effects of macrolide antimicrobials on the performance of anaerobic ...

WEFTEC®.06

EFFECTS OF MACROLIDE ANTIMICROBIALS ON THE PERFORMANCE OF ANAEROBIC TREATMENT SYSTEMS Toshio Shimada*, Julie Zilles*, Eberhard Morgenroth*, Lutgarde Raskin** * University of Illinois at Urbana-Champaign, 205 N Mathews Ave, Urbana, IL 61801, USA ** University of Michigan, 1351 Beal Ave, Ann Arbor, MI 48109, USA ABSTRACT An anaerobic sequencing batch reactor (ASBR) was operated to study the effects of tylosin, a macrolide antimicrobial commonly used in swine production, on treatment performance and the development of antimicrobial resistance. The addition of 1 mg/l tylosin to the reactor had no effects on the biogas production and chemical oxygen demand (COD) removal efficiency. Specific methanogenic activity (SMA) batch tests showed a significant (p < 0.05) decrease of the maximum methane production rate in the presence of 100 mg/l of tylosin for the substrate acetate and 1 mg/l and 100 mg/l of tylosin for the substrates propionate and butyrate. Tylosin addition to the ASBR would affect the utilization of acetate, propionate, and butyrate. Tylosin may exert inhibitory effects on saturated fatty acid beta-oxidizing syntrophic bacteria and gram-positive homoacetogenic bacteria. KEYWORDS anaerobic, antimicrobial, macrolide, methanogenesis INTRODUCTION Several classes of veterinary antimicrobials used for therapy, prophylaxis, and/or growth promotion in confined animal operations have been detected in manure, anaerobic lagoons, and amended soil samples (Campagnolo et al. 2002; Hamscher et al. 2002; Snow et al. 2002; Zilles et al. 2005). We recently determined that the treatment performance of an anaerobic lagoon at a swine production facility was acceptable even though antimicrobials were present at levels that inhibit the growth of sensitive bacteria (Zilles et al. 2005). Furthermore, we evaluated anaerobic treatment performance in a laboratory-scale anaerobic sequencing batch reactor (ASBR) simulating the treatment of a pharmaceutical waste stream containing the macrolide antimicrobial erythromycin (Amin et al. 2004). After addition of the antimicrobial, only slight decreases in substrate removal and biogas production were observed. Specific methanogenic activity (SMA) batch experiments suggested the partial inhibition of fatty acid-oxidizing syntrophic bacteria (Amin et al. 2004). These studies did not establish whether antimicrobial resistance of microbial populations in these treatment systems is necessary to allow substantial biological activity and acceptable treatment performance in the presence of antimicrobial agents. Macrolides (e.g., erythromycin and tylosin) consist of macrocyclic lactone rings with sugars linked by glycosidic bonds. This group of antimicrobials inhibits protein synthesis by interacting with the 50S subunit of the bacterial ribosome. Although macrolides do not target methanogenic archaea, the inhibition of fermentative and/or syntrophic bacteria may result in adverse effects on methanogenesis. Copyright ©2006 Water Environment Foundation. All Rights Reserved

