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Human Mitochondrial DNA and Endogenous Bacterial Surrogates for Risk Assessment of Graywater Reuse Brian D. Zimmerman,†,‡ Nicholas J. Ashbolt,‡,§ Jay L. Garland,‡ Scott Keely,‡ and David Wendell*,† †

Department of Biomedical, Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio 45221, United States ‡ Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, United States S Supporting Information *

ABSTRACT: Previous graywater risk assessment studies have focused on fecal contamination, yet the low density of fecal indicators may not provide the most useful approach to assess pathogen removal during graywater treatment. In this study, we employed high throughput bacterial sequencing and qPCR to elucidate potential microbial surrogates in wastewater sourced from an industrial laundry. In addition, we explored human mitochondrial DNA (HmtDNA) as a new, potentially more reliable molecular marker, because it can be unambiguously sourced, has a high copy number per cell, and is persistent when released from cells with no self-replication in graywater. Pyrosequencing and qPCR revealed that laundry water microbiota was dominated by the skin-associated bacteria Staphylococcus, Corynebacterium, and Propionibacterium (6.5, 5.7, 5.4 log10 copies/100 mL, respectively). While HmtDNA was less abundant (2.8 log10 copies/100 mL), it showed a strong positive correlation with the opportunistic pathogen Staphylococcus aureus (r = 0.54, P = 3.2 × 10−4) and closely followed a first-order exponential decay model (R2 = 0.98), remaining detectable in stored laundry graywater for up to 6 days at 20 °C. Based on abundance and persistence, we propose HmtDNA and total Staphylococcus as future laundry graywater treatment surrogates to potentially assess a wide dynamic range of pathogen removal.

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INTRODUCTION Graywater can be broadly defined as any wastewater within a home or residential setting that does not include toilet flush water.1 Sources can include dishwashing, shower, bath, laundry, and sink water; of these, the most abundant by volume is laundry.2 Recycling domestic graywater can assist water distribution systems by creating a decentralized water source capable of reducing potable demand on stressed supplies in arid regions, during periods of water shortage, and where population increase outpaces available supplies.1,3 However, due to varying but significant levels of traditional wastewater parameters such as COD, BOD, TSS, VSS, turbidity, pH, and electrical conductivity,4−6 graywater quality has been described as containing contaminants between that of raw wastewater and secondary effluent.7 In addition, opportunistic and/or enteric bacterial, viral, and protozoan pathogens have been found in graywater,6−10 suggesting a health risk for reuse of untreated graywater. © 2014 American Chemical Society

Of these possible contaminants, microbial pathogens of enteric origin transferred via fecal material are considered to present one of the greatest public health risks for graywater reuse.9 Thus, biological water quality techniques targeting fecal indicator bacteria (FIB) have been the focus of previous graywater microbial risk assessments.5,8,10 However, traditional biological indicators, such as total coliforms, fecal coliforms, and enterococci are known to grow in graywater,11 and more specific fecal indicators, such as E. coli and Clostridium perf ringens/sulphite-reducing clostridia, are not likely to vary quantitatively with pathogen presence.12 Given the potential pathogen presence and downstream human exposure to graywater, it has been suggested that Received: Revised: Accepted: Published: 7993

