Degradation of veterinary antibiotics during anaerobic digestion of dairy manure

Large amounts of antibiotics are used routinely in veterinary therapy for the prevention and control of disease. It is estimated that approximately 75% of antibiotics are not absorbed by animals and are excreted in waste (Chee-Sanford et al. 2009). Zhao et al. (2010) reported that 27.6 mg/kg chlortetracycline (CTC) could be detected in cow dung. Antibiotic residues in livestock manure may cause environmental contamination. In agricultural drainage, tetracycline antibiotics were detected at microgram-per-litter levels (Matsui et al. 2008). The presence of antibiotics leads to concerns with the spread of antibiotic-resistant micro-organisms in soil and water. Some elimination processes of antibiotic substances are known such as biodegradation, sorption, hydrolysis, photolysis and oxidation. In surface water, sunlight plays an important role in eliminating the antibiotics. However, photo-decomposition may not occur when antibiotics are present in turbid water, soil, sewage (Kümmerer, 2009), and some antibiotics are not light sensitive. Halling-Sørensen estimated the half-lives for CTC was from 25 to 34 days. Gilbertson et al. (1990) reported that the half-lives of ceftiofur sodium, a cephalosporin, were above 22.2 days. Since even small amounts of antibiotics is associated with the selection of resistance in bacteria (Kümmerer, 2009), a fast and certain degradation process of antibiotics which are contained in animal manure is required.

Anaerobic digestion is an established technology for the treatment of animal manure and produces biogas contains methane. Land application of treated sludge or manure as a supplement to fertilizer is common in many countries. If they contain antibiotic residues, antibiotics can be released into the environment. It is required to reveal degradation property of veterinary antibiotics during anaerobic digestion of animal manure. Several researchers have shown the effect of antibiotics on anaerobic digestion of manure. Although most research has focused on antibiotic inhibition of biogas production, a few studies have reported antibiotic degradation rates during anaerobic digestion. We evaluated the potential for degradation of CTC and cefazolin (CEZ) during anaerobic digestion. CTC and CEZ are common veterinary antibiotics and active against broad bacteria. They have different modes of action on prokaryotic systems. CTC is a tetracycline antibiotic and widely used for veterinary therapy. CEZ, cephalosporin, safest antimicrobial agent (Karageorgou & Samanidou 2010) and mainly used for cow infections. The research findings for the treatment of CEZ are limited except for that reported by Beneragama et al. (2013), despite the fact that cephalosporin antibiotics are one of the highest consumption antibiotics.

The objective of this work was to study the degradation property and the effect on biogas production of CTC and CEZ added to dairy manure during anaerobic digestion to reduce the amount and impact of antibiotics which are released into the environment.

CTC hydrochloride and acetonitrile (HPLC grade) were obtained from Sigma (St. Louis, MO, USA). Methanol (HPLC grade) and other reagents were purchased from Wako (Osaka, Japan).

Dairy manure was collected from a livestock farm located in Kansai, Japan. The anaerobic digested slurry used as inoculums was collected from a full-scale anaerobic digested plant in Hokkaido.

The batch experiments of anaerobic digestion were performed in a reactor at 37 ̊C for 20 days. Each reactor was connected to a gas holder and biogas was collected by water replacement. The reactor volume was 500 mL, and 300 mL of the mixture of dairy manure and inoculum (6:4) was added. The reactor and gas holder was filled with nitrogen gas. Prior to the experiment, the manure sample were filtered to remove large solids. Antibiotic concentration was 10 or 50 mg/L in all experiments. The antibiotic concentrations were chosen in consideration of the actual concentration in manure. CTC concentrations in animal manure samples have been detected in the range from μg/L to several tens mg/L level (Álvarez et al. 2010; Zhao et al. 2010). The report of occurrence of CEZ could not be found since β-lactams has not covered frequently (Kümmerer 2009).

The manure is collected from the dairy cow which was not mediated for at least 6 months before sampling. CTC and CEZ were not detected in the original manure (detection limit of HPLC is 0.1 mg/L).

The anaerobic digested slurry samples were collected every few days, and analysed CTC and CEZ concentration by HPLC. Methane concentration was measured using a gas chromatograph equipped with a thermal conductivity detector.

The control solution which contains only distilled water and 10 mg/L antibiotic were also performed to evaluate the stability of antibiotics in anaerobic warm water.

According to Blackwell et al. (2004), sample was extracted by ultrasonication and centrifugation. The supernatant was applied to an SPE cartridge (SAX and Oasis HLB). An aliquot was injected into the HPLC equipped with a UV detector (Shimadzu, Japan).

The separation and determination of CTC were carried out using a C18 column (L-column, 150 × 4.6 mm I.D., particle size 5 μm, Chemicals Evaluation and Research Institute). The mobile phase was a mixture of tetrahydrofuran, acetonitrile and 0.05% trifluoroacetic acid. The detection wavelength was set at 365 nm (Blackwell et al. 2004).

According to Cagnardi et al. (2010), chromatographic separations of CEZ were carried out using a Waters Nova-Pack Phenyl column (150 × 3.9 mm I.D., particle size 4 μm, Waters). The separations were performed at 20°C. The mobile phase was a mixture of 0.005 M octane sulfonic acid solution (pH2.52) acetonitrile, and methanol. The detection wavelength was set at 270 nm.

