Mechanisms of Pathogenic Virus Removal in a Full-Scale Membrane

Article pubs.acs.org/est

Mechanisms of Pathogenic Virus Removal in a Full-Scale Membrane Bioreactor Rabia M. Chaudhry,§,‡ Kara L. Nelson,*,§,‡ and Jörg E. Drewes† §

ReNUWIt (Reinventing the Nation’s Urban Water Infrastructure) Engineering Research Center, University of California, Berkeley, California, 94720, United States ‡ Department of Civil and Environmental Engineering, University of California, Berkeley, California 94720, United States † Chair of Urban Water Systems Engineering, Technische Universität München, 85748 Garching, Germany S Supporting Information *

ABSTRACT: Four pathogenic virus removal mechanisms were investigated in a full-scale membrane bioreactor (MBR; nominal pore size 0.04 μm): (i) attachment of virus to mixed liquor solids; (ii) virus retention by a just backwashed membrane; (iii) virus retention by the membrane cake layer; and (iv) inactivation. We quantified adenovirus, norovirus genogroup II (GII), and F+ coliphage in the influent wastewater, the solid and liquid fractions of the mixed liquor, return flow, and permeate using quantitative PCR (adenovirus and norovirus GII) and infectivity assays (F+ coliphage). Permeate samples were collected 4−5 days, 1 day, 3 h, and immediately after chlorine enhanced backwashes. The MBR achieved high log removals for adenovirus (3.9 to 5.5), norovirus GII (4.6 to 5.7), and F+ coliphage (5.4 to 7.1). The greatest contribution to total removal was provided by the backwashed membrane, followed by inactivation, the cake layer, and attachment to solids. Increases in turbidity and particle counts after backwashes indicated potential breakthrough of particles, but virus removal following backwashes was still high. This study demonstrates the ability of the MBR process to provide over 4 logs of removal for adenovirus and norovirus GII, even after a partial loss of the cake layer, and provides evidence for assigning virus disinfection credit to similar MBRs used to reclaim wastewater for reuse.

1. INTRODUCTION

environmental barrier are currently being discussed and will likely require similarly strict removal criteria for viruses.4 The assignment of appropriate pathogen reduction credit for MBRs within water reuse treatment trains requires a mechanistic understanding of virus removal. As the smallest pathogens (20 to 100 nm), viruses are comparable in size to the pores of typical MBR membranes and are not likely to be removed sufficiently by size exclusion alone. Bacteria and protozoan cysts are considerably larger in size, and they are considered to be reliably removed by MBRs.5 The viral protein capsid has isolated hydrophobic and charged regions, which can impact viruses’ behavior in solution, their interaction with other particles and surfaces, and consequently their removal. Operational changes that impact the composition of surfaces, such as bacterial flocs or membrane fouling, may affect virus removal. Morphological differences among viruses may also impact their removal. Therefore, pathogenic viruses should be studied in addition to indicator viruses (bacteriophages or

Reuse of treated wastewater can increase the water security and sustainability of urban centers by expanding the portfolio of water sources and increasing resource recovery. Membrane bioreactors (MBRs) are an attractive treatment technology for water reuse applications.1 MBRs consist of a biological treatment process similar to conventional activated sludge but use microfiltration or ultrafiltration membranes to separate the solids from the effluent, rather than settling. This allows MBRs to produce better quality effluent within a smaller footprint by selection of microbial communities most suited to breaking down wastewater organics and through operation at higher solids concentrations.1 Sufficient reduction of pathogenic microorganisms is a regulatory focus for reuse due to their acute impact on human health. Within the United States, California has the most comprehensive water reuse regulations, requiring a 5 log reduction in viruses from postsecondary treatment for unrestricted use of recycled water in irrigation.2 For indirect potable reuse via groundwater recharge, viruses have the highest treatment requirement of 12 log reduction compared to a 10 log reduction for Cryptosporidium oocysts and Giardia cysts.3 Regulations for direct potable reuse without an © 2015 American Chemical Society

Received: Revised: Accepted: Published: 2815

October 31, 2014 January 28, 2015 February 2, 2015 February 2, 2015 DOI: 10.1021/es505332n Environ. Sci. Technol. 2015, 49, 2815−2822

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Environmental Science & Technology

using indigenous pathogenic viruses (adenovirus, norovirus GII, hepatitis A, and rotavirus) and to (2) assess the suitability of indigenous F+ coliphages as indicator organisms for pathogenic virus removal. This study also aims to understand the time required for the cake layer to redevelop so that the permeate stream can be handled appropriately following cleaning procedures to protect public health.