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logical presence and highest microbial risk associated with graywater reuse.6 All grab samples were placed on ice immediately after collection and transported to the laboratory for sample concentration within 2 h. Sample Concentration and DNA Extraction. Samples were vacuum filtered through 47 mm diameter, 0.45 μm mixed cellulose ester (nitrocellulose) membranes (Pall Corporation, Inc.). Filtrate volume ranged from 70 to 380 mL to ensure adequate DNA yields. Filters were removed from the filter cone apparatus using sterile forceps and placed into 47 mm Petri dishes (Pall Corp., Inc.) and processed immediately or stored at −70 °C until DNA extraction. DNA was extracted using the PowerWater DNA Extraction Kit (MoBio Laboratories, Inc.) according to the manufacturer’s instructions. DNA extracts were quantified using a ND-1000 nanodrop spectrophotometer (NanoDrop Technology, Wilmington, DE) and stored at −70 °C. High Throughput Sequencing. To identify the bacterial community dominant in graywater, a portion (n = 24) of samples were sequenced on the 454 pyrosequencing platform (Roche, Inc.) using primers 454B_27F 5′-CTATCCCCTGTGTGCCTTGGCAGTCTCAGAGAGTTTGATCCTGGCTCAG-3′ and 454A_534R 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGATTACCGCGGCTG-CTGG-3′ corresponding to the V1 through V3 regions of the 16S rRNA gene.21 The PCR reaction contained 1 μL of 10 mM dNTP mix, 1 μL of DMSO, 5 μL of 10X FastStart Buffer containing 1.8 mM MgCl2, 3 μL of 10 μM forward and reverse primers, 5 units/μL FastStart HiFi enzyme, 10 ng of DNA, and PCRgrade water to a final volume of 50 μL. PCR cycling was run on the MJ 7000 cycler (Bio Rad) under the following conditions: 95 °C for 2 min followed by 25 cycles of 95 °C for 40-s, 56 °C for 30-s, and 72 °C for 1 min followed by a final extension of 72 °C for 7 min. No template controls (NTCs) were run with each reaction and, along with the PCR products, visualized on precast E-gel 2% agarose ethidium bromide gels (Applied Biosystems, Inc.) for amplification confirmation. Triplicate PCR was pooled by volume (20 μL per replicate) for 454 pyrosequencing. Over the course of the experiment, all NTC’s (n = 32) were negative. Raw sequences were trimmed using the Ribosomal Database Project’s (RDP) pipeline initial process using the suggested parameters with a Q-score of 25 and minimum sequence length of 150 base pairs. Processed reads were run through RDP’s UCHIME chimera slayer,22 aligned according to the rRNA secondary structure,23 and classified using the 16S rDNA training set 9 within RDP.24 Reads were visualized in MEGAN version 4.7 with the minimum lowest common ancestor parameter of 80.25 Quantitative PCR Standards. Bacterial species used for standards included Bacteroides f ragilis (HE608156),26,27 Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (S. aureus) (ATCC 25923), Escherichia coli (NCTC 12923), and Enterococcus faecalis (NCTC 12697). Genomic DNA was provided (see Acknowledgments) and extracted using the DNEasy Blood and Tissue Kit (Qiagen, Inc.) according to the manufacturer’s instructions and quantified via Nanodrop 1000 (NanoDrop Technology). Bacterial gene copies were estimated based on DNA yield and the approximated genome mass and GC content, available in the Supporting Information (Table S1). The HmtDNA standard curve method was obtained via serial dilution of a purified amplicon as described previously,28 with the exception that the HmtDNA amplicon came from a

graywater undergo biological treatment and disinfection prior to reuse.5 USEPA water reuse guidelines suggest the use of fecal coliforms as the treatment surrogate,13 but this target can exhibit growth in graywater11 and has been unreliable when predicting viral and protozoan removal/inactivation.13 Hence there is a need for new (metabiomic) data that may assist with identifying novel treatment surrogates for monitoring treatment performance in reuse scenarios.3 The decentralized approach of graywater reuse likely renders external surrogate spiking uneconomical, resulting in a need for endogenous molecular metrics indicative of treatment efficiency.14 Given the public’s perception of health risks associated with graywater reuse,15 a scientifically conservative and tolerable infection risk of nonpotable reuse could be 1:10,000, which necessitates a 4-log removal/inactivation of surface water viruses for potable water treatment in the US.16,17 Similar log-reductions have been sought for stormwater reuse in Australia, but performance evaluation there had to rely upon spiking in of surrogate microorganisms to assess such removals.18 Therefore, molecular targets of consistency and high abundance found endogenously in the graywater matrix were the target of the current investigation. This approach allows for the widest dynamic range of pathogen reduction in future risk assessments. In this investigation, we employ high-throughput pyrosequencing to identify the metabiomic profile of laundry graywater and utilize qPCR to quantify abundant and consistent targets that may be useful to evaluate pathogen removal and human health risk during graywater treatment. Human mitochondrial DNA (HmtDNA) was evaluated specifically as a potential surrogate given its human-specificity, high copy number per cell,19 and ability to persist longer than the larger human chromosomal DNA when released from cells.19,20 The results highlight the potential of high-throughput sequencing and qPCR to aid in graywater risk assessment and identify endogenously abundant and consistent molecular targets such as HmtDNA and other skin-associated bacteria (total Staphylococcus) as novel candidate surrogates potentially suited to evaluate microbial removal/inactivation in future laundry graywater treatment performance studies.

