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Tylosin effect on methanogenesis in an anaerobic biomass from swine wastewater treatment Liliana García-Sánchez, Marco Antonio Garzón-Zúñiga, Gerardo Buelna and Edson Baltazar Estrada-Arriaga
ABSTRACT The effect of different concentrations of tylosin on methane production was investigated: first methanogenesis in a biomass without contact with the antibiotic, and later the ability of the sludge to adapt to increasing concentrations of tylosin. Results showed that, for biomass that had no contact with the antibiotic, the presence of tylosin inhibits the generation of methane even at concentrations as small as 0.01 mg L1, and samples at concentrations above 0.5 mg L1 produced practically no methane, whereas, in the digesters acclimated in the presence of tylosin at a concentration of 0.01 to 0.065 mg L1, methanogenesis is not inhibited in the presence of antibiotic and the generation of methane is improved. This behaviour suggests the microorganisms have developed not only resistance to the antibiotic but also an ability to metabolize it. Key words
| specific methanogenic activity (SMA), swine anaerobic sludge, tylosin
Liliana García-Sánchez Faculty of Engineering, National Autonomous University of Mexico, Campus Morelos, Paseo Cuauhnahuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, Mexico Marco Antonio Garzón-Zúñiga National Polytechnic Institute, CIIDIR IPN Unidad Durango, Sigma 119, Fracc. 20 de Noviembre II, Durango, Durango C.P. 34220, Mexico Gerardo Buelna Industrial Research Center of Quebec, 333 rue Franquet, Quebec, Canada Edson Baltazar Estrada-Arriaga (corresponding author) Mexican Institute of Water Technology, Paseo Cuauhnahuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, Mexico E-mail: [email protected]
INTRODUCTION Nowadays, there is growing interest in the study of emerging contaminants, including antibiotics and other drugs present in wastewaters. There is a large amount of information about fate and transformations of drugs released by humans, but regarding drugs for veterinary use, the information is scarce. Specifically regarding antibiotics, many problems have been detected: (1) bacterial resistance, (2) inappropriate use, (3) they are metabolized partially, (4) they affect the efficiency of wastewater treatment systems, (5) low removal in wastewater treatment systems and (6) improper disposal. The reason that pharmaceuticals such as antibiotics are of interest in wastewater treatment is because they are substances that interfere with the active part of the bacteria, affecting the removal efficiencies of the biological treatments. In addition, they are persistent compounds that are developed to prevent the substance becoming inactive before having a curative effect, tending to bioaccumulate and to cause adverse effects when discharged into aquatic and soil ecosystems. Some antibiotics are not persistent but have slow biodegradation and so doi: 10.2166/wst.2015.507
their constant introduction into the environment can cause a similar inhibitory effect which affects the environment. One of most used veterinary antibiotics worldwide is tylosin. Tylosin is an antibiotic whose main use is as a growthpromoting agent in pig farms in Mexico and the USA (92%). However, over 70% of tylosin administered is excreted from faeces and urine of animals in its original form: reason why the use of this antibiotic becomes an environmental problem. Concentrations of tylosin are detected in different matrixes around the world: soils (2 μg L1) (Zilles et al. ), manure (0.000002–0.0019 μg L1) (Huang et al. ; Zilles et al. ), surface water (0.00029–0.28 μg L1) (Kolpin et al. ; Watkinson et al. ) and wastewater (0.060–2 μg L1) (Zilles et al. ; Watkinson et al. ). García-Sánchez et al. () detected concentrations of tylosin from 20.2 to 32.4 μg L1 in swine wastewater in Mexico. In the European Union, this antibiotic was prohibited, because it generates bacterial resistance in the environment and in animals, which could be transferable to humans. In Mexico, there are no studies of this antibiotic, despite being widely used. In addition,
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existing research on the effect of tylosin on the performance of anaerobic wastewater treatment systems seems contradictory, since the inhibition of methanogenesis has been described from insignificant (Massé et al. ; Chelliapan et al. ) to considerable (Sanz et al. ; Lotfin et al. ). The specific methanogenic activity (SMA) test is one of the most commonly used tests to determine the ability of an anaerobic biomass to degrade complex substrates in methane (McHugh et al. ; Sumino et al. ). The SMA test carried out with known amounts of biomass under defined conditions and substrates can evaluate the methane production of biomass per unit over a long period of time. It is important to carry out the SMA test to determine the behaviour of sludge under the effect of potentially toxic or inhibitory compounds and to determine the threshold value (Souto et al. ). During the degradation of toxic compounds in biological reactors, usually at the beginning, there is a period when there is no transformation of said compounds. This period is called acclimatization, adaptation, or delay. Acclimatization is a selection and multiplication of specialized microorganisms, which after a certain adaptation time, are capable of biodegrading or cometabolizing the compound or only surviving in the presence of the relevant compound. The acclimatization phase is considered vital for the biodegradation of toxic compounds, because after that occurs, the speed at which the compound is metabolized is faster. Many countries, including Mexico, have implemented programmes to reduce emissions of greenhouse gas, having as incentive the purchase of carbon credits. Therefore, anaerobic digesters have been installed to treat integrally residual liquids and solids (herefafter termed ‘dalys’, its acronym in Spanish (digestor anaerobio de líquidos y sólidos)) generated in pig farms with the purpose of producing a greater amount of biogas (Garzón-Zúñiga & Buelna ). However, there are no reports of the efficiency or operation of the process, or of the results obtained when the antibiotics are used on farms. The aim of this study was to evaluate the effect of tylosin on the methanogenesis of anaerobic sludge, the product of dalys digesters operated with different hydraulic residence times (HRT).