1543

WEFTEC®.06

The purpose of this work is to study the effect of tylosin, a macrolide antimicrobial commonly used in swine production facilities, on the development of antimicrobial resistance and the treatment performance in a laboratory-scale ASBR. METHODOLOGY A 7-liter jacketed bioreactor (Applikon Instruments Co, Schiedam, The Netherlands) was inoculated using granular sludge and operated as an ASBR at 35oC with 24-h cycles and intermittent mixing. Glucose was fed to the reactor at an organic loading rate (OLR) of 3.5 kgCOD m-3 day-1. On day 750 of reactor operation, tylosin solution was added to the reactor at a loading rate of 1 g m-3 day-1. The hydraulic retention time (HRT) and the target solids retention time (SRT) were 1.67 and 70 days, respectively. Total solids (TS), volatile solids (VS), total suspended solids (TSS), volatile suspended solids (VSS), chemical oxygen demand (COD), and nutrient concentrations (NH3-N and total-P) were determined according to standard methods (American Public Health Association 2000). Total volatile fatty acids (VFA) and bicarbonate alkalinity were determined through a two point titration (Anderson and Yang 1992). Analysis for individual VFAs was conducted through high performance liquid chromatography (Waters, Milford, MA). Biogas generation was measured using wet-test gas meters (Schlumberger Industries, Dordrecht, The Netherlands). The methane concentration in the biogas was determined through gas chromatography (Perkin Elmer, Norwalk, CT). SMA batch tests were conducted in triplicates combining three levels of tylosin dosage (0 mg/l, 1 mg/l, and 100 mg/l) with five substrate conditions (no substrate, glucose, acetate, propionate, and butyrate). The batch tests were conducted in 125 ml glass bottles using a 16-cell AER-216 aerobic/anaerobic respirometer (Challenge Environmental Systems, Springdale, AZ, USA) and a 15-position magnetic stirrer with intermittent mixing. The SMA tests were run for 48 hours. Following the period of operation with low-level antimicrobial addition, tylosin will be dosed to the ASBR at inhibitory concentrations and then will be removed from the influent to the reactor. The levels of macrolide-lincosamide-streptogramin B (MLSB) resistant bacteria will be determined through a recently developed fluorescence in situ hybridization (FISH) technique (Zhou et al. 2004). RESULTS AND DISCUSSION The ASBR showed good treatment performance throughout the operational period (Figure 1) with COD removal efficiency above 95%. Biogas production has been at approximately 10 liters per day for the target OLR of 3.5 kg-COD m-3 day-1. Monitoring during a 24-h cycle indicated a rapid conversion of glucose into propionate and acetate, similar to observations in a previous ASBR study (Shizas and Bagley 2002). Biogas production and COD removal in the ASBR were not affected by the addition of low levels of tylosin (1 mg/l).

Copyright ©2006 Water Environment Foundation. All Rights Reserved

1544

WEFTEC®.06

1600

A

Concentration, mg-COD l

15

Propionate -1

-3

target OLR obs OLR

3

10

5

Glucose

B

Gas Volume

-1

Gas Volume (dm ) and OLR (kg m d )

20

0

Acetate

1200

Total VFA

800

400

0 0

100

200

300

400

500

600

700

0

Time, days

6

12

18

24

Time, h

Figure 1. ASBR Performance. (A) Daily biogas production during period without antimicrobial addition. (B) Individual volatile fatty acids concentration during a 24-h cycle (day 483 in operational period). Butyrate and valerate Were below the detection limit of 500 μg/l. Before day 750 of reactor operation, SMA batch tests showed a significant (p < 0.05) decrease of the maximum methane production rate in the presence of 100 mg/l of tylosin for the substrate acetate and 1 mg/l and 100 mg/l of tylosin for the substrates propionate and butyrate (data not shown). There were no significant differences (p < 0.05) between the different tylosin conditions for the substrate glucose and the tests without substrate. Figure 2 shows the residual VFA concentrations at the end of the SMA batch tests. Acetate and propionate were completely degraded at the end of the SMA experiments for all test conditions. Butyrate was only present at the end of the SMA experiments in those bottles that received tylosin and were fed butyrate as the substrate. Butyrate degradation began after a 26-h lag phase, but was inhibited in the presence of 1 mg/l and 100 mg/l tylosin with removal efficiencies of 90 and 75%, respectively. These results support previous work in which inhibition of butyrate degradation was observed in the presence of the macrolide erythromycin (Amin et al. 2004). The macrolide tylosin used in the present study similarly may exert inhibitory effects on saturated fatty acid beta-oxidizing syntrophic bacteria (i.e., bacteria that degrade butyrate and higher saturated fatty acids). The tests using acetate as substrate suggest that tylosin may have an impact on other bacteria (in particular on gram-positive homoacetogenic bacteria, i.e., bacteria that form syntrophic interactions with hydrogenotrophic methanogens in order to degrade acetate).