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METHODS AND MATERIALS Laundry Sampling. The University of Cincinnati’s equipment facility in Cincinnati, Ohio, USA washes approximately 10−30 University student-athletes’ sportswear at a time in a commercial washing machine with a capacity of 0.43 m3 (Pellermin Milnor Corporation, model 36026 V5J). The washer is run daily using the following conditions: a 3 min rinse (21 °C), 6 min wash (29 °C), 12 min wash (29 °C), followed by two 2 min rinse cycles at (27 °C) and (21 °C) discharging approximately 250 L of water per cycle, and a final spin-only extract. The same concentration of detergent (Tide 2X concentrate, Procter & Gamble Co.) is automatically injected for each wash cycle. Twenty samples were collected from each of the first two cycles (first rinse−no detergent added; first wash−detergent added) from February to May, 2013. Each grab sample was collected in sterile bottles halfway through the 40-s discharge period, directly from the exit pipe, before the graywater entered into a drainage trough leading to the blackwater system. Collection of the first two cycles was based on the expectation that a majority of microbes from clothing would be removed during these first two cycle discharges, thereby representing the highest predicted bio7994

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Figure 1. Top 25 most abundant genera classified in laundry graywater following 454 sequencing.

well as a variety of other PCR conditions, we were unable to obtain amplification from a pure S. aureus culture (ATCC 25923) DNA extract using the primer sequences specified. As a result, another taqman assay targeting the nuc gene of S. aureus was successfully adopted and used.37 Duplicate qPCR was carried out in 30 μL reactions consisting of 15 μL of Taq Environmental Master Mix 2.0 (Life Technologies, Inc.), 500 nM of each primer, 100 nM of each probe, 2 μL of DNA template, and water to volume. All primers and probes were synthesized by Life Technologies, Inc. Cycling was run on ABI Prism 7000 (Applied Biosystems, Inc.) at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15-s and 60 °C for 1 min. qPCR Quality Control and Data Analysis. Each qPCR setup and amplification step occurred in separate designated laboratories to avoid contamination. No template controls (NTC’s) were run with all samples. DNA template dilutions of 1:10 and 1:25 were employed to check for PCR inhibitors. PCR inhibition criterion was defined as the expected environmental sample ΔCq ± the standard deviation of the assay39 for the expected Cq + 0.5 cycles for PCR variation recommended previously40 resulting in an approximate one cycle variation from the expected ΔCq. For inhibited samples, original copy numbers were back-calculated based on the log−linear relationship observed between subsequent uninhibited 1:10 and 1:25 dilutions. The Enterococcus spp. assay gene copy number was divided by four to account for the fact that

cheek swab of a human volunteer as opposed to an environmental sample. The Corynebacterium and Propionibacterium standard curves were generated in a similar manner, adapting the method of Gao et al., 201029 by replacing plasmid dilutions with amplicon dilutions. Amplicons were purified using the MinElute PCR Purification Kit (Qiagen, Inc.), and DNA yield was assessed via Nanodrop 1000 spectrophotometry (NanoDrop Technology). HmtDNA, Corynebacterium, and Propionibacterium gene copies were calculated based off DNA yield, base pair content, and amplicon size.28 Standard curves for targets were generated using 10-fold serial dilutions in PCRgrade water ranging from 101 to 108 gene copies per reaction. qPCR standards were run in triplicate, and replicates were used to create a master standard curve for each assay. Amplification efficiency (E) was calculated using the equation E = (10−1/slope) − 1. Data analysis was completed in R version 2.15.230 or Microsoft Excel 2011 using evaluation criteria of goodness-of-fit linear regression (R2) and slope to evaluate each assay. Samples below the lower limit of quantification (LLOQ), which was defined as the mean Cq of the lowest detectable gene copy number from the calibration curve qPCR reactions, were assigned a value of 1/2 the LLOQ for subsequent data analysis. qPCR Assay. Most qPCR primers and probes used for this study have previously established sensitivity and target specificity (Table S2).29,31−37 The one exception to this specificity was the S. aureus assay initially reported by Lee et al.38 which targeted the sec gene. Using reported parameters, as 7995

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Figure 2. Mean log10 copies ± SD of qPCR targets (Hmt = HmtDNA, Bac = total Bacteroides spp., hBac = human-specific Bacteroides, Pse = Pseudomonas spp., Cory = Corynebacterium, Propi = Propionibacterium, Staph = Staphylococcus spp., StaphA = Staphylococcus aureus) in laundry graywater.