Degradation profiles for CTC were shown in Figure 1. The CTC levels in the manure were removed 86.3% and 82.8% at initial concentrations of 10.0 mg/L and 50.0 mg/L, respectively. The degradation properties were similar between 10.0 and 50.0 mg/L. In first 8 days, CTC showed a minor reduction to residue of 5.17 mg/L (48.3%) in control solution compared with that of 2.28 mg/L (77.2% removal) in manure. In control solution, it took as twice as in manure to reach half CTC concentration. CTC was degraded during anaerobic digestion process. It is supposed that biological, chemical, and the thermal components of anaerobic digestion process contribute to degradation of the antibiotics (Mitchell et al. 2013). However, the CTC concentration showed small reduction from eighth day to 20th day. The reason for this behavior may be strong adsorption property of CTC. Tetracycline antibiotics have high adsorption coefficient and strongly adsorb to manure particles such as metal ion, proteins and organic matter (Loke et al. 2002).

Degradation profiles for CEZ were shown in Figure 2. The CEZ levels in the manure decreased from 10.0 to 0.089 mg/L in 6 days (77.2% removal). The degradation rate constant of CEZ in manure (0.887 day⁻¹) was much higher than that of CEZ in control solution (0.109 day⁻¹). Gilbertson et al. (1990) reported that hydrolysis rate of ceftiofur sodium, a cephalosporin antibiotic, extremely increased when solution pH was increased from 5 to 7. The hydrolysis of CEZ may be promoted in manure since pH of manure is around 7.5. It is possible that the reason for initial rapidly removal of CEZ was due to increased pH. The removal rate of CEZ is larger than that of CTC. It is well known that β-lactam ring is unstable (Gurkan et al. 2012; Kümmerer, 2009). Antibiotics have several modes of action on prokaryotic systems. The mode of CEZ is blocking cell wall synthesis, while that of CTC is inhibiting protein biosynthesis. Methanogens are archaea, and then lack murein. The antibiotics directed against murein biosynthesis, such as b-lactam antibiotics, have no growth inhibiting effect against archaea (Kandler & König 1998). Then, biodegradation of CEZ may be more active.

The inherent strain of β-lactam ring is commonly thought to be the essential factor for the high antibiotic activity of this compound (Gurkan et al. 2012). Gilbertson et al. (1990) reported that fecal materials which contain micro-organisms could degrade ceftiofur to nonmicrobiologically active materials through fission of the β-lactam ring and hydrolysis. The by-products of CEZ degradation may not be an environmental concern.

It was found that the anaerobic digestion was effective for CTC and CEZ elimination. The fast degradation process of antibiotic residues in dairy manure at the initial stage may contribute to prevent the growth promotion of antibiotic-resistant micro-organisms.

Cumulative biogas productions were shown in Figure 3. Methane gas rate in biogas was 56.9–62.1% in all reactors. The reactors which contain 10 or 50 mg/L CTC indicate similar property regardless of the antibiotic concentration. The data suggested that CTC did not effect on the anaerobic digestion of dairy manure seriously. Gartiser et al. (2007) reported that EC₅₀ values of CTC were between 25.4 and 102 mg/L in ISO test schemes. Therefore, the biogas inhibition could not be serious with 10 mg/L or 50 mg/L CTC in this study. Stone et al. (2011) reported that 20 and 40 mg/L CTC treatments resulted in either slight or no differences, while the 80 mg/L CTC treatment inhibited reactor performance.

In comparison with CTC reactors, the reactor containing CEZ produced less biogas and lag phase for biogas and methane gas was observed. However, total biogas volume reached the same amount with a CTC reactor after 20 days. The reason for this behavior may be acclimatization of anaerobic bacteria. In addition, it may relate to the decrease of CEZ concentration. CEZ was degraded to 0.089 mg/L in 6 days. Beneragama et al. (2013) reported that 60 and 90 mg/L CEZ did not significantly affect methane production but a lag phase of approximately 1 day was observed during anaerobic digestion at 55 °C. The methane production property in this study was similar to their results though digestion temperature and CEZ concentration were different.

The reactor containing 5 mg/L CTC and CEZ produced the least biogas in the four reactors. The lag phase was also observed. CTC and CEZ may mutually effect the biogas production.

The data shows that less than 50 mg/L CTC do not impact biogas production while 10 mg/L CEZ showed short lag phase during anaerobic digestion of dairy manure. Other concentration and antibiotics which represent difference modes of action should be also tested to attain safe and sustainable use of antibiotic.

The degradation property of CEZ and CTC during anaerobic digestion at 37 °C was tested. More than 83% removal of CTC and 99% removal of CEZ were achieved in 20 days. Compared to CTC, CEZ had an inhibitory effect on the biogas production. The results indicate that anaerobic digestion is an effective method for elimination of CTC and CEZ residue in manure. Anaerobic digestion may become solution for reducing the amount of antibiotics released into the environment.

This work was partially supported by JSPS KAKENHI 26292131.