phages) for a range of MBR operating conditions to understand removal and better inform water reuse regulations. Successful operation of the MBR process requires proper management of membrane fouling to maintain acceptable permeate flux,6,7 which may have implications for virus removal as the dynamic fouling layer has been shown to play a role in bacteriophage removal in some bench8−12 and pilot-scale studies.13,14 Even though viruses are typically smaller than the nominal membrane pore sizes, high removals (up to 5.3 logs) have been reported for bacteriophages at full-scale MBRs under long-term steady operating conditions, suggesting that the membrane cake layer contributes to removal.5,15,16 Studies of pathogenic viruses at full-scale plants have also demonstrated high log removals under steady-state conditions (up to 5, 6.8, and 5.1 for adenovirus, norovirus genogroup II (GII), and enterovirus, respectively),17−20 but the effect of chemical enhanced membrane backwashes (CEB) has not been quantified. Terminology for describing membrane fouling varies in the literature, but it can be classified according to cleaning processes: reversible, irreversible, and irrecoverable fouling. Reversible fouling can be removed from the membrane by physical cleaning such as air bubble scour, relaxation of the membrane, or backflushing with permeate. Irreversible fouling must be removed by chemical or mechanical cleaning. This may take the form of regular CEBs, which involve backflushing the membranes with a solution of sodium hypochlorite or acid, and recovery cleans, which involve soaking the membranes in a cleaning solution when membrane flux is no longer adequate. Irrecoverable fouling cannot be removed by any cleaning and determines the lifetime of the membrane.7 MBR facilities may undertake CEBs a few times every month, and it is important to understand their impact on pathogenic virus removal. The component of fouling removed by regular CEBs (a type of irreversible fouling) will be referred to as the cake layer in this study. The only mechanistic studies on virus removal in an MBR process have utilized bacteriophages in bench and pilot scale systems.8,9,11 All studies noted poor log removal (0.1−0.6) by a clean membrane with a nominal pore size of 0.45 μm, and the experiments utilizing real wastewater attributed over 2 logs of removal to attachment of phage to solids biomass.8,11 The contribution of the fouling cake layer ranged from 0.9 to 3.6 logs. A phage decay estimate of 0.5 h−1 was determined through incubation of somatic coliphage with mixed liquor in the laboratory.11 For reliable treatment of viruses by MBRs for reuse applications, it is critical to understand whether the decrease in log removal after CEBs occurs for pathogenic viruses at full-scale facilities. In addition, because the previous mechanistic studies were conducted with 0.45 μm pore-size membranes, it is important to also characterize the removal mechanisms for tighter ultrafiltration membranes, which are in widespread use. In this study, virus removal is hypothesized to occur via four mechanisms: (i) incorporation of viruses into the mixed liquor suspended solids, which are excluded by the membrane; (ii) retention of viruses by the clean membrane immediately following a chemical enhanced backwash; (iii) retention of viruses by the cake layer formed on the membrane surface after a period of use; and (iv) inactivation of the viruses due to predation or enzymatic breakdown.21,22 The objectives of this research were to (1) determine the relative contributions of the main virus removal mechanisms at a full-scale MBR facility

2. MATERIALS AND METHODS 2.1. Experimental Approach. A full-scale municipal wastewater plant (American Canyon, California) employing a submerged MBR treatment process was selected for this study. The plant serves a population of 20 000 and has the capacity to treat 14 200 m3 of wastewater per day. The plant has four parallel treatment trains, each containing ten 10-year old ZeeWeed 500 hollow fiber membrane cassettes with a nominal pore size of 0.04 μm. One train treats industrial wastewater and was isolated from the municipal trains whenever sampling was conducted. Typical dry weather municipal flows are 5700 m3/d, and the plant is operated at a mixed liquor suspended solids (MLSS) concentration of 8 g/L. Waste sludge from the aeration tanks is settled in a holding pond, and the supernatant is returned to the influent line. Membrane fouling is managed by relaxing the membranes every hour by turning off the vacuum pump for 1 min and chemical enhanced backwashing the membranes in each train with sodium hypochlorite solution on a staggered schedule twice a week. The impact of CEB on virus removal was assessed by collecting permeate samples from treatment trains at different stages of the membrane cake layer development after backwash events. A schematic of the wastewater treatment system and sampling locations is shown in Figure 1. Samples were collected

