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High performance concentration method for viruses in drinking water
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
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Short communication
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High performance concentration method for viruses in drinking water
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Q1
Andreas Kunze, Lu Pei, Dennis Elsässer, Reinhard Niessner, Michael Seidel ∗ Institute of Hydrochemistry, Chair of Analytical Chemistry, Technische Universität München, Marchioninistr. 17, 81377 Munich, Germany
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a b s t r a c t
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Article history: Received 17 February 2015 Received in revised form 21 May 2015 Accepted 14 June 2015 Available online xxx
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Keywords: Large-volume filtration Water hygiene Ultrafiltration Bacteriophage MS2 Drinking water Virus concentration
According to the risk assessment of the WHO, highly infectious pathogenic viruses like rotaviruses should not be present in large-volume drinking water samples of up to 90 m3 . On the other hand, quantification methods for viruses are only operable in small volumes, and presently no concentration procedure for processing such large volumes has been reported. Therefore, the aim of this study was to demonstrate a procedure for processing viruses in-line of a drinking water pipeline by ultrafiltration (UF) and consecutive further concentration by monolithic filtration (MF) and centrifugal ultrafiltration (CeUF) of viruses to a final 1-mL sample. For testing this concept, the model virus bacteriophage MS2 was spiked continuously in UF instrumentation. Tap water was processed in volumes between 32.4 m3 (22 h) and 97.7 m3 (72 h) continuously either in dead-end (DE) or cross-flow (CF) mode. Best results were found by DE-UF over 22 h. The concentration of MS2 was increased from 4.2 × 104 GU/mL (genomic units per milliliter) to 3.2 × 1010 GU/mL and from 71 PFU/mL to 2 × 108 PFU/mL as determined by qRT-PCR and plaque assay, respectively. © 2015 Published by Elsevier B.V.
The numerous routes of virus transmission to drinking water and the high infectious potential of waterborne viruses cause a great hazard potential to the public health (Haas et al., 1993; Griffin et al., 2003). Therefore, a more frequent analysis of viral pollutants in raw and drinking water and reduction efficiencies of applied water filters is recommended (Grabow et al., 2001; Gibson et al., 2012). By risk assessment, the WHO has increased the volume of drinking water that should not contain more than one rotavirus in 32 m3 (WHO, 2004) to 90 m3 (WHO, 2011). The analysis of viruses in such large volumes is very laborious. Considering the detection limits of methods for virus quantification, methods, which are able to measure viruses in drinking water samples of much more than 1 m3 , are recommended. Most concentration methods of large-volume water samples were established in the 1980s and have so far not undergone large changes (Ikner et al., 2012). Positively or negatively charged filter membranes, glass wool filtration, as well as ultrafiltration are the typical methods applied to concentrate various viruses in water samples up to ∼2 m3 (Table 1). In combination with various secondary enrichment steps, the volumetric concentration factors for these methods can range from 103 to 105 . It has been shown in a meta-analysis, that the filter type, the water type and the water volume had no significant influence on virus recovery (Cashdollar
∗ Corresponding author. Tel.: +49 89 2180 78238; fax: +49 89 2180 78255. E-mail address: [email protected] (M. Seidel).
and Wymer, 2013). Despite the potential of all these techniques, the ability to meet the WHO’s demand for a concentration limit of one virus in 90 m3 remains presently unsolved. In comparison to charged filter membranes and glass wool, ultrafiltration is an attractive choice, as various targets can be concentrated at the same time without preconditioning of the sample (Hill et al., 2009). Moreover, racks of UF modules are already used for removal of particles, microorganisms, and viruses in drinking water treatment with capacities above 100,000 m3 /d (Laine et al., 2000). Nowadays, the price of UF modules is decreasing considerably due to an increase in the number of water treatment plants. Hollow fiber modules are especially in use. The possibility of backwashing reduces membrane fouling (Nakatsuka et al., 1996) and it can also be used for the elution of the concentrated fraction inside the hollow fiber module. It has been shown in a previous study, that UF could be very effectively combined with monolithic filtration for drinking water samples with volumes of 10 L (Pei et al., 2012). As a secondary concentration step, monolithic filtration was proven to be a powerful tool not only for selective enrichment of viruses, but also for sample purification. Therefore the monolithic filtration was adapted for the filtration of large sample volumes. The aim of this work was to demonstrate in a proof-of-principle study a procedure for concentrating viruses like MS2 in drinking water samples of 90 m3 to a final volume of 1 mL (Fig. 1). The measured enrichment factors (EF) were compared with the maximal volumetric concentration factor of 9 × 107 by plaque assay and qRT-PCR.
http://dx.doi.org/10.1016/j.jviromet.2015.06.007 0166-0934/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Kunze, A., et al., High performance concentration method for viruses in drinking water. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.007
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Table 1 Published literature for the concentration of microorganisms in sample volumes of more than 1 m3 . Initial Volume
Primary concentration method
Secondary concentration method
Water type
2010 L
1MDS filter (positively charged) Cellulose membrane filter Nanoceram filter (positively charged) Glass wool
Celite
Well
Membrane filter
1900 L
1500–1900 L
1500 L
1500 L
1MDS filter (positively charged)
1000 L
Membrane filter with aluminum chloride
600 L
UF (negatively charged) HA filter (negatively charged) Fiberglass depth filter
100–532 L
19–1000 L
Volumetric concentration factor
Target
Analysis
Recovery
Reference
∼104
Norovirus
PCR
n.d.
