Foulum, Aarhus University for assistance in this work. This work was financially supported by Innovation fund Denmark (1377-
00040A, ‘Nomigas’ project) and DCE project “identification of metabolites and metabolic pathways in removing organic micro-pollutants from wastewater and waste”. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.05.280.
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L. Feng et al. / Science of the Total Environment 603–604 (2017) 219–225 Hirsch, R., Ternes, T., Haberer, K., Kratz, K.L., 1999. Occurrence of antibiotics in the aquatic environment. Sci. Total Environ. 225, 109–118. Hjorth, M., Christensen, K.V., Christensen, M.L., Sommer, S.G., 2010. Solid—liquid separation of animal slurry in theory and practice. A review. Agron. Sustain. Dev. 30, 153–180. Insam, H., Gómez-Brandón, M., Ascher, J., 2015. Manure-based biogas fermentation residues – friend or foe of soil fertility? Soil Biol. Biochem. 84, 1–14. Kemper, N., 2008. Veterinary antibiotics in the aquatic and terrestrial environment. Ecol. Indic. 8, 1–13. Kim, Y.-H., Heinze, T.M., Beger, R., Pothuluri, J.V., Cerniglia, C.E., 2004. A kinetic study on the degradation of erythromycin A in aqueous solution. J Pharm.]–>Int. J. Pharm. 271, 63–76. Knapp, C.W., Dolfing, J., Ehlert, P.A., Graham, D.W., 2009. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environ. Sci. Technol. 44, 580–587. Kümmerer, K., 2008. Pharmaceuticals in the Environment: Sources, Fate, Effects and Risks. Springer Verlag Berlin Heidelberg, Germany. Levy, S.B., 2013. The Antibiotic Paradox: How Miracle Drugs are Destroying the Miracle. Springer. Llorca, M., Rodríguez-Mozaz, S., Couillerot, O., Panigoni, K., de Gunzburg, J., Bayer, S., Czajac, R., Barceló, D., 2015. Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap. Chemosphere 119, 90–98. Luostarinen, S., 2013. Energy potential of manure in the Baltic Sea region: biogas potential & incentives and barriers for implementation. Baltic Forum for Innovative Technologies for Sustainable Manure Management. Saatavissa:. http://www.balticmanure.eu/ download/Reports/bm_energy_potentials_web.pdf (2013). Maron, D.F., Smith, T.J., Nachman, K.E., 2013. Restrictions on antimicrobial use in food animal production: an international regulatory and economic survey. Glob. Health 9, 48. McArdell, C.S., Molnar, E., Suter, M.J.F., Giger, W., 2003. Occurrence and fate of macrolide antibiotics in wastewater treatment plants and in the Glatt Valley watershed, Switzerland. Environ. Sci. Technol. 37, 5479–5486. Mohring, S.A.I., Strzysch, I., Fernandes, M.R., Kiffmeyer, T.K., Tuerk, J., Hamscher, G., 2009. Degradation and elimination of various sulfonamides during anaerobic fermentation: a promising step on the way to sustainable pharmacy? Environ. Sci. Technol. 43, 2569–2574. Moset, V., Poulsen, M., Wahid, R., Højberg, O., Møller, H.B., 2015. Mesophilic versus thermophilic anaerobic digestion of cattle manure: methane productivity and microbial ecology. Microb. Biotechnol. 8, 787–800. Nakada, N., Shinohara, H., Murata, A., Kiri, K., Managaki, S., Sato, N., Takada, H., 2007. Removal of selected pharmaceuticals and personal care products (PPCPs) and