Figure 1. Schematic of the municipal wastewater treatment portion of the American Canyon treatment plant. Stars represent the locations of samples collected during the study. All samples from the oxic zone of the tanks were separated into solid and liquid fractions before quantification.

from February to August 2013 at four locations: municipal raw influent wastewater, return flow, activated sludge mixed liquor from each municipal train, and the corresponding permeate from each train. The influent samples were 24 h composites, and mixed liquor, return flow, and permeate were taken as grab samples. Solid and liquid fractions of all mixed liquor samples were quantified separately to determine the association of viruses with mixed liquor biomass. Virus concentrations in the municipal influent were lower after rainfall events due to 2816

DOI: 10.1021/es505332n Environ. Sci. Technol. 2015, 49, 2815−2822

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mengovirus (RNA) were seeded into 21 control samples before any processing and then quantified after all processing steps with quantitative PCR. Details can be found in Supporting Information Section S3 and Table S2. Nontemplate controls (NTC) were also included for each 96-well qPCR plate and were negative for all reported results. 2.4.2. PCR Inhibition. Inhibitory compounds found in environmental samples can disrupt PCR enzymes and result in inaccurate quantification. Two different dilutions of all samples were tested in triplicate; inhibition controls were conducted for each dilution of each sample type during each PCR run by the addition of standard RNA or DNA for that assay. Results from a particular dilution were included in the analysis if the recovery of the standard addition was greater than 50%. 2.4.3. Selection of Samples for Analysis. 57 samples were collected during 10 sampling events. Dedicated adenovirus process controls comprised 9 of the total samples, and they were collected during two sampling events. Actual samples also doubled as mengovirus process controls, and these were collected during three events. Samples were considered independent if they were taken on separate days or from separate treatment trains on the same day. Quality control criteria for inclusion in analysis required that the dilution with the higher process efficiency for each virus assay was chosen and that the recovery from the inhibition control for that dilution was greater than 50%. Additionally, samples were reextracted and requantified by PCR a second time if PCR amplification efficiency was less than 90% or greater than 110%. The numbers of samples included in the analysis for each assay after all criteria were satisfied are shown in Table 1.

dilution by stormwater, so samples were only collected if there was no rainfall during the preceding week. The industrial influent was separated from the municipal trains for at least 24 h before each sampling event. 2.2. Concentration of Samples. All samples were concentrated to a smaller volume so that low concentrations of indigenous viruses could be quantified. For each permeate sample, approximately 200 L was concentrated using electropositive filters following the methods in Chapter 14 of the USEPA Manual of Methods for Virology23 except that NanoCeram (Argonide, Sanford, Florida) filters were used, which have been shown to be an acceptable alternative to the Virosorb 1MDS (Cuno, Meriden, Connecticut) filters described in the USEPA protocol24 (Section S1 in the Supporting Information). All samples other than MBR permeate were concentrated by elution of viruses from particles, precipitation, and then resuspension into a smaller volume. 40 mL of mixed liquor samples was separated into solid and liquid components by centrifugation at 10 000g for 1 h. This allowed a theoretical particle size cutoff of 340 nm based on Stokes settling.25 The solid fraction was suspended in 40 mL of 10% tris-glycine beef extract buffer (0.1 M tris base, 0.05 M glycine, and 10% w/v beef extract at pH 9.5) and mixed thoroughly. 40 mL each of influent, return flow, and the mixed liquor liquid fraction was mixed with 10 mL of 10% tris-glycine beef extract buffer. After thorough mixing, samples were sonicated for 10 min to elute viruses and then centrifuged at 10 000g for 30 min. The supernatants were carefully harvested, and 2 mL was retained from each sample for the F+ coliphage infectivity assay within 6 h of sample collection. The supernatants were then mixed with 10 mL of 50% polyethylene glycol 6000 (Alfa Aesar) solution and sodium chloride (Fisher) to a final concentration of 0.2 M, vortexed, and allowed to precipitate overnight without stirring at 4 °C. After 12 to 16 h, the samples were centrifuged at 10 000g for 1 h. The pellets were carefully suspended in 2−4 mL of RNase-free water; the final concentrated volumes were recorded, and the samples were stored at −80 °C. 2.3. Quantitative PCR. Nucleic acids were extracted from 500 μL of the final concentrated samples using a PureLink Viral RNA/DNA Mini Kit (Invitrogen) and eluted into a volume of 15 μL. The extracted nucleic acids were then tested for adenovirus using qPCR and for norovirus GII, hepatitis A virus, and rotavirus using reverse transcriptase RT-qPCR. Mastermix reagents were purchased from Applied Biosystems (TaqMan Fast Universal PCR Master Mix for qPCR and TaqMan RNAto-Ct 1-Step Kit for RT-qPCR), and the plates were processed on a StepOnePlus instrument (Applied Biosystems). Primers and probes were purchased from Integrated DNA Technologies (Coralville, Iowa), and freeze−thaw cycles were minimized for primers, probes, and all samples by aliquoting into single-use volumes. Nucleic acids were extracted immediately before conducting PCR to prevent loss of signal due to a freeze−thaw cycle. Details about the molecular assays (Table S1, Supporting Information) and generation of external standards are provided in the Supporting Information (Section S2). 2.4. Virus Quantification Quality Control. 2.4.1. Process Controls. Controls were analyzed for all sample types to quantify overall virus process recoveries across the different concentration and quantification methods. DNA virus recoveries during concentration by electropositive filters have been reported to be lower than those for RNA viruses.24,26 Therefore, known quantities of adenovirus-2 (DNA) and