Tap
∼105
Poliovirus
Culture
92%
Parshionikar et al. (2003) Wallis et al. (1972)
Flocculation
Ground
∼104
Poliovirus Norovirus
PCR Culture
20% (PCR) 30% (PCR)
Cashdollar et al. (2013)
Flocculation
Well
∼105
Poliovirus Adenovirus
PCR
56% 28%
Flocculation (acidification) Flocculation (PEG) Adsorption to aluminum hydroxide flocs UF
Ground
∼105
PCR
n.d.
Tap
∼104
Enterovirus Human Adenovirus Rotavirus Norovirus Poliovirus
Lambertini et al. (2008) Borchardt et al. (2004)
Culture
70%
Farrah et al. (1978)
River
∼103 –104
Enterovirus
PCR
n.d.
Rutjes et al. (2005)
2. HA filter 3. CeUF
Tap
∼104 –105
Norovirus
PCR
n.d.
Flocculation
Tap Sea Sewage
∼102
Poliovirus
Culture
50%
Haramoto et al. (2004) Gerba et al. (1978)
Fig. 1. The three-step concentration process consisting of dead-end/cross-flow ultrafiltration (DE-/CF-UF), monolithic filtration (MF), and centrifugal ultrafiltration (CeUF).
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A metagenomic study at the Institute of Virology, University of Bonn, was carried by means of next generation sequencing prior to large-volume experiments. No pathogenic viruses were found in 30 m3 of Munich tap water which was concentrated by the threestep concentration process consisting of DE-UF, MF, and CeUF (data not shown). Therefore the proof-of-principle study was conducted with tap water spiked with bacteriophage MS2 as a model virus. Technical studies with waterborne human pathogenic viruses like rotaviruses or noroviruses are currently not manageable. Bacteriophage MS2 is a good candidate to serve as virus surrogate for the evaluation of technical concentration processes because of the small size, non-pathogenicity to humans, and the link to fecal contaminations (Leclerc et al., 2000; Grabow, 2004). UF as first level of concentration was performed using a polyethersulfone multibore hollow fiber module (dizzer multibore
0.9, Inge AG, Germany), connected to a stainless steel pipe system and mounted on a steel frame for easier transportation (Fig. 2a). The nominal pore size of the membrane was 20 nm, the membrane area was 6 m2 and the permeability was about 1000 L h−1 bar−1 m−2 . Two filtration modes can be performed. In cross-flow (CF) UF the sampled water is passed across the filter membrane tangentially which prevents the development of a filter cake and membrane fouling. Particles larger than the pore size are concentrated at the feed site of the membrane and in the closed loop of the UF system. In dead-end (DE) filtration the sampled water is pumped against the membrane. A filter cake is formed but compared to crossflow filtration shearing forces are reduced. Particles larger than the pore size are concentrated at the feed site on the membrane. The instrument (see Fig. 2b) was driven by a rotary pump with a maximum frequency of 50 Hz (Koch, Germany) producing filtration flow rates between 0.7 and 1.7 m3 h−1 . The process modes (i) conditioning/degassing, (ii) cross-flow/dead-end filtration, (iii) elution and (iv) disinfection were controlled by three manual ball valves. A nylon filter (pore size 25 m, Apic GmbH, Germany) was placed between tap water pipeline and UF instrumentation. Tap water was provided from Stadtwerke München GmbH (Munich, Germany). The tap water analysis was performed at our institute and gave the following results: pH 7.6, <0.01 NTU of turbidity, 0.461 ± 0.032 mg/L of total organic carbon, 5.26 mg/L chloride and 433 S/cm of conductivity. As the sampled tap water was free of human pathogenic viruses, a suspension of model organism bacteriophage MS2 (DSM 13767, DSMZ, Germany) was injected continuously (flow rate – 0.1 mL/min, concentration according to Table 2) by a peristaltic pump (Ismatec GmbH, Germany) and an integrated T-connector before the concentration loop of the UF instrumentation. After filtration, the concentrated viruses were eluted by a backflush within a volume of 20 L. The backflush was
Please cite this article in press as: Kunze, A., et al., High performance concentration method for viruses in drinking water. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.007
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Fig. 2. (a) Schematic diagram of the ultrafiltration setup (not in scale). All process steps are manually controlled (sample valve, SV; conditioning valve, CV; dead-end filtration valve, DV and elution valve, EV). The filtrated volume and the transmembrane pressure can be observed by a volume flow meter and three manometers (P 1–3). (b) Picture of the ultrafiltration setup. 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158
performed with a stainless steel pressure tank filled with 24 L of filtrated water at 2.5 bar. Tap water was processed in volumes between ∼32 m3 (22–24 h) and ∼98 m3 (69–72 h) continuously either by DE-UF or CF-UF. 20-L eluates were analyzed by plaque assay and qRT-PCR as published elsewhere (Pei et al., 2012). Table 2 summarizes the UF process. Low recoveries of MS2 (0.1% and 4%) were determined for each CF-UF process quantified with plaque assay and qRT-PCR which may be due to higher shearing stress in the concentration loop. Best recoveries (31.88% ± 0.03% for qRT-PCR and 11.0% ± 5.0% for plaque assay) were determined in 20-L eluates with DE-UF and a filtration process not longer than 1 day (39.4 m3 ). DE-UF over 3 days resulted in comparable recoveries for qRT-PCR (21.38% ± 0.02%) but lower activity determined by plaque assay (0.8% ± 0.3%). The dwell time of the phages in the instrument was enlarged to 69 h for processing 98 m3 of tap water with DE-UF. Therefore, inactivation processes in the concentration loop of the UF instrumentation may result from agglomeration and physical stresses of phages. Additionally to the recovery, an enrichment factor (EF) was calculated by comparing recovered and spiked concentrations of MS2. Enrichment factors