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endocrine-disrupting chemicals (EDCs) during sand filtration and ozonation at a municipal sewage treatment plant. Water Res. 41, 4373–4382. Radjenović, J., Petrović, M., Barceló, D., 2009. Fate and distribution of pharmaceuticals in wastewater and sewage sludge of the conventional activated sludge (CAS) and advanced membrane bioreactor (MBR) treatment. Water Res. 43, 831–841. Roth, H.J., Fenner, H., 1994. Struktur-Bioreaktivität-Wirkungsbezogene Eigenschaften. 2nd ed. Georg Thieme Verlag, Stuttgart, pp. 65–68. Schlüsener, M.P., von Arb, M.A., Bester, K., 2006. Elimination of macrolides, tiamulin, and salinomycin during manure storage. Arch. Environ. Contam. Toxicol. 51, 21–28. Sengeløv, G., Agersø, Y., Halling-Sørensen, B., Baloda, S.B., Andersen, J.S., Jensen, L.B., 2003. Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Environ. Int. 28, 587–595. Spielmeyer, A., Breier, B., Groissmeier, K., Hamscher, G., 2015. Elimination patterns of worldwide used sulfonamides and tetracyclines during anaerobic fermentation. Bioresour. Technol. 193, 307–314. Spielmeyer, A., Stahl, F., Petri, M.S., Zerr, W., Brunn, H., Hamscher, G., 2017. Transformation of sulfonamides and tetracyclines during anaerobic fermentation of liquid manure. J. Environ. Qual. 46, 160–168. Thiele-Bruhn, S., 2003. Pharmaceutical antibiotic compounds in soils–a review. J. Plant Nutr. Soil Sci. 166, 145–167. Udo, H.M.J., Aklilu, H.A., Phong, L.T., Bosma, R.H., Budisatria, I.G.S., Patil, B.R., Samdup, T., Bebe, B.O., 2011. Impact of intensification of different types of livestock production in smallholder crop-livestock systems. Livest. Sci. 139, 22–29. Van Boeckel, T.P., Brower, C., Gilbert, M., Grenfell, B.T., Levin, S.A., Robinson, T.P., Teillant, A., Laxminarayan, R., 2015. Global trends in antimicrobial use in food animals. Proc. Natl. Acad. Sci. U. S. A. 112, 5649–5654. Widyasari-Mehta, A., Hartung, S., Kreuzig, R., 2016. From the application of antibiotics to antibiotic residues in liquid manures and digestates: a screening study in one European center of conventional pig husbandry. J. Environ. Manag. 177, 129–137. Wolters, B., Ding, G.-C., Kreuzig, R., Smalla, K., 2016. Full-scale mesophilic biogas plants using manure as C-source: bacterial community shifts along the process cause changes in the abundance of resistance genes and mobile genetic elements. FEMS Microbiol. Ecol. 92, 163. Yang, S.W., Cha, J.M., Carlson, K., 2006. Trace analysis and occurrence of anhydroerythromycin and tylosin in influent and effluent wastewater by liquid chromatography combined with electrospray tandem mass spectrometry. Anal. Bioanal. Chem. 385, 623–636. Zhu, Y.G., Johnson, T.A., Su, J.Q., Qiao, M., Guo, G.X., Stedtfeld, R.D., Hashshamet, S.A., Tiedje, J.M., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. U. S. A. 110, 3435–3440 (2013).
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Removal of antibiotics during the anaerobic digestion of pig manure Lu Feng a,1, Mònica Escolà Casas b,1, Lars Ditlev Mørck Ottosen a, Henrik Bjarne Møller a, Kai Bester b,⁎ a b
Department of Engineering, Aarhus University, Blichers Allé 20, DK 8830 Tjele, Denmark Department of Environmental Science, Aarhus University, Frederiksborgvej 399, DK 4000 Roskilde, Denmark
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Digestion is able to remove some antibiotics while others are persistent. • Sulfadiazine and sulfamethizole are persistent during manure digestion. • Sulfamethoxazole, erythromycin and trimethoprim are degraded rapidly. • Some transformation products are characterized.
