Environ. Sci. Technol. 2010, 44, 9128–9133
Effect of Temperature on the Fate of Genes Encoding Tetracycline Resistance and the Integrase of Class 1 Integrons within Anaerobic and Aerobic Digesters Treating Municipal Wastewater Solids DAVID L. DIEHL† AND TIMOTHY M. LAPARA* Department of Civil Engineering, University of Minnesota, 500 Pillsbury Drive SE, Minneapolis, Minnesota 55455-0116, United States
Received August 12, 2010. Revised manuscript received October 25, 2010. Accepted October 28, 2010.
The objective of this research was to investigate the ability of anaerobic and aerobic digesters to reduce the quantity of antibiotic resistant bacteria in wastewater solids. Labscale digesters were operated at different temperatures (22 °C, 37 °C, 46 °C, and 55 °C) under both anaerobic and aerobic conditions and fed wastewater solids collected from a fullscale treatment facility. Quantitative PCR was used to track five genes encoding tetracycline resistance (tet(A), tet(L), tet(O), tet(W), and tet(X)) and the gene encoding the integrase (intI1) of class 1 integrons. Statistically significant reductions in the quantities of these genes occurred in the anaerobic reactors at 37 °C, 46 °C, and 55 °C, with the removal rates and removal efficiencies increasing as a function of temperature. The aerobic digesters, in contrast, were generally incapable of significantly decreasing gene quantities, although these digesters were operated at much shorter mean hydraulic residence times. This research suggests that high temperature anaerobic digestion of wastewater solids would be a suitable technology for eliminating various antibiotic resistance genes, an emerging pollutant of concern.
Introduction Since their discovery, antibiotics have been used to treat and control many bacterial infections and are partially responsible for the rise in human life expectancy that has occurred during the last century. In recent years, however, there has been substantial concern regarding the rapid pace by which bacteria are becoming resistant to antibiotics (1, 2). Different measures have been taken in an effort to slow the spread of antibiotic resistance. In healthcare, steps have been taken to limit inappropriate usage of antibiotics, such as not prescribing antibiotics for viral infections. In agriculture, the subtherapeutic use of antibiotics is under intense scrutiny; some countries have even banned this practice. The global hypothesis driving our research is that municipal wastewater treatment could be used as a potential * Corresponding author phone: (612)624-6028; fax: (612)626-7750; e-mail:
[email protected]. † Current address: Applied Technologies, Inc., Brookfield, WI 53005. 9128
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tool to slow the proliferation of antibiotic resistance. Antibiotic resistant bacteria are selected and/or evolve wherever antibiotics are used. It is plausible, therefore, that the gastrointestinal tracts of humans taking antibiotics would be a location where antibiotic resistant bacteria originate. These resistant bacteria are then expelled from the body during defecation and collected by sanitary sewers. Thus, municipal wastewater contains substantial quantities of antibiotic resistant bacteria (3-12), and municipal wastewater treatment plant operations could be used to systematically eliminate resistant bacteria. Considering the processes commonly used to treat municipal wastewater, the majority of antibiotic resistant bacteria in municipal wastewater undoubtedly reside in the residual wastewater solids. Wastewater solids are comprised of a mixture of primary solids (i.e., solids collected from untreated wastewater via gravitational sedimentation) and secondary solids (i.e., excess bacteria grown in the aeration tanks). These wastewater treatment solids are currently treated by numerous processes (e.g., anaerobic digestion, drying, application to agricultural soils) that likely reduce the presence of antibiotic resistant bacteria by some degree. Additional research is needed, therefore, to determine the fate of antibiotic resistant bacteria in these processes so that the design of wastewater treatment facilities can be optimized to limit the spread of resistance. Temperature is the principal variable used to control the inactivation of pathogenic microorganisms in aerobic and anaerobic digesters treating municipal wastewater solids (13, 14). By extension, temperature should be a critical variable for the destruction and inactivation of antibiotic resistant bacteria and antibiotic resistance genes during the digestion of wastewater solids. Our prior research, which demonstrated that a full-scale anaerobic thermophilic digester outperformed a full-scale mesophilic anaerobic digester in reducing several antibiotic resistance determinants (15), supports this hypothesis. The goal of the present research study, therefore, was to investigate the effect of temperature on the reduction of antibiotic resistance determinants under tightly controlled laboratory conditions while yet mimicking many of the characteristics of full-scale anaerobic and aerobic digestion processes. The fate of five different tetracycline resistance determinants was tracked during these experiments, including representatives of each of the three classes of tetracycline resistance genes (encoding: tetracycline efflux (tet(A), tet(L)), ribosomal protection (tet(O), tet(W)), and tetracycline transformation (tet(X))), genes specific for Gramnegative (tet(A)) and for Gram negative-bacteria (tet(L)), and genes specific for aerobic (tet(O)) and for anaerobic bacteria (tet(W)) (16). We also quantified the integrase gene (intI1) of Class 1 integrons, which are genetic elements believed to substantially contribute to the evolution and proliferation of multiple antibiotic resistant bacteria (17).
