Effect of Different Treatment Technologies on the Fate of Antibiotic

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The Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil Tucker Burch, Michael J. Sadowsky, and Timothy M. LaPara Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04760 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Environmental Science & Technology

The Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil Tucker R. Burch,1 Michael J. Sadowsky,2, 3, 4 and Timothy M. LaPara1,2* 1

University of Minnesota Department of Civil, Environmental, and Geo- Engineering Minneapolis, MN 55455 2

University of Minnesota Biotechnology Institute St. Paul, MN 55108

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University of Minnesota Department of Soil, Water, and Climate St. Paul, MN 55108 4

University of Minnesota Department of Plant and Microbial Biology St. Paul, MN 55108

Current affiliation: USDA-ARS, Environmentally Integrated Dairy Management Research Unit, Marshfield, WI 54449 *

Correspondence: Timothy M. LaPara, University of Minnesota, Department of Civil, Environmental, and Geo- Engineering, 500 Pillsbury Drive SE, Minneapolis, MN 55455. Tel: (612) 624-6028. Fax: (612) 626-7750. Email: [email protected] Word Count: 3,092 + two figures (300+300) and three tables (300+300+600) = 5,092

ACS Paragon Plus Environment


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ABSTRACT

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Residual wastewater solids are a significant reservoir of antibiotic resistance genes

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(ARGs). While treatment technologies can reduce ARG levels in residual wastewater solids, the

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effects of these technologies on ARGs in soil during subsequent land-application are unknown.

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In this study we investigated the use of numerous treatment technologies (air drying, aerobic

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digestion, mesophilic anaerobic digestion, thermophilic anaerobic digestion, pasteurization, and

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alkaline stabilization) on the fate of ARGs and class 1 integrons in wastewater solids-amended

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soil microcosms. Six ARGs (erm(B), qnrA, sul1, tet(A), tet(W), and tet(X)), the integrase gene

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of class 1 integrons (intI1), and 16S rRNA genes were quantified using quantitative PCR. The

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quantities of ARGs and intI1 decreased in all microcosms, but thermophilic anaerobic digestion,

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alkaline stabilization, and pasteurization led to the most extensive decay of ARGs and intI1,

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often to levels similar to the control microcosms to which no wastewater solids had been applied.

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In contrast, the rates by which ARGs and intI1 declined using the other treatment technologies

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were generally similar, typically varying by less than 2-fold. These results demonstrate that

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wastewater solids treatment technologies can be used to decrease the persistence of ARGs and

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intI1 during their subsequent application to soil.

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TOC ART

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INTRODUCTION Bacterial resistance to antibiotics is a major public health dilemma with serious health

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and financial consequences.1,2 Numerous strategies are being pursued to mitigate the effects of

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antibiotic resistance, including the development of new or alternative antibiotics, the

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development of alternatives to antibiotics, and the reduction of antibiotic use for purposes other

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than treating diseases in humans.3-8 An alternative and complementary strategy consists of

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identifying and mitigating antibiotic resistance genes (ARGs) as environmental contaminants.9

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This has led to the identification of numerous environmental reservoirs of ARGs, including

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surface waters, aquaculture facilities, and agricultural waste.9-23 One of the largest reservoirs,

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however, appears to be untreated municipal wastewater and the residual municipal wastewater

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solids produced from the wastewater treatment process.9, 14, 24-34 The treatment of residual

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wastewater solids, therefore, may be an excellent opportunity to mitigate the release of ARGs to

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the environment.

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Previous research has demonstrated the potential for existing treatment technologies to

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remove ARGs from residual solids. These technologies, referred to as Processes to Significantly

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Reduce Pathogens (PSRPs) and Processes to Further Reduce Pathogens (PFRPs),36 are currently

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designed to reduce the water, organic carbon, and pathogen content of untreated residual

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municipal wastewater solids.37 However, PSRPs and PFRPs have also been demonstrated to

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remove ARGs from untreated residual municipal wastewater solids,38-41 both by reducing the

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overall number of microorganisms in the residual solids and by creating environmental

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conditions that fail to select for particular ARGs based on their ecological niches.41-42 Aerobic

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digestion, air-drying beds, and anaerobic digestion have all been demonstrated to remove ARGs

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from residual municipal wastewater solids to varying degrees. Moreover, ARG removal can be

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optimized by adding pretreatment steps, increasing treatment temperatures, and inducing feast-