a r t i c l e
i n f o
Article history: Received 20 March 2017 Received in revised form 31 May 2017 Accepted 31 May 2017 Available online xxxx Editor: Simon Pollard Keywords: Macrolide Sulfonamide Erythromycin Thermophilic Psychrophilic
a b s t r a c t Antibiotics are frequently used in animals to treat sickness and prevent infection especially in industrial meat production. Some of the antibiotics cannot be completely metabolized and, as an unavoidable result, are excreted and thus end up in manure which is then spread in the environment. Currently increasing amounts of manure is used in biogas production before spreading the residuals on agricultural fields. In this study, the removal patterns of sulfonamides (sulfadiazine, sulfamethizole, sulfamethoxazole) and macrolides (clarithromycin, erythromycin), as well as trimethoprim, were investigated during the anaerobic digestion of pig manure. Batch kinetic tests were conducted both at thermophilic and psychrophilic condition for 40 days. Some of the antibiotics (clarithromycin, sulfadiazine, sulfamethizole) were persistent in all experiments. Thus, no biodegradation was found for sulfadiazine and sulfamethizole in this study. From the studied compounds, only erythromycin was clearly removed and probably degraded during anaerobic digestion with 99% and 20% removal under thermophilic and psychrophilic condition. The removal of erythromycin was fitted to a single first-order kinetic reaction function, giving reaction rate constant of 0.29 day−1 and 0.005 day−1, respectively. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics are frequently used in humans and animals to treat and prevent infections (Kümmerer, 2008). The conventional livestock ⁎ Corresponding author. E-mail address: [email protected] (K. Bester). 1 Joint first author.
http://dx.doi.org/10.1016/j.scitotenv.2017.05.280 0048-9697/© 2017 Elsevier B.V. All rights reserved.
husbandry has been changed into intensive livestock production due to population growth and economic progress (Hjorth et al., 2010; Udo et al., 2011). The raise of intensive livestock production is inevitably linked with higher demands on animal health thus more antibiotics are used to treat and prevent infections (Kemper, 2008; Widyasari-Mehta et al., 2016). Additionally, another major use of antibiotics is to enhance growth and feed efficiency in animals (Levy, 2013). However this practice is legally outphased in the EU in 2005
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2.2. Instruments
(European Parliament and Council, 2003)), while in the United States, the Food and Drug Administration (FDA) has initiated a voluntary withdrawal of antibiotics for growth promotion by the pharmaceutical companies (Van Boeckel et al., 2015), which have been argued by U.S. food animal and pharmaceutical industries that such restriction have been detrimental to their food animal production. Canada, China, Australia, Brazil and Ukraine do not have any formal national restrictions on antimicrobial use for the purposes of growth promotion (Maron et al., 2013). Depending on the compound used, 50% to 90% of the antibiotics will be absorbed quickly but are excreted via urine and feces after several days (Spielmeyer et al., 2017) resulting in high concentrations in manure (Hamscher et al., 2005) and thus high exposure to antibiotics via dust (Hamscher et al., 2003). Additionally, high concentrations of antibiotics in other compartments of the environment, such as soil and water have been described (Kemper, 2008) as well as their fate in soil (Thiele-Bruhn, 2003) or in treatment systems, such as a biogas plants (Widyasari-Mehta et al., 2016; Wolters et al., 2016). In Denmark, for instance, there are approximately 80 agricultural biogas plants digesting 2.5 million tons of manure (6–7% of all manure produced) (Luostarinen, 2013). Insam et al. (2015) reported advantages of utilizing digestates from fermenters as organic fertilizer. However, the application of fertilizer containing antibiotics has a risk of altering soil microbial constitution and function, and increased occurrence and abundance of antibiotic resistant genes in various soil bacteria (Knapp et al., 2009). Sengeløv et al. (2003) observed higher occurrence of tetracycline resistance after spreading of pig manure containing 48–698 μg/L of tetracycline. Zhu et al. (2013) also found that the genes potentially conferring resistance to sulfonamide, florfenicol and quaternary ammonium compounds were also found to be enriched broadly in farm samples. The occurrence of antibiotic resistant genes are not only diverse but also offers a higher statistical probability of transfer to the environment (Zhu et al., 2013). Therefore, it is essential to determine reaction rates and residual concentrations of antibiotics in anaerobic digestion to assess the risk connected to the use of this material as fertilizer. In the present study, batch kinetic tests were conducted to determine the bio-degradability of antibiotics at anaerobic digestion of pig manure as a model scenario. Both, thermophilic and psychrophilic (as manure storage) anaerobic digestion were investigated. As a mechanistic and not a merely monitoring study was intended, we focusing on several groups of antibiotics with little isomers to gain clear data: Six commonly used antibiotics (clarithromycin, erythromycin, sulfadiazine, sulfamethizole, sulfamethoxazole, and trimethoprim) were spiked into pig manure which was then digested while sampling during the incubation for quantification.