for 34 m3 of processed tap water were 524 and 182 determined with qRT-PCR and plaque assay, respectively. EF was increased for 97.6 m3 of processed tap water (EF = 1021), but was decreased by quantification with plaque assay (EF = 36). The envelopes of MS2 phages might be damaged during the DE-UF process of 3 days. However, nucleic acids could be eluted more effectively than with CF-UF (EF = 82 for 97.7 m3 ). A secondary concentration method was applied to further concentrate the 20-L UF eluate to a final volume in the milliliter range. Monolithic filtration was chosen, as it is capable of selectively separating viruses from the matrix. The synthesis of the epoxy based macroporous monolith was adapted from a previously published protocol (Peskoller et al., 2009). Modifications to increase the flow rate, binding capacity and cost effectiveness were achieved by synthesizing the monolith in disk shape PTFE molds. The selfpolymerization of polyglycerol-3-glycidyl ether was carried out using toluene and tert-butyl methyl ether as porogen (3:2, w/v) at room temperature for 1 h. After washing in methanol, the synthesized monolith was activated with sulfuric acid (0.5 M) at 60 ◦ C for 3 h. Thus, hydroxyl groups on the monolithic surface are formed. The acid was removed with ultrapure water and the monolithic
disks were stored at 4 ◦ C until use. Monolithic disks were assembled in a 50-mL plastic dispenser tip (PD-tip, Brand GmbH, Germany) to form the filtration module (Fig. S1b). A PTFE support plate with bore holes of 2 mm in diameter and O-rings were put in the module followed by monolithic disks and a PTFE fitting for connection to silicone tubes. To characterize the monolithic disks, tap water samples spiked with MS2 were equilibrated with hydrochloric acid to pH 3 prior to monolithic filtration. Since the isoelectric point of most viruses is above pH 3 (Langlet et al., 2007; Michen and Graule, 2010) positively charged viruses were adsorbed on the surface of the monolith via ionic and hydrophobic interactions. The dimensions of the monolithic disk were optimized by filtration of 10-L tap water samples spiked with bacteriophage MS2 (6.4 × 106 –1.5 × 107 PFU/mL) at a flow rate of 1 L/min. After filtration, adsorbed viruses were eluted with 20 mL of elution buffer (pH 9.5) containing 3% beefextract and 0.5 M glycine (BEG buffer). Filtration modules with a monolithic disk of 3.86 cm in diameter and 1.0 cm in height (total volume: 46.8 cm3 ) was characterized using 10-L tap water samples, which contained MS2 in amounts between 2 ± 0.5 × 103 PFU and 1.9 ± 0.2 × 108 PFU. Within the given concentration range, 102% ± 23% of added MS2 could be recovered. Details are shown in Fig. S2. The complete 3-step concentration procedure for the enrichment of MS2 in spiked tap water samples of ∼32 m3 and ∼97 m3 was carried out by combining ultrafiltration, monolithic filtration and CeUF. During ultrafiltration, the initial sample volume was concentrated to 20 L. The 20-L UF eluate was conditioned to pH 3 and applied to monolithic filtration for further concentration to a final volume of 20 mL. The module for monolithic filtration contained two monolithic disks in series. The sealing of the module was guaranteed by an O-ring between the disks. If a high particle fraction of the UF eluate would clog the upper disk, the increasing back pressure would squeeze the monoliths, thereby sealing the interface between the monolithic disks and the dispenser wall. Finally centrifugal ultrafiltration with a molecular weight cut-off of 50 kDa (amicon ultra-15, Millipore, Germany) was applied to achieve a final volume of ∼1 mL. As summarized in Table 3, the concentration of MS2 after ultrafiltration/monolithic filtration/centrifugal ultrafiltration was quantified by qRT-PCR and plaque assay, respectively.
Please cite this article in press as: Kunze, A., et al., High performance concentration method for viruses in drinking water. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.007
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UF
Flow m3 h−1
Spiked c(MS2) PFU mL−1
32.4 33.9 97.7 97.6
CF DE CF DE
1.35 1.54 1.36 1.42
184 71 119 185
± ± ± ±
30 10 6 16
Recovery1 %
Recovered c(MS2) PFU mL−1 3.2 × 103 1.3 × 104 5.0 × 102 6.7 × 103
± ± ± ±
3 × 102 4 × 103 5.0 × 102 2 × 103
1.2 11.0 0.1 0.8
± ± ± ±
0.3 5.0 0.1 0.3
EF1
Spiked c(MS2) GU mL−1
17 182 4 36
4.3 × 104 4.2 × 104 1.1 × 104 5.3 × 104
Recovery2 %
Recovered c(MS2) GU mL−1 ± ± ± ±
9.2 × 102 7.2 × 102 3.8 × 102 6.8 × 102
2.5 × 106 2.2 × 107 8.6 × 105 5.4 × 107
± ± ± ±
1.6 × 106 2.6 × 106 6.5 × 105 6.7 × 106
3.87 31.88 1.75 21.38
± ± ± ±
0.04 0.03 0.01 0.02
EF2
57 524 82 1021
Table 3 Results for the 3-step enrichment procedure consisting of cross-flow (CF-UF) or dead-end (DE-UF)/monolithic filtration/centrifugal ultrafiltration for large-volume tap water samples determined by 1 plaque assay and 2 qRT-PCR (m = 1, n = 3). V m3
UF
Flow m3 h−1
Spiked c(MS2) PFU mL−1
32.4 33.9 97.7 97.6
CF DE CF DE
1.35 1.54 1.36 1.42
184 71 119 185
± ± ± ±
30 10 6 16
Recovery1 %
Recovered c(MS2) PFU mL−1 4.3 × 107 1.9 × 108 2.3 × 107 1.6 × 108
± ± ± ±
8 × 106 1 × 108 5 × 106 1 × 108
0.6 13 0.2 1.2
± ± ± ±
0.3 12 0.1 0.9
EF1
2 × 105 3 × 106 2 × 105 9 × 105
Spiked c(MS2) GU mL−1 4.3 × 104 4.2 × 104 1.1 × 104 5.3 × 104
Recovery2 %
Recovered c(MS2) GU mL−1 ± ± ± ±
9.2 × 102 7.2 × 102 3.8 × 102 6.8 × 102
1.5 × 1010 3.2 × 1010 8.4 × 109 9.2 × 109
± ± ± ±
2.5 × 108 9.6 × 108 7.7 × 108 1.8 × 108
0.90 3.55 1.16 0.18
± ± ± ±
0.03 0.02 0.01 0.01
EF2
3 × 105 8 × 105 8 × 105 2 × 105
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Table 2
Q3 Ultrafiltration (UF) study of continuously spiked MS2 in tap water of 32–98 m3 analyzed by 1 plaque assay and 2 qRT-PCR (m = 1, n = 3). The enrichment factor (EF) was calculated by comparing recovered and spiked concentrations Q4 of MS2.
