- Email: [email protected]
Adsorption of bacteriophage MS2 to colloids: Kinetics and particle interactions
Colloids and Surfaces A xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Adsorption of bacteriophage MS2 to colloids: Kinetics and particle interactions Yun Xinga, Ashlee Ellisa, Matthew Magnusonb, Willie F. Harper Jr.a,* a b
Air Force Institute of Technology, Department of Systems Engineering and Management, Environmental Engineering and Science Program, Wright-Patterson AFB, Ohio, US US Environmental Protection Agency, National Homeland Security Research Center, Water Infrastructure Protection Division, Cincinnati, Ohio, US
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Keywords: Adsorption Biocontaminant Bacteriophage MS2 XDLVO Atomic force microscopy
Virus adsorption to colloidal particles is an important issue in the water quality community. Namely, if viruses can quickly and strongly associate to colloids, this can potentially lead to significant implications for the management of biohazardous wastes at water reclamation facilities. This research evaluated the adsorption of bacteriophage MS2 to colloidal suspensions of kaolinite (KAO) and fiberglass (FG). Observed pseudo first-order MS2 removal rate constants were between 0.53 and 5.1 min−1 and between 2.4 and 3.5 min−1 for KAO and FG, respectively. These kinetics were at least an order of magnitude faster than previously reported values when compared to data retrieved at similar colloid concentrations. Fluorescent and bright field microscopic images showed clusters of MS2 on and around the edges of the colloids, and the majority of the bound MS2 was not readily removed during a vigorous wash step, suggesting comparatively strong, operationally relevant adsorption. MS2 aggregation was observed experimentally and predicted on the basis of interaction energies calculated with XDLVO models. When virus-containing biohazardous wastes are introduced into wastewater treatment plants, removing colloids is essential.
1. Introduction Improving water infrastructure security continues to be a high priority for environmental professionals. One ongoing security
⁎
initiative concerns high-consequence biocontaminants, including a wide range of dangerous bacteria, viruses, proteins, and metabolites. These biological agents can pose a very serious threat to the public when unusually large loads are discharged by hospitals [1], accidents,
Corresponding author. E-mail address: [email protected] (W.F. Harper).
https://doi.org/10.1016/j.colsurfa.2019.124099 Received 4 September 2019; Received in revised form 8 October 2019; Accepted 8 October 2019 Available online 25 October 2019 0927-7757/ Published by Elsevier B.V.
Please cite this article as: Yun Xing, et al., Colloids and Surfaces A, https://doi.org/10.1016/j.colsurfa.2019.124099
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
or terrorist attacks [2]. To protect public health and the environment, water and wastewater treatment facilities must be prepared for situations that involve the introduction of biocontaminants into their unit processes [3–5]. An important aspect of this issue concerns adsorption of biocontaminants to colloidal materials common in wastewaters. Colloids can protect pathogens by quenching the oxidants that would otherwise cause inactivation, and they can also transport pathogens through the wastewater treatment process [6]. Colloid concentrations in untreated domestic wastewater are typically < 50 mg/L [7], however values as high as 5900 mg/L have been reported [8]. Elevated levels of colloidal kaolinite and silica can be caused by significant volumetric contributions from industrial sources such as pulp and paper mills [9,10]. The presence of colloids is also problematic at small drinking water treatment plants (i.e. those serving < 10,000 people), because these facilities are more likely to violate turbidity standards [11,12]. Pathogens adsorbed to colloids in drinking water are more difficult to inactivate at the point of use and can harm public health [13,14]. The focus of this study is bacteriophage MS2, a 25 nm diameter, icosahedral, single-stranded RNA virus, and a commonly used surrogate for Ebola, human enteric viruses, and as an indicator of fecal contamination in recreational waters [15–19]. Kaolinite (KAO) and fiberglass (FG) colloids are used in this study to simulate colloids found in wastewater [20]. Virus adsorption to colloidal particles is wellknown and documented (e.g [21–23].); however, the observed kinetics of adsorption vary greatly (e.g. 0.02 day−1 to 0.04 min−1 for MS2 adsorption to kaolin; [13,24]). Many of the previous studies were carried out before modern DNA-labeling techniques which were available to permit more accurate kinetic measurements. Additionally, most of the previous studies did not characterize binding strength, which has a profound impact on the fate of pathogens. Virus-to-virus aggregation has been often overlooked in previous adsorption studies but should be studied because viruses may interact with colloidal surfaces as clusters, as opposed to individual virions. The objectives of this research are to: 1) determine the observed pseudo first-order rate constants associated with the removal of bacteriophage MS2 by attachment to colloid surfaces over a range of concentrations of KAO and FG colloid suspensions; 2) experimentally characterize MS2 aggregation and the strength of the surface attachment; and 3) use XDLVO modeling to comparatively assess surface attachment and aggregation.
pore size, Fischer Scientific Catalog No. 09 720 004) to remove the E. coli debris. This method generally produced 5 to ∼9 × 1010 PFU/ml. An additional purification step was performed to remove the organic compounds, as these small molecules may interfere the phage-colloids adsorption experiments. This was achieved by centrifuging the sample through a centrifugal unit with a molecular weight cutoff of 100 K (Amicon Ultra-15, MWCO 100 K). The phages were resuspended in sterile PBS and stored at 4 °C for future use. Purified MS2 was then incubated with SYTO-9 green fluorescent nucleic acid stain (Thermo Fisher, S34854) for 30–60 minutes in dark at a dye concentration of 4 μM (1:1250 dilution of the original stock). The fluorescently labeled phages were used in all of the following experiments. 2.3. Colloidal suspensions KAO (Fisher Chemical, Catalog# K2-500) colloidal suspensions were prepared at 23 °C by adding measured amounts (i.e., between 0.1 and 1 g) to 1 L of DI water, stirring vigorously with a magnetic stirrer for 30 min, and settling for at least 2 h. The liquid phase was decanted, and used as the colloid suspension. The FG colloidal suspensions were prepared at 23 °C by mixing 1 F G filter (GE Healthcare, Catalog# 1827042) with DI water (i.e. between 150 and 500 mL) in a blender (Waring Commercial, Model #HGBEGYG4, Torrington, CT) for 2–3 minutes at approx. 10,000 rpm. The mixed suspension was allowed to settle overnight, and the liquid phase was decanted and used as the colloids suspension. The pH of each suspension was typically 6.5–7.2. Colloid concentrations were measured gravimetrically [25]. Colloid sizes are documented in the illustrations 2.4. Batch tests A 20 mL sample of each colloidal solution was added to a 50-mL sterilized beaker with a plastic magnetic stirrer on a stirring plate. Next, 1 ml of the fluorescently-labeled MS2 (∼ 109 pfu/ml) was added to each beaker, which was then covered with foil to prevent photobleaching. The mixtures were constantly stirred for 120 min and 1 ml samples were withdrawn immediately after addition of colloids, and at 15-, 30-, 60-, and (in some cases) 120-minute time points. For each time point, 1 ml samples were taken in triplicate. The samples were centrifuged at 4000 rpm for 15 min to separate supernatant from the colloids. The supernatant, containing free floating MS2, was then measured for SYTO 9 green fluorescence using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Walthan, MA) using blue light (470 nm) excitation. To investigate the strength of the association between MS2 phages and colloids, the pellets were washed as follows. Briefly, pellet samples were centrifuged at 4000 rpm for 15 min to remove residual supernatant. Afterwards, 1 ml of DI water was added to each pellet sample, the mixture was vigorously vortexed for 1 min at approx. 2500 rpm (Daigger Votex, Genie 2), and then centrifuged again to separate the washed pellets from the washing solution. The washing solution was then measured with Qubit 3.0 Fluorometer for SYTO 9 fluorescence; viruses that were recovered from the pellet washing step were classified as “weakly-bound” and the “strongly-bound” viruses were calculated by mass balance. Additional batch tests were done with colloid centrate (instead of colloids); this was prepared by centrifuging the colloid suspensions for approx. 15 min at 4000 rpm and removing the pellets; the remaining solution was used as the colloid centrate (i.e. a solution containing ions or supramolecular materials that were extracted from the colloids via centrifugation). Controls were also carried out without colloids or colloid centrate.