Materials and Methods Reactor Set-up. Conical 26.2 L fermenters (BrewCity; Milwaukee, WI) were used as bench-scale digesters. Each reactor was maintained at the desired reactor temperature using 120 V silicone rubber heating tape (OEM Heaters; Edina, MN). Duplicate anaerobic digesters were initiated using 10 L of solids from the anaerobic digesters at the Empire Wastewater Treatment Facility (Farmington, MN) and 5 L of untreated wastewater solids (a mixture of primary and secondary solids). To achieve a mean hydraulic residence time of 15 days, 5 L of reactor contents was removed every 10.1021/es102765a
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fifth day and replaced with 5 L of untreated wastewater solids. Digesters were mixed by recirculating gas from the headspace to the bottom of the reactor using a peristaltic pump. Digester pH was monitored daily and kept above a minimum of 6.0 by the periodic addition of a 1 M sodium carbonate solution; pH control was needed only during the start-up of the reactor such that no sodium carbonate was added to the digesters after the first 40 days of the experiment. Biogas was continuously collected in Tedlar bags (Keika Ventures; Chapel Hill, NC). Anaerobic digesters were operated until process performance stabilized with respect to biogas production and with respect to total solids concentrations. Occasional analysis of the biogas revealed that the methane content was typically 50-70% of the total biogas. Bench-scale aerobic digesters were initiated using 8 L of undigested wastewater solids and imposing aeration (1 L/min) using fine bubble air diffusers. Every day, 2 L of digested solids was removed from each reactor and replaced with 2 L of undigested wastewater solids to provide a mean hydraulic residence time of 4 days. Deionized water was added daily to compensate for evaporation losses and to maintain a constant volume within each reactor. Aerobic digesters were operated until process performance stabilized with respect to total solids removal. Chemical and Physical Analyses. In the anaerobic reactors, gas production and pH were measured daily. Gas production was measured by using a peristaltic pump to empty the 16 L Tedlar bags at a known flow rate. The pH was measured using a Corning 340 pH meter (Corning INC.; Corning, NY). The total solids content of all digester samples was determined by collecting approximately 80 mL of sample, determining its mass, and then determining its mass following drying at 105 °C. Temperature was continuously monitored in all digesters and was typically within 1 °C of the targeted value. Genomic DNA Extraction. Samples (approximately 100 µL) were collected in triplicate from each of the digesters to which 500 µL of lysis buffer (120 mM sodium phosphate, 5% sodium dodecyl sulfate, pH 8) was added. Samples then underwent three consecutive freeze-thaw cycles and a 90 min incubation at 70 °C. Genomic DNA extraction and purification was performed using the FastDNA Spin Kit (MP Biomedicals; Solon, OH) according to manufacturer’s instructions. DNA extractions were stored at -20 °C until needed. Quantitative Real Time PCR. Quantitative real time PCR (qPCR) was used to quantify the presence of five genes encoding tetracycline resistance (tet(A), tet(L), tet(O), tet(W), and tet(X)), the integrase