Enterococcus spp. contains approximately four 23S rRNA gene copies per genome.41 Results were normalized to 10 ng of DNA per PCR reaction per 100 mL of graywater. Pearson’s correlation coefficient (r) was used to assess the validity of statistical relationships between targets.42 Statistical strength was determined at α = 0.01, 1-β = 0.8.43 All qPCR results are reported as log10 gene copies ± the standard deviation (SD) per 100 mL of graywater. Laundry Graywater Storage. Since on-site graywater recycling systems are likely to have a temporary collection or storage tank to equalize flow to the treatment process, an experiment was conducted to assess the decay of qPCRdetectable surrogates (HmtDNA and Staphylococcus) and pathogen (S. aureus) in stored laundry graywater. Prerinse and first-wash samples (3-L each) were sampled in 1-L Nalgene bottles as described previously, then were combined (1.5-L each) into a 3-L glass bioreactor (Bellco Glass, Inc.) and stored in the dark at 4 and 20 °C. A stir bar was automatically programmed to gently mix the solution for 1 min every 2 h, with a 556 multiparameter probe (YSI, Inc.) measuring dissolved oxygen (DO), pH, specific conductivity, oxygen reduction potential, and salinity every 15 min during storage, starting at the commencement of mixing. Temperature control was based on the assumption that microbial activity would be limited at 4 °C, thereby attributing most of the decay due to

nonactive biological parameters within the matrix, whereas 20 °C would include the additional degradation due to microbial activity. Due to the lack of influences on degradation from any pipe biofilms and/or collection tank microbiota from an existing system, this study set out to gauge the stability of the surrogate or pathogen in the graywater matrix itself and is expected to be a conservative estimate of the degradation in a real-world system. Subsamples were processed (100 mL) as described previously, and replicate qPCR assays quantified the decay following 0, 1, 2, 3, 4, 6, and 8 days storage. HmtDNA Sanger Sequencing. To confirm the specificity of the HmtDNA primer to human origin, all qPCR HmtDNA amplicons (n = 40) targeting the 195 base pair region of the NADH5 gene36 were purified with the MinElute PCR Purification Kit (Qiagen, Inc.) according to the manufacturer’s instructions and Sanger sequenced by Genewizz, Inc., according to the manufacturer’s instructions, using both HmtDNA forward and reverse primers (Table S2). NCBI BLAST searches of the Sanger sequences (n = 80) were performed to verify sequence identity.

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RESULTS Pyrosequencing. A portion of prerinse samples (n = 12) and wash samples (n = 12) were pyrosequenced using the V1− V3 variable regions of the 16S rRNA gene to identify the 7996

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Figure 3. A) qPCR correlation between HmtDNA vs S. aureus (r = 0.54, P = 3.2 × 10−4) (n = 40) and B) qPCR fluctuations between select HmtDNA (blue) and S. aureus (orange) laundry wash samples (n = 14).

Figure 4. Decay of HmtDNA (blue) and total Staphylococcus (purple) and S. aureus (orange) in laundry graywater at 20 °C (n = 3). Error bars represent standard deviation.

Acinetobacter, Propionibacterium, and Paracoccus, which accounted for over 46% of the total reads classified in this study (Figure 1). Of interest was a sample from late March with approximately 46% of reads identified in the genus Vibrio in the first rinse and about 40% in the corresponding wash sample. No other sample contained identifiable Vibrio.

metabiomic bacterial profile in laundry graywater samples. After data processing and chimera removal, a total of 268,672 reads were available for input to the RDP Classifier, which equated to an average of over 11,000 classified sequences per sample. The top seven most abundant genera identified in laundry graywater were Staphylococcus, Corynebacterium, Micrococcus, Lactobacillus, 7997

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approximately 24 h of storage at 20 °C losing 2-logs and 0.5logs, respectively (Figure S3). At 4 °C, the Staphylococcus and S. aureus drop was delayed until day 4 (Figure S3) where a similar decline rate was observed, which also plummeted below detection limits, consistent with the drop in DO and pH levels observed around this time (Figure S1). HmtDNA Sanger Sequencing. Purified HmtDNA amplicons (n = 80) targeting the 195 base pair region of NADH5 gene28,36 were Sanger-sequenced and subjected to NCBI BLAST searches to check for species cross-reactivity with other mammals. All sequences exhibited ≥99% match identities except one, which exhibited a 97% match identity. All accession e-values were 1.0 × E−60 or lower (Table S7) to Homo sapiens mitochondrion complete genome (accession# KC994161.1), thereby confirming no nonhuman contamination or crossreactivity to nonhuman mitochondria.