2. Materials and methods 2.1. Experimental overview Colloidal suspensions of KAO and FG were mixed with bacteriophage MS2 in laboratory-scale batch tests carried out in triplicate. Time series samples were collected to measure the concentration of MS2 which remained in solution. Liquid and pellet samples were collected and prepared from microscopic analysis. Pellets obtained from the adsorption experiments consisted of MS2 and colloids and were used for binding strength experiments. XDLVO modeling was carried out to help investigate MS2 aggregation and surface interactions with colloids. Two-tailed, student T-tests were used to determine statistical significance at the 95% confidence level (α = 0.05). 2.2. Preparation of bacteriophage MS2 MS2 stock (108 PFU/ml, a gift from EPA, Cincinnati, OH) was added to an overnight growth stock of host bacteria, E. coli Famp, with a final concentration of 400 PFU/ml or higher. The E. coli stock was then allowed to grow in TSB with streptomycin/ampicillin for 24–48 hours at 35 °C in an incubator (Lab-Line, Imperial III) for the phage to propagate. Afterwards, the liquid suspension was centrifuged (4000 rpm for 20 min at 4 °C) and syringe filtered (MCE membrane, 33 mm diameter, 0.22 μm
2.5. Rate constant calculations Curve fitting was carried out in order to retrieve the observed pseudo first-order rate constants associated with MS2 removal. MATLAB R2016 was used as the computational platform. The 2
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 1. Supernatant fluorescence at different KAO colloid concentrations. Curve fits based on Eq. (2) are shown.
mathematical basis of the curve fit was the well-established Lagergren pseudo first-order model [26]:
dq = k(qe dt
q)
dC = k'(C - Ce) dt
deflection of the cantilever. The AFM cantilever carried a silicon nitride tip, with a spring constant of ∼ 0.4 N/m and tip radius of 2 nm. Each sample was scanned multiple times to collect data that was representative and statistically valid. Each scan in the peak force imaging mode returned information on spore height and peak force error.
(1) (2)
2.7. X-ray photoelectron spectroscopy (XPS)
where C is liquid phase concentration of unbound MS2 at time t; Ce is liquid phase concentration of unbound MS2 at equilibrium; q is the concentration of bound MS2 at time t; qe is the concentration of bound MS2 at equilibrium; k and k’ are observed pseudo first-order rate constants with respect to the solid and liquid phase concentrations respectively. Curve fits were retrieved by minimizing the root mean square error. All liquid phase MS2 concentrations are reported as fluorescence a.u. as described in section 2.4.
The spectrometer was an Omicron Nanotechnology Multiprobe S (Denver, CO) based system equipped with both monochromatic and achromatic X-ray sources. The hemispherical analyzer had a mean radius of 125 mm with a 128 channel detector. The monochromatic X-ray source uses an Al Kα radiation, which is produced by a XM1000 monochromator with a 500 mm Rowland Circle that produces an X-ray line width of less than 0.2 eV. The achromatic source is a DAR400 twinanode (Al/Mg) X-ray source. The X-ray sources were operated at 225 or 300 W. The base pressure of this instrument was between 5 × 10- 9 to 5 × 10-10 mBar during data collection. The spectrometer’s energy scale was calibrated using silver and copper spectra. All spectra were referenced against the internal calibration of C1s of 285.0 eV advantageous hydrocarbon. XPS analysis was done on selected colloid surfaces to confirm the presence of cations.