gene of class 1 integrons (intI1), and the 16S rRNA gene. The qPCR analysis was conducted using an Eppendorf Mastercycler ep realplex thermal cycler (Eppendorf; Westbury, NY). Each qPCR run consisted of 10 min initial denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, and anneal and extension for 1 min. A 25 µL reaction mixture contained 12.5 µL of 2× Power SYBR Green Master Mix (Applied Biosystems; Foster City, CA), 25 µg of bovine serum albumin (Roche Applied Science; Indianapolis, IN), optimized quantities of forward and reverse primers, and ∼1 ng of template DNA. Additional information on the qPCR primer and their associate products are provided in the Supporting Information. The quantity of target DNA in unknown samples was calculated based on a standard curve generated using known quantities of template DNA. Standards for qPCR were prepared by PCR amplification of genes from positive controls, followed by ligation into pGEM-T Easy vectors following manufacturer’s instructions (Promega; Madison, WI), and transformation into E. coli DH5R. Plasmids were
TABLE 1. Quantities of Genes Encoding Five Tetracycline Resistance Determinants and Quantities of Genes Encoding the Integrase of Class 1 Integrons in Untreated Wastewater Solids Fed to Bench-Scale Anaerobic and Aerobic Bioreactorsa anaerobic digester feed (copies/µL) tet(A) 6.0 × 104 (s.d. ) 2.8 tet(L) 4.2 × 105 (s.d. ) 1.1 tet(O) 1.8 × 105 (s.d. ) 6.5 tet(W) 8.4 × 101 (s.d. ) 7.2 tet(X) 3.3 × 104 (s.d. ) 1.4 intI1 1.0 × 106 (s.d. ) 6.8
× 104; n ) 12) × 106; n ) 11) × 104; n ) 12) × 101; n ) 12) × 104; n ) 12) × 105; n ) 12)
aerobic digester feed (copies/µL) 1.5 × 105 (s.d. ) 9.1 5.6 × 104 (s.d. ) 1.5 1.4 × 105 (s.d. 4.1 × 5.0 × 101 (s.d. ) 4.8 4.9 × 104 (s.d. ) 1.7 1.3 × 106 (s.d. ) 3.7
× 104; n ) 6) × 104; n ) 3 104; n ) 6) × 101; n ) 5) × 104; n ) 6) × 105; n ) 6)
a
Quantities are presented as the arithmetic means of gene copies per microliter; standard deviations and the number of replicates are shown in parentheses.
purified using the alkaline lysis procedure (18). Plasmid DNA was quantified by staining with Hoechst 33258 dye and measured on a TD-700 fluorometer (Turner Designs; Sunnyvale, CA) using calf thymus as a DNA standard. 10-fold serial dilutions of plasmid DNA were prepared and run on the thermal cycler to generate standard curves (r2 > 0.99). Following qPCR, melting curves were generated and analyzed to verify that nonspecific amplification did not occur. In this study, gene quantities are presented on a per reactor volume basis, because our view is that the total quantity of genes is of greatest importance. An alternative approach would be to normalize gene quantities by the number of 16S rRNA genes, which is a measure of total bacterial biomass. Although both sets of data reveal similar patterns regarding the effect of temperature, we have included the gene quantity data normalized to 16S rRNA genes in the Supporting Information. Data Analysis. The quantities of genes in each of the digesters were fit to a first-order kinetic model as is often assumed for the decay of bacteria in wastewater treatment facilities (19). These kinetic rates (and the associated confidence that these rates were different than zero) were determined using SigmaPlot ver. Ten (Systat Software Inc.; San Jose, CA).