Table 1. Number of Independent Samples Processed for Each Sample Category and Target Organisma sample type influent return sludge mixed liquor solids mixed liquor liquids permeate, after backwash permeate, 3 h after backwash permeate, 1 day after backwash permeate, 4−5 days after backwash permeate, 10−11 days after backwash

adenovirus samples

norovirus GII samples

F+ coliphage samples

6 4 8 7 5 (ND:2/5) 3 (ND:1/3)

4 3 8 6 5 (ND:1/5) 3

4 7 (ND:7/7) 6 4 4 3

4

4 (ND:1/4, BLOQ:1/4) 3 (ND:1/3, BLOQ:1/3) 2

4

3 2

3 0

a ND specifies the number of samples for which the target was not detected. If the target was detected below the limit of quantification for that sample, it is specified as BLOQ. The limit of quantification was used for samples that were ND or BLOQ.

2.5. F+ Coliphage Infectivity Assay. Indigenous F+ coliphage was quantified to evaluate its usefulness as an indicator of pathogenic virus removal using the double agar layer (DAL) method. Briefly, top agar at about 50°C was mixed with antibiotics, 50 μL of exponential phase bacteria host (E. coli ATCC 700891 or Famp), and the appropriate dilution of sample and poured onto Petri plates with prepared bottom agar. Plates were incubated overnight at 37°C, and the virus plaques were counted after 18−24 h. Modified LB agar contains 2817


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Environmental Science & Technology ⎛ ∂y ⎞2 ⎛ ∂y ⎞2 ⎛ ∂y ⎞2 δy 2 = ⎜ δ1⎟ + ⎜ δ2⎟ , ..., +⎜ δn⎟ ⎝ ∂x 2 ⎠ ⎝ ∂x1 ⎠ ⎝ ∂xn ⎠

tryptone (10 g), sodium chloride (8 g), yeast extract (1 g), dextrose (1 g), calcium chloride (0.3 g), and BactoAgar (BD) (15 g for the bottom layer and 7.5 g for the top layer). Antibiotics streptomycin sulfate and ampicillin sodium salt were used in both layers, each at a final concentration of 0.015 g/L. Indicator virus infectivity assays were completed within 8 h of sample collection. The final concentrated volume of permeate samples was plated after all concentration steps. The supernatants from the first centrifugation for all other samples were directly plated as described in Section 2.2. The appropriate dilutions for all samples were determined ahead of time, and 1 mL of sample was plated in all cases. 2.6. Permeate Turbidity and Particle Counts. To provide insight into particle breakthrough after hypochlorite backwashes, particles in the permeate line were counted using a portable MetOne (WGS 267) Water Grab Sampler on three different sampling dates. The instrument was programmed to sample every minute. Detection limits were from 2 to 100 μm, and data were reported for six size ranges. The American Canyon plant staff kindly provided turbidity data for the same periods. 2.7. Virus Decay Modeling. A number balance over a single treatment train was used to estimate the virus decay constant k in the system. The control volume is shown in Figure S1, Supporting Information. It was assumed that the system was at steady state, the mixed liquor tank was well mixed, the decay followed first-order kinetics, and the decay rate was the same for viruses in the liquid phase and associated with solids. An estimate for k was calculated on the basis of plant process flow data, and the average measured virus concentrations as shown in eq 1.