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The 1-day and 3-day UF-processes show comparable recoveries of MS2. Highest recovery was found for a tap water volume of 33.9 m3 and by concentrating MS2 with dead-end ultrafiltration/monolithic filtration/centrifugal ultrafiltration. 3.55% ± 0.02% and 13% ± 12% of MS2 were recovered with qRT-PCR and plaque assay, respectively. Applying the 3-step concentration method for tap water over 3 days, the EF could not be increased by DE-UF (Table 3). Changes of the EF from 1-step (UF) to 3-step concentration result from the fact, that adsorption on the monolithic disk works better for intact MS2 particles than for MS2 fragments or free nucleic acids. Therefore, a fragmentation by physical stresses during UF reduced the efficiency of the concentration process. Since plaque assay quantifies only infectious MS2, a higher EF was determined (EF = 9 × 105 ) than with qRT-PCR (EF = 2 × 105 ). We have shown that the analysis of viruses in large-volume drinking water samples (>1 m3 ) is manageable on-site of water pipelines of house installations or drinking water. Hollow fiber UF modules allowed to process drinking water up to a volume of 97.7 m3 in DE and CF mode without backwashing during the process. Tap water could directly be concentrated to transportable sample volume without adjustments of pH or conditioning with additives as demanded for negatively or positively charged membranes. The 20-L eluate of the UF was transported to the lab and used for further concentration by monolithic filtration and centrifugal ultrafiltration and analysis of viruses either by plaque assay or qRT-PCR. The comparative measurements by plaque assay and qRT-PCR showed that sampling of viruses should be done in 1 day by DE-UF. Regarding an increase of the enrichment factor, UF modules with larger membrane area might be more efficient than processing DE-UF over more than 1 day. For analysis of relevant pathogens in large-volume drinking water, the development of an effective secondary concentration method was necessary. The module for monolithic filtration was able to collect bacteriophage MS2 highly efficient at flow rates of 1 L/min. In combination with CeUF, the UF eluate was concentrated from 20 L to 1 mL in 1 h. The new procedure for concentration of viruses from largevolume drinking water samples is now ready for many applications. Other viruses like X174, human adenovirus (hAdV) and murine norovirus were successfully tested by monolithic filtration (Pei et al., 2012) and can be further investigated by the concentration process in future studies. Besides the monitoring of pathogenic viruses in drinking water, the cell culture independent analysis of multiple waterborne pathogens by sequencing (Duhaime et al., 2012) or microarray-based detection methods (Lengger et al., 2014) are favorable research tasks. In combination with the UF process, our presented concentration method can also be important for the understanding of filtration processes and the development of new membrane systems for the reduction of waterborne pathogens in water treatment plants with 4 or 6 log steps (Madaeni, 1999). Much more than 90 m3 of drinking water has to be concentrated by the 3-step concentration procedure to quantify single rotaviruses in 90 m3 as recommended by the WHO. For practical reasons, water treatment plants should achieve a virus reduction of 6 log steps and the analysis of smaller water sample volumes of 1–10 m3 should be repeated in shorter intervals.
Acknowledgement The authors like to thank Sebastian Wiesemann and Roland Hoppe for producing the UF instrumentation, Dr. Martin Rieger for first tests, and Susanne Mahler for performing the plaque assays. We thank Dr. Nils Hartmann and Dr. Hans-Christoph Selinka from the German Federal Environment Agency (Umweltbundesamt, UBA) for the monolithic filtration experiments with MNV, hAdV and bacteriophage PhiX174. We like to thank Prof.