Results and Discussion Bench-scale anaerobic and aerobic digesters were operated at 22 °C, 37 °C, 46 °C, and 55 °C to discern the effect of temperature on the quantities of five different tetracycline resistance determinants as well as the integrase gene of class 1 integrons. The anaerobic and aerobic digesters had mean hydraulic residence times of 15 days and 4 days, respectively, to approximate the residence times that typically occur at full-scale. Digesters were operated in a semicontinuous fashion, whereby a portion of the digester contents (33% and 25% for the anaerobic and aerobic digesters, respectively) was replaced at discrete time intervals (once every 5 days and once each day for the anaerobic and aerobic digesters, respectively). Digesters were fed untreated wastewater solids from a full-scale wastewater treatment facility. The quantities of the five different tetracycline resistance determinants as well as the integrase gene of class 1 integrons in the untreated wastewater solids are shown in Table 1. The performance of all duplicate anaerobic digesters required >50 days to stabilize with respect to biogas producVOL. 44, NO. 23, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. The quantities of five genes encoding tetracycline resistance and the gene encoding the integrase of class 1 integrons per volume of bench-scale reactor fluid in duplicate bench-scale anaerobic digesters operated at different temperatures. Digesters were operated in semicontinuous fashion, whereby 33% of the digester contents were replaced every 5 days with undigested wastewater solids. The data shown represent a discrete time interval between the introduction of undigested wastewater solids. Solid and dashed lines represent best fits to a first-order kinetic model. Gene quantities in the untreated wastewater solids are shown in Table 1. tion and total solids concentrations (Supporting Information). Temperature had substantial impact on the performance of these digesters, as higher rates of biogas production were measured at 37 and 46 °C than at 22 and 55 °C. Total solids concentrations were similar at 37 °C, 46 °C, and 55 °C, corresponding to a reduction of total solids concentrations of 35-40%. The total solids concentrations in the digester operated at 22 °C were slightly higher, corresponding to removal efficiencies of approximately 30%. Results from the duplicate anaerobic digesters were generally similar. The quantities of tetracycline resistance determinants and integrase genes from class 1 integrons were also substantially affected by temperature (Figure 1; for the same data normalized to 16S rRNA genes, see the Supporting Information). Within a discrete feed cycle, the quantities of each gene were relatively constant as a function of time at 22 °C. As the temperature of the anaerobic digesters increased, however, the quantities of tet(A), tet(O), tet(W), tet(X), and intI1 noticeably declined over time, often by more than an order of magnitude - generally consistent with reductions that we previously observed in full-scale treatment facilities (15). These reductions in gene quantities were fit to a firstorder kinetic model (Table 2), which also suggested that higher temperatures resulted in more rapid declines in gene quantities. In fact, the first-order kinetic rates were much more likely to be statistically significant (P < 0.05) as the temperature increased. Furthermore, the extent to which the quantities of these genes declined was also affected by temperature. For each of the genes, except for tet(L), the gene quantities at the end of the 5-day treatment period gradually declined as temperature increased. Elevated digester temperatures had the greatest impact on the quantities of intI1 genes, which were approximately 3 orders of magnitude lower in the 55 °C digester compared to the 22 °C digester. The performance of the aerobic digesters stabilized within 30 days as indicated by the total solids concentrations (data not shown). The efficiencies by which total solids were removed in the digesters operated at 37 °C, 46 °C, and 55 °C were similar, ranging between 35 and 45%. In contrast, the 9130
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total solids concentration in the digester operated at 22 °C was reduced by approximately 30%. Aerobic digestion of the wastewater solids had a less substantial effect on the quantities of tetracycline resistance determinants and integrase genes from class 1 integrons, regardless of the operating temperature (Figure 2; for the same data normalized to 16S rRNA genes, see the Supporting Information). The quantities of each gene were relatively constant as a function of time regardless of operating temperature. In fact, only three of the aerobic digesters exhibited a statistically significant rate of change in gene quantities, two of which were increases - only the tet(X) gene at 55 °C declined significantly (Table 3). The quantities of these genes, however, generally declined as temperatures increased, typically by 1-2 orders of magnitude. This research shows that