2.6. Microscopy Both fluorescence microscopy and atomic force microscopy (AFM) were used to obtain visual images. Fluorescence microscopy distinguished the SYTO 9-labeled MS2 from non-fluorescent colloidal particles while AFM images provided high-resolution images of the colloids and MS2 aggregates. Washed pellets samples were prepared for fluorescent microscopic imaging on a Zeiss Axioskop fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY) with a filter set of excitation: 488 nm and emission: 520 nm. Five representative regions of interest (ROI) were randomly picked for each sample to image. Two types of images were captured for each ROI: a bright field image and a fluorescence image. The camera exposure time and the excitation light intensity were kept identical for all samples. For AFM imaging, a small drop (5–10 μl) of the pellet slurry was placed on a freshly-cleaved mica substrate and then left to air dry in a biosafety cabinet. The samples were then loaded on the sample stage of Dimension Icon AFM (Bruker Corporation, Billerica, MA) and scanned under ScanAsyst in air mode. The technique is based on PeakForce Tapping, which oscillates the Z piezo at a rate well below the resonance of the probe. As the probe periodically taps the sample, the interaction force is measured by the
2.8. XDLVO modeling XDLVO theory was used to predict the relative favorability of adsorption and aggregation. The model includes van der Waals forces (attractive energy), repulsive electrostatic forces (electrostatic repulsion energy), and Lewis acid-base forces to calculate interaction energy as a function of the separation distance between particles. XDLVO modeling was as described previously [27]. See Supplementary Materials, Part B for a detailed description of XDLVO theory and the equations. Model parameters (i.e. MS2 ζ-potential = -0.02 V, KAO ζ-potential = -0.04 V, FG ζ-potential = -0.02 V, ionic strength = 8.8 × 10−7 M) were retrieved from published sources [27–30]. 3
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
experiments, a finding that implicates MS2 aggregation (Fig. 3A – supplementary materials). Both aggregation and surface interaction occur when MS2 is exposed to colloids. Interestingly, MS2 removal was faster in the KAO-centrate controls than it was in the MS2-only or FG centrate controls; this finding implicates weakly-bound ions (e.g. calcium) or supramolecular materials that can disassociate from KAO and then act to enhance MS2 aggregation. KAO colloids consist mainly of SiO2, with small amounts of other inorganic oxide compounds [27], and it is possible that some of cations were present in the centrate [32]. XPS was used to confirm the presence of numerous cations on the surface of the KAO colloids used in this study (see Fig. 4A - Supplementary materials). The rate constants observed in the current study are at least an order-of-magnitude faster than comparable results from previous studies. [13] mixed 1.9 × 105 PFU/mL MS2 with 35 mg/L of clay particles in deionized water and reported an observed pseudo first-order adsorption rate constant of 0.04 min−1; the current study observed a pseudo first-order adsorption rate constant of 0.76 min−1 at a KAO concentration of 28 mg/L. [24] mixed 103 – 109 PFU/mL of MS2 with 10 mg/mL of KAO suspended in a buffer solution in tubes mounted to a vertical rotator and observed a pseudo first-order rate constant of ∼ 0.02 day−1, much smaller than the rate constants shown in Table 1. The current results also stand in sharp contrast with those of [22], who mixed 60 PFU/mL of bacteriophage f2, a virus that is very similar to MS2, with 16 mg/L of KAO in deionized water and found negligible adsorption after 30 min; they also reported similar results in the case of adsorption to bentonite. The current results show that MS2 adsorption to colloidal particles has the potential to be much faster than previously suggested in the peer-reviewed literature. While this may be a function of the particle clay used, it has practical significance in that natural environments contain many types of clay, with a range potential adsorption rates, some high like the ones shown here. The adsorption and aggregation were qualitatively confirmed with microscopy. Fluorescent and bright field images show examples of MS2 present along the surface of KAO particles (Fig. 2 and 4A – supplementary materials). Similar qualitative observations are shown for MS2
Table 1 Observed pseudo first order rate constants for MS2 removal in the presence of colloids. KAO sample
KAO Concentration (mg/L)
k’ (min−1)
FG sample
FG Concentration (mg/L)
k’ (min−1)
K1 K2 K3 K4 K5 K6
450 220 110 56 28 14
5.1 5.1 4.8 4.5 0.76 0.53
FG1 FG2 FG3 FG4 FG5 FG6
83 42 21 10 5.2 2.6
2.4 3.0 4.2 3.5 4.7 3.4
3. Results and discussion 3.1. MS2 adsorption kinetics Measured data points, first-order adsorption model curve fits, and control data for KAO experiments are shown in Fig. 1. In each experiment, SYTO 9 fluorescence intensity dropped rapidly after mixing with colloids, but the speed of MS2 removal was a function of the experimental condition (Table 1). The observed first-order rate constants increased as the KAO colloid concentration increased as expected due to the additional surface area for adsorption. These findings were confirmed in replicate trials (Fig. 1A - supplementary materials). The results from the FG experiments also showed a rapid decrease in the SYTO 9 fluorescence, but the observed first-order rate constants were not correlated with the colloid concentrations (Table 1, Fig. 2A - supplementary materials). The differences between KAO- and FG-related MS2 removal rates are likely due to dissimilar material properties [31]. For instance, the FG colloids, unlike KAO, were heterogeneous, filamentous, and irregular in shape. These, along with differences in their chemical make-up may cause a number of changes in the kinetics and mechanisms through which MS2 binding occurs. The concentration of MS2 also decreased during MS2-only control
Fig. 2. AFM images of KAO colloids with and without the presence of MS2: a) Kaolin particle without MS2; note the angular edges that are characteristic of the multilayered structures kaolin particle and the clean smooth surface; b) Kaolin particles with MS2 adsorption both fully covered (red arrow) and peripherally covered (blue arrow). The white arrow indicates MS2 aggregates not associated with a kaolin particle; c) High resolution images of an MS2 aggregate showing that it consists of smaller particles (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 4
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 3. Bond strength profile for K4-K6 experiments. K1- 450 mg KAO/L; K4 – 56 mg KAO/L; K6 – 14 mg KAO/L.
co-settled with colloids. This finding has important operational implications. In a full-scale water reclamation facility (which may include less vigorous mixing than what was used in the washing steps reported in this study), the fate of such strongly-bound MS2 viruses will likely be very different from unbound or weakly-bound MS2 viruses. Stronglybound MS2 will likely end up in the sludge or possibly in the effluent, associated with particles and likely viable. To the author’s knowledge, this study appears to be the first to present evidence for binding strength characteristics for MS2 in the presence of colloidal materials.