MATERIALS AND METHODS Tests with biomass without previous contact with tylosin The methane production was evaluated for an anaerobic biomass coming from a liquid and solid digester (dalys) at
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450 L pilot scale, with 300 d of operation, and operated with HRT of 30 d, used for the treatment of swine wastewater in Morelos, Mexico. Said biomass had no previous contact with tylosin. The biomass was used as a target to evaluate the effect of different concentrations of antibiotic (0.01, 0.05, 0.10, 0.50, 1.0, 10, 30, 50, 100, 400 and 1,000 mg L1) on the generation of methane. This process is known as specific methanogenic activity. SMA test The SMA test was performed according to the procedure described by Souto et al. (). The test consisted of filling serological flasks in an anaerobic environmental chamber (70/30, v/v, N2/CO2) with ratio of 9.03 L: 1 L:1 L:10 mL of carbon source (sodium acetate solution), inoculum, macronutrients and micronutrients respectively, maintaining 10% of the total volume of the flask for the gas phase. The test was performed in triplicate. Subsequently, the flasks were sealed with a rubber stopper and aluminium, and removed from the anaerobic chamber. The flasks were stirred in an orbital shaker at 100 rpm and at temperature of 30 C in a warm room. The determination of the methane production was performed with a volumetric method by liquid displacement, since according to Souto et al. () there is no difference between volumetric, manometric, and chromatographic methods in this test’s results. To determine the real behaviour of the effect of tylosin during experimentation, tylosin tartrate commercial grade was used. Tylosin tartrate is used as a growth promoter in pig farms. W
Adaptation of biomass to tylosin In order to determine if the biomass of the dalys systems can be adapted to increasing concentrations of tylosin and to determine the HRT effect on the removal efficiency of antibiotic, three anaerobic digesters (D1, D2 and D3) were evaluated. The digesters used had an effective volume of 4.5 L and were operated at a constant temperature of 30 C with orbital shaking and inoculated with 10% of anaerobic biomass, without previous contact with tylosin. The digesters were fed with swine wastewater without separation of solids and with a constant concentration of tylosin equal to the first test, in which the concentration showed the lowest inhibition of the methanogenic activity (0.01 mg L1). Digesters were operated with a different HRT (Table 1). The monitoring of the digester operation was carried out taking as a control parameter the removal of chemical oxygen demand (COD). W
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Table 1
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Tylosin concentrations and HRT used during the adaptation of the biomass to the antibiotic
Stage Digester
Tylosin (mg L1) HRT (d)
D1
1
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(VS) according to Standard Methods (APHA ) and ammonia nitrogen (NH4-N) according to the Hach procedure.
2
3
4
0.01 30
0.05 30
0.05 15
0.065 15
RESULTS AND DISCUSSION Methanogenesis inhibition in unacclimated biomass
Tylosin (mg L ) HRT (d)
0.01 40
0.05 40
0.05 20
0.065 20
D3
Tylosin (mg L1) HRT (d)
0.01 60
0.05 60
0.05 25
0.065 25
D4
Tylosin (mg L1) HRT (d)
0 30
0 30
0 15
0 15
Once the digesters showed stable removal efficiencies 60% of the COD, a sample of the biomass was taken. With this biomass, a new test of methanogenic activity was carried out and later the tylosin concentration was increased in the digesters until the biomass adapted to a greater tylosin concentration. This procedure was repeated with tylosin values of 0.05 and 0.065 mg L1, this last one being the maximum concentration found in swine wastewaters in Mexico (GarcíaSánchez et al. ). During the development of this experimental stage, a fourth digester was used as a control (D4), without adding tylosin.
Wastewater characteristics The wastewater used was from a growth-finishing area in a pig farm. The wastewater was collected once a week during the experiment and stored at 4 C to prevent its degradation. The wastewater was brought back to room temperature, previous to their feeding in digesters about 2 h. Characteristics of the swine wastewater are shown in Table 2. W
Analytical methods Samples of both influent and effluent of anaerobic digesters were analyzed for COD, total solids (TS) and volatile solids |
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Table 2
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When an SMA test was carried out to a biomass without initial contact with tylosin, it was found that the addition of this antibiotic inhibits methane production even at concentrations as small as 0.01 mg L1 (Figure 1). After 28 d of monitoring SMA, samples at concentrations above 0.5 mg L1 produce virtually no methane. In contrast to the samples with tylosin, the sludge without addition of antibiotic generated methane at a 100% in a period of 21 d. Taken as 100% methane generated by a sample without addition of tylosin, all concentrations showed a methane inhibition percentage greater than 97%, except for the lower concentrations (0.01, 0.05 and 0.1 mg L1) in which an inhibition of 49 to 87% was obtained. The results agree with the ones reported by Shimada et al. () for concentrations such as 167 mg L1 in a sequencing batch reactor. However, for concentrations lower than 16.7 mg L1 the results are different than the ones reported by Shimada et al. (), Angenent et al. () and Poels et al. (), who reported that there is no inhibition in the presence of the tylosin in anaerobic digestion systems. This difference is due to the fact that the results reported by these authors relate to the generation of methane in anaerobic digesters in operation and not to an SMA test. The only experiment that was evaluated in batch corresponds to Stone et al. (), who found that there is no methanogenesis inhibition in a concentration of 1.1 mg L1 of tylosin. However, the results are reported only for 216 d. Such a long period of experimentation could allow the biomass to adapt to the presence of tylosin because the disappearance of the antibiotic was reported after 216 d, which probably had an impact on adaptation of the biomass to tylosin.
Characteristics of swine wastewater
Total COD 1
Stage
mg L
1
17,958 ± 1,571
Soluble COD 1
mg L
4,763 ± 223
TS
VS 1
1
mg L
mg L
17 ± 1
12 ± 1
NH4-N
1
mg L
656 ± 195
2
16,293 ± 3,359
4,823 ± 453
16 ± 2
11 ± 2
1,322 ± 417
3
20,497 ± 1,459
15,182 ± 1,364
18 ± 2
11 ± 1
1,061 ± 268
4
20,007 ± 2,478
13,334 ± 2,356
17 ± 3
11 ± 2
1,038 ± 216
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Figure 1
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Methane production and inhibition of SMA for unacclimated biomass [TYL]: tylosin concentration.