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famine (i.e. batch) cycles.38-42

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The degree to which all of these technologies affect the fate of ARGs in soil following

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land-application of treated residual solids, however, is unknown. This is a significant gap in the

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body of knowledge, because approximately 50% of all treated residual municipal wastewater

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solids are land-applied in the United States.43 Furthermore, previous work has indicated that

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ARGs originating in anaerobically-digested (35°C) residual solids can persist for months at

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relatively high concentrations in soil following land-application.44 This leads to the potential

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opportunity for off-site transport of, and ultimately human exposure to, ARGs with stormwater

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or airborne particulate matter.45-46 Many current PSRP and PFRP technologies are widely used

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in the U.S.43 or could be retrofitted to existing infrastructure with relative ease. Thus, their

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implementation for controlling the quantities of ARGs circulating in the environment could

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represent a tractable management strategy for preventing antibiotic resistant infections.

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The objective of this work was to determine how different technologies used to treat

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residual wastewater solids affected the persistence of ARGs and class 1 integrons in soil

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following land-application of treated residual solids. The treatment technologies examined

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included aerobic digestion at 17°C, air drying, alkaline stabilization, pasteurization, and

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anaerobic digestion at 38°C, 55°C, 63°C and 69°C. In addition, untreated residual municipal

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wastewater solids were examined to represent a “worst-case scenario” control for the application

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of ARGs to soil. Residual solids from each of the nine sources were added to soil microcosms,

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and the quantities of several ARGs and the integrase gene of class 1 integrons were determined

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in each microcosm via quantitative PCR over a six-month time series.

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MATERIALS AND METHODS Experimental Design. Soil microcosms were constructed as described previously.44

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Briefly, triplicate microcosms were used for each experiment were comprised of a mixture of

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200 g of agricultural soil (Waukegan silt loam; fine-silty, mixed, mesic Typic Hapludoll; initial

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moisture content of 12.1% ± 0.3%) thoroughly mixed with 8 g (wet mass) of residual wastewater

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solids. No manure or wastewater solids had been previously applied to the soil. This mass

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loading rate of residual wastewater solids to soil is typical of common practices.37 All

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microcosms were stored at room temperature (approximately 20°C). Evaporative water losses

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for all microcosms were estimated by weighing the microcosms at approximately monthly

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intervals and assuming that all mass loss was due to evaporation of water.

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Untreated wastewater solids were obtained from a municipal wastewater treatment

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facility in south-central Minnesota. These solids were then treated by various laboratory-scale

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simulations of full-scale processes commonly used to treat wastewater solids. Air-dried solids,41

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aerobically-digested solids 40 and anaerobically digested solids42 were collected from the

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laboratory-scale treatment units described previously. To produce alkaline stabilized residual

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solids, the pH of untreated residual solids was increased to above 12 for a period of two hours

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using sodium hydroxide. The pH was then neutralized using hydrochloric acid. To produce

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pasteurized residual solids, the temperature of untreated residual solids was increased to more

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than 70°C for a period of 30 minutes.

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Sample Collection and Genomic DNA Extraction. Microcosms were mixed prior to

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each sample collection event and then triplicate samples of ~0.5 g were collected from each the

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triplicate soil microcosms. Samples were stored at -20 °C until DNA was extracted and purified

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using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) and a BIO 101 Thermo

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Savant FastPrep FP120 Cell Disruptor (Qbiogene, Inc., Carlsbad, CA).

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Real-time PCR. The forward and reverse primer sequences, expected amplicon sizes,

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and primer annealing temperatures for all gene targets are provided in the Supporting

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Information (Table S1). The genes targeted in this study were selected based on prior studies that

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demonstrated each of these genes is easily quantifiable in untreated wastewater solids.38, 40-42, 44

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The number of standards, slope, intercept, amplification efficiency, r2, and quantification limit

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for each standard curve are also provided in the Supporting Information (Table S2). Of the 58

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assays performed, 26 (45%) had amplification efficiencies of 100% ± 10% (maximum deviation

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from 100%) with r2 ≥ 0.99 for a minimum of five points on each standard curve. The absolute

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values by which amplification efficiencies for the remaining 32 assays deviated from 100% were

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between 11% and 27% (median of 15%) with r2 ≥ 0.98 for a minimum of five points on each

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standard curve. No template controls were performed during all qPCR assays. The amplification

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efficiencies of experimental samples were visually inspected to ensure that their amplification

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efficiency matched that of the standards.