(3)

Thus, the standard error for log removal values was calculated using eq 4, which is based on eq 3. ⎛ δCfinal ⎞2 ⎛ δCinitial ⎞2 δ LRV 2 = ⎜ ⎟ +⎜ ⎟ ⎝ ln10 Cfinal ⎠ ⎝ ln10 C initial ⎠

(4)

All other uncertainties reported in this study were also propagated on the basis of eq 3.

3. RESULTS AND DISCUSSION 3.1. Total Virus Removal Across the MBR Process. Adenovirus, norovirus GII, and F+ coliphage were consistently detected in all influent samples, and the results are summarized in Figure 2. Rotavirus was not detected in any samples, and

Q influentC influent + Q returnCreturn = Q wasteCwaste + Q permeateC permeate − kV (Csolids + C liquids)

(1)

Q represents typical dry weather process flows, and V is the volume of the bioreactor; values were obtained from plant records. About 80% of the wasted mixed liquor flow was reintroduced into the system as return flow after being allowed to settle in a holding pond. C represents the virus concentrations, which were measured as described above (qPCR for pathogenic viruses and plaque assay for F+ coliphage). Cwaste was assumed to be equal to Csolids + Cliquid. 2.8. Mechanism Contribution to Log Removal. Pathogen log removal values (LRV) were calculated using eq 2. LRV = −log10(Cfinal /C initial)

Figure 2. Average virus concentrations at the American Canyon facility were quantified from February through August 2013. Error bars represent standard error, and details of the number of independent samples used to calculate average concentrations are given in Table 1. Adenovirus and norovirus GII were quantified by quantitative PCR and are shown in units of genome copies per milliliter (gc/mL). Indigenous F+ coliphage was quantified by infectivity assay and is shown in units of plaque-forming units per milliliter (PFU/mL). The detection limit of 1.25 PFU/mL is shown for F+ coliphage in the return flow.

hepatitis A was only detected in two samples (Section 3.3). No seasonal trends were observed over the duration of sampling, and the concentrations shown are averages with their associated standard errors. Table 1 shows the number of samples analyzed for each category, including those that were not detected (ND; no CT value recorded by instrument) and those that were detected below the limit of quantification (BLOQ), which is defined as the lowest calibration standard for that qPCR run. The LOQ was substituted for samples that were either ND or BLOQ. Fortunately, most samples were above the limit of quantification, so the bias introduced by this substitution was minimal. The MBR process achieved high log removal of adenovirus (3.9 to 5.5) and norovirus GII (4.6 to 5.7) across the treatment plant (from influent to permeate) at different levels of cake development. F+ coliphage log removal values were higher than

(2)

Details for how the LRV was determined for each removal mechanism are provided in the Supporting Information (Figure S2). For LRV contribution due to solids, Creturn was ignored because its magnitude was insignificant relative to Cinfluent. The LRV contribution due to decay was estimated by setting Cfinal to the measured mixed liquor concentration (Csolids + Cliquid) and setting Cinitial to the hypothetical concentration of Cwaste calculated from eq 1 when k was set to zero. 2.9. Quantification of Uncertainty. For directly measured concentrations, standard deviation was calculated and the standard error is reported. The error for calculated values was propagated as follows. For a function y dependent on variables x1, x2, ..., xn with associated uncertainties δ1, δ2, ..., δn, the function uncertainty (δy) can be propagated using eq 3. 2818


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Figure 3. (a) Mechanism contributions toward log removal from decay, mixed liquor solids, membrane only, and membrane cake layer at different stages of development. The membrane only LRV for F+ coliphage is high due to the disinfection by residual chlorine in the permeate line after a backwash. Total LRV from the raw influent to permeate can be obtained by adding the appropriate cake layer LRV and the Solids & Decay LRV component. (b) Contributions of individual mechanisms toward the total log removal from influent to permeate (see Figure S2 in the Supporting Information for details). The membrane and subsequent cake layer LRV contributions for F+ coliphage were calculated using the permeate concentration 3 h after backwash.