10 mL of manure were centrifuged for 15 min with 6000 G (6000 rpm) for the quantification of antibiotics during the anaerobic digestion. Subsequently 100 μL of the aqueous phase were taken and placed into an HPLC vial, together with 1000 μL of distilled water. 20 μL of labelled internal standard of erythromycin 13C D3, sulfadiazine 13 C6 and trimethoprim D3 were added to reach a concentration of 1 ng/mL in the samples. 10 μL of each prepared sample were injected into the HPLC-MS/MS in MRM mode. For the identification of transformation products, separate incubations for the individual compounds were conducted: 5–10 mL of manure (containing 1 mg of single antibiotics) were centrifuged for 20 min with 6000 G (6000 rpm). Then, 1 mL of the aqueous phase was transferred to an HPLC vial. First, 10 μL were injected into the HPLC-MS/MS in full-scan mode. After identifying possible transformation products, 5 μL were injected in the HPLC-MS/MS in product ion scan and MRM mode in order to verify and quantify them.
2. Materials and methods
2.4. Incubations
2.1. Substrate and chemicals
Three batch assays were conducted to 1) determine the influence of antibiotics on biogas production from pig manure; 2) investigate the reaction kinetics; and to 3) identify the transformation product. Table 1 shows the concentration (μg/L) of antibiotics in the original unspiked pig manure used for the experiments. It can easily be seen that the conventional operation from which the manure originated, used not only one but several antibiotics. However, since our spike level was at mg/L and not at μg/L as the background, the quantitation and kinetic results should be uninfluenced by the background values. However, the microbial communities will to some extent be adapted to these antibiotics.
40 L of fresh pig manure was collected from a pig farm (Bjørnkærvej 1, Øster Velling, Denmark) on October 2015. The operations at this pig farm mostly produce 7–30 kg pigs. Total solids (TS) and volatile solids (VS) for the manure were 3.14 ± 0.04% and 2.16 ± 0.04%, respectively. It had a pH of 7.05 ± 0.1. After sampling, the coarse particles contained in pig manure were screened out by a 2 mm mesh (Spielmeyer et al., 2015). Half of the pig manure was pre-incubated at 52 °C for 2 weeks serving as inoculum in both biogas yield test and biodegradation kinetic test. The rest was stored at −18 °C prior to using. Clarithromycin, erythromycin, sulfamethoxazole and trimethoprim were obtained from Sigma-Aldrich while sulfadiazine and sulfamethizole were obtained from Dr. Ehrenstorfer, (Wesel, Germany). HPLCgrade methanol and formic acid were obtained from Merck (Darmstadt, Germany) and HPLC-MS-grade water was obtained from Sigma-Aldrich (Brøndby, Denmark).
Gas chromatography (Agilent technologies 7890A, Santa Clara, CA 95051, USA) equipped with a thermal conductivity detector (TCD), an Alltech® CTR 1 double column (Grace, MD, USA), and helium as the carrier gas was used to determine the biogas composition. The temperature of oven, injector port, and detector was 120, 150, and 150 °C, respectively. Biodegradation experiments were conducted by using an incubation unit (Bioreactor simulator, Bioprocess Control, Lund, Sweden) consisting of 2 L reactors with rubber plug, mechanical agitation (100 rpm) and a temperature control system. HPLC-MS/MS was used to quantify the antibiotics in the manure. The HPLC was equipped with dual low-pressure mixing ternary-gradient system Ultimate 3000 (Dionex, Germering, Germany). The system had a pump of the 3000 series (DGP-3600 M), a 3000 TSL autosampler (WPS 3000 TSL) and a column oven and degasser also from the Dionex 3000 series. The HPLC operated with two ten-port Valco valves. The mass spectrometer was an API 4000 (ABSciex, Framingham, MA, USA). The API 4000 was operated with an ESI source in positive mode which was set at 400 °C with a capillary voltage of 5500 V. The method used methanol and water, both containing 0.2% of formic acid, as mobile phases. The column used was a Synergy 4 μm Polar-RP (150 × 2 mm) (Phenomenex, Torrance, CA, USA). Further details of the HPLC-MS/MS conditions are described in (Escolà-Casas et al., 2015). The details of the mass spectrometrical operation including the conditions for the multi reaction monitoring (MRMs) are shown in the supplementary material Table S1. The pH value was measured using portable pH meter (Portamess 911, Knick, Germany). 2.3. Sampling and sample preparation