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Christian Drosten from the Institute of Virology (University of Bonn) for the next generation sequencing of our tap water after applying our concentration method. The DFG (SE 1722/2-1), the Max- Q2 Buchner-Forschungsstiftung (MBFSt-2907), BMBF-FONA project EDIT (WO 033W010E) and the China Scholarship Council are gratefully acknowledged for financial support.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2015. 06.007
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Rutjes, S.A., Italiaander, R., van den Berg, H.H.J.L., Lodder, W.J., Husman, A.M.D., 2005. Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 71, 3734–3740. Wallis, C., Henderson, M., Melnick, J.L., 1972. Enterovirus concentration on cellulose membranes. Appl. Microbiol. 23, 476–480. WHO, 2004. Guidelines for Drinking Water Quality, 3 ed. WHO, 2011. Guidelines for Drinking Water Quality, 4 ed.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
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Short communication
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High performance concentration method for viruses in drinking water
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Q1
Andreas Kunze, Lu Pei, Dennis Elsässer, Reinhard Niessner, Michael Seidel ∗ Institute of Hydrochemistry, Chair of Analytical Chemistry, Technische Universität München, Marchioninistr. 17, 81377 Munich, Germany
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a b s t r a c t
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Article history: Received 17 February 2015 Received in revised form 21 May 2015 Accepted 14 June 2015 Available online xxx
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21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Keywords: Large-volume filtration Water hygiene Ultrafiltration Bacteriophage MS2 Drinking water Virus concentration
According to the risk assessment of the WHO, highly infectious pathogenic viruses like rotaviruses should not be present in large-volume drinking water samples of up to 90 m3 . On the other hand, quantification methods for viruses are only operable in small volumes, and presently no concentration procedure for processing such large volumes has been reported. Therefore, the aim of this study was to demonstrate a procedure for processing viruses in-line of a drinking water pipeline by ultrafiltration (UF) and consecutive further concentration by monolithic filtration (MF) and centrifugal ultrafiltration (CeUF) of viruses to a final 1-mL sample. For testing this concept, the model virus bacteriophage MS2 was spiked continuously in UF instrumentation. Tap water was processed in volumes between 32.4 m3 (22 h) and 97.7 m3 (72 h) continuously either in dead-end (DE) or cross-flow (CF) mode. Best results were found by DE-UF over 22 h. The concentration of MS2 was increased from 4.2 × 104 GU/mL (genomic units per milliliter) to 3.2 × 1010 GU/mL and from 71 PFU/mL to 2 × 108 PFU/mL as determined by qRT-PCR and plaque assay, respectively. © 2015 Published by Elsevier B.V.
The numerous routes of virus transmission to drinking water and the high infectious potential of waterborne viruses cause a great hazard potential to the public health (Haas et al., 1993; Griffin et al., 2003). Therefore, a more frequent analysis of viral pollutants in raw and drinking water and reduction efficiencies of applied water filters is recommended (Grabow et al., 2001; Gibson et al., 2012). By risk assessment, the WHO has increased the volume of drinking water that should not contain more than one rotavirus in 32 m3 (WHO, 2004) to 90 m3 (WHO, 2011). The analysis of viruses in such large volumes is very laborious. Considering the detection limits of methods for virus quantification, methods, which are able to measure viruses in drinking water samples of much more than 1 m3 , are recommended. Most concentration methods of large-volume water samples were established in the 1980s and have so far not undergone large changes (Ikner et al., 2012). Positively or negatively charged filter membranes, glass wool filtration, as well as ultrafiltration are the typical methods applied to concentrate various viruses in water samples up to ∼2 m3 (Table 1). In combination with various secondary enrichment steps, the volumetric concentration factors for these methods can range from 103 to 105 . It has been shown in a meta-analysis, that the filter type, the water type and the water volume had no significant influence on virus recovery (Cashdollar
∗ Corresponding author. Tel.: +49 89 2180 78238; fax: +49 89 2180 78255. E-mail address: [email protected] (M. Seidel).
and Wymer, 2013). Despite the potential of all these techniques, the ability to meet the WHO’s demand for a concentration limit of one virus in 90 m3 remains presently unsolved. In comparison to charged filter membranes and glass wool, ultrafiltration is an attractive choice, as various targets can be concentrated at the same time without preconditioning of the sample (Hill et al., 2009). Moreover, racks of UF modules are already used for removal of particles, microorganisms, and viruses in drinking water treatment with capacities above 100,000 m3 /d (Laine et al., 2000). Nowadays, the price of UF modules is decreasing considerably due to an increase in the number of water treatment plants. Hollow fiber modules are especially in use. The possibility of backwashing reduces membrane fouling (Nakatsuka et al., 1996) and it can also be used for the elution of the concentrated fraction inside the hollow fiber module. It has been shown in a previous study, that UF could be very effectively combined with monolithic filtration for drinking water samples with volumes of 10 L (Pei et al., 2012). As a secondary concentration step, monolithic filtration was proven to be a powerful tool not only for selective enrichment of viruses, but also for sample purification. Therefore the monolithic filtration was adapted for the filtration of large sample volumes. The aim of this work was to demonstrate in a proof-of-principle study a procedure for concentrating viruses like MS2 in drinking water samples of 90 m3 to a final volume of 1 mL (Fig. 1). The measured enrichment factors (EF) were compared with the maximal volumetric concentration factor of 9 × 107 by plaque assay and qRT-PCR.
http://dx.doi.org/10.1016/j.jviromet.2015.06.007 0166-0934/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Kunze, A., et al., High performance concentration method for viruses in drinking water. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.06.007
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Table 1 Published literature for the concentration of microorganisms in sample volumes of more than 1 m3 . Initial Volume
Primary concentration method
Secondary concentration method
Water type
2010 L
1MDS filter (positively charged) Cellulose membrane filter Nanoceram filter (positively charged) Glass wool
Celite
Well
Membrane filter
1900 L
1500–1900 L
1500 L
1500 L
1MDS filter (positively charged)
1000 L
Membrane filter with aluminum chloride
600 L
UF (negatively charged) HA filter (negatively charged) Fiberglass depth filter
100–532 L
19–1000 L
Volumetric concentration factor
Target
Analysis
Recovery
Reference
∼104
Norovirus
PCR
n.d.