Regarding the value of SMA calculated for biomass without tylosin, it was 0.34 gCOD gVS1 d1. This value is slightly higher than the one reported by López et al. () for anaerobic digesters (0.02 to 0.2 gCOD gVS1 d1), which suggests that the biomass that is not in contact with the antibiotic carried out its complete metabolic cycle. On the other hand, the biomass that was in contact with the antibiotic concentrations between 0.1 and 1,000 mg L1 had SMA values of between 0.002 and 0.019 gCOD gVS1 d1, values lower than the one mentioned above, suggesting that methanogenic activity of biomass is being inhibited by the presence of the tylosin. For tylosin concentrations of 0.01 mg L1 and 0.05 mg L1, SMA values were 0.155 gCOD gVS1 d1 and 0.068 gCOD gVS1 d1, respectively. These results, in spite of being in an acceptable range, are lower than the ones found for biomass without tylosin. These results coincide with what was mentioned by Chelliapan et al. (): ‘It is impossible to predict the effect of antibiotics in anaerobic treatment systems because each system requires a specific investigation.’ Therefore,
contradictions regarding the inhibitory effect of tylosin on methanogenesis could be attributed to the type of biomass used, tylosin concentration, evaluation conditions (for example TS concentration and type of mixture), and type of water evaluated, among others. Adaptation of biomass to tylosin During the four experimental stages, it was found that the digester without presence of tylosin (D4) showed the lower removal of organic matter, compared to the other three digesters (62 ± 12%, 51 ± 11%, 58 ± 6% and 49 ± 20% in stage 1, 69 ± 10%, 66 ± 14%, 67 ± 12% and 66 ± 12% in stage 2, 57 ± 10%, 62 ± 7%, 64 ± 8% and 48 ± 10% in stage 3 and 72 ± 6%, 75 ± 4%, 71 ± 6% and 61 ± 11% in stage 4 for D1, D2, D3 and D4, respectively). This would probably indicate that swine wastewater contains pollutants not measured, which negatively affected non-acclimatized biomass. And contrary to expectations, when increasing the tylosin concentration at each stage, a better efficiency in the removal of
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organic matter was obtained. This could be explained by a protocooperation or cooperation between the microorganisms in the presence of the antibiotic. Another explanation for this behaviour might be as Ortiz () mentions: the reduced competition for nutrients between microorganisms or the reduction of microbial metabolites or both processes causes the antibiotic to function as a growth promoter in specialized microorganisms such as in the intestinal microbiota of pigs. According to the tylosin degradation pathway, it is decomposed into two fractions: sugar and desmycosin. Desmycosin is a factor B of tylosin, whose properties are similar to factor A (tylosin). Mycarose and mycaminose are neutral saccharide compounds generated in the degradation of desmycosin. These compounds are sugars of short chain which are easily biodegradable by microorganisms. Tylosin metabolites are not found to be toxic. Regarding the results of solids during the four experimental stages, the digester effluents were very stable, confirming that the biomass acclimated to the HRT change and to the presence of the antibiotic. The average removal rates of TS during stage 1 were 69 ± 3%, 67 ± 4%, 66 ± 2%, and 63 ± 8%, during stage 2 were 72 ± 7%, 70 ± 6%, 70 ± 6%, and 67 ± 8%, during stage 3 were 71 ± 6%, 71 ± 4%, 68 ± 8%, and 67 ± 5%, and during stage 4 were 67 ± 3%, 69 ± 6%, 70 ± 5%, and 66 ± 6% for digesters D1, D2, D3 and D4, respectively for each stage. The average percentages of VS removal during stage 1 were 75 ± 5%, 72 ± 5%, 71 ± 3%, and 67 ± 9%, during stage 2 were 78 ± 7%, 79 ± 5%, 76 ± 6%, and 74 ± 8%, during stage 3 were 69 ± 5%, 69 ± 5%, 67 ± 9%, and 65 ± 5%, and during stage 4 were 66 ± 8%, 69 ± 8%, 68 ± 6%, and 58 ± 8% for digesters D1, D2, D3 and D4, respectively, for each stage. It was observed that similarly to the removal of organic matter during the biomass acclimatization to tylosin, the digester without added antibiotic (D4) presented efficiencies slightly lower of solids removal, during the four experimental phases. The concentrations of ammonia nitrogen in the effluents were very similar to those of the influent for all the digesters, except for stage 2, in which the average values of ammonia nitrogen in the influent outweighed the effluent; however, this behaviour is not held for the final two stages. The lower values of ammonia nitrogen were obtained in stage 1 with average values of 740 ± 196 mg L1, 654 ± 102 mg L1, 737 ± 165 mg L1, and 684 ± 46 mg L1 for digesters D1, D2, D3, and D4, respectively. Methanogenesis in acclimated biomass For stages 1 and 2, the test was performed on day 18 and 50 respectively, with biomass from the four experimental
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digesters at a concentration of tylosin corresponding to the experimental stage (Figure 2). It was observed that during the first stage, the digesters with presence of tylosin needed only 2 d to accomplish a full methanogenesis of the added carbon source, while the digester D4 took 10 d to produce the expected 100% methane. These results could indicate that part of the biomass has specialized in using the antibiotic as a carbon source. For stage 2 (0.05 mg L1, HRT ¼ 30, 40, and 60 d), D1 and D3 digesters again generated 100% of the methane within 2 d, whereas the D2 and D4 took about 5 d generating it. For D3, this behaviour is expected since the load of volumetric tylosin is less when operating with greater amount. Stage 3 was carried out under the same concentration of antibiotic (0.05 mg L1 of tylosin) but the HRT was reduced from 30, 40, and 60 d to 15, 20 and 25 d, respectively for D1, D2, and D3. In addition, for stages 3 and 4, it was decided to test the SMA only with the biomass of digester D1, thinking that having the shortest HRT and therefore the bigger load of organic matter and tylosin, it could be expected to have a greater negative impact on the performance of the digester. This was verified with respect to organic matter as it had the lowest percentages of removal of such. However, it was the opposite with the generation of methane. The SMA test was performed with different concentrations of tylosin (and not only with the concentration of tylosin corresponding to the experimental stage as performed in stages 1 and 2) in order to compare the negative effect and the adaptability of the biomass to the antibiotic. The results are shown in Figure 3. It was observed that the biomass of the digester D1 acclimatized to 0.05 mg L1 of tylosin and an HRT of 15 d; not adding tylosin (0 mg L1 of tylosin added for the SMA testing) did produce the expected 100% of methane within 5 d. It was also noted that the generation of methane reached 100% only in those samples in which tylosin was added in a lower or equal concentration than that of a stage of acclimatization in progress (0.05 mg L1), whereas with higher concentrations, the production of methane was inhibited by 33%, 43%, and 78% for the concentration of 0.075 mg L1, 0.01 mg L1, and 0.15 mg L1 of tylosin, respectively. Although some inhibition for some tylosin concentrations was still found, this was less than the one found for the biomass of the pilot digester (not acclimatized to tylosin), in which an inhibition of over 44% for tylosin concentrations equal to or higher than 0.05 mg L1 was found.
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Methane production for biomass acclimated to 0.01 and 0.05 mg tylosin L
In the experimental stage 4, the antibiotic concentration was increased (0.065 mg L1 of tylosin). Under these conditions, the D1 with HRT of 15 d showed a positive displacement of the curves corresponding to methane generation, since the biomass without antibiotic reached 100% of the methane generated in approximately 12 d. While the samples of 0.05 mg L1 of tylosin (the closest concentration to the acclimation) reached 100% of the methane generation in a shorter time of 5 d, samples with higher concentrations (0.075 and 0.10 mg L1) did not reach 100% of methane generation, thus presenting some inhibition but with much lower values (33 and 44% inhibition) than those found in non-acclimated biomass (87% inhibition at a concentration of 0.1 mg L1 of tylosin, Figure 1), which shows a clear tendency of biomass acclimatization at higher concentrations of tylosin. The results indicate that the biomass in stage four is acclimated to concentrations of tylosin similar to those found by Kolz et al. () in swine wastewater treatment systems (0.4 mg L1 of tylosin).
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(HRT ¼ 30, 40 and 60 d).