2.4.1. Determining the influence of antibiotics on biogas production Anaerobic incubations were performed to determine the influence of antibiotic on biogas production (bio-methane yield). The experiments were carried out following the procedures suggested by Moset et al. (2015). Incubation bottles (1 L) containing a mixture of pig manure and pre-incubated inoculum at a mixing ratio of approximately 1:1 based on volume, were dosed with all targeted antibiotics to reach
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Table 1 Concentrations of antibiotics in fresh unspiked pig manure. Antibiotics
Sulfadiazine (SDZ)
Sulfamethizole (SMZ)
Sulfamethoxazole (SMX)
Clarithromycin (CLA)
Erythromycin (Eryc)
Trimethoprim (TMP)
3.1
3.2
1.0
b0.0016
0.2
0.2
Concentration [μg/L]
a concentration of 1 mg/Lmanure. As a control, manure and inoculum were incubated without adding antibiotics. A blank control with only inoculum was also included. All bottles were tightly sealed with rubber stoppers and screw caps and then purged with nitrogen gas for two minutes to create anaerobic conditions. All bottles were incubated at 52 °C for 90 d in triplicates. Biogas yield and composition was periodically measured at day 4, 7, 10, 20, 30, 60 and 90. Biomethane production from each sample was calculated by subtracting the volume of methane produced from the blank control. The specific methane yield was adjusted to standard conditions (0 °C and 1.013 bar). 2.4.2. Antibiotics kinetic degradation experiment Anaerobic incubations were performed to measure the biodegradation kinetics of the antibiotics, which were conducted in triplicates at 52 °C. The bottle contained a mixture of pig manure and inoculum (pre-incubated pig manure) at a mixing ratio of approximately 1:1 based on volume, and all aimed antibiotics at concentration of 1 mg/Lmanure. The working volume for each unit was 1.5 L. The headspace was filled with nitrogen gas for 15 min prior to securing the rubber plug and septum cap as well as after sampling. During the batch assay, 10 mL samples were taken from each anaerobic unit at predefined time from 24 h to 40 days. Additional bottles were prepared in parallel with the same mixture of pig manure, inoculum and aimed antibiotics at the same concentration but incubated at 15 °C (psychrophilic condition) without agitation. The samples obtained from these sets were used to mimic the bio-degradation potential during storage process. All bottles were re-flushed with nitrogen gas after sampling. 2.4.3. Characterization of transformation products Antibiotics for which significant bio-degradation was determined in batch assay 2 (clarithromycin, erythromycin, sulfamethoxazole and trimethoprim) were used in a second spiked batch test to identify the transformation products during anaerobic digestion. The batch assay was conducted in triplicates using 500 mL glass bottles fitted with rubber plug and septum cap and controlled at the same condition with batch assay 2. The bottles contained mixture of pig manure and inoculum at a ratio of approximately 1:1 based on volume and single antibiotics at a concentration of 1 mg/Lmanure spiked into the incubations. During the batch assay, 10 mL samples were taken from each replica at predefined time from 1 h to 20 days. All samples were preserved in a freezer at −18 °C and transported under cool condition when the experiment was finished. All bottles were re-flushed with nitrogen gas for 2 min after sampling.