Tap
∼105
Poliovirus
Culture
92%
Parshionikar et al. (2003) Wallis et al. (1972)
Flocculation
Ground
∼104
Poliovirus Norovirus
PCR Culture
20% (PCR) 30% (PCR)
Cashdollar et al. (2013)
Flocculation
Well
∼105
Poliovirus Adenovirus
PCR
56% 28%
Flocculation (acidification) Flocculation (PEG) Adsorption to aluminum hydroxide flocs UF
Ground
∼105
PCR
n.d.
Tap
∼104
Enterovirus Human Adenovirus Rotavirus Norovirus Poliovirus
Lambertini et al. (2008) Borchardt et al. (2004)
Culture
70%
Farrah et al. (1978)
River
∼103 –104
Enterovirus
PCR
n.d.
Rutjes et al. (2005)
2. HA filter 3. CeUF
Tap
∼104 –105
Norovirus
PCR
n.d.
Flocculation
Tap Sea Sewage
∼102
Poliovirus
Culture
50%
Haramoto et al. (2004) Gerba et al. (1978)
Fig. 1. The three-step concentration process consisting of dead-end/cross-flow ultrafiltration (DE-/CF-UF), monolithic filtration (MF), and centrifugal ultrafiltration (CeUF).
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
A metagenomic study at the Institute of Virology, University of Bonn, was carried by means of next generation sequencing prior to large-volume experiments. No pathogenic viruses were found in 30 m3 of Munich tap water which was concentrated by the threestep concentration process consisting of DE-UF, MF, and CeUF (data not shown). Therefore the proof-of-principle study was conducted with tap water spiked with bacteriophage MS2 as a model virus. Technical studies with waterborne human pathogenic viruses like rotaviruses or noroviruses are currently not manageable. Bacteriophage MS2 is a good candidate to serve as virus surrogate for the evaluation of technical concentration processes because of the small size, non-pathogenicity to humans, and the link to fecal contaminations (Leclerc et al., 2000; Grabow, 2004). UF as first level of concentration was performed using a polyethersulfone multibore hollow fiber module (dizzer multibore
0.9, Inge AG, Germany), connected to a stainless steel pipe system and mounted on a steel frame for easier transportation (Fig. 2a). The nominal pore size of the membrane was 20 nm, the membrane area was 6 m2 and the permeability was about 1000 L h−1 bar−1 m−2 . Two filtration modes can be performed. In cross-flow (CF) UF the sampled water is passed across the filter membrane tangentially which prevents the development of a filter cake and membrane fouling. Particles larger than the pore size are concentrated at the feed site of the membrane and in the closed loop of the UF system. In dead-end (DE) filtration the sampled water is pumped against the membrane. A filter cake is formed but compared to crossflow filtration shearing forces are reduced. Particles larger than the pore size are concentrated at the feed site on the membrane. The instrument (see Fig. 2b) was driven by a rotary pump with a maximum frequency of 50 Hz (Koch, Germany) producing filtration flow rates between 0.7 and 1.7 m3 h−1 . The process modes (i) conditioning/degassing, (ii) cross-flow/dead-end filtration, (iii) elution and (iv) disinfection were controlled by three manual ball valves. A nylon filter (pore size 25 m, Apic GmbH, Germany) was placed between tap water pipeline and UF instrumentation. Tap water was provided from Stadtwerke München GmbH (Munich, Germany). The tap water analysis was performed at our institute and gave the following results: pH 7.6, <0.01 NTU of turbidity, 0.461 ± 0.032 mg/L of total organic carbon, 5.26 mg/L chloride and 433 S/cm of conductivity. As the sampled tap water was free of human pathogenic viruses, a suspension of model organism bacteriophage MS2 (DSM 13767, DSMZ, Germany) was injected continuously (flow rate – 0.1 mL/min, concentration according to Table 2) by a peristaltic pump (Ismatec GmbH, Germany) and an integrated T-connector before the concentration loop of the UF instrumentation. After filtration, the concentrated viruses were eluted by a backflush within a volume of 20 L. The backflush was
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Fig. 2. (a) Schematic diagram of the ultrafiltration setup (not in scale). All process steps are manually controlled (sample valve, SV; conditioning valve, CV; dead-end filtration valve, DV and elution valve, EV). The filtrated volume and the transmembrane pressure can be observed by a volume flow meter and three manometers (P 1–3). (b) Picture of the ultrafiltration setup. 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158