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Data Analysis. Prior experience with qPCR analysis of ARGs suggested that the data

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are best described by a log10-normal distribution.41-42, 44 In this current study, therefore, the data

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were log10-transformed prior to statistical analysis. Tukey’s honest significance difference test

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was performed using JMP Pro. Ver. 12.0.1 (SAS Institute, Inc.; Cary, NC).

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ARG data from the soil microcosms were fit to a modified Collins-Selleck model47-48 using the following equation:

= −

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In this equation, N is the number of ARG copies at time t, N0 is the initial quantity of

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ARG, ΛCS is the specific lethality coefficient, and b is the lag coefficient. This modified Collins-

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Selleck model has been previously used to fit ARG decay data in anaerobic digestors42 and in

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soil microcosms.49 ARG data from the soil microcosms were also fit to a first-order model, as

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described previously,44 except that the initial time point was excluded from analysis.

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RESULTS Untreated wastewater solids were collected from a full-scale municipal wastewater

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treatment facility and processed using numerous bench-scale simulations of full-scale

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technologies used to treat wastewater solids (air drying, aerobic digestion, mesophilic anaerobic

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digestion, thermophilic anaerobic digestion, pasteurization, and alkaline stabilization). These

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approaches generated treated wastewater solids that contained relatively similar quantities of

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bacterial biomass (i.e., within one log10 unit), but markedly different levels of ARGs and class 1

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integrons (Table 1).

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Negative control soil microcosms (i.e., microcosms to which no wastewater solids were

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applied) were described previously.44 Briefly, sul1 and intI1 were detected in about 50% of the

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samples in the negative control microcosms at concentrations near the detection limit (~103 gene

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copies per gram of wet soil), whereas erm(B) and tet(X) were detected less frequently. In

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contrast, tet(A) and tet(W) were never detected in the negative control soil microcosms.

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The quantities of bacterial biomass, measured as 16S rRNA gene copy number, generally

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followed similar trends for all experimental treatments. The quantities of 16S rRNA genes were

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statistically unchanged (P > 0.05) throughout the experiment for most experimental treatments,

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ranging in concentration from 1.5 1010 gene copies per g of soil (alkaline stabilization) to 9.3

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1010 gene copies per g of soil (anaerobic digestion at 38°C). In the soil microcosms receiving

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untreated wastewater solids or wastewater solids treated by aerobic digestion or pasteurization,

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the quantities of 16S rRNA genes declined (P ≤ 0.05) at relatively slow rates, with half-lives

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varying between 120 day (aerobic digestion) and 230 days (wastewater solids without treatment).

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Three common trends were observed in the quantities of ARGs and intI1 after treated

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wastewater solids had been applied (Figs. 1-2; Figs. S1-S5): (1) a pattern in which the quantities

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of ARGs rapidly declined initially (i.e., < 30 days), but then subsequently declined at a much

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slower rate. This trend could be be modeled using a modified Collins-Selleck model or using a

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first-order model that neglected the initial time point.; (2) the quantities of ARGs and intI1

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followed a first-order model throughout the experiment; and (3) the quantities of ARGs (intI1 did

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not exhibit this trend) were initially detectable but then declined such that they could no longer

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be detected. This last trend was not fit to any kinetic model.

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The rates at which different ARGs and intI1 decayed in soils exhibited a myriad of

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effects with respect to the technology used to treat the wastewater solids prior to soil application,

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independent of which regression model was applied (Table 2). For many of the combinations of

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genes and treatment technologies, decay coefficients could not be computed because gene

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quantities declined below the detection limits of the assays. Presumably, these treatment

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technologies either significantly reduced the quantities of these genes such that the quantities

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were at or below the detection limits at the initiation of the soil microcosm experiments or these

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genes decayed very rapidly such that decay coefficients could not be computed. This inability to

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regress decay coefficients occurred most commonly with wastewater solids treated by

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thermophilic anaerobic digestion, which achieved substantial removal of all of these genes prior

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to application to soils.42 In contrast, pasteurization and alkaline stabilization did not achieve

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substantial removal of ARGs (Fig. S6) even though it is likely that both of these technologies

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were very effective at killing the bacteria in the wastewater solids that harbored these genes;

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thus, the cells almost certainly died, but their DNA remained intact and susceptible to

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amplification via PCR. In these cases, the inability to compute a decay rate was likely due to

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rapid decay rather than a low initial quantity of ARGs in the soils. Decay coefficients could be

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computed for intI1 and sul1, regardless of the wastewater solids treatment technology. With

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both of these genes, the decay coefficients were relatively similar, varying by less than an order

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of magnitude, independent of the technology used to treat wastewater solids.