Additionally, there is a likely relationship beyond the single survival of microorganisms in the presence of tylosin, since the methanogenic activity was slower in the absence of the antibiotic, while in its presence, a higher speed was observed in the methanogenic phase, which could indicate that the biomass has acquired the ability of using the antibiotic as a carbon source.
CONCLUSIONS It was determined that at concentrations of tylosin of 0.01 mg L1, the production of methane is inhibited in anaerobic sludge that has not had contact with the antibiotic by as much as 49%, and concentrations of 1,000 mg L1 inhibit methanogenesis up to 98%. However, it was found that the percentage of inhibition was affected not only by the antibiotic concentration but also by other factors such as the type of biomass used, testing conditions, the type of
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Figure 3
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Methane production for biomass acclimated to 0.05 and 0.065 mg tylosin L
water evaluated, the configuration of the evaluated reactor and the type of mix used. During the acclimatization stage, the biomass decreased the HRT in the anaerobic digesters from 30 to 15 d (D1), from 40 to 20 d (D2) and from 60 to 25 d (D3) with a concentration of tylosin of 0.05 mg L1. It was found that this decrease in HRT did not impact negatively the efficiency of the systems. It was found that the concentration of tylosin does have an effect on removal of organic matter during the acclimation of the biomass to the antibiotic. However, contrary to expectations, it was determined that when the concentration of tylosin was increased at every stage of operation, the efficiency of removal of organic matter increased. The results above indicate that tylosin affects biomass in very small concentrations but a continuous contact of the antibiotic generates acclimatization, and not only that but it acts to improve the generation of methane, which suggests not only antibiotic resistance but also the capacity to
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for anaerobic digester D1.
metabolize it. For this reason, it is important to conduct more studies about this subject matter.
ACKNOWLEDGEMENTS Financial support for the realization of this research was granted from Industrial Research Center of Quebec (CRIQ), Canada, and Mexican Institute of Water Technology.
REFERENCES Angenent, L., Mau, M., George, U., Zahn, J. & Raskin, L. Effect of the presence of the antimicrobial tylosin in swine waste on anaerobic treatment. Water Research 42, 2377–2384. APHA (American Public Health Association) Standards Methods for the Examination of Water and Wastewater. 22nd edn, American Public Health Association/American Water
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Works Association/Water Environment Federation, Washington, DC. Chelliapan, S., Wilby, T. & Sallis, P. Performance of an upflow anaerobic stage reactor (UASR) in the treatment of pharmaceutical wastewater containing macrolide antibiotics. Water Research 40, 507–516. Chelliapan, S., Wilby, T., Sallis, P. J. & Yuzir, A. Tolerance of the antibiotic tylosin on treatment performance of an up-flow anaerobic stage reactor (UASR). Water Science and Technology 63 (8), 1599–1606. García-Sánchez, L., Garzón-Zúñiga, M. A., Buelna, G., MoellerChávez, G. E., Noyola, A., Avilez-Flores, M. & EstradaArriaga, E. B. Occurrence of tylosin in swine wastewater in Mexico. Water Science and Technology 68 (4), 894–900. Garzón-Zúñiga, M. A. & Buelna, G. Caracterización de aguas residuales porcinas y su tratamiento por diferentes procesos en México (Characterization of pig wastewaters and its treatment by different processes in Mexico). Revista Internacional de Contaminación Ambiental 30 (1), 65–79. Huang, C. H., Renew, J. E., Smeby, K. L., Pinkston, K. & Sedlak, D. L. Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis. Journal of Contemporary Water Research and Education 120 (1), 30–40. Kolpin, D., Furlong, E., Meyer, M., Thurman, M., Zaugg, S., Barber, L. & Buxton, H. Pharmaceuticals, hormones and other organic wastewater contaminants in U.S. streams, 1999–2000: a national reconnaissance. Environmental Science and Technology 36 (6), 1202–1211. Kolz, A., Ong, S. & Moorman, T. Sorption of tylosin onto swine manure. Chemosphere 60, 284–289. López, M., Villa, P. & Escobedo, R. Estudio del comportamiento de reactores anaerobios de residuos sólidos a través de ensayos microbiológicos. Revista CENIC. Centro Nacional de Investigaciones Científicas Cuba 35 (3), 179–183. Lotfin, K. A., Henny, C., Adams, C. D., Surampali, R. & Mormile, M. R. Inhibition of microbial metabolism in anaerobic lagoons by selected sulfonamides, tetracyclines, lincomycin and tylosin tartrate. Environmental Toxicology and Chemistry 24 (4), 782–788. Massé, D., Lu, D., Massé, L. & Droste, R. Effect of antibiotics on psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors. Bioresource Technology 75, 205–211.
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McHugh, S., Carton, M., Collins, G. & O’Flaherty, V. Reactor performance and microbial community dynamics during anaerobic biological treatment of wastewaters at 16–37 C. FEMS Microbiology Ecology 48, 369–378. Ortiz, M. R. Bases fisiológicas para el uso de antibióticos promotores de crecimiento y preventivo en enfermedades bacterianas intestinales (Physiological basis for the use of antibiotic growth promoters and preventive intestinal bacterial diseases). http://www.webveterinaria.com/virbac/ news22/cerdos.pdf. Poels, J., Van Assche, P. & Verstraete, W. Effects of disinfectants and antibiotics on the anaerobic digestion of piggery waste. Agricultural Wastes 9, 239–247. Sanz, J. L., Rodríguez, N. & Amils, R. The action of antibiotics on the anaerobic digestion process. Applied Microbiology and Biotechnology 46 (5–6), 587–592. Shimada, T., Zilles, J., Morgenroth, E. & Raskin, L. Inhibitory effects of the macrolide antimicrobial tylosin on anaerobic treatment. Biotechnology and Bioengineering 101 (1), 73–82. Souto, T., Aquino, S., Silva, S. & Chernicharo, C. Influence of incubation conditions on the specific methanogenic activity test. Biodegradation 21, 411–424. Stone, J., Clay, S., Zhu, Z., Wong, K., Porath, L. & Spellman, G. Effect of antimicrobial compounds tylosin and chlortetracycline during batch anaerobic swine manure digestion. Water Research 43 (18), 4740–4750. Sumino, H., Takahashi, M., Yamaguchi, T., Abe, K., Araki, N., Yamazaki, S., Shimozaki, S., Nagano, A. & Nishio, N. Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature. Bioresource Technology 98, 177–182. Watkinson, A., Murby, E., Kolpin, D. & Costanzo, S. The occurrence of antibiotics in an urban watershed: from wastewater to drinking water. Science of the Total Environment 407, 2711–2723. Zilles, J., Shimada, T., Jindal, A., Robert, M. & Raskin, L. Presence of macrolide-lincosamide-streptogramin B and tetracycline antimicrobials in swine waste treatment processes and amended soil. Water Environment Research 77 (1), 57–62.