1545

WEFTEC®.06

250 0 mg-tylosin/l 1 mg-tylosin/l 100 mg-tylosin/l

150

-1

ml CH 4 d g-VS

-1

200

100

50

0 Acetate

Propionate

Butyrate

Glucose

Control

Figure 2. Maximum methane production rate for different substrate conditions in SMA batch tests. Experiments were performed in triplicate and average results with standard deviations (indicated by error bars) are reported. Based on the results of the SMA batch tests, it is expected that the addition of tylosin to the ASBR would affect the utilization of acetate, propionate, and butyrate. With continued operation, this inhibition could result in a decrease in biogas production and COD removal. The reactor performance with the presence of 1 mg/l tylosin and the individual VFA ASBR cycle profile suggested that butyrate is not an important intermediate in this reactor. Operation of the reactor with higher levels of tylosin (100 mg/l) would show deleterious effects on reactor performance. SMA tests performed with biomass from the ASBR during tylosin addition will help elucidate which populations are affected most by tylosin addition. This study expands previous work in several ways. (i) Since glucose is used as the substrate, the effect of the presence of tylosin on fermentation will be evaluated, not just the effect on VFA conversion. (ii) The development of antimicrobial resistance will be monitored over time and bacteria that develop resistance will be identified through the use of FISH probes that target MLSB resistant bacteria in combination with FISH probes that identify relevant populations. (iii) Finally, we will evaluate whether the removal of tylosin from the reactor influent will allow the system to return to conditions (i.e., biogas production and COD removal efficiency) prior to tylosin addition. ACKNOWLEDGEMENTS Toshio Shimada was supported by a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico. This research was partially supported by the U.S. Department of Agriculture under Cooperative Agreement AG 58-3620-1-179.


1546

WEFTEC®.06

REFERENCES American Public Health Association (2000). Standard Methods for the Examination of Water and Wastewater. Amin, M.; Zilles, J.; Grainer, J.; Charbonneau, S.; Raskin, L.; Morgenroth, E. (2004). Influence of the antibiotic erythromycin on anaerobic treatment of a pharmaceutical wastewater. 10th IWA World Congress on Anaerobic Digestion, Montreal, Canada, IWA. Anderson, G. K.; Yang, G. (1992). "Determination of Bicarbonate and Total Volatile Acid Concentration in Anaerobic Digesters Using a Simple Titration." Water Environment Research 64(1): 53-59. Campagnolo, E. R.; Johnson, K. R.; Karpati, A.; Rubin, C. S.; Koplin, D. W.; Meyer, M. T.; Esteban, J. E.; Currier, R. W.; Smith, K.; Thu, K. M.; McGeehin, M. (2002). "Antimicrobial residues in animal waste and water resources proximal to large-scale swine poultry feeding operations." The Science of the Total Environment 299: 89-95. Hamscher, G.; Sczesny, S.; Hoper, H.; Nau, H. (2002). "Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry." Analytical Chemistry 74(7): 1509-1518. Shizas, L.; Bagley, D. M. (2002). "Improving anaerobic sequencing batch reactor performance by modifying operational parameters." Water Research 36(1): 363-367. Snow, D. D.; Cassada, D. A.; Monson, S. J.; Zhu, J.; Spalding, R. F. (2002). Trace analysis of tetracycline and macrolide antibiotics using phase extraction and liquid chromatographytandem mass spectrometry. Analysis of Emerging Contaminants using LC/MS/MS, Orlando, FL, Division of Environmental Chemistry, American Chemical Society. Zhou, Z.; Jindal, A.; Wagoner, M.; Raskin, L.; Zilles, J. (2004). FISH analysis of MLSB antimicrobials in swine waste. 104th General Meeting of the American Society for Microbiology, New Orleans, LA, ASM. Zilles, J.; Shimada, T.; Jindal, A.; Robert, M.; Raskin, L. (2005). "Presence of Macrolidelincosamide-streptogramin B and Tetracycline Antimicrobials in Swine Waste Treatment Processes and Amended Soil." Water Environment Research 77(1): 57-62.


1547