qPCR Standards. The standard curves for Corynebacterium, Propionibacterium, and Staphylococcus spp. assays exhibited linear relationships from 108 to 103 gene copies per reaction, with linearity for the remaining targets from 106 to 101 gene copies per reaction. PCR efficiencies ranged from 96.8% to 109%, slopes ranged from −3.13 to −3.40 with all assays exhibiting correlation coefficients (R2) ≥ 0.99, except for the S. aureus assay which exhibited a correlation coefficient of 0.98. Calibration equations for the standard curves can be found in SI Table S3. PCR Inhibition. The HmtDNA, universal Bacteroides, human-specific Bacteroides, and Pseudomonas spp. assays showed inhibition in 10% (4/40) of samples; E. coli had 5% (2/40) inhibited samples and 2.5% (1/40) of the Propionibacterium and Corynebacterium assays, and no inhibition was observed for the Enterococcus and S. aureus assays. The Staphylococcus assay showed inhibition in 88% (35/40) samples. Some samples showed inhibition for multiple targets, while other samples were only inhibitory for a particular target. To compensate for the variable inhibition, qPCR reactions were optimized by dilution. Expected ΔCq values were observed between 1:10 and 1:25 dilutions of inhibited samples, suggesting that the samples were potentially free of PCR inhibitors after the 1:10 DNA template dilution. Original gene copy numbers were back-calculated using the ΔCq of the linear relationship between the noninhibitory 1:10 and 1:25 dilutions. PCR Results for Surrogate and Pathogenic Targets. E. coli, Enterococcus spp., and S. aureus were detected in 10 (50%), 19 (95%), and 17 (85%) of prerinse samples and 8 (40%), 20 (100%), and 15 (75%), respectively, of first wash samples (Table S4). All other targets tested for in this study were detected in all samples. Staphylococcus spp., Propionibacterium, and Corynebacterium ranked highest in abundance of qPCR targets (mean = 6.54 ± 0.54, 5.73 ± 0.68, 5.44 ± 0.78, respectively) log10 copies per 100 mL of graywater, while HmtDNA was present (mean = 2.75 ± 0.52) in all samples (Figure 2). Human-specific Bacteroides comprised roughly 80% of the total Bacteroides spp. present. When present, an opportunistic pathogen, S. aureus, was detected (1.67 ± 0.82) log10 copies, comprising approximately 25% of total Staphylococcus spp. Several significant correlations were found between qPCR targets. A summary of all statistical correlations and significance between targets is available in Table S5. Of note, significant positive correlations were observed between the presence of HmtDNA and skin-associated organisms Staphylococcus, Propionibacterium, and Corynebacterium (r ≥ 0.45, P ≤ 1.4 × 10−3) and the skin pathogen S. aureus (r = 0.54, P = 3.2 × 10−4) (Figure 3A, B). Interestingly, none of the FIB assayed in this study showed any significant correlation with each other or to HmtDNA (P ≥ 0.01) (Table S5). Laundry Graywater Storage. A summary of the physiochemical results can be found in SI, Table S6. Dissolved oxygen (DO) levels dropped to anoxic levels after ∼24 h of storage at 20 °C and 6−8.5 days at 4 °C (Figure S1A-B). The pH trend mimicked the DO by dropping around the same time, remaining stable around a pH of 7.0. HmtDNA was detectable until day 8 (1.79 ± 0.14) at 4 °C and day 6 (1.08 ± 0.37) at 20 °C in stored laundry graywater (Figure S3). HmtDNA degraded 7% at 20 °C and 4% at 4 °C after 24 h (Figure 4). HmtDNA experienced a strong first-order exponential decay curve at both temperatures with R2 values ≥0.98 (Figure S3). Both total Staphylococcus and S. aureus rapidly decayed within