The removal (%) of antibiotics was calculated by comparing concentrations according to Eq. (3): Removal ð%Þ ¼
Concentration ðt Þ 100 1− Concentration0
ð3Þ
where Concentration (t) represents the concentration of the respective antibiotic at time t and Concentration0 is the initial concentration. Considering experimental uncertainties, degradation was considered significant when the numerical value exceeded 20% (Escolà-Casas et al., 2015). JMP 13.0 (SAS Insititute Inc., Cary, NC, USA) was used for graphing and modelling. 3. Results and discussion 3.1. Influence of antibiotics on bio-methane production As shown in Fig. 1, the final bio-methane yield from pig manure and pig manure with antibiotics were determined to be 443.67 ± 11.01 and 452 ± 1.82 L/kgVS. The bio-methane yield of pig manure observed in this study was similar to those estimated by the IPCC as ultimate biomethane yield in developed countries (Eggleston et al., 2006). There is no significant difference on bio-methane yield from pig manure with/ without antibiotics at the given concentrations. The result indicates that there is no significant toxic or inhibition effect with mixture of antibiotics at concentration of 1 mg/Lmanure on the biogas producing communities. 3.2. Removal of antibiotics The removal of six antibiotics during anaerobic digestion is shown in Table.2. In Fig. 2 the concentrations of antibiotics in the spiked incubations are shown over time. All incubations start at the spiked level (considering an uncertainty of 10%) thus demonstrating the capability of the used methods to determine the concentrations correctly. Different
2.5. Calculations and statistics The concentrations of all the compounds during anaerobic digestion of pig manure were plotted for each unit and compound. A first-order reaction equation was fitted with no weighting Eq. (1): C ¼ ðC 0 Þ e−kt
ð1Þ
where C0 and C represents the initial and concentration at a given point in time, respectively; k is the removal rate constant, and t stands for time. The half-life of antibiotics was calculated based on Eq. (2)
t1 ¼ 2
ln 2 k
ð2Þ
Fig. 1. Bio-methane yield from pig manure with (1 mg/L) and without antibiotics.
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Table 2 Removal of antibiotics during anaerobic digestion of pig manure (40 days). Removal [%]
Current research Thermophilic Psychrophilic Literature (Mohring et al., 2009; Spielmeyer et al., 2015)
Sulfadiazine (SDZ)
Sulfamethizole (SMZ)
Sulfamethoxazole (SMX)
Clarithromycin (CLA)
Erythromycin (Eryc)
Trimethoprim (TMP)
ns ns 33%–99.9%
ns 26 na
98.5 ± 0.5 99.88 98.7–99.9%
36 ± 12 33 na
99.0 ± 0.2 20 40%
99.97 ± 0.05 99.94 Rapidly removed
ns: not significant; na – not available.
removal patterns were observed as some antibiotics were removed rapidly, while others reacted slowly or were persistent. Concerning thermophilic and psychrophilic anaerobic digestion processes, temperature generally influenced the reaction rate constants. However, there was no general trend towards whether higher or lower temperatures resulted in higher reaction rates. Sulfamethoxazole and trimethoprim were quantitatively removed (degraded) in one day under both conditions. 3.2.1. Fast removing compounds: sulfamethoxazole (SMX) and trimethoprim (TMP) During the kinetic experiment, a rapid decrease leading to removal close to 100% was determined from sulfamethoxazole and trimethoprim (Table 2). This might be connected to the fact that the operation from which the manure was taken is using some of the compounds as demonstrated by the concentrations found in unspiked manure (Table 1). Thus the microbial community in the manure is adapted to some of the antibiotics in question. The concentration of sulfamethoxazole and trimethoprim from first sample was found to be 1.21 ± 0.20 and 0.16 ± 0.03 μg/L, corresponding to close to 100% removal. The results from this study agree to the findings of Mohring et al. (2009) who also detected a rapid removal of sulfamethoxazole and
Fig. 2. Measured concentrations of antibiotics during batch test 2 (anaerobic thermophilic and psychrophilic digestion). The lines are just a smooth connection between dots to guide the eye. (Erythromycin (Eryc), Clarithromycin (CLA), Sulfadiazine (SDZ), Trimethoprim (TMP), Sulfamethoxazole (SMX), Sulfamethizole (SMZ)).