performed with a stainless steel pressure tank filled with 24 L of filtrated water at 2.5 bar. Tap water was processed in volumes between ∼32 m3 (22–24 h) and ∼98 m3 (69–72 h) continuously either by DE-UF or CF-UF. 20-L eluates were analyzed by plaque assay and qRT-PCR as published elsewhere (Pei et al., 2012). Table 2 summarizes the UF process. Low recoveries of MS2 (0.1% and 4%) were determined for each CF-UF process quantified with plaque assay and qRT-PCR which may be due to higher shearing stress in the concentration loop. Best recoveries (31.88% ± 0.03% for qRT-PCR and 11.0% ± 5.0% for plaque assay) were determined in 20-L eluates with DE-UF and a filtration process not longer than 1 day (39.4 m3 ). DE-UF over 3 days resulted in comparable recoveries for qRT-PCR (21.38% ± 0.02%) but lower activity determined by plaque assay (0.8% ± 0.3%). The dwell time of the phages in the instrument was enlarged to 69 h for processing 98 m3 of tap water with DE-UF. Therefore, inactivation processes in the concentration loop of the UF instrumentation may result from agglomeration and physical stresses of phages. Additionally to the recovery, an enrichment factor (EF) was calculated by comparing recovered and spiked concentrations of MS2. Enrichment factors for 34 m3 of processed tap water were 524 and 182 determined with qRT-PCR and plaque assay, respectively. EF was increased for 97.6 m3 of processed tap water (EF = 1021), but was decreased by quantification with plaque assay (EF = 36). The envelopes of MS2 phages might be damaged during the DE-UF process of 3 days. However, nucleic acids could be eluted more effectively than with CF-UF (EF = 82 for 97.7 m3 ). A secondary concentration method was applied to further concentrate the 20-L UF eluate to a final volume in the milliliter range. Monolithic filtration was chosen, as it is capable of selectively separating viruses from the matrix. The synthesis of the epoxy based macroporous monolith was adapted from a previously published protocol (Peskoller et al., 2009). Modifications to increase the flow rate, binding capacity and cost effectiveness were achieved by synthesizing the monolith in disk shape PTFE molds. The selfpolymerization of polyglycerol-3-glycidyl ether was carried out using toluene and tert-butyl methyl ether as porogen (3:2, w/v) at room temperature for 1 h. After washing in methanol, the synthesized monolith was activated with sulfuric acid (0.5 M) at 60 ◦ C for 3 h. Thus, hydroxyl groups on the monolithic surface are formed. The acid was removed with ultrapure water and the monolithic
disks were stored at 4 ◦ C until use. Monolithic disks were assembled in a 50-mL plastic dispenser tip (PD-tip, Brand GmbH, Germany) to form the filtration module (Fig. S1b). A PTFE support plate with bore holes of 2 mm in diameter and O-rings were put in the module followed by monolithic disks and a PTFE fitting for connection to silicone tubes. To characterize the monolithic disks, tap water samples spiked with MS2 were equilibrated with hydrochloric acid to pH 3 prior to monolithic filtration. Since the isoelectric point of most viruses is above pH 3 (Langlet et al., 2007; Michen and Graule, 2010) positively charged viruses were adsorbed on the surface of the monolith via ionic and hydrophobic interactions. The dimensions of the monolithic disk were optimized by filtration of 10-L tap water samples spiked with bacteriophage MS2 (6.4 × 106 –1.5 × 107 PFU/mL) at a flow rate of 1 L/min. After filtration, adsorbed viruses were eluted with 20 mL of elution buffer (pH 9.5) containing 3% beefextract and 0.5 M glycine (BEG buffer). Filtration modules with a monolithic disk of 3.86 cm in diameter and 1.0 cm in height (total volume: 46.8 cm3 ) was characterized using 10-L tap water samples, which contained MS2 in amounts between 2 ± 0.5 × 103 PFU and 1.9 ± 0.2 × 108 PFU. Within the given concentration range, 102% ± 23% of added MS2 could be recovered. Details are shown in Fig. S2. The complete 3-step concentration procedure for the enrichment of MS2 in spiked tap water samples of ∼32 m3 and ∼97 m3 was carried out by combining ultrafiltration, monolithic filtration and CeUF. During ultrafiltration, the initial sample volume was concentrated to 20 L. The 20-L UF eluate was conditioned to pH 3 and applied to monolithic filtration for further concentration to a final volume of 20 mL. The module for monolithic filtration contained two monolithic disks in series. The sealing of the module was guaranteed by an O-ring between the disks. If a high particle fraction of the UF eluate would clog the upper disk, the increasing back pressure would squeeze the monoliths, thereby sealing the interface between the monolithic disks and the dispenser wall. Finally centrifugal ultrafiltration with a molecular weight cut-off of 50 kDa (amicon ultra-15, Millipore, Germany) was applied to achieve a final volume of ∼1 mL. As summarized in Table 3, the concentration of MS2 after ultrafiltration/monolithic filtration/centrifugal ultrafiltration was quantified by qRT-PCR and plaque assay, respectively.