membrane, and together with the associated cake layer, these two mechanisms accounted for over 50% of the total reported LRV for the three viruses studied. The backwashed membrane LRVs (2.0 for adenovirus, 3.1 for norovirus GII, and 2.7 for F+ coliphage) are much higher than those reported by previous bench-scale studies8,9,11 (0.1−0.6 logs for bacteriophages), demonstrating that a tight membrane provides reliable virus removal even after CEBs. The nominal pore size of ZeeWeed membranes (used in this study) is 0.04 μm, which is very similar to the sizes of the target viruses, whereas previous mechanistic studies used membranes with nominal pore sizes that were about 10 times larger. Three hours after a CEB, the virus log removal contribution from the membrane and cake layer improved for all three viruses and continued to do so for 5 days (Figure 3). Adenovirus removal was influenced more by the development of the cake layer, demonstrating an additional 1.6 logs of removal beyond the membrane only contribution compared to norovirus GII, whose removal only improved by 0.5 logs. Total process log removals for pathogenic viruses declined slightly (4.3 for adenovirus and 4.6 for norovirus GII) when 10−11 days had passed since the last CEB (data not shown). This may have occurred due to the sloughing of the membrane cake layer or degradation of the dynamic cake layer. On the basis of these data, the ideal CEB schedule for MBR treatment trains is once a week. However, it should be noted that it was only possible to collect two permeate samples 10−11 days after CEB (normally, the plant backwashed twice per week), so these results should be interpreted with caution. Further research is necessary to determine whether pathogenic virus log removal consistently decreases 10−11 days after a CEB. The F+ coliphage permeate concentration data provide the clearest evidence for improvement in log removal due to the buildup of the cake layer (Figure 2). The average F+ coliphage concentration after CEB is unusually low due to inactivation of the viruses by the residual chlorine in the permeate line (5 × 10−4 PFU/mL) but increases within 3 h (1 × 10−2 PFU/mL). Each subsequent measurement of the permeate concentration

those for the pathogenic viruses under the same conditions (5.4 to 7.1). F+ coliphage do not appear to be a conservative indicator organism for pathogenic viruses enumerated by qPCR. Adenovirus had the lowest removals overall, particularly immediately after the CEB (LRV 3.9). These removals are comparable to findings that have previously been reported.17−19 Some of the differences between pathogenic and indicator virus log removals may be due to the methods used for quantification. F+ coliphages were quantified using an infectivity assay rather than the nucleic acid-based methods used for adenovirus and norovirus GII. PCR-based methods cannot distinguish between infectious and noninfectious viruses and often report concentrations that are several orders of magnitude higher than those determined from infectivity assays. In particular, log removals of infective adenovirus and norovirus GII immediately after a hypochlorite enhanced backwash may be higher than those reported in this study because inactivated viruses are also quantified in PCR assays. Note that a qPCR assay that captures the wide diversity of viruses present in the F + coliphage group is not available, nor is a culture-based method for norovirus, so a direct comparison of the three virus groups using the same methods was not possible. 3.2. Mechanism Contribution to Virus Log Removal Values. The log removal contributions by virus decay, mixed liquor solids, the clean (i.e. just backwashed) membrane, and the fouling cake layer at different levels of development are shown in Figure 3. Part (a) shows LRVs directly calculated from measured concentrations while part (b) breaks down total log removal contributions by individual mechanisms using the number balance and estimation approaches described in section 2.7. The membrane bioreactor process consistently achieved high removals of pathogenic viruses (>4 logs). The clean membrane itself (immediately after a CEB) provided the largest contribution to removal for all three viruses. The next most important mechanism was virus decay, followed by either solids removal or retention by the fouling cake layer. 3.2.1. Retention by Cleaned Membrane and Cake Layer. The largest LRV contribution was due to the backwashed 2819