trimethoprim in swine manure at 37 °C. The longer degradation time in that study maybe due to the lower digestion temperature compared to our study. Haller et al. (2002) were only able to detect trimethoprim in very selected samples and discussed selective sorption and rapid degradation. The fate of sulfamethoxazole and trimethoprim could vary in terms of the treatment system. No kinetic fit was applied to sulfamethoxazole and trimethoprim due to the high speed of the degradation which generated a lack of data between initial concentrations and the 100% degradation. No transformation products could be observed for sulfamethoxazole or trimethoprim in our study. 3.2.2. Sulfamethizole (SMZ) and sulfadiazine (SDZ) No-removal of sulfadiazine and sulfamethizole was found from the kinetic experiments. Mathematically a non-significant production of sulfadiazine and sulfamethizole is detected, which is probably artefact and easily covered by the stated uncertainty. In detail: an insignificantly increase from the initial measured concentration of 822 μg/L to 938 ± 124 μg/L at day 1 was observed, followed by a period of 40 days of no further change in concentration (Fig. 2). Similar observations were determined for sulfadiazine: A non-significant productions followed by a 40 d period of non-changed concentrations (Fig. 2). García Galán et al. (2012) reported that sulfamethizole was the most recalcitrant sulfonamide among nine investigated sulfonamide antibiotics when using conventional activated sludge and membrane bioreactor to determine the removal of sulfonamide antibiotics. This is an indication that sulfamethizole is recalcitrant at both aerobic and anaerobic condition. Oppositely, Mohring et al. (2009) found similar results for sulfamethizole while these authors reported rapid degradation of sulfadiazine. Mohring et al. (2009) detected the hydroxy metabolite of sulfadiazine. This difference in sulfadiazine degradation may be attributed to the different microorganisms in the two systems which might be connected to different incubation temperatures or different substrates. In our study, all microorganisms were from (anaerobically pre-incubated) pig manure and no inoculum from digesters under long-term operations were used. 3.2.3. Macrolides: clarithromycin (CLA) and erythromycin (Eryc) The removal of the macrolides (clarithromycin and erythromycin) were determined to be 36% and 99% in the kinetic experiment. As shown in Fig. 2, clarithromycin is slowly degraded from initial concentration of 557 μg/L to 356 ± 65 μg/L, with a total degradation rate of around 36% during 40 days. There is no information found about degradation of clarithromycin during anaerobic digestion of animal manure. However, the removal rate of clarithromycin was in agreement with that found in wastewater treatment. Significant adsorption and low biodegradability of clarithromycin to sewage sludge had been reported by Abegglen et al. (2009) using single-house MBR treatment. EstradaArriaga et al. (2016) also found that clarithromycin was one of the most persistent compounds from effluent of different WWTPs, with degradation rates between 0 and 50%. Higher degradation rate could be expected with longer solids retention time in the activated sludge reactor of the wastewater treatment plant. Results reported in the
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to that, there were differences considering erythromycin, which showed an exponential degradation curve at thermophilic conditions (Fig. 3), while a slower degradation rate was determined at psychrophilic conditions. The (first order) removal rate constant of erythromycin at thermophilic and psychrophilic condition was 0.287 day−1 (R2 = 0.9834) and 0.005 day− 1 (R2 = 0.8995), respectively. This is corresponding to a half-life of 3 and 142 days. 3.4. Fate of antibiotic compounds during anaerobic degradation of pig manure (transformation)
Fig. 3. Concentrations of erythromycin under thermophilic and psychrophilic conditions fitted with first-order reaction rates.