First received 3 July 2015; accepted in revised form 22 September 2015. Available online 5 October 2015
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© IWA Publishing 2016 Water Science & Technology
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Tylosin effect on methanogenesis in an anaerobic biomass from swine wastewater treatment Liliana García-Sánchez, Marco Antonio Garzón-Zúñiga, Gerardo Buelna and Edson Baltazar Estrada-Arriaga
ABSTRACT The effect of different concentrations of tylosin on methane production was investigated: first methanogenesis in a biomass without contact with the antibiotic, and later the ability of the sludge to adapt to increasing concentrations of tylosin. Results showed that, for biomass that had no contact with the antibiotic, the presence of tylosin inhibits the generation of methane even at concentrations as small as 0.01 mg L1, and samples at concentrations above 0.5 mg L1 produced practically no methane, whereas, in the digesters acclimated in the presence of tylosin at a concentration of 0.01 to 0.065 mg L1, methanogenesis is not inhibited in the presence of antibiotic and the generation of methane is improved. This behaviour suggests the microorganisms have developed not only resistance to the antibiotic but also an ability to metabolize it. Key words
| specific methanogenic activity (SMA), swine anaerobic sludge, tylosin
Liliana García-Sánchez Faculty of Engineering, National Autonomous University of Mexico, Campus Morelos, Paseo Cuauhnahuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, Mexico Marco Antonio Garzón-Zúñiga National Polytechnic Institute, CIIDIR IPN Unidad Durango, Sigma 119, Fracc. 20 de Noviembre II, Durango, Durango C.P. 34220, Mexico Gerardo Buelna Industrial Research Center of Quebec, 333 rue Franquet, Quebec, Canada Edson Baltazar Estrada-Arriaga (corresponding author) Mexican Institute of Water Technology, Paseo Cuauhnahuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, Mexico E-mail: [email protected]
INTRODUCTION Nowadays, there is growing interest in the study of emerging contaminants, including antibiotics and other drugs present in wastewaters. There is a large amount of information about fate and transformations of drugs released by humans, but regarding drugs for veterinary use, the information is scarce. Specifically regarding antibiotics, many problems have been detected: (1) bacterial resistance, (2) inappropriate use, (3) they are metabolized partially, (4) they affect the efficiency of wastewater treatment systems, (5) low removal in wastewater treatment systems and (6) improper disposal. The reason that pharmaceuticals such as antibiotics are of interest in wastewater treatment is because they are substances that interfere with the active part of the bacteria, affecting the removal efficiencies of the biological treatments. In addition, they are persistent compounds that are developed to prevent the substance becoming inactive before having a curative effect, tending to bioaccumulate and to cause adverse effects when discharged into aquatic and soil ecosystems. Some antibiotics are not persistent but have slow biodegradation and so doi: 10.2166/wst.2015.507
their constant introduction into the environment can cause a similar inhibitory effect which affects the environment. One of most used veterinary antibiotics worldwide is tylosin. Tylosin is an antibiotic whose main use is as a growthpromoting agent in pig farms in Mexico and the USA (92%). However, over 70% of tylosin administered is excreted from faeces and urine of animals in its original form: reason why the use of this antibiotic becomes an environmental problem. Concentrations of tylosin are detected in different matrixes around the world: soils (2 μg L1) (Zilles et al. ), manure (0.000002–0.0019 μg L1) (Huang et al. ; Zilles et al. ), surface water (0.00029–0.28 μg L1) (Kolpin et al. ; Watkinson et al. ) and wastewater (0.060–2 μg L1) (Zilles et al. ; Watkinson et al. ). García-Sánchez et al. () detected concentrations of tylosin from 20.2 to 32.4 μg L1 in swine wastewater in Mexico. In the European Union, this antibiotic was prohibited, because it generates bacterial resistance in the environment and in animals, which could be transferable to humans. In Mexico, there are no studies of this antibiotic, despite being widely used. In addition,
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existing research on the effect of tylosin on the performance of anaerobic wastewater treatment systems seems contradictory, since the inhibition of methanogenesis has been described from insignificant (Massé et al. ; Chelliapan et al. ) to considerable (Sanz et al. ; Lotfin et al. ). The specific methanogenic activity (SMA) test is one of the most commonly used tests to determine the ability of an anaerobic biomass to degrade complex substrates in methane (McHugh et al. ; Sumino et al. ). The SMA test carried out with known amounts of biomass under defined conditions and substrates can evaluate the methane production of biomass per unit over a long period of time. It is important to carry out the SMA test to determine the behaviour of sludge under the effect of potentially toxic or inhibitory compounds and to determine the threshold value (Souto et al. ). During the degradation of toxic compounds in biological reactors, usually at the beginning, there is a period when there is no transformation of said compounds. This period is called acclimatization, adaptation, or delay. Acclimatization is a selection and multiplication of specialized microorganisms, which after a certain adaptation time, are capable of biodegrading or cometabolizing the compound or only surviving in the presence of the relevant compound. The acclimatization phase is considered vital for the biodegradation of toxic compounds, because after that occurs, the speed at which the compound is metabolized is faster. Many countries, including Mexico, have implemented programmes to reduce emissions of greenhouse gas, having as incentive the purchase of carbon credits. Therefore, anaerobic digesters have been installed to treat integrally residual liquids and solids (herefafter termed ‘dalys’, its acronym in Spanish (digestor anaerobio de líquidos y sólidos)) generated in pig farms with the purpose of producing a greater amount of biogas (Garzón-Zúñiga & Buelna ). However, there are no reports of the efficiency or operation of the process, or of the results obtained when the antibiotics are used on farms. The aim of this study was to evaluate the effect of tylosin on the methanogenesis of anaerobic sludge, the product of dalys digesters operated with different hydraulic residence times (HRT).