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Environmental Science & Technology is statistically lower due to the buildup of the cake layer (2 × 10−3 PFU/mL for 1 day after wash and 3 × 10−4 PFU/mL for 4−5 days after wash). The permeate concentration measured 3 h after a CEB was used to calculate LRV contributions for F+ coliphage (details in the Supporting Information Figure S2). No statistically significant differences in permeate concentrations were observed for pathogenic viruses at various levels of membrane cake development. The quantification method used for F+ coliphage allowed greater power to distinguish differences in permeate concentrations due to the ability to assay larger volumes of the concentrated eluent compared to the small-volume molecular-based methods used for adenovirus and norovirus GII. 3.2.2. Virus Inactivation in Mixed Liquor. Overall virus decay, due to inactivation processes such as predation and enzymatic breakdown,21,27 was estimated using a number balance approach, and detailed results are shown in Table S3, Supporting Information. The estimated half-lives of adenovirus, norovirus GII, and F+ coliphage were 1.6, 0.5, and 0.3 h, respectively. Given the MBR hydraulic residence time of approximately 10 h, decay was a relevant loss process over the time scale of treatment. Decay processes were estimated to contribute 1.1 logs for adenovirus, 1.6 logs for norovirus and 1.7 logs for F+ coliphage toward total removal by the MBR process. The decay constants for both norovirus GII and indigenous F + coliphage were higher than the decay constant for somatic coliphage (0.5 h−1) determined by Wu et. al in a bench-scale study,11 suggesting that their laboratory study may not have replicated all relevant inactivation processes. Furthermore, the decay constants for the pathogenic viruses are likely underestimated because the qPCR methods can detect inactivated viruses that contain intact RNA or DNA. In contrast, the decay constant for F+ coliphages may be overestimated, because all return flow samples were below the detection limit for F+ coliphage and the detection limit (1.25 PFU/mL) was used to estimate virus inactivation in the number balance. A drawback of our approach is that we could not differentiate between inactivation rates within the solid and liquid phases of the mixed liquor. Nonetheless, these results provide strong evidence that indigenous virus inactivation mechanisms occur within the MBR process and are important contributors toward overall virus removal. 3.2.3. Attachment to Mixed Liquor Solids. Indigenous viruses often exist as aggregates or are associated with larger particles in wastewaters, which has implications for treatment processes.28,29 In an MBR, viruses attached to mixed liquor flocs would be unlikely to pass through the membrane pores. The mixed liquor solid and liquid separation we used was not perfect for differentiating mechanisms as viruses attached to particles smaller than the theoretical size cutoff of 340 nm remained suspended and were quantified as a part of the liquid fraction, thus underestimating the fraction of viruses attached to the solids. Despite the limitations of the method, 95% of adenovirus, 91% of norovirus GII, and 97% of F+ coliphage were found to be attached to the solids in the mixed liquor. The apparent contributions of solids attachment toward log removal (0.8 for adenovirus, 0.5 for norovirus GII, and 1.0 F+ coliphage) were small relative to the removal provided by the presence of the membrane, its cake layer, and indigenous decay. Thus, increasing the mixed liquor solids concentration would be unlikely to significantly improve virus removal in MBRs through the solids attachment mechanism.

3.3. Hepatitis A Virus and Rotavirus. Four pathogenic viruses (adenovirus, norovirus GII, hepatitis A virus, and rotavirus) were quantified after sampling events, but hepatitis A and rotavirus were not consistently detected, precluding any analysis of removal mechanisms. Hepatitis A virus was detected in the raw influent on only two occasions (2 × 104 and 5 × 104 gc/mL). Rotavirus was never detected in any samples. These four viruses were chosen as viral targets, because they are ubiquitous in wastewaters and have previously been detected in US waters.30,31 The typical detection limit for the viral assays in influent samples was 103 gc/mL. Hepatitis A virus may not have been consistently quantified because its concentrations were lower than those for adenovirus and norovirus GII. Rotavirus may not be circulating within the contributing population due to the routine use of a live, oral, human-bovine rotavirus vaccine, RotaTeq (Merck & Company, Whitehouse Station, New Jersey) since February 2006. Inquiries at two major hospitals in the area provided no reported cases of rotavirus during the period of sampling. Surveillance data collected by the Centers for Disease Control since 2006 also demonstrate a decrease in the number of cases by over 50%.32,33 3.4. Particle Counts after a Hypochlorite Enhanced Backwash. Typical particle size and turbidity data collected on July 30, 2013 immediately after a hypochlorite enhanced backwash are shown in Figure 4. In both cases, there was a large

Figure 4. Turbidity (top) and particle counts (bottom) in permeate immediately after the end of a CEB of a treatment train on July 30, 2013. Membrane relaxation occurred every hour. Turbidity data has an upper detection limit of 10 NTU. Particle count data represents cumulative particles counted within a size range of 2 and 100 μm. The smallest particle size range (2 to 4.9 μm) and the largest particle size range (15 to 100 μm) are also shown for comparison.

initial increase followed by gradual decreases, with additional small spikes coinciding with programmed hourly relaxations of the membranes. Similar results were obtained during two additional sampling events (data not shown). The concentration of the largest particles (15 to 100 μm) decreased within the first 15 min after a CEB. If the effect of hourly relaxations is ignored, there appears to be no significant further decrease in the concentration of the largest particles after the first hour. 2820