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DISCUSSION The most abundant bacterial genera identified by pyrosequencing in laundry graywater were Staphylococcus, Corynebacterium, Micrococcus, Lactobacillus, Acinetobacter, and Propionibacterium spp., representing over 46% of the reads classified in our study. The human microbiome project identified these same genera as some of the main inhabitants on human skin, concluding that Staphylococcus, Propionibacterium, and Corynebacterium were the most abundant organisms on human skin.44,45 Traditional FIB genera such as Bacteroides spp. and Enterococcus spp. were present but represented less than 9.0 × 10−4 % of classified reads. This data suggests that the majority of the bacterial load in the University athletic laundry graywater is of skin origin. Since our study was limited to industrial laundry graywater only, future work should also examine the degree of similarity in the microbial composition of various sources of graywater. One sample set (prerinse and wash) occurred with an anomalously large number of reads assigned to the genus Vibrio, which could have been associated with an infection. In general, various Vibrio spp. exist naturally in marine and estuarine waters,46 and while many may be nonpathogenic, seven including V. parahemolyticus and V. cholerae are human enteric pathogens.47 Additionally, V. alginolyticus and V. vulnif icus have the ability to infect skin abrasions, with V. vulnif icus causing wound infections and septicemia, accompanied by edematous skin damage.47,48 While Vibrio seemed a somewhat unexpected genus, the particular laundry graywater sample was derived from the return of several athletic teams from coastal destinations. Given the potentially high 16S gene copy number per genome of some species,49 we hypothesize that the Vibrio contribution could be explained by cells transferred to laundry graywater via fecal, oral, or dermal desquamations deposited in clothing from an infected individual and/or by an athlete’s clothing. The qPCR analysis revealed that FIB: E. coli, human-specific Bacteroides, and fecal enterococci were present in laundry graywater, which is congruent with previous investigations;8−10 however, these FIB were 2- to 5-logs lower in abundance and showed less consistency when compared to putative skin microbiome members (Figure 2). This suggests that their use as a surrogate to demonstrate a wide dynamic range of log removal during treatment is inadequate. In addition, no significant correlations could be drawn between any FIB and the observed opportunistic pathogen S. aureus (P ≥ 0.01), supporting previous conclusions that the use of FIB as an indicator of pathogen presence in laundry graywater is also 7998

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necessary to determine the importance of skin pathogens and associated enterotoxins in graywater and assess their significance for measuring pathogen removal and risk during treatment and reuse. The observed decreases in DO observed after 24−29 h of storage confirm previous investigations that aesthetic nuisances (smell) may play a role in governing acceptance of these systems,2,67 while insinuating that both HmtDNA and total Staphylococcus could have use in storage applications. The slower decay rates observed for all targets during storage at 4 °C when compared to 20 °C is likely explained by temperaturedependent enzymatic nucleases and general antagonistic microbial activity at the higher temperature, both previously identified factors influencing enzyme DNA degradation.68,69 The apparent relationship between rapid staphylococci degradation with decreased DO and pH levels in stored, untreated laundry graywater indicates that total Staphylococcus could be used as an indicator of “fresh” laundry graywater. However, this rapid degradation could become problematic for measuring microbial removal during subsequent treatment. In contrast, HmtDNA was quantifiable for the duration of the study which was 8 days of storage at 20 °C, which is similar to previous investigations that were able to isolate HmtDNA from wastewater after 15 days of storage at 20 °C.70 This increased signal longevity provides evidence that HmtDNA may be a useful treatment metric for transiently stored laundry graywater. This research supports previous investigations that biological treatment of graywater prior to reuse is probably necessary but dependent upon the likelihood of exposure to microbial hazards. Where direct human exposure is likely, an infection risk target of 1:10,00071 may necessitate at least a 4-log removal/inactivation of pathogens as currently required for surface waters intended for potable use in the U.S.16 Based on the endogenous targets identified in unstored laundry graywater (Figure 2), such a log-reduction could be undertaken with HmtDNA or skin-associated organisms such as total Staphylococcus, once relationships with specific pathogens have been identified. Despite the abundance advantage of the staphylococcal bacteria, HmtDNA is an attractive alternative due to its superior persistence in the University’s laundry graywater at ambient temperatures. Although the focus of this study was limited to laundry graywater, the human signature observed in our study suggests that the presence of human-associated surrogates in other graywater types could be expected to yield similar results and application, but further research is necessary to warrant confirmation. Since significant treatment correlations have been found between relatively dissimilar surrogates such as C. perf ringens with viral/Eukaryotic pathogens in previous water treatment evaluations,72 there exists motivation for future studies to elucidate the utility of the bacterial and HmtDNA surrogates described herein for graywater treatment evaluation.