MATERIALS AND METHODS Tests with biomass without previous contact with tylosin The methane production was evaluated for an anaerobic biomass coming from a liquid and solid digester (dalys) at
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450 L pilot scale, with 300 d of operation, and operated with HRT of 30 d, used for the treatment of swine wastewater in Morelos, Mexico. Said biomass had no previous contact with tylosin. The biomass was used as a target to evaluate the effect of different concentrations of antibiotic (0.01, 0.05, 0.10, 0.50, 1.0, 10, 30, 50, 100, 400 and 1,000 mg L1) on the generation of methane. This process is known as specific methanogenic activity. SMA test The SMA test was performed according to the procedure described by Souto et al. (). The test consisted of filling serological flasks in an anaerobic environmental chamber (70/30, v/v, N2/CO2) with ratio of 9.03 L: 1 L:1 L:10 mL of carbon source (sodium acetate solution), inoculum, macronutrients and micronutrients respectively, maintaining 10% of the total volume of the flask for the gas phase. The test was performed in triplicate. Subsequently, the flasks were sealed with a rubber stopper and aluminium, and removed from the anaerobic chamber. The flasks were stirred in an orbital shaker at 100 rpm and at temperature of 30 C in a warm room. The determination of the methane production was performed with a volumetric method by liquid displacement, since according to Souto et al. () there is no difference between volumetric, manometric, and chromatographic methods in this test’s results. To determine the real behaviour of the effect of tylosin during experimentation, tylosin tartrate commercial grade was used. Tylosin tartrate is used as a growth promoter in pig farms. W
Adaptation of biomass to tylosin In order to determine if the biomass of the dalys systems can be adapted to increasing concentrations of tylosin and to determine the HRT effect on the removal efficiency of antibiotic, three anaerobic digesters (D1, D2 and D3) were evaluated. The digesters used had an effective volume of 4.5 L and were operated at a constant temperature of 30 C with orbital shaking and inoculated with 10% of anaerobic biomass, without previous contact with tylosin. The digesters were fed with swine wastewater without separation of solids and with a constant concentration of tylosin equal to the first test, in which the concentration showed the lowest inhibition of the methanogenic activity (0.01 mg L1). Digesters were operated with a different HRT (Table 1). The monitoring of the digester operation was carried out taking as a control parameter the removal of chemical oxygen demand (COD). W
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Table 1
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Tylosin concentrations and HRT used during the adaptation of the biomass to the antibiotic
Stage Digester
Tylosin (mg L1) HRT (d)
D1
1
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(VS) according to Standard Methods (APHA ) and ammonia nitrogen (NH4-N) according to the Hach procedure.
2
3
4
0.01 30
0.05 30
0.05 15
0.065 15
RESULTS AND DISCUSSION Methanogenesis inhibition in unacclimated biomass
Tylosin (mg L ) HRT (d)
0.01 40
0.05 40
0.05 20
0.065 20
D3
Tylosin (mg L1) HRT (d)
0.01 60
0.05 60
0.05 25
0.065 25
D4
Tylosin (mg L1) HRT (d)
0 30
0 30
0 15
0 15
Once the digesters showed stable removal efficiencies 60% of the COD, a sample of the biomass was taken. With this biomass, a new test of methanogenic activity was carried out and later the tylosin concentration was increased in the digesters until the biomass adapted to a greater tylosin concentration. This procedure was repeated with tylosin values of 0.05 and 0.065 mg L1, this last one being the maximum concentration found in swine wastewaters in Mexico (GarcíaSánchez et al. ). During the development of this experimental stage, a fourth digester was used as a control (D4), without adding tylosin.
Wastewater characteristics The wastewater used was from a growth-finishing area in a pig farm. The wastewater was collected once a week during the experiment and stored at 4 C to prevent its degradation. The wastewater was brought back to room temperature, previous to their feeding in digesters about 2 h. Characteristics of the swine wastewater are shown in Table 2. W
Analytical methods Samples of both influent and effluent of anaerobic digesters were analyzed for COD, total solids (TS) and volatile solids |
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D2
Table 2
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When an SMA test was carried out to a biomass without initial contact with tylosin, it was found that the addition of this antibiotic inhibits methane production even at concentrations as small as 0.01 mg L1 (Figure 1). After 28 d of monitoring SMA, samples at concentrations above 0.5 mg L1 produce virtually no methane. In contrast to the samples with tylosin, the sludge without addition of antibiotic generated methane at a 100% in a period of 21 d. Taken as 100% methane generated by a sample without addition of tylosin, all concentrations showed a methane inhibition percentage greater than 97%, except for the lower concentrations (0.01, 0.05 and 0.1 mg L1) in which an inhibition of 49 to 87% was obtained. The results agree with the ones reported by Shimada et al. () for concentrations such as 167 mg L1 in a sequencing batch reactor. However, for concentrations lower than 16.7 mg L1 the results are different than the ones reported by Shimada et al. (), Angenent et al. () and Poels et al. (), who reported that there is no inhibition in the presence of the tylosin in anaerobic digestion systems. This difference is due to the fact that the results reported by these authors relate to the generation of methane in anaerobic digesters in operation and not to an SMA test. The only experiment that was evaluated in batch corresponds to Stone et al. (), who found that there is no methanogenesis inhibition in a concentration of 1.1 mg L1 of tylosin. However, the results are reported only for 216 d. Such a long period of experimentation could allow the biomass to adapt to the presence of tylosin because the disappearance of the antibiotic was reported after 216 d, which probably had an impact on adaptation of the biomass to tylosin.
Characteristics of swine wastewater
Total COD 1
Stage
mg L
1
17,958 ± 1,571
Soluble COD 1
mg L
4,763 ± 223
TS
VS 1
1
mg L
mg L
17 ± 1
12 ± 1
NH4-N
1
mg L
656 ± 195
2
16,293 ± 3,359
4,823 ± 453
16 ± 2
11 ± 2
1,322 ± 417
3
20,497 ± 1,459
15,182 ± 1,364
18 ± 2
11 ± 1
1,061 ± 268
4
20,007 ± 2,478
13,334 ± 2,356
17 ± 3
11 ± 2
1,038 ± 216
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Figure 1
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Methane production and inhibition of SMA for unacclimated biomass [TYL]: tylosin concentration.