adsorption to FG (Figs. 5A, 6A and 7A – supplementary materials). MS2 aggregates ranged from 20 to 30 nm in diameter. These images may be impacted by the drying step during sample preparation for microscopy. In principle, agglomeration is possible but may not be significant in suspensions containing irregularly shaped colloidal particles subject to DLVO interactions [33,34]. This topic is appropriate for future study. 3.2. Binding strength Previous adsorption studies have characterized the strength of sorbent binding for a variety of sorbates [35–37], but there is a need to do so for bacteriophages adsorbed to colloidal particles. As described in the methods section, binding strength was assessed by collecting and washing the pellets and measuring the resulting fluorescence in the supernatants. MS2 viruses that were washed off of the pellet were characterized as “weakly-bound”. The strongly-bound fraction was calculated by mass balance. The binding strength profile for MS2 in the presence of KAO is shown in Figs. 3 and 8A (supplementary materials). The y-axis is the percentage of MS2 that is associated with each binding characteristic. The percentage of strongly-bound (or entrapped) MS2 was typically 70% (or greater) immediately (i.e. 30 s) after injection of MS2, and it was typically 80% (or greater) after 1 h. The percentage of weakly-bound MS2 was less than 10% throughout the experiment. The differences between the strongly- and weakly- bound MS2 were statistically significant (p < 0.05) at all time points. The binding profile for the FG experiments showed that the percentage of strongly-bound MS2 increased from approx. 60% at 0.5 min to approx. 75% at 60 min; the percentage of weakly-bound MS2 was approx. 5% (or less) throughout the experiment (Figs. 9A and 10A – supplementary materials). The differences between the strongly- and weakly-bound MS2 were statistically significant (p < 0.05) at all time points. The experimental protocol used to determine the aforementioned binding strength characteristics also permitted MS2 aggregates to be separated, or co-settled, with colloids. Such aggregates may have been either unattached or weakly-bound to colloids. Thus, the stronglybound fraction likely included viruses that were entrapped within aggregates. In a dynamic water treatment system, such aggregates could either remain in the bulk liquid or reattach to other small particles. The data in this study show that most of the MS2 was either strongly-bound (i.e. bound to colloidal particles) or enmeshed within aggregates that
3.3. XDLVO analysis XDLVO modeling facilitates an analysis of the favorability of interactions between MS2 and the colloidal surfaces included in this study. XDLVO models account for: 1) van der Waals forces caused by the interaction of electric dipoles; 2) electrostatic forces; 3) Lewis acidbase forces which may reflect electron transfer properties and/or hydrophobicity. As the separation distance approaches zero, the dimensionless potential energy is lowest (i.e. most favorable) for KAO, followed by FG, and then MS2-MS2 interaction (Fig. 4); this trend is consistent with the three experimental findings from this study, namely: 1) the percentage of strongly-bound MS2 was higher for KAO than for FG; 2) the maximum MS2 removal rate in the presence of KAO was higher than that of FG: and 3) the kinetics of MS2 aggregation were significantly (p < 0.05) slower than MS2 removal in the presence of either colloid. While the XDLVO results support the current results, the modeling framework should be improved to account for the interactions between MS2 aggregates and colloid surfaces. 4. Conclusions To the best of our knowledge, this is the first study to use modern DNA-labeling techniques and XDLVO models to examine bacteriophage MS2 adsorption to kaolinite and fiberglass colloids over a wide range of operationally relevant colloid concentrations. MS2 adsorption kinetics are much faster than previously reported, even when compared to results retrieved with similar colloid concentrations. The majority (i.e. 75% or greater after 60 min) of the MS2 was strongly-bound or entrapped, suggesting important operations implications for those interested in understanding and controlling the fate of MS2 (and other 5
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 4. XDLVO interaction energy profiles: MS2-MS2, FG-MS2, and KAO-MS2 interactions in DI water. A) Energy primary maximum for aggregation/adsorption. (MS2 ζ-potential = -0.02 V, KAO ζ-potential = -0.04 V, FG ζ-potential = -0.02 V, ionic strength = 8.8 × 10−7 M, sphere-sphere for MS2-MS2 XDLVO calculations and sphere-plate formulas for the FGand KAO-MS2 XDLVO calculations).
References
viruses) in full-scale water reclamation facilities. MS2 aggregates quickly and strongly adsorb to colloids, which in turn provide protection from oxidants and govern their final fate. If strongly-bound viruses are neglected, there is great potential to underestimate the environmental and public health impact of colloidal particles in wastewater effluents. The current work also showed the merit associated with applying XDLVO theory, which helped explain the relative speed of MS2 adsorption to KAO and FG as well as the strong tendency to generate MS2 aggregates. XDLVO models should be revised to include interactions between MS2 aggregates and colloid surfaces and future studies should employ these research techniques to further elucidate the intricacies of the viral adsorption to colloids.
[1] Q. Wang, P. Wang, Q. Yang, Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci. Total Environ. 621 (15) (2018) 990–999. [2] R. Roffey, K. Lantorp, A. Tegnell, F. Elgh, Biological weapons and bioterrorism preparedness: importance of public-health awareness and international cooperation, Clin. Microbiol. Infect. 8 (8) (2002) 522–528. [3] EPA, Progress on Water Sector Decontamination Recommendation and Proposed Strategic Plan, EPA Office of Water, Washington, D.C, 2015 Report Number 817-R15-001. [4] WERF, Collaborative Workshop on Handling, Management, and Treatment of Biocontaminated Wastewater by Water Resource Recovery Facilities, Final Report No.WERF7W15 Water Environmental Research Foundation, Alexandria, VA, 2016. [5] C. Zoli, L. Steinbuerg, M. Grabowski, M. Hermann, Terrorist critical infrastructures, organizational capacity and security risk, Saf. Sci. 110 (Part C) (2018) 121–130. [6] A. Sakoda, Y. Sakai, K. Hayakawa, M. Suzuki, Adsorption of viruses in water environment onto solid surfaces, Water Sci. Technol. 35 (7) (1997) 107–114. [7] Rickert and Hunter, Rapid fractionation and materials balance of solids fractions in wastewater and wastewater effluent, Journal of Water Pollution Control Federation 39 (9) (1967) 1475–1486. [8] Y. Huang, W. li, X. Xie, B. Gao, A. Li, Distribution of endocrine-disrupting chemicals in colloidal and soluble phases in municipal secondary effluents and their removal by different advanced treatment processes, Chemosphere 219 (2019) 730–739. [9] M. Nasser, F. Twaiq, S. Onaizi, Effect of polyelectrolytes on the degree of flocculation of papermaking suspensions, Sep. Purif. Technol. 103 (2013) 43–52. [10] C. Dykstra, H. Giles, S. Banerjee, S. Pavlostathis, Fate and biotransformation of phytosterols during treatment of pulp and paper wastewater in a simulated aerated stabilization basin, Water Res. 68 (2015) 589–600. [11] L. Bennear, S. Olmstead, The impacts of the “right to know”: information disclosure and the violation of drinking water standards, J. Environ. Econ. Manage. 56 (2) (2008) 117–130. [12] EPA, Drinking Water: EPA Needs to Take Additional Steps to Ensure Small Community Water Systems Designated As Serious Violators Achieve Compliance, EPA Office of Inspector General, Washington, D.C, 2016 Report Number 16-P-0108. [13] C.H. Stagg, C. Wallis, C.H. Ward, Inactivation of clay-associated bacteriophage MS2 by chlorine, Appl. Environ. Microbiol. 33 (2) (1977) 385–391. [14] M. Templeton, R. Andrews, R. Hofmann, Inactivation of particle-associated viral surrogates by ultraviolet light, Water Res. 39 (15) (2005) 3487–3500. [15] C. Gerba, Applied and theoretical aspects of virus adsorption to surfaces, Advanced in applied microbiology 30 (1984) 133–168. [16] E. Skripkin, M. Adhin, M. de Smit, J. van Duin, Secondary structure of the central region of bacteriophage MS2 RNA: conservation and biological significance, J. Mol. Biol. 211 (2) (1990) 447–463. [17] K. Toropova, G. Basnak, P. Twarock Stockley, N. Ranson, The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly, J. Mol. Biol. 375 (3) (2008) 824–836. [18] M. Shishovs, J. Rumnieks, C. Diebolder, K. Jauzems, L. Andreas, J. Stanek, A. Kazaks, S. Kotelovica, I. Akopjana, G. Pintacuda, R. Koning, K. Tars, Structure of AP205 coat protein reveals circular permutation in ssRNA bacteriophages, J. Mol. Biol. 428 (21) (2016) 4267–4279. [19] B. McMinn, N. Ashbolt, A. Korajkic, Bacteriophages as indicators of faecal pollution and enteric virus removal, Lett. Appl. Microbiol. 65 (2017) 11–26. [20] M.F. Pouet, N. Azema, E. Touraud, O. Thomas, Physical and aggregate properties, Chapter 6, in: O. Thomas, C. Burgess (Eds.), UV-Visible Spectrophotometry of Water and Wastewater, Elsevier Publishers, London, UK, 2007, pp. 145–161. [21] G. Carlson, F. Woodard, D. Wentworth, O. Sproul, Virus inactivation on clay particle in natural waters, Journal of the Water Pollution Control Federation 40 (2) (1968) R89–R106. [22] B. Moore, B. Sagik, J.F. Malina, Viral association with suspended solids, Water Res.