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UF
Flow m3 h−1
Spiked c(MS2) PFU mL−1
32.4 33.9 97.7 97.6
CF DE CF DE
1.35 1.54 1.36 1.42
184 71 119 185
± ± ± ±
30 10 6 16
Recovery1 %
Recovered c(MS2) PFU mL−1 3.2 × 103 1.3 × 104 5.0 × 102 6.7 × 103
± ± ± ±
3 × 102 4 × 103 5.0 × 102 2 × 103
1.2 11.0 0.1 0.8
± ± ± ±
0.3 5.0 0.1 0.3
EF1
Spiked c(MS2) GU mL−1
17 182 4 36
4.3 × 104 4.2 × 104 1.1 × 104 5.3 × 104
Recovery2 %
Recovered c(MS2) GU mL−1 ± ± ± ±
9.2 × 102 7.2 × 102 3.8 × 102 6.8 × 102
2.5 × 106 2.2 × 107 8.6 × 105 5.4 × 107
± ± ± ±
1.6 × 106 2.6 × 106 6.5 × 105 6.7 × 106
3.87 31.88 1.75 21.38
± ± ± ±
0.04 0.03 0.01 0.02
EF2
57 524 82 1021
Table 3 Results for the 3-step enrichment procedure consisting of cross-flow (CF-UF) or dead-end (DE-UF)/monolithic filtration/centrifugal ultrafiltration for large-volume tap water samples determined by 1 plaque assay and 2 qRT-PCR (m = 1, n = 3). V m3
UF
Flow m3 h−1
Spiked c(MS2) PFU mL−1
32.4 33.9 97.7 97.6
CF DE CF DE
1.35 1.54 1.36 1.42
184 71 119 185
± ± ± ±
30 10 6 16
Recovery1 %
Recovered c(MS2) PFU mL−1 4.3 × 107 1.9 × 108 2.3 × 107 1.6 × 108
± ± ± ±
8 × 106 1 × 108 5 × 106 1 × 108
0.6 13 0.2 1.2
± ± ± ±
0.3 12 0.1 0.9
EF1
2 × 105 3 × 106 2 × 105 9 × 105
Spiked c(MS2) GU mL−1 4.3 × 104 4.2 × 104 1.1 × 104 5.3 × 104
Recovery2 %
Recovered c(MS2) GU mL−1 ± ± ± ±
9.2 × 102 7.2 × 102 3.8 × 102 6.8 × 102
1.5 × 1010 3.2 × 1010 8.4 × 109 9.2 × 109
± ± ± ±
2.5 × 108 9.6 × 108 7.7 × 108 1.8 × 108
0.90 3.55 1.16 0.18
± ± ± ±
0.03 0.02 0.01 0.01
EF2
3 × 105 8 × 105 8 × 105 2 × 105
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Table 2
Q3 Ultrafiltration (UF) study of continuously spiked MS2 in tap water of 32–98 m3 analyzed by 1 plaque assay and 2 qRT-PCR (m = 1, n = 3). The enrichment factor (EF) was calculated by comparing recovered and spiked concentrations Q4 of MS2.
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255
256 257 258 259 260 261 262
The 1-day and 3-day UF-processes show comparable recoveries of MS2. Highest recovery was found for a tap water volume of 33.9 m3 and by concentrating MS2 with dead-end ultrafiltration/monolithic filtration/centrifugal ultrafiltration. 3.55% ± 0.02% and 13% ± 12% of MS2 were recovered with qRT-PCR and plaque assay, respectively. Applying the 3-step concentration method for tap water over 3 days, the EF could not be increased by DE-UF (Table 3). Changes of the EF from 1-step (UF) to 3-step concentration result from the fact, that adsorption on the monolithic disk works better for intact MS2 particles than for MS2 fragments or free nucleic acids. Therefore, a fragmentation by physical stresses during UF reduced the efficiency of the concentration process. Since plaque assay quantifies only infectious MS2, a higher EF was determined (EF = 9 × 105 ) than with qRT-PCR (EF = 2 × 105 ). We have shown that the analysis of viruses in large-volume drinking water samples (>1 m3 ) is manageable on-site of water pipelines of house installations or drinking water. Hollow fiber UF modules allowed to process drinking water up to a volume of 97.7 m3 in DE and CF mode without backwashing during the process. Tap water could directly be concentrated to transportable sample volume without adjustments of pH or conditioning with additives as demanded for negatively or positively charged membranes. The 20-L eluate of the UF was transported to the lab and used for further concentration by monolithic filtration and centrifugal ultrafiltration and analysis of viruses either by plaque assay or qRT-PCR. The comparative measurements by plaque assay and qRT-PCR showed that sampling of viruses should be done in 1 day by DE-UF. Regarding an increase of the enrichment factor, UF modules with larger membrane area might be more efficient than processing DE-UF over more than 1 day. For analysis of relevant pathogens in large-volume drinking water, the development of an effective secondary concentration method was necessary. The module for monolithic filtration was able to collect bacteriophage MS2 highly efficient at flow rates of 1 L/min. In combination with CeUF, the UF eluate was concentrated from 20 L to 1 mL in 1 h. The new procedure for concentration of viruses from largevolume drinking water samples is now ready for many applications. Other viruses like X174, human adenovirus (hAdV) and murine norovirus were successfully tested by monolithic filtration (Pei et al., 2012) and can be further investigated by the concentration process in future studies. Besides the monitoring of pathogenic viruses in drinking water, the cell culture independent analysis of multiple waterborne pathogens by sequencing (Duhaime et al., 2012) or microarray-based detection methods (Lengger et al., 2014) are favorable research tasks. In combination with the UF process, our presented concentration method can also be important for the understanding of filtration processes and the development of new membrane systems for the reduction of waterborne pathogens in water treatment plants with 4 or 6 log steps (Madaeni, 1999). Much more than 90 m3 of drinking water has to be concentrated by the 3-step concentration procedure to quantify single rotaviruses in 90 m3 as recommended by the WHO. For practical reasons, water treatment plants should achieve a virus reduction of 6 log steps and the analysis of smaller water sample volumes of 1–10 m3 should be repeated in shorter intervals.
Acknowledgement The authors like to thank Sebastian Wiesemann and Roland Hoppe for producing the UF instrumentation, Dr. Martin Rieger for first tests, and Susanne Mahler for performing the plaque assays. We thank Dr. Nils Hartmann and Dr. Hans-Christoph Selinka from the German Federal Environment Agency (Umweltbundesamt, UBA) for the monolithic filtration experiments with MNV, hAdV and bacteriophage PhiX174. We like to thank Prof.
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Christian Drosten from the Institute of Virology (University of Bonn) for the next generation sequencing of our tap water after applying our concentration method. The DFG (SE 1722/2-1), the Max- Q2 Buchner-Forschungsstiftung (MBFSt-2907), BMBF-FONA project EDIT (WO 033W010E) and the China Scholarship Council are gratefully acknowledged for financial support.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2015. 06.007
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