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A substantial variation was observed in the quantities of ARGs after the soil microcosms

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had been incubated for six months (Table 3). In many cases, the quantities of ARGs in the soils

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were below the detection limits, similar to the control experiments. This result was most

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common for ARGs following thermophilic anaerobic digestion. Only two genes (intI1 and sul1)

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were quantifiable in all microcosms, independent of the treatment technology. Thermophilic

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anaerobic digestion (55°C and 69°C), pasteurization, and alkaline stabilization led to the

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statistically lowest quantities of intI1. Similarly, the same upstream treatment technologies led

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to the statistically lowest quantities of sul1.

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DISCUSSION

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There is a growing body of literature that has identified numerous reservoirs of antibiotic

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resistant bacteria and ARGs in the environment.50-54 Untreated municipal wastewater appears to

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be a particularly large reservoir of ARGs,30, 55 of which more than 90% leave municipal

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wastewater treatment facilities with the residual wastewater solids.30 Because wastewater solids

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are commonly applied to agricultural soils, it is critically important to understand the fate of

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ARGs in soils after land-application. While previous studies have explored the fate of ARGs

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following the application of wastewater solids to soil,44, 56 the present study provides new

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knowledge by delineating the role of the various treatment technologies on the rate and extent by

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which ARGs decay in soil. This research demonstrates that treatment technologies already

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known to aggressively inactivate pathogens in wastewater solids, such as thermophilic anaerobic

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digestion, alkaline stabilization, and pasteurization (most of these are PFRPs), lead to more

222

extensive decay of ARGs than less aggressive treatment technologies, such as aerobic digestion,

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mesophilic anaerobic digestion, and air drying.

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The various technologies used to treat wastewater solids had only a marginal effect on the

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rates (usually less than 2-fold) of decay of ARGs and intI1 after residual wastewater solids were

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applied to soil. Similarly, pasteurization and alkaline stabilization often led to comparatively

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rapid decay rates when treated wastewater solids were applied to soil, albeit these rates are likely

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an artifact of qPCR. We failed to observe substantial decay of ARGs during the pasteurization

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and alkaline stabilization of wastewater solids (Fig. S6), even though both of these technologies

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are known to aggressively inactivate bacteria.57 We assumed that pasteurization and alkaline

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stabilization processes successfully inactivated most bacteria in the wastewater solids during the

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relatively short, but lethal, incubations at high temperature and pH, respectively. Their DNA,

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however, remained intact, thus leading to artificially high quantifications of ARGs and intI1 at

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the initiation of our soil microcosm experiments. The quantities of these genes then decayed at

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comparatively rapid rates in soil because they were harbored by dead organisms. In contrast,

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thermophilic anaerobic digestion was able to achieve substantial ARG and intI1 removal prior to

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soil application, such that the subsequent decay of ARGs and intI1 were slower because they

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were harbored by viable organisms.

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The various technologies used to treat wastewater solids had a substantial effect on the

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extent of decay of ARGs and intI1 after residual wastewater solids were applied to soil. The

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treatment technologies that are known to aggressively inactivate human pathogens (thermophilic

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anaerobic digestion, pasteurization, and alkaline stabilization) usually resulted in either ARG

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levels that were below the detection limit of our qPCR assays (i.e., similar to our negative

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control microcosms) or were statistically the lowest levels of our experimental comparisons

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(albeit higher than our negative control microcosms). This research, therefore, makes a seminal

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contribution to new knowledge by identifying a potentially sustainable approach to managing

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ARGs and intI1 genes during wastewater treatment. These results are substantially different than

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what was previously reported for the application of untreated animal manure in which ARG

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levels in soils did not return to pre-manure-application levels within six months.49, 58 Thus, our

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research also suggests that the treatment of animal manure prior to application to soil could be

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beneficial for limiting the spread of antibiotic resistance from animal feeding operations.