literature show that the degradation of clarithromycin increased to 87– 90% with a solid retention time of 60–80 days compared to the lower solid retention times (Göbel et al., 2007). No transformation products from clarithromycin could be identified in our study. Erythromycin was the only antibiotic that showed an exponential degradation curve, with a half-life of 3 days. A quantitative removal of 89.9% was determined at day 8 and 99.9% at day 40. The degradation pattern of erythromycin was similar as reported by Schlüsener et al. (2006) which has a typical first-order degradation curve during storage of pig manure in manure tanks. In contrast, the poor removal of erythromycin has been reported for advanced membrane bioreactor (MBR) and conventional activated sludge (CAS) (Göbel et al., 2007; Radjenović et al., 2009). 3.3. Influence of temperature The degradation of sulfonamides (sulfadiazine, sulfamethizole, sulfamethoxazole), clarithromycin, and trimethoprim antibiotics followed similar patterns at thermophilic and psychrophilic condition. Opposite
In the kinetic biodegradation experiment, some antibiotics degraded in anaerobic digestion of pig manure. Thus in the last batch assay, selected antibiotics were incubated in individual assays for eventual identification of transformation products. For these experiments, clarithromycin, erythromycin, sulfamethoxazole and trimethoprim were used as they were degraded in the initial biodegradation experiment. The identification of transformation products of the tested compounds was made by full-scan analysis using Q1 scans on the HPLCMS. Only for the samples of the degradation of erythromycin transformation products could be identified. For the other antibiotics, no peaks could be identified as being originated by potential transformation products in these experiments. There are two possible reasons for that: 1) there was not enough amount of parent compound degraded (e.g. clarithromycin) or 2) the degradation of the parent compound was fast and probably giving small transformation products due to the relatively small molecular size of the parent compounds (e.g. trimethoprim and sulfamethoxazole) or 3) the degradation of the transformation products was occurring with a similar speed as their formation. The metabolite of erythromycin was detected with mass 716 Da (Fig.4). This mass would correspond to a structure of erythromycin minus a water molecule, and the metabolite was denominated erythromycin-H2O. To further elucidate the identity of the metabolite, Q3 fragmentation assessment was performed. This process proved that the loss of water was in the lactone ring (Fig.5). In the literature, erythromycin-H2O has been inconsistently linked to the structures of anhydroerythromycin (CAS 23893-13-2) (McArdell et al., 2003; Yang et al., 2006) and erythromycin enol ether (CAS 33396-29-1) (McArdell et al., 2003; Nakada et al., 2007). However, none of these structures was the one matching with the erythromycinH2O identified in our manure samples. On the one hand, the Q3
Fig. 4. HPLC Chromatogram with electrospray (+) ionization and MS detection in Q1 fullscan mode of the erythromycin- H2O metabolite at RT 23.20 min giving the mass signal of 716.8 Da. As a comparison the peak of Erythromycin (m/z) [M + 1] = 734.8 Da is superimposed as TIC.
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Fig. 5. Comparison erythromycin-H2O product ion spectra (ESI+). a) of the suspect compound in the manure samples and b) of the standard Anhydroerythromycin A obtained from Sigma-Aldrich.
fragmentation pattern of the erythromycin-H2O in our samples did not coincide with the one of anhydroerythromycin standard. On the other hand, erythromycin enol ether had a somehow similar Q3 fragmentation pattern as the erythromycin-H2O in the samples, but the retention times did not coincide. Alternatively, other literature suggests pseudoerythromycin enol ether (also 716 Da) as a possible metabolite (Kim et al., 2004). Also, other structures implying a loss of water in the lactone ring may be possible. Furthermore, other transformation products of erythromycin with different masses than 716 Da have been described (Llorca et al., 2015). Thus the real structure of the erythromycin-H2O found in this study remains unresolved. Some loss of water from erythromycin has been reported by Hirsch et al., 1999 and Roth and Fenner, 1994 as simple hydrolysis of erythromycin at low pH. As the used manure contained relative high concentrations of sulfadiazine, sulfamethizole, sulfamethoxazole, and traces of erythromycin and trimethoprim, but no clarithromycine, the selectivity cannot be linked to the known preconditioning. 4. Conclusion In this study, sulfamethoxazole, erythromycin and trimethoprim were removed rapidly during anaerobic digestion of pig manure, concerning these compounds digestion provides a functional barrier against dissipation into the environment. For sulfadiazine, sulfamethizole, and clarithromycine this barrier was less pronounced (or nonexistent). No adverse effects of the presence of antibiotic compounds on biogas/methane yield during digestion was observed during the batch tests. Erythromycin is hydrolysed during digestion to form an unknown derivative of erythromycine-H2O. Acknowledgement The authors wish to thank the post-doc, Radziah Wahid at Biogas Plant, Foulum, Aarhus University for assistance in this work. This work was financially supported by Innovation fund Denmark (1377-
00040A, ‘Nomigas’ project) and DCE project “identification of metabolites and metabolic pathways in removing organic micro-pollutants from wastewater and waste”. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.05.280.
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