Disclaimer The views expressed in this article are those of the authors and do not reflect the official policy or position of the Air Force Institute of Technology, the United States Air Force, the Department of Defense, or the United States government. The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development partially funded and collaborated in the research described here under Interagency Agreement DW-057-92440901-3. It has been subjected to the EPA’s review and has been approved for publication. Note that approval does not signify that the contents necessarily reflect the views of EPA. Any mention of trade names, products, or services does not imply an endorsement by the EPA or the U.S. Government. EPA does not endorse any commercial products, services, or enterprises. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank Dr. Daniel Felker (AFIT) and John Hixenbaugh (AFIT) for assistance handling hazardous material. The authors also thank Brian McMinn (EPA) for providing bacteriophage MS2 stocks. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2019.124099. 6
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al. 9 (197) (1975) 203. [23] M. Tong, Y. Shen, H. Yang, H. Kim, Deposition kinetics of MS2 bacteriophages on clay mineral surfaces, Colloids Surf. B Biointerfaces 92 (2012) 340–347. [24] M.I. Bellou, V.I. Syngouna, M.A. Tselepi, P.A. Kokkinos, S.C. Paparrodopoulos, A. Vantarakis, C.V. Chrysikopoulos, Interaction of human adenoviruses and coliphages with kaolinite and bentonite, Sci. Total Environ. 517 (2015) 86–95. [25] APHA, Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA, 2016. [26] S. Douven, C. Paez, C. Gommes, The range of validity of sorption kinetic models, J. Colloid Interface Sci. 448 (2015) 437–450. [27] C.V. Chrysikopoulos, V.I. Syngouna, Attachment of bacteriophages MS2 and ΦX174 onto kaolinite and montmorillonite: Extended-DLVO interactions, Colloids Surf. B Biointerfaces 92 (2012) 74–83. [28] J. Ryan, Effects of ionic strength and flow rate on colloid release: relating kinetics to intersurface potential energy, J. Colloid Interface Sci. 164 (1994) 21–34. [29] H. Schwegmann, J. Ruppert, F. Frimmel, Influence of the pH-value on the photocatalytic disinfection of bacteria with TiO2-explnanation by DLVO and XDLVO theory, Water Res. 47 (2013) 1503–1511.
[30] W. Zhang, X. Zhang, Adsorption of MS2 on oxide nanoparticles affects chlorine disinfection and solar inactivation, Water Res. 69 (2015) 59–67. [31] M. Haerifar, S. Azizian, Fractal-like adsorption kinetics on heterogeneous solid surfaces, J. Phys. Chem. C 118 (2014) 1129–1134. [32] A. Palomino, J. Santamarina, Fabric map for kaolinite: effect of pH and ionic concentration on behavior, Clays Clay Miner. 53 (3) (2005) 209–222. [33] P. Yunker, T. Still, M. Lohr, A. Yodh, Suppression of the coffee-ring effect by shapedependent capillary interactions, Nature 476 (2011) 308–311. [34] V. Dugyala, M. Basavaraj, Control over coffee-ring formation in evaporating liquid drops containing ellipsoids, Langmuir 30 (29) (2014) 8680–8686. [35] S. Vaishanav, K. Chandraker, J. Korram, R. Nagwanshi, K. Ghosh, M. Satnami, Protein nanoparticle interaction: a spectrophotometric approach for adsorption kinetics and binding studies, J. Mol. Struct. 1117 (2016) 300–310. [36] J. Ciejka, K. Wolski, M. Nowakowska, K. Pyrc, K. Szczubiałka, Biopolymeric nano/ microspheres for selective and reversible adsorption of coronaviruses, Mater. Sci. Eng. C 76 (July (1)) (2017) 735–742 2017. [37] A. Julbe, M. Drobek, A. Ayral, About the role of adsorption in inorganic and composite membranes, Curr. Opin. Chem. Eng. 24 (2019) 88–97.
7
Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Adsorption of bacteriophage MS2 to colloids: Kinetics and particle interactions Yun Xinga, Ashlee Ellisa, Matthew Magnusonb, Willie F. Harper Jr.a,* a b
Air Force Institute of Technology, Department of Systems Engineering and Management, Environmental Engineering and Science Program, Wright-Patterson AFB, Ohio, US US Environmental Protection Agency, National Homeland Security Research Center, Water Infrastructure Protection Division, Cincinnati, Ohio, US
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Keywords: Adsorption Biocontaminant Bacteriophage MS2 XDLVO Atomic force microscopy
Virus adsorption to colloidal particles is an important issue in the water quality community. Namely, if viruses can quickly and strongly associate to colloids, this can potentially lead to significant implications for the management of biohazardous wastes at water reclamation facilities. This research evaluated the adsorption of bacteriophage MS2 to colloidal suspensions of kaolinite (KAO) and fiberglass (FG). Observed pseudo first-order MS2 removal rate constants were between 0.53 and 5.1 min−1 and between 2.4 and 3.5 min−1 for KAO and FG, respectively. These kinetics were at least an order of magnitude faster than previously reported values when compared to data retrieved at similar colloid concentrations. Fluorescent and bright field microscopic images showed clusters of MS2 on and around the edges of the colloids, and the majority of the bound MS2 was not readily removed during a vigorous wash step, suggesting comparatively strong, operationally relevant adsorption. MS2 aggregation was observed experimentally and predicted on the basis of interaction energies calculated with XDLVO models. When virus-containing biohazardous wastes are introduced into wastewater treatment plants, removing colloids is essential.
1. Introduction Improving water infrastructure security continues to be a high priority for environmental professionals. One ongoing security
⁎
initiative concerns high-consequence biocontaminants, including a wide range of dangerous bacteria, viruses, proteins, and metabolites. These biological agents can pose a very serious threat to the public when unusually large loads are discharged by hospitals [1], accidents,
Corresponding author. E-mail address: [email protected] (W.F. Harper).
https://doi.org/10.1016/j.colsurfa.2019.124099 Received 4 September 2019; Received in revised form 8 October 2019; Accepted 8 October 2019 Available online 25 October 2019 0927-7757/ Published by Elsevier B.V.