substantial quantities of antibiotic resistance genes are present in untreated municipal wastewater solids (Table 1) and that different digestion conditions can have substantially different performance efficiencies. Previous research has shown that antibiotic resistant genes are prevalent in wastewater biosolids (3, 11, 12, 15), but little research has been explicitly directed to determine how different treatment technologies and their associated operating conditions affect the quantities of antibiotic resistant bacteria and antibiotic resistance genes. Because these stabilized biosolids can be applied to land for use as a fertilizer and as a soil conditioner (19), it is important to limit the spread of antibiotic resistance genes from these solids to soil. The present research, therefore, provides important and novel information about the application of anaerobic and aerobic digestion for reducing the quantities of antibiotic resistant bacteria and antibiotic resistance genes in wastewater solids. The present research provides more detailed kinetic information over a broader ranger of temperatures compared to our prior work in which a full-scale, thermophilic anaerobic digester (operating temperature )55-60 °C) was more effective than a full-scale, mesophilic anaerobic digester (operating temperature ) 35-37 °C) (15). The present study clearly demonstrates that increasing the temperature of anaerobic digesters leads to smaller quantities
TABLE 2. First-Order Rates by Which Tetracycline Resistance Determinants and Integrase Genes of Class 1 Integrons Decrease in Quantity in Bench-Scale Anaerobic Digesters Treating Municipal Wastewater Solidsa tet(A)
tet(L)
Digester 1 -1
Digester 2
Digester 1
-1
-1
Digester 2
k (d )
r
k (d )
r
k (d )
r2
0.28 *0.15 *0.47 *0.44
0.59 0.85 0.98 0.81
*0.261 0.037 0.118
0.79 0.07 0.16
0.02 0.14 -0.11 0.008
0.01 0.50 0.51 0.001
temp (°C)
k (d )
r
22 37 46 55
-0.02 *0.42 0.22 *0.43
0.01 0.98 0.59 0.88
2
-1
2
2
tet(O)
tet(X)
Digester 1
Digester 2
Digester 1
Digester 2
temp (°C)
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
22 37 46 55
0.11 -0.12 0.14 *0.15
0.31 0.22 0.39 0.68
-0.10 *0.22 0.039 *0.33
0.22 0.84 0.07 0.86
0.183 *0.351 *0.431 *1.488
0.612 0.903 0.858 0.987
-0.014 -0.019 0.211 *1.092
0.022 0.011 0.418 0.986
tet(W) Digester 1
intI1 Digester 2
Digester 1
Digester 2
temp (°C)
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
22 37 46 55
0.15 0.14 0.21 *0.59
0.36 0.47 0.36 0.91
*-0.2 -0.015 -0.004 *0.849
0.73 0.02 0.007 0.89
0.19 *0.99 *1.7 *2.9
0.48 0.99 0.98 0.99
0.008 *0.60 *0.96 *2.1
0.27 0.95 0.93 0.99
a Statistically significant decreases (P < 0.05) in gene quantities are identified with an asterisk. Negative values represent a first-order increase in gene quantities.
FIGURE 2. The quantities of five genes encoding tetracycline resistance and the gene encoding the integrase of class 1 integrons per volume of bench-scale aerobic digestion fluid operated at different temperatures. Digesters were operated in semicontinuous fashion, whereby 25% of the digester contents were replaced each day with undigested wastewater solids. The data shown represent a discrete time interval between the introduction of undigested wastewater solids. Solid lines represent best fits to a first-order kinetic model. Gene quantities in the untreated wastewater solids are shown in Table 1. of five tetracycline resistance determinants as well as class 1 integrons. A secondary goal of this research was to determine if operating conditions would have differing impacts on the various tetracycline resistance genes. The genes that were targeted, therefore, coded for each of the known mechanisms of tetracycline resistance (efflux: tet(A) and tet(L); ribosomal protection: tet(O) and tet(W); enzymatic modification: tet(X))
(16). Furthermore, these specific genes were analyzed because they confer tetracycline resistance via the same mechanism but for different types of bacteria - that is, tet(A) and tet(L) are specific for Gram negative and Gram positive bacteria, respectively, and tet(O) and tet(W) are specific for aerobic and anaerobic bacteria, respectively. In the anaerobic digesters, increases in temperature seemed to have had a parallel effect on each of these genes (except tet(L)), regardless VOL. 44, NO. 23, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. First-Order Rates by Which Tetracycline Resistance Determinants and Integrase Genes of Class 1 Integrons Decrease in Quantity in Bench-Scale Aerobic Digesters Treating Municipal Wastewater Solidsa tet(A)
tet(L)
tet(O)
tet(W)
tet(X)
intI1
temp (°C)
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
k (d-1)
r2
22 37 46 55
0.13 0.34 0.05 *-0.85
0.07 0.59 0.01 0.59
-0.35 -0.35 0.14 1.1
0.11 0.10 0.13 0.28
-0.09 0.38 -0.54 -0.26
0.02 0.18 0.32 0.08
0.26 -0.06 *-0.81 0.13
0.26 0.02 0.74 0.03
0.21 0.48 0.36 *5.3
0.05 0.40 0.07 0.93
-0.01 0.96 0.30 0.53
0.01 0.54 0.15 0.12
a Statistically significant decreases (P < 0.05) in gene quantities are identified with an asterisk. Negative values represent a first-order increase in gene quantities.