Regarding the value of SMA calculated for biomass without tylosin, it was 0.34 gCOD gVS1 d1. This value is slightly higher than the one reported by López et al. () for anaerobic digesters (0.02 to 0.2 gCOD gVS1 d1), which suggests that the biomass that is not in contact with the antibiotic carried out its complete metabolic cycle. On the other hand, the biomass that was in contact with the antibiotic concentrations between 0.1 and 1,000 mg L1 had SMA values of between 0.002 and 0.019 gCOD gVS1 d1, values lower than the one mentioned above, suggesting that methanogenic activity of biomass is being inhibited by the presence of the tylosin. For tylosin concentrations of 0.01 mg L1 and 0.05 mg L1, SMA values were 0.155 gCOD gVS1 d1 and 0.068 gCOD gVS1 d1, respectively. These results, in spite of being in an acceptable range, are lower than the ones found for biomass without tylosin. These results coincide with what was mentioned by Chelliapan et al. (): ‘It is impossible to predict the effect of antibiotics in anaerobic treatment systems because each system requires a specific investigation.’ Therefore,
contradictions regarding the inhibitory effect of tylosin on methanogenesis could be attributed to the type of biomass used, tylosin concentration, evaluation conditions (for example TS concentration and type of mixture), and type of water evaluated, among others. Adaptation of biomass to tylosin During the four experimental stages, it was found that the digester without presence of tylosin (D4) showed the lower removal of organic matter, compared to the other three digesters (62 ± 12%, 51 ± 11%, 58 ± 6% and 49 ± 20% in stage 1, 69 ± 10%, 66 ± 14%, 67 ± 12% and 66 ± 12% in stage 2, 57 ± 10%, 62 ± 7%, 64 ± 8% and 48 ± 10% in stage 3 and 72 ± 6%, 75 ± 4%, 71 ± 6% and 61 ± 11% in stage 4 for D1, D2, D3 and D4, respectively). This would probably indicate that swine wastewater contains pollutants not measured, which negatively affected non-acclimatized biomass. And contrary to expectations, when increasing the tylosin concentration at each stage, a better efficiency in the removal of
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organic matter was obtained. This could be explained by a protocooperation or cooperation between the microorganisms in the presence of the antibiotic. Another explanation for this behaviour might be as Ortiz () mentions: the reduced competition for nutrients between microorganisms or the reduction of microbial metabolites or both processes causes the antibiotic to function as a growth promoter in specialized microorganisms such as in the intestinal microbiota of pigs. According to the tylosin degradation pathway, it is decomposed into two fractions: sugar and desmycosin. Desmycosin is a factor B of tylosin, whose properties are similar to factor A (tylosin). Mycarose and mycaminose are neutral saccharide compounds generated in the degradation of desmycosin. These compounds are sugars of short chain which are easily biodegradable by microorganisms. Tylosin metabolites are not found to be toxic. Regarding the results of solids during the four experimental stages, the digester effluents were very stable, confirming that the biomass acclimated to the HRT change and to the presence of the antibiotic. The average removal rates of TS during stage 1 were 69 ± 3%, 67 ± 4%, 66 ± 2%, and 63 ± 8%, during stage 2 were 72 ± 7%, 70 ± 6%, 70 ± 6%, and 67 ± 8%, during stage 3 were 71 ± 6%, 71 ± 4%, 68 ± 8%, and 67 ± 5%, and during stage 4 were 67 ± 3%, 69 ± 6%, 70 ± 5%, and 66 ± 6% for digesters D1, D2, D3 and D4, respectively for each stage. The average percentages of VS removal during stage 1 were 75 ± 5%, 72 ± 5%, 71 ± 3%, and 67 ± 9%, during stage 2 were 78 ± 7%, 79 ± 5%, 76 ± 6%, and 74 ± 8%, during stage 3 were 69 ± 5%, 69 ± 5%, 67 ± 9%, and 65 ± 5%, and during stage 4 were 66 ± 8%, 69 ± 8%, 68 ± 6%, and 58 ± 8% for digesters D1, D2, D3 and D4, respectively, for each stage. It was observed that similarly to the removal of organic matter during the biomass acclimatization to tylosin, the digester without added antibiotic (D4) presented efficiencies slightly lower of solids removal, during the four experimental phases. The concentrations of ammonia nitrogen in the effluents were very similar to those of the influent for all the digesters, except for stage 2, in which the average values of ammonia nitrogen in the influent outweighed the effluent; however, this behaviour is not held for the final two stages. The lower values of ammonia nitrogen were obtained in stage 1 with average values of 740 ± 196 mg L1, 654 ± 102 mg L1, 737 ± 165 mg L1, and 684 ± 46 mg L1 for digesters D1, D2, D3, and D4, respectively. Methanogenesis in acclimated biomass For stages 1 and 2, the test was performed on day 18 and 50 respectively, with biomass from the four experimental
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digesters at a concentration of tylosin corresponding to the experimental stage (Figure 2). It was observed that during the first stage, the digesters with presence of tylosin needed only 2 d to accomplish a full methanogenesis of the added carbon source, while the digester D4 took 10 d to produce the expected 100% methane. These results could indicate that part of the biomass has specialized in using the antibiotic as a carbon source. For stage 2 (0.05 mg L1, HRT ¼ 30, 40, and 60 d), D1 and D3 digesters again generated 100% of the methane within 2 d, whereas the D2 and D4 took about 5 d generating it. For D3, this behaviour is expected since the load of volumetric tylosin is less when operating with greater amount. Stage 3 was carried out under the same concentration of antibiotic (0.05 mg L1 of tylosin) but the HRT was reduced from 30, 40, and 60 d to 15, 20 and 25 d, respectively for D1, D2, and D3. In addition, for stages 3 and 4, it was decided to test the SMA only with the biomass of digester D1, thinking that having the shortest HRT and therefore the bigger load of organic matter and tylosin, it could be expected to have a greater negative impact on the performance of the digester. This was verified with respect to organic matter as it had the lowest percentages of removal of such. However, it was the opposite with the generation of methane. The SMA test was performed with different concentrations of tylosin (and not only with the concentration of tylosin corresponding to the experimental stage as performed in stages 1 and 2) in order to compare the negative effect and the adaptability of the biomass to the antibiotic. The results are shown in Figure 3. It was observed that the biomass of the digester D1 acclimatized to 0.05 mg L1 of tylosin and an HRT of 15 d; not adding tylosin (0 mg L1 of tylosin added for the SMA testing) did produce the expected 100% of methane within 5 d. It was also noted that the generation of methane reached 100% only in those samples in which tylosin was added in a lower or equal concentration than that of a stage of acclimatization in progress (0.05 mg L1), whereas with higher concentrations, the production of methane was inhibited by 33%, 43%, and 78% for the concentration of 0.075 mg L1, 0.01 mg L1, and 0.15 mg L1 of tylosin, respectively. Although some inhibition for some tylosin concentrations was still found, this was less than the one found for the biomass of the pilot digester (not acclimatized to tylosin), in which an inhibition of over 44% for tylosin concentrations equal to or higher than 0.05 mg L1 was found.
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Methane production for biomass acclimated to 0.01 and 0.05 mg tylosin L
In the experimental stage 4, the antibiotic concentration was increased (0.065 mg L1 of tylosin). Under these conditions, the D1 with HRT of 15 d showed a positive displacement of the curves corresponding to methane generation, since the biomass without antibiotic reached 100% of the methane generated in approximately 12 d. While the samples of 0.05 mg L1 of tylosin (the closest concentration to the acclimation) reached 100% of the methane generation in a shorter time of 5 d, samples with higher concentrations (0.075 and 0.10 mg L1) did not reach 100% of methane generation, thus presenting some inhibition but with much lower values (33 and 44% inhibition) than those found in non-acclimated biomass (87% inhibition at a concentration of 0.1 mg L1 of tylosin, Figure 1), which shows a clear tendency of biomass acclimatization at higher concentrations of tylosin. The results indicate that the biomass in stage four is acclimated to concentrations of tylosin similar to those found by Kolz et al. () in swine wastewater treatment systems (0.4 mg L1 of tylosin).