Please cite this article as: Yun Xing, et al., Colloids and Surfaces A, https://doi.org/10.1016/j.colsurfa.2019.124099
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
or terrorist attacks [2]. To protect public health and the environment, water and wastewater treatment facilities must be prepared for situations that involve the introduction of biocontaminants into their unit processes [3–5]. An important aspect of this issue concerns adsorption of biocontaminants to colloidal materials common in wastewaters. Colloids can protect pathogens by quenching the oxidants that would otherwise cause inactivation, and they can also transport pathogens through the wastewater treatment process [6]. Colloid concentrations in untreated domestic wastewater are typically < 50 mg/L [7], however values as high as 5900 mg/L have been reported [8]. Elevated levels of colloidal kaolinite and silica can be caused by significant volumetric contributions from industrial sources such as pulp and paper mills [9,10]. The presence of colloids is also problematic at small drinking water treatment plants (i.e. those serving < 10,000 people), because these facilities are more likely to violate turbidity standards [11,12]. Pathogens adsorbed to colloids in drinking water are more difficult to inactivate at the point of use and can harm public health [13,14]. The focus of this study is bacteriophage MS2, a 25 nm diameter, icosahedral, single-stranded RNA virus, and a commonly used surrogate for Ebola, human enteric viruses, and as an indicator of fecal contamination in recreational waters [15–19]. Kaolinite (KAO) and fiberglass (FG) colloids are used in this study to simulate colloids found in wastewater [20]. Virus adsorption to colloidal particles is wellknown and documented (e.g [21–23].); however, the observed kinetics of adsorption vary greatly (e.g. 0.02 day−1 to 0.04 min−1 for MS2 adsorption to kaolin; [13,24]). Many of the previous studies were carried out before modern DNA-labeling techniques which were available to permit more accurate kinetic measurements. Additionally, most of the previous studies did not characterize binding strength, which has a profound impact on the fate of pathogens. Virus-to-virus aggregation has been often overlooked in previous adsorption studies but should be studied because viruses may interact with colloidal surfaces as clusters, as opposed to individual virions. The objectives of this research are to: 1) determine the observed pseudo first-order rate constants associated with the removal of bacteriophage MS2 by attachment to colloid surfaces over a range of concentrations of KAO and FG colloid suspensions; 2) experimentally characterize MS2 aggregation and the strength of the surface attachment; and 3) use XDLVO modeling to comparatively assess surface attachment and aggregation.
pore size, Fischer Scientific Catalog No. 09 720 004) to remove the E. coli debris. This method generally produced 5 to ∼9 × 1010 PFU/ml. An additional purification step was performed to remove the organic compounds, as these small molecules may interfere the phage-colloids adsorption experiments. This was achieved by centrifuging the sample through a centrifugal unit with a molecular weight cutoff of 100 K (Amicon Ultra-15, MWCO 100 K). The phages were resuspended in sterile PBS and stored at 4 °C for future use. Purified MS2 was then incubated with SYTO-9 green fluorescent nucleic acid stain (Thermo Fisher, S34854) for 30–60 minutes in dark at a dye concentration of 4 μM (1:1250 dilution of the original stock). The fluorescently labeled phages were used in all of the following experiments. 2.3. Colloidal suspensions KAO (Fisher Chemical, Catalog# K2-500) colloidal suspensions were prepared at 23 °C by adding measured amounts (i.e., between 0.1 and 1 g) to 1 L of DI water, stirring vigorously with a magnetic stirrer for 30 min, and settling for at least 2 h. The liquid phase was decanted, and used as the colloid suspension. The FG colloidal suspensions were prepared at 23 °C by mixing 1 F G filter (GE Healthcare, Catalog# 1827042) with DI water (i.e. between 150 and 500 mL) in a blender (Waring Commercial, Model #HGBEGYG4, Torrington, CT) for 2–3 minutes at approx. 10,000 rpm. The mixed suspension was allowed to settle overnight, and the liquid phase was decanted and used as the colloids suspension. The pH of each suspension was typically 6.5–7.2. Colloid concentrations were measured gravimetrically [25]. Colloid sizes are documented in the illustrations 2.4. Batch tests A 20 mL sample of each colloidal solution was added to a 50-mL sterilized beaker with a plastic magnetic stirrer on a stirring plate. Next, 1 ml of the fluorescently-labeled MS2 (∼ 109 pfu/ml) was added to each beaker, which was then covered with foil to prevent photobleaching. The mixtures were constantly stirred for 120 min and 1 ml samples were withdrawn immediately after addition of colloids, and at 15-, 30-, 60-, and (in some cases) 120-minute time points. For each time point, 1 ml samples were taken in triplicate. The samples were centrifuged at 4000 rpm for 15 min to separate supernatant from the colloids. The supernatant, containing free floating MS2, was then measured for SYTO 9 green fluorescence using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Walthan, MA) using blue light (470 nm) excitation. To investigate the strength of the association between MS2 phages and colloids, the pellets were washed as follows. Briefly, pellet samples were centrifuged at 4000 rpm for 15 min to remove residual supernatant. Afterwards, 1 ml of DI water was added to each pellet sample, the mixture was vigorously vortexed for 1 min at approx. 2500 rpm (Daigger Votex, Genie 2), and then centrifuged again to separate the washed pellets from the washing solution. The washing solution was then measured with Qubit 3.0 Fluorometer for SYTO 9 fluorescence; viruses that were recovered from the pellet washing step were classified as “weakly-bound” and the “strongly-bound” viruses were calculated by mass balance. Additional batch tests were done with colloid centrate (instead of colloids); this was prepared by centrifuging the colloid suspensions for approx. 15 min at 4000 rpm and removing the pellets; the remaining solution was used as the colloid centrate (i.e. a solution containing ions or supramolecular materials that were extracted from the colloids via centrifugation). Controls were also carried out without colloids or colloid centrate.
2. Materials and methods 2.1. Experimental overview Colloidal suspensions of KAO and FG were mixed with bacteriophage MS2 in laboratory-scale batch tests carried out in triplicate. Time series samples were collected to measure the concentration of MS2 which remained in solution. Liquid and pellet samples were collected and prepared from microscopic analysis. Pellets obtained from the adsorption experiments consisted of MS2 and colloids and were used for binding strength experiments. XDLVO modeling was carried out to help investigate MS2 aggregation and surface interactions with colloids. Two-tailed, student T-tests were used to determine statistical significance at the 95% confidence level (α = 0.05). 2.2. Preparation of bacteriophage MS2 MS2 stock (108 PFU/ml, a gift from EPA, Cincinnati, OH) was added to an overnight growth stock of host bacteria, E. coli Famp, with a final concentration of 400 PFU/ml or higher. The E. coli stock was then allowed to grow in TSB with streptomycin/ampicillin for 24–48 hours at 35 °C in an incubator (Lab-Line, Imperial III) for the phage to propagate. Afterwards, the liquid suspension was centrifuged (4000 rpm for 20 min at 4 °C) and syringe filtered (MCE membrane, 33 mm diameter, 0.22 μm
2.5. Rate constant calculations Curve fitting was carried out in order to retrieve the observed pseudo first-order rate constants associated with MS2 removal. MATLAB R2016 was used as the computational platform. The 2
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 1. Supernatant fluorescence at different KAO colloid concentrations. Curve fits based on Eq. (2) are shown.
mathematical basis of the curve fit was the well-established Lagergren pseudo first-order model [26]:
dq = k(qe dt
q)
dC = k'(C - Ce) dt
deflection of the cantilever. The AFM cantilever carried a silicon nitride tip, with a spring constant of ∼ 0.4 N/m and tip radius of 2 nm. Each sample was scanned multiple times to collect data that was representative and statistically valid. Each scan in the peak force imaging mode returned information on spore height and peak force error.