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The present study demonstrated that the fate of ARGs and intI1 in wastewater solids

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applied to soils can be kinetically-modeled, as has been observed previously when animal

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manure was applied to soil.49 The ability to model the decay of ARGs is of importance because

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it allows different studies and experiments to be compared and because it allows for predictions

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on the fate of ARGs in future applications. Although the utility of kinetic modeling in

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understanding and predicting the fate of ARGs is clear, relatively few studies thus far have

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performed kinetic modeling.

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The kinetic modeling used herein suggests that there are multiple mechanisms by which

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ARGs decay after wastewater solids are applied to soil. In many of our experiments, ARGs

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initially declined rapidly but then slowed substantially. In this study, we modeled these

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phenomena with a modified Collins-Selleck model, similar to prior experiments on the fate of

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ARGs during thermophilic anaerobic digestion42 and when animal manure was applied to soils.49

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Alternatively, the data could likely be modeled using two first-order models, as previously used

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to describe aerobic digestion of wastewater solids,40 although this approach was not generally

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feasible in this study due to the lack of data during the initial period during which ARG decay

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was rapid. We hypothesize that the initial rapid decay of ARGs occurs because inactivated cells

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and bacteria incapable of living in soil perish and release their DNA, which is then rapidly turned

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over via normal biogeochemical cycling. Additional research is needed to test this hypothesis as

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well as to understand the mechanisms by which ARGs appear to slowly decay in soil after this

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initial period of rapid decay.

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This study identified approaches to better remove class 1 integrons from wastewater

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solids. Integrons are believed to be particularly pertinent for the spread of antibiotic resistance

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because they enable both horizontal gene transfer and the accumulation of multiple ARGs in an

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individual bacterium.59-60 In fact, integrons have been shown to contain multiple ARGs in

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environmental samples.61-65 Similar to the ARGs, intI1 genes were best removed by treatment

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technologies known to aggressively inactivate human pathogens (thermophilic anaerobic

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digestion, pasteurization, and alkaline stabilization). Curiously, the application of untreated

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wastewater solids to soils resulted in a greater extent of intI1 decay than if air drying, aerobic

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digestion, or mesophilic anaerobic digestion was used to treat the wastewater solids prior to soil-

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application.

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Although the experiments described here uncovered important information regarding the

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fate of ARGs and class 1 integrons when wastewater solids are applied to soil, our study also

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suffered from several limitations due to our experimental design. The qPCR assays used in this

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work cannot distinguish between genes in viable cells from extracellular DNA or from DNA of

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dead but intact bacteria. This is pertinent because a substantial fraction of ARGs can be

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extracellular66-67 or in inactivated cells.57 Another limitation is that the microbial hosts of the

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ARGs and intI1 genes remain unknown. This is pertinent because ARGs are more worrisome in

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pathogens than in bacteria benign to humans. Similarly, ARGs have been found in bacteria

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lacking the capability of expressing these genes.68 Our study was also limited because the soil

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microcosms used were kept under the controlled conditions of a laboratory rather than being

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exposed to highly variable field conditions, which include direct sunlight, natural fluctuations in

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moisture content and temperature, and the presence of other organisms. Finally, the work

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presented in this study investigates only a small fraction of known ARGs. Other researchers

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have quantified ARGs by shotgun metagenomics,69-70 although the sensitivity of shotgun

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metagenomics is poor compared to qPCR. Similarly, other researchers have developed assays to

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simultaneously track more than 250 ARGs and mobile genetic elements by PCR,23, 56 although

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we question the validity of these PCR arrays to provide quantitative information because they

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typically do not use external standards for more than a single gene.

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SUPPORTING INFORMATION The Supporting Information contains data regarding the qPCR primers, conditions, and

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performance (Tables S1-S2), a table of the extent of ARG and intI1 removal in the soil

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microcosms (Table S3), the quantities of other ARGs and intI1 in all experimental microcosms

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(Figures S1-S5), and the quantities of 16S rRNA genes, intI1, and tet(X) during pasteurization

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and alkaline stabilization experiments (Figure S6). This information is available free of charge

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via the Internet at http://pubs.acs.org/.

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ACKNOWLEDGEMENTS This work was supported by National Science Foundation grant 0967176.