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(HRT ¼ 30, 40 and 60 d).
Additionally, there is a likely relationship beyond the single survival of microorganisms in the presence of tylosin, since the methanogenic activity was slower in the absence of the antibiotic, while in its presence, a higher speed was observed in the methanogenic phase, which could indicate that the biomass has acquired the ability of using the antibiotic as a carbon source.
CONCLUSIONS It was determined that at concentrations of tylosin of 0.01 mg L1, the production of methane is inhibited in anaerobic sludge that has not had contact with the antibiotic by as much as 49%, and concentrations of 1,000 mg L1 inhibit methanogenesis up to 98%. However, it was found that the percentage of inhibition was affected not only by the antibiotic concentration but also by other factors such as the type of biomass used, testing conditions, the type of
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Figure 3
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Methane production for biomass acclimated to 0.05 and 0.065 mg tylosin L
water evaluated, the configuration of the evaluated reactor and the type of mix used. During the acclimatization stage, the biomass decreased the HRT in the anaerobic digesters from 30 to 15 d (D1), from 40 to 20 d (D2) and from 60 to 25 d (D3) with a concentration of tylosin of 0.05 mg L1. It was found that this decrease in HRT did not impact negatively the efficiency of the systems. It was found that the concentration of tylosin does have an effect on removal of organic matter during the acclimation of the biomass to the antibiotic. However, contrary to expectations, it was determined that when the concentration of tylosin was increased at every stage of operation, the efficiency of removal of organic matter increased. The results above indicate that tylosin affects biomass in very small concentrations but a continuous contact of the antibiotic generates acclimatization, and not only that but it acts to improve the generation of methane, which suggests not only antibiotic resistance but also the capacity to
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for anaerobic digester D1.
metabolize it. For this reason, it is important to conduct more studies about this subject matter.
ACKNOWLEDGEMENTS Financial support for the realization of this research was granted from Industrial Research Center of Quebec (CRIQ), Canada, and Mexican Institute of Water Technology.
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Works Association/Water Environment Federation, Washington, DC. Chelliapan, S., Wilby, T. & Sallis, P. Performance of an upflow anaerobic stage reactor (UASR) in the treatment of pharmaceutical wastewater containing macrolide antibiotics. Water Research 40, 507–516. Chelliapan, S., Wilby, T., Sallis, P. J. & Yuzir, A. Tolerance of the antibiotic tylosin on treatment performance of an up-flow anaerobic stage reactor (UASR). Water Science and Technology 63 (8), 1599–1606. García-Sánchez, L., Garzón-Zúñiga, M. A., Buelna, G., MoellerChávez, G. E., Noyola, A., Avilez-Flores, M. & EstradaArriaga, E. B. Occurrence of tylosin in swine wastewater in Mexico. Water Science and Technology 68 (4), 894–900. Garzón-Zúñiga, M. A. & Buelna, G. Caracterización de aguas residuales porcinas y su tratamiento por diferentes procesos en México (Characterization of pig wastewaters and its treatment by different processes in Mexico). Revista Internacional de Contaminación Ambiental 30 (1), 65–79. Huang, C. H., Renew, J. E., Smeby, K. L., Pinkston, K. & Sedlak, D. L. Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis. Journal of Contemporary Water Research and Education 120 (1), 30–40. Kolpin, D., Furlong, E., Meyer, M., Thurman, M., Zaugg, S., Barber, L. & Buxton, H. Pharmaceuticals, hormones and other organic wastewater contaminants in U.S. streams, 1999–2000: a national reconnaissance. Environmental Science and Technology 36 (6), 1202–1211. Kolz, A., Ong, S. & Moorman, T. Sorption of tylosin onto swine manure. Chemosphere 60, 284–289. López, M., Villa, P. & Escobedo, R. Estudio del comportamiento de reactores anaerobios de residuos sólidos a través de ensayos microbiológicos. Revista CENIC. Centro Nacional de Investigaciones Científicas Cuba 35 (3), 179–183. Lotfin, K. A., Henny, C., Adams, C. D., Surampali, R. & Mormile, M. R. Inhibition of microbial metabolism in anaerobic lagoons by selected sulfonamides, tetracyclines, lincomycin and tylosin tartrate. Environmental Toxicology and Chemistry 24 (4), 782–788. Massé, D., Lu, D., Massé, L. & Droste, R. Effect of antibiotics on psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors. Bioresource Technology 75, 205–211.
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McHugh, S., Carton, M., Collins, G. & O’Flaherty, V. Reactor performance and microbial community dynamics during anaerobic biological treatment of wastewaters at 16–37 C. FEMS Microbiology Ecology 48, 369–378. Ortiz, M. R. Bases fisiológicas para el uso de antibióticos promotores de crecimiento y preventivo en enfermedades bacterianas intestinales (Physiological basis for the use of antibiotic growth promoters and preventive intestinal bacterial diseases). http://www.webveterinaria.com/virbac/ news22/cerdos.pdf. Poels, J., Van Assche, P. & Verstraete, W. Effects of disinfectants and antibiotics on the anaerobic digestion of piggery waste. Agricultural Wastes 9, 239–247. Sanz, J. L., Rodríguez, N. & Amils, R. The action of antibiotics on the anaerobic digestion process. Applied Microbiology and Biotechnology 46 (5–6), 587–592. Shimada, T., Zilles, J., Morgenroth, E. & Raskin, L. Inhibitory effects of the macrolide antimicrobial tylosin on anaerobic treatment. Biotechnology and Bioengineering 101 (1), 73–82. Souto, T., Aquino, S., Silva, S. & Chernicharo, C. Influence of incubation conditions on the specific methanogenic activity test. Biodegradation 21, 411–424. Stone, J., Clay, S., Zhu, Z., Wong, K., Porath, L. & Spellman, G. Effect of antimicrobial compounds tylosin and chlortetracycline during batch anaerobic swine manure digestion. Water Research 43 (18), 4740–4750. Sumino, H., Takahashi, M., Yamaguchi, T., Abe, K., Araki, N., Yamazaki, S., Shimozaki, S., Nagano, A. & Nishio, N. Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature. Bioresource Technology 98, 177–182. Watkinson, A., Murby, E., Kolpin, D. & Costanzo, S. The occurrence of antibiotics in an urban watershed: from wastewater to drinking water. Science of the Total Environment 407, 2711–2723. Zilles, J., Shimada, T., Jindal, A., Robert, M. & Raskin, L. Presence of macrolide-lincosamide-streptogramin B and tetracycline antimicrobials in swine waste treatment processes and amended soil. Water Environment Research 77 (1), 57–62.
First received 3 July 2015; accepted in revised form 22 September 2015. Available online 5 October 2015
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