(1) (2)
2.7. X-ray photoelectron spectroscopy (XPS)
where C is liquid phase concentration of unbound MS2 at time t; Ce is liquid phase concentration of unbound MS2 at equilibrium; q is the concentration of bound MS2 at time t; qe is the concentration of bound MS2 at equilibrium; k and k’ are observed pseudo first-order rate constants with respect to the solid and liquid phase concentrations respectively. Curve fits were retrieved by minimizing the root mean square error. All liquid phase MS2 concentrations are reported as fluorescence a.u. as described in section 2.4.
The spectrometer was an Omicron Nanotechnology Multiprobe S (Denver, CO) based system equipped with both monochromatic and achromatic X-ray sources. The hemispherical analyzer had a mean radius of 125 mm with a 128 channel detector. The monochromatic X-ray source uses an Al Kα radiation, which is produced by a XM1000 monochromator with a 500 mm Rowland Circle that produces an X-ray line width of less than 0.2 eV. The achromatic source is a DAR400 twinanode (Al/Mg) X-ray source. The X-ray sources were operated at 225 or 300 W. The base pressure of this instrument was between 5 × 10- 9 to 5 × 10-10 mBar during data collection. The spectrometer’s energy scale was calibrated using silver and copper spectra. All spectra were referenced against the internal calibration of C1s of 285.0 eV advantageous hydrocarbon. XPS analysis was done on selected colloid surfaces to confirm the presence of cations.
2.6. Microscopy Both fluorescence microscopy and atomic force microscopy (AFM) were used to obtain visual images. Fluorescence microscopy distinguished the SYTO 9-labeled MS2 from non-fluorescent colloidal particles while AFM images provided high-resolution images of the colloids and MS2 aggregates. Washed pellets samples were prepared for fluorescent microscopic imaging on a Zeiss Axioskop fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY) with a filter set of excitation: 488 nm and emission: 520 nm. Five representative regions of interest (ROI) were randomly picked for each sample to image. Two types of images were captured for each ROI: a bright field image and a fluorescence image. The camera exposure time and the excitation light intensity were kept identical for all samples. For AFM imaging, a small drop (5–10 μl) of the pellet slurry was placed on a freshly-cleaved mica substrate and then left to air dry in a biosafety cabinet. The samples were then loaded on the sample stage of Dimension Icon AFM (Bruker Corporation, Billerica, MA) and scanned under ScanAsyst in air mode. The technique is based on PeakForce Tapping, which oscillates the Z piezo at a rate well below the resonance of the probe. As the probe periodically taps the sample, the interaction force is measured by the
2.8. XDLVO modeling XDLVO theory was used to predict the relative favorability of adsorption and aggregation. The model includes van der Waals forces (attractive energy), repulsive electrostatic forces (electrostatic repulsion energy), and Lewis acid-base forces to calculate interaction energy as a function of the separation distance between particles. XDLVO modeling was as described previously [27]. See Supplementary Materials, Part B for a detailed description of XDLVO theory and the equations. Model parameters (i.e. MS2 ζ-potential = -0.02 V, KAO ζ-potential = -0.04 V, FG ζ-potential = -0.02 V, ionic strength = 8.8 × 10−7 M) were retrieved from published sources [27–30]. 3
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
experiments, a finding that implicates MS2 aggregation (Fig. 3A – supplementary materials). Both aggregation and surface interaction occur when MS2 is exposed to colloids. Interestingly, MS2 removal was faster in the KAO-centrate controls than it was in the MS2-only or FG centrate controls; this finding implicates weakly-bound ions (e.g. calcium) or supramolecular materials that can disassociate from KAO and then act to enhance MS2 aggregation. KAO colloids consist mainly of SiO2, with small amounts of other inorganic oxide compounds [27], and it is possible that some of cations were present in the centrate [32]. XPS was used to confirm the presence of numerous cations on the surface of the KAO colloids used in this study (see Fig. 4A - Supplementary materials). The rate constants observed in the current study are at least an order-of-magnitude faster than comparable results from previous studies. [13] mixed 1.9 × 105 PFU/mL MS2 with 35 mg/L of clay particles in deionized water and reported an observed pseudo first-order adsorption rate constant of 0.04 min−1; the current study observed a pseudo first-order adsorption rate constant of 0.76 min−1 at a KAO concentration of 28 mg/L. [24] mixed 103 – 109 PFU/mL of MS2 with 10 mg/mL of KAO suspended in a buffer solution in tubes mounted to a vertical rotator and observed a pseudo first-order rate constant of ∼ 0.02 day−1, much smaller than the rate constants shown in Table 1. The current results also stand in sharp contrast with those of [22], who mixed 60 PFU/mL of bacteriophage f2, a virus that is very similar to MS2, with 16 mg/L of KAO in deionized water and found negligible adsorption after 30 min; they also reported similar results in the case of adsorption to bentonite. The current results show that MS2 adsorption to colloidal particles has the potential to be much faster than previously suggested in the peer-reviewed literature. While this may be a function of the particle clay used, it has practical significance in that natural environments contain many types of clay, with a range potential adsorption rates, some high like the ones shown here. The adsorption and aggregation were qualitatively confirmed with microscopy. Fluorescent and bright field images show examples of MS2 present along the surface of KAO particles (Fig. 2 and 4A – supplementary materials). Similar qualitative observations are shown for MS2
Table 1 Observed pseudo first order rate constants for MS2 removal in the presence of colloids. KAO sample
KAO Concentration (mg/L)
k’ (min−1)
FG sample
FG Concentration (mg/L)
k’ (min−1)
K1 K2 K3 K4 K5 K6
450 220 110 56 28 14
5.1 5.1 4.8 4.5 0.76 0.53
FG1 FG2 FG3 FG4 FG5 FG6
83 42 21 10 5.2 2.6
2.4 3.0 4.2 3.5 4.7 3.4
3. Results and discussion 3.1. MS2 adsorption kinetics Measured data points, first-order adsorption model curve fits, and control data for KAO experiments are shown in Fig. 1. In each experiment, SYTO 9 fluorescence intensity dropped rapidly after mixing with colloids, but the speed of MS2 removal was a function of the experimental condition (Table 1). The observed first-order rate constants increased as the KAO colloid concentration increased as expected due to the additional surface area for adsorption. These findings were confirmed in replicate trials (Fig. 1A - supplementary materials). The results from the FG experiments also showed a rapid decrease in the SYTO 9 fluorescence, but the observed first-order rate constants were not correlated with the colloid concentrations (Table 1, Fig. 2A - supplementary materials). The differences between KAO- and FG-related MS2 removal rates are likely due to dissimilar material properties [31]. For instance, the FG colloids, unlike KAO, were heterogeneous, filamentous, and irregular in shape. These, along with differences in their chemical make-up may cause a number of changes in the kinetics and mechanisms through which MS2 binding occurs. The concentration of MS2 also decreased during MS2-only control
Fig. 2. AFM images of KAO colloids with and without the presence of MS2: a) Kaolin particle without MS2; note the angular edges that are characteristic of the multilayered structures kaolin particle and the clean smooth surface; b) Kaolin particles with MS2 adsorption both fully covered (red arrow) and peripherally covered (blue arrow). The white arrow indicates MS2 aggregates not associated with a kaolin particle; c) High resolution images of an MS2 aggregate showing that it consists of smaller particles (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 4
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 3. Bond strength profile for K4-K6 experiments. K1- 450 mg KAO/L; K4 – 56 mg KAO/L; K6 – 14 mg KAO/L.