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518

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Table 1. Quantities of genes encoding 16S rRNA, five different antibiotic resistance determinants, and the integrase of class 1 integrons in wastewater solids treated in different ways prior to being applied to soil microcosms. Quantities are presented as the arithmetic means (± standard deviation; n = 3) of log10-transformed gene copies per gram of wet sludge. No Treatment

Air dried

Aerobic Anaerobic Anaerobic Anaerobic Anaerobic Alkaline Pasteurization Digestion Digestion Digestion Digestion Digestion Stabilization (17°°C) (38°°C) (55°°C) (63°°C) (69°°C)

16S rRNA

9.4 ± 0.2

9.2 ± 0.4

9.8 ± 0.1

9.4 ± 0.3

9.5 ± 0.1

9.3 ± 0.1

9.0 ± 0.1

10.2 ± 0.03

10.1 ± 0.05

erm(B)

7.5 ± 0.5

4.9 ± 1.1

6.7 ± 0.1

6.7 ± 0.3

6.0 ± 0.2

6.3 ± 0.2

6.6 ± 0.1

8.8 ± 0.05

8.9 ± 0.05

intI1

7.2 ± 0.2

6.8 ± 0.4

8.3 ± 0.1

6.7 ± 0.2

6.6 ± 0.1

6.8 ± 0.1

6.4 ± 0.1

8.8 ± 0.03

8.9 ± 0.03

sul1

7.6 ± 0.1

6.6 ± 0.4

8.0 ± 0.1

7.0 ± 0.3

6.5 ± 0.1

6.6 ± 0.1

6.2 ± 0.1

9.2 ± 0.07

8.7 ± 0.02

tet(A)

6.0 ± 0.2

4.3 ± 1.0

7.2 ± 0.1

5.5 ± 0.3

5.8 ± 0.1

5.8 ± 0.1

5.4 ± 0.1

7.4 ± 0.01

7.7 ± 0.04

tet(W)

7.3 ± 0.2

4.6 ± 0.3

6.2 ± 0.1

6.5 ± 0.3

6.4 ± 0.1

6.4 ± 0.2

6.5 ± 0.1

8.1 ± 0.07

8.4 ± 0.03

tet(X)

5.5 ± 0.3

5.6 ± 0.5

8.0 ± 0.1

4.3 ± 0.4

4.1 ± 0.1

4.3 ± 0.1

3.9 ± 0.3

7.4 ± 0.1

7.6 ± 0.02

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Table 2. Kinetic coefficients regressed when the decay of ARGs and intI1 were fit to a modified CollinsSelleck model (ΛCS, b) and a first-order model (k). n.d. = not determined due to a poor fit to the CollinsSelleck model or due to insufficient data associated with gene levels below the quantification limit.

Gene

Treatment

ΛCS

b (d)

k (d)

t1/2

erm(B)

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

-1.6 n.d. -1.3 -1.6 n.d. n.d. n.d. -1.4 -1.0

0.3 n.d. 16 0.3 n.d. n.d. n.d. 0.9 0.07

0.02 n.d. 0.01 0.02 n.d. n.d. n.d. 0.02 0.01

41 n.d. 49 30.2 n.d. n.d. n.d. 46 64.8

intI1


-1.0 -0.5 -0.8 -0.8 -0.3 -0.2 -0.6 -0.4 -0.4

15 21 6.4 93 1.8 0.2 14 0.003 0.2

0.01 0.006 0.008 0.009 0.004 0.002 0.007 0.005 0.004

52 124 86 75 187 330 99 151 169

sul1

No Treatment Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization

-0.8 -0.5 -0.7 -0.9 -0.3 -0.07 -0.6 -0.7

10 20 10 27 0.03 500 103 90

Alkaline stabilization

-0.5

0.02

0.005

151

tet(A)


-1.1 n.d. -0.5 -1.3 n.d. n.d. n.d. -0.2

0.4 n.d. 5.3 230 n.d. n.d. n.d. 1.4

0.01 n.d. 0.006 0.02 n.d. n.d. n.d. 0.002 0.003

60 n.d. 122 46 n.d. n.d. n.d. 408 203

tet(W)

No Treatment

-1.3

16.6

0.01

60

22



tet(X)

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Air drying Aerobic Digestion (17°C) Anaerobic Digestion (38°C) Anaerobic Digestion (55°C) Anaerobic Digestion (63°C) Anaerobic Digestion (69°C) Pasteurization Alkaline stabilization

n.d. -1.4 -0.2 n.d. n.d. n.d.

n.d. 8.0 1.4 n.d. n.d. n.d.

n.d. 0.015 0.002 n.d. n.d. n.d.

n.d. 47 346 n.d. n.d. n.d.