of whether the genes were from aerobic or from anaerobic organisms. In contrast, the aerobic digester experiments had a less substantial effect on gene quantities, although the residence time was much shorter than the anaerobic digesters, perhaps leading to a smaller impact. We speculate that thermophilic aerobic digestion operated at longer residence times (>15 days) would achieve similar removal efficiencies of antibiotic resistance genes compared to the anaerobic digester experiments described herein (note: due to cost considerations, full-scale thermophilic aerobic digester operated at longer residence times is likely impractical). High-temperature anaerobic digestion appears to be particularly effective at reducing the quantities of class 1 integrons in wastewater solids. Integrons are genetic elements that allow bacteria to incorporate exogenous gene cassettes and modulate their expression (17). As such, integrons are believed to be key genetic elements involved in the exchange and integration of numerous resistance genes, leading to the development of multiple antibiotic resistance (17, 20). Although several researchers have previously reported the presence of integrons in wastewater solids and wastewater effluents (7, 12, 15) as well as animal manure (21, 22), relatively little research has explored the fate of integrons in various engineered treatment systems. Our present research, therefore, makes critically important contribution to the technical literature by demonstrating that thermophilic anaerobic digestion can eliminate more than 99.9% of class 1 integrons. Additional research is needed to determine the mechanisms by which the quantities of resistance genes decline, are sustained, or (possibly) increase during the digestion of wastewater solids. Wastewater solids are a complex mixture of particulate substrates, bacteria, and other material collected from the primary and secondary clarifiers at municipal wastewater treatment facilities. The quantity of resistance genes, therefore, could decline as their bacterial hosts perish or as their bacterial hosts jettison genetic information (e.g., via plasmid curing, transposition, etc.). Resistance genes could be sustained in digesters if their bacterial hosts survive or if the genes are transferred to other organisms at a rate equaling the decay rate of their original host(s). Finally, the quantity of resistance genes could increase in a digester if a lateral gene transfer event occurred in which genetic material was transferred to an organism that propagated/ thrived under the specific conditions imposed during digestion. This latter scenario is particularly worrisome because full-scale digesters are intentionally designed to be dense microbial suspensions, which are simultaneously conducive to bacterial conjugation. A substantial limitation of this research is the method used to quantify antibiotic resistant bacteria. Quantitative PCR characterizes the presence of gene fragments, regardless of whether the host is viable or the gene is functional within that host. The alternative approach - cultivating resistant bacteria - is known to represent only a small fraction of the total bacterial community (23). The quantitative PCR approach is also advantageous for studying antibiotic resistance 9132
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because it targets the genotype, which can be potentially shared among bacteria via lateral gene transfer, rather than targeting the phenotype - as would be the case with a cultivation-based approach. Indeed, previous researchers have proposed that antibiotic resistance genes be considered an emerging pollutant of concern (24). In conclusion, anaerobic digesters operated at elevated temperatures can substantially reduce the quantities of tetracycline resistance determinants as well as class 1 integrons in wastewater solids. Because municipal wastewater is a substantial reservoir of antibiotic resistance, wastewater treatment processes offer an excellent opportunity to actively target and eliminate antibiotic resistant bacteria. The present research demonstrates that thermophilic anaerobic digestion can be very effective at eliminating various antibiotic resistance genes, which should lead to a substantial decrease in the number of resistant bacteria being introduced into the environment.
Acknowledgments This research was financially supported by the Minnesota Environment and Natural Resources Trust Fund.
Supporting Information Available qPCR primers and conditions (Table S1), additional results regarding tetracycline resistance and intI1 genes normalized to 16S rRNA genes (Figures S1 and S2), the production of biogas from the anaerobic digesters (Figure S3), and the total solids concentrations in the anaerobic digesters (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.
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