limited. In contrast, HmtDNA was consistently present in laundry graywater (2.8 ± 0.5 log10) gene copies per 100 mL suggesting that it could measure a wider dynamic range of pathogen log removal than FIB in this laundry graywater. HmtDNA significantly positively correlated with all skinassociated bacteria (P ≤ 0.01) but not with any FIB (P ≥ 0.01), suggesting the HmtDNA contribution in our study is of nonfecal origin. In addition, HmtDNA also exhibited a significant correlation with S. aureus (r = 0.54, P = 3.2 × 10−4) indicating that HmtDNA could be a valuable marker of pathogen presence as well as a useful indicator of treatment performance. The presence of S. aureus in laundry graywater is somewhat contradictory to previous investigations that were unable to isolate S. aureus from graywaters7 but confirmatory from other studies that did find S. aureus in graywater.50 This could be caused by the methodology used for detection, the source of the graywater, or that samples that could not detect S. aureus in the previous study were taken from a holding tank, and we have shown that S. aureus does not appear to persist for long periods of time in laundry graywater at 20 °C (Figure S4), a factor that may limit this marker if graywater is stored for extended periods before treatment. Nonetheless, it was not surprising that S. aureus was found in laundry graywater, given that S. aureus has been documented on the human skin or in the nasal passages of approximately 30−36% of healthy individuals in the United States,51,52 and graywater is expected to contain organisms from body orifices and nasal passages.53,54 The presence of S. aureus is of potential concern due to its ability to infect oral, dermal, or respiratory tracts of individuals with weakened immune systems, children, or the elderly and its antibiotic-resistant form, Methicillin-resistant S. aureus (MRSA).55 MRSA is known to cause severe skin infections among contact sport athletes56,57 and has more recently infected otherwise healthy people having no association to hospitals or sporting facilities, which are typical MRSA infection hotspots.55 While MRSA was not specifically tested for in this study, the potential for an infected individual to contribute MRSA to laundry graywater seems plausible based on the prevalent skin contribution (Figure 2) and salinity (Table S6) of laundry graywater as well as MRSA’s persistence in halophilic waters.58,59 Even without the Methicillin-resistant form, skin-associated S. aureus can still colonize and infect the human skin60 and has the ability to produce staphylococcal enterotoxins (SE) that have been responsible for an estimated 185,000 cases of foodborne gastroenteritis per year in the U.S.61 Humanassociated S. aureus has been shown to harbor SE genes gene(s) encoding for at least one of the 20+ currently identified enterotoxins.62,63 The SEs produced are more resistant to environmental stresses than the bacteria itself,64 making the toxins themselves an additional treatment concern. In addition, a recent U.S. Department of Defense study determined that a specific SE, identified as SEB, remained stable in deionized water (pH 7.0, 10 mM PO4) for at least 30 days, which was the duration of the study.65,66 Therefore, water reuse scenarios in which there is oral ingestion, respiratory inhalation, or dermal contact of untreated laundry graywater may produce an increased human health risk to opportunistic pathogens as previous research has suggested.50,54 However, the myriad of reuse scenarios and inevitable downstream human contact makes it difficult to quantify the risks attributable to skin pathogens during reuse.53 Therefore, future research is

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ASSOCIATED CONTENT

S Supporting Information *

A description of the bacterial culture genome sizes, primers, and probes used in this study, qPCR standard curve equations, qPCR sample detections, qPCR correlations, storage affects on select chemical parameters, HmtDNA, Staphylococcus, and S. aureus decay detections, and HmtDNA Sanger sequence information. This material is available free of charge via the Internet at http://pubs.acs.org. 7999

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

School of Public Health, University of Alberta, Edmonton, AB, Canada. Author Contributions

The manuscript was produced through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

Funding for this research was provided by the USEPA. Notes

Disclosure: The views expressed in this article are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. Mention of trade names, products, or services does not convey, and should not be interpreted as conveying, official EPA approval, endorsement, or recommendation. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dennis Lye, Rachel Logsdon, Laura Boczek, Asja Korajkic, and Rich Haugland of the US EPA for generously providing genomic bacterial DNA for this study as well as the University of Cincinnati Athletics Program for granting graywater access. Sequences will be uploaded to tp:// www.ncbi.nlm.nih.gov/.

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