-0.2 -0.3

< 0.001 < 0.001

0.004 0.003

182 203


-2.1 -0.7 -1.6 n.d. n.d. n.d. n.d. n.d. n.d.

6.1 65.5 17.8 n.d. n.d. n.d. n.d. n.d. n.d.

0.02 0.008 0.02 n.d. n.d. n.d. n.d. n.d. n.d.

30 87 39 n.d. n.d. n.d. n.d. n.d. n.d.

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Table 3. Quantities of genes encoding 16S rRNA, five different antibiotic resistance determinants, and the integrase of class 1 integrons in soils to which wastewater solids had been applied and then subsequently incubated for 6 months. Wastewater solids were treated in different ways prior to being applied to soil microcosms. Quantities are presented as the arithmetic means (± standard deviation; n = 3) of log10-transformed gene copies per gram of wet soil. Superscripted letters denote groups that statistically clustered with each other (P < 0.05). All gene quantities were below the detection limit quantities in the control soil microcosms (to which no wastewater solids were applied) for all genes except for 16S rRNA genes (7.7 ± 0.1). b.d. = below detection.

No Treatment

Air drying

Aerobic Digestion (17°°C)

Anaerobic Digestion (38°°C)




Pasteurization

Alkaline Stabilization

16S rRNA

8.0 ± 0.2A

7.4 ± 0.03B,C

7.7 ± 0.04A,B

7.7 ± 0.5A,B

7.5 ± 0.1B,C

7.7 ± 0.02A,B

7.8 ± 0.1A,B

7.1 ± 0.2C

7.1 ± 0.03C

erm(B)

3.2 ± 0.2B

b.d.

4.0 ± 0.1A

b.d.

b.d.

b.d.

b.d.

3.0 ± 0.1B

3.1 ± 0.1B

intI1

3.4 ± 0.01C,D

5.1 ± 0.4A

5.0 ± 0.1A

4.3 ± 0.1B

3.7 ± 0.1C,D

3.9 ± 0.1B,C

3.3 ± 0.1D

3.9 ± 0.4B,C,D

3.9 ± 0.01B,C

sul1

4.6 ± 0.1B,C

5.7 ± 0.5A

5.0 ± 0.04A,B

4.1 ± 0.01B,C,D

3.2 ± 0.1D,E

3.6 ± 0.1D,E

2.9 ± 0.4E

3.7 ± 0.8C,D,E

3.5 ± 0.1D,E

tet(A)

b.d.

b.d.

4.2 ± 0.1A

4.0 ± 0.3A

b.d.

b.d.

b.d.

b.d.

2.8 ± 0.1B

tet(W)

4.3 ± 0.01A

b.d.

2.8 ± 0.1B

b.d.

b.d.

2.4 ± 0.1C

2.2 ± 0.1C

b.d.

b.d.

tet(X)

4.5 ± 0.03A

4.1 ± 0.6A

4.6 ± 0.1A

b.d.

b.d.

b.d.

b.d.

b.d.

b.d.

24



Figure 1. Quantities of ARGs and intI1 following the application of untreated wastewater solids to soil. Solid circles represents individual qPCR quantifications performed in triplicate from each of triplicate microcosms. The solid line represents the best fit to a modified Collins-Selleck model; the dashed line represents the best fit to a first-order kinetic model, excluding the initial time point. The regressed coefficients for the kinetic models are shown in Table 2.

25


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Figure 2

Figure 2. The quantities of sul1 genes following the application of treated wastewater solids to soils. Solid circles represents individual qPCR quantifications performed in triplicate from each of triplicate microcosms. The solid line represents the best fit to a modified Collins-Selleck model; the dashed line represents the best fit to a first-order kinetic model, excluding the initial time point. The regressed coefficients for the kinetic models are shown in Table 2. Analogous plots for the quantities of erm(B) (Fig. S1), intI1 (Fig. S2), tet(A) (Fig. S3), tet(W) (Fig. S4), and tet(X) (Fig. S5) are provided in the Supporting Information.

26



TOC graphic 338x190mm (300 x 300 DPI)


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Effect of Different Treatment Technologies on the Fate of Antibiotic

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