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Environmental Science & Technology Smaller particles (2 to 4.9 μm) persisted, and their concentrations continued to decrease significantly over the course of 3 h. Unfortunately, it is not possible to unambiguously distinguish whether these turbidity and particle increases are due to breakthrough of particles from the mixed liquor or sloughing of particles or biofilm from the permeate side of the membrane. If they are due to breakthrough of particles, it follows that the decreasing patterns are due to rapid pore clogging (e.g., of membrane tears, imperfections, or leaks in seals) after the CEB, with faster clogging of the largest pores. It may require several hours to achieve clogging of smaller pores, including in the size range of viruses. This finding would appear to be troubling for virus retention by MBRs. However, recall that permeate samples collected immediately after a CEB did not indicate significant breakthrough of adenovirus or norovirus GII. There was also no statistically significant difference between the permeate concentrations collected just after a CEB and up to 4−5 days later for the pathogenic viruses (Figure 2). It should be noted that, to sample sufficient volume for concentrating the pathogenic viruses, it was necessary to collect permeate for about 30 min; thus, it was not possible to detect any variation in virus concentrations during the first 30 min. Further research to distinguish breakthrough of particles from sloughing on the permeate side is required before measurement of turbidity or particle counts can be confirmed as useful surrogates for virus removal. 3.5. Implications for MBR Operation and Design. Even though this study demonstrated over 4 logs of removal for pathogenic viruses in the 30 min period after CEBs, the turbidity and particle count data raise concerns about membrane integrity. Fortunately, existing operational practices such as staggering the timing of cleaning for treatment trains, blending permeates from cleaned and caked trains, and limiting CEBs to once a week per train are likely to mitigate any short duration breakthroughs of viruses, if they occur. These results are from a system with membranes that had been in operation for about 10 years and investigated the role of the cake layer (irreversible fouling) toward virus removal. We did not address the contribution of irrecoverable fouling, which has built up over the lifetime of the membrane and remains in place after CEB, toward virus removal. Membranes that have been in operation for many years may have more integrity problems, but they may provide additional benefits for virus removal due to the buildup of irrecoverable fouling. It would be prudent for MBR facilities using membranes with less run time to interpret our findings with care until more research is done on newer membranes, with different pore sizes and materials, using the approaches in this paper to isolate the contribution of different mechanisms and the impact of membrane maintenance activities. Another finding from this study is the important contribution of indigenous virus decay toward overall removal. We hypothesize that virus inactivation within the solid phase may be greater than in the liquid phase inactivation due to a greater density of enzymes and predators. Given that attachment of viruses to biomass also contributed directly to removal, operation at higher MLSS concentrations and longer residence times may improve virus removal via two different mechanisms (size exclusion and inactivation). 3.6. Implications for Water Reuse. Current California water reuse regulations for unrestricted nonpotable irrigation were originally implemented for conventional biological

treatment and require that the 5 log virus reduction be demonstrated entirely during tertiary treatment, which typically consists of granular media filtration and disinfection with chlorine or UV254 irradiation. Similarly, indirect potable reuse regulations for groundwater replenishment require a 12 log reduction for viruses. MBRs occupy an unusual space within these regulations because, even though they primarily provide biological treatment, the presence of the membrane confers additional pathogen removal benefits. Under the current regulations, MBRs do not receive additional virus removal credit beyond conventional biological wastewater treatment. This study demonstrates the ability of the MBR process to reliably provide more than 4 logs of removal for adenovirus and norovirus GII in a full-scale process, even immediately after CEBs, and provides evidence to motivate a re-evaluation of the reuse regulations to provide additional virus removal credit to similar MBRs.

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ASSOCIATED CONTENT

S Supporting Information *

Additional explanation of methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +1 (510) 643-5023. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to the American Canyon Wastewater Treatment Plant staff (Peter Lee, Jay Atkinson, and Joshua McCauley); Trussell Technologies Inc. for input during research design and for loaning the particle counter; Ali Boehm (Stanford University) for sharing qPCR standard plasmids; and William Bradford and Matthew Blair for fieldwork assistance. Financial support was provided by a National Science Foundation (NSF) Graduate Research Fellowship to R.M.C. and the NSF Engineering Research Center for Reinventing the Nation’s Water Infrastructure (ReNUWIt) under cooperate agreement EEC-1028968.

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REFERENCES

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