co-settled with colloids. This finding has important operational implications. In a full-scale water reclamation facility (which may include less vigorous mixing than what was used in the washing steps reported in this study), the fate of such strongly-bound MS2 viruses will likely be very different from unbound or weakly-bound MS2 viruses. Stronglybound MS2 will likely end up in the sludge or possibly in the effluent, associated with particles and likely viable. To the author’s knowledge, this study appears to be the first to present evidence for binding strength characteristics for MS2 in the presence of colloidal materials.
adsorption to FG (Figs. 5A, 6A and 7A – supplementary materials). MS2 aggregates ranged from 20 to 30 nm in diameter. These images may be impacted by the drying step during sample preparation for microscopy. In principle, agglomeration is possible but may not be significant in suspensions containing irregularly shaped colloidal particles subject to DLVO interactions [33,34]. This topic is appropriate for future study. 3.2. Binding strength Previous adsorption studies have characterized the strength of sorbent binding for a variety of sorbates [35–37], but there is a need to do so for bacteriophages adsorbed to colloidal particles. As described in the methods section, binding strength was assessed by collecting and washing the pellets and measuring the resulting fluorescence in the supernatants. MS2 viruses that were washed off of the pellet were characterized as “weakly-bound”. The strongly-bound fraction was calculated by mass balance. The binding strength profile for MS2 in the presence of KAO is shown in Figs. 3 and 8A (supplementary materials). The y-axis is the percentage of MS2 that is associated with each binding characteristic. The percentage of strongly-bound (or entrapped) MS2 was typically 70% (or greater) immediately (i.e. 30 s) after injection of MS2, and it was typically 80% (or greater) after 1 h. The percentage of weakly-bound MS2 was less than 10% throughout the experiment. The differences between the strongly- and weakly- bound MS2 were statistically significant (p < 0.05) at all time points. The binding profile for the FG experiments showed that the percentage of strongly-bound MS2 increased from approx. 60% at 0.5 min to approx. 75% at 60 min; the percentage of weakly-bound MS2 was approx. 5% (or less) throughout the experiment (Figs. 9A and 10A – supplementary materials). The differences between the strongly- and weakly-bound MS2 were statistically significant (p < 0.05) at all time points. The experimental protocol used to determine the aforementioned binding strength characteristics also permitted MS2 aggregates to be separated, or co-settled, with colloids. Such aggregates may have been either unattached or weakly-bound to colloids. Thus, the stronglybound fraction likely included viruses that were entrapped within aggregates. In a dynamic water treatment system, such aggregates could either remain in the bulk liquid or reattach to other small particles. The data in this study show that most of the MS2 was either strongly-bound (i.e. bound to colloidal particles) or enmeshed within aggregates that
3.3. XDLVO analysis XDLVO modeling facilitates an analysis of the favorability of interactions between MS2 and the colloidal surfaces included in this study. XDLVO models account for: 1) van der Waals forces caused by the interaction of electric dipoles; 2) electrostatic forces; 3) Lewis acidbase forces which may reflect electron transfer properties and/or hydrophobicity. As the separation distance approaches zero, the dimensionless potential energy is lowest (i.e. most favorable) for KAO, followed by FG, and then MS2-MS2 interaction (Fig. 4); this trend is consistent with the three experimental findings from this study, namely: 1) the percentage of strongly-bound MS2 was higher for KAO than for FG; 2) the maximum MS2 removal rate in the presence of KAO was higher than that of FG: and 3) the kinetics of MS2 aggregation were significantly (p < 0.05) slower than MS2 removal in the presence of either colloid. While the XDLVO results support the current results, the modeling framework should be improved to account for the interactions between MS2 aggregates and colloid surfaces. 4. Conclusions To the best of our knowledge, this is the first study to use modern DNA-labeling techniques and XDLVO models to examine bacteriophage MS2 adsorption to kaolinite and fiberglass colloids over a wide range of operationally relevant colloid concentrations. MS2 adsorption kinetics are much faster than previously reported, even when compared to results retrieved with similar colloid concentrations. The majority (i.e. 75% or greater after 60 min) of the MS2 was strongly-bound or entrapped, suggesting important operations implications for those interested in understanding and controlling the fate of MS2 (and other 5
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al.
Fig. 4. XDLVO interaction energy profiles: MS2-MS2, FG-MS2, and KAO-MS2 interactions in DI water. A) Energy primary maximum for aggregation/adsorption. (MS2 ζ-potential = -0.02 V, KAO ζ-potential = -0.04 V, FG ζ-potential = -0.02 V, ionic strength = 8.8 × 10−7 M, sphere-sphere for MS2-MS2 XDLVO calculations and sphere-plate formulas for the FGand KAO-MS2 XDLVO calculations).
References
viruses) in full-scale water reclamation facilities. MS2 aggregates quickly and strongly adsorb to colloids, which in turn provide protection from oxidants and govern their final fate. If strongly-bound viruses are neglected, there is great potential to underestimate the environmental and public health impact of colloidal particles in wastewater effluents. The current work also showed the merit associated with applying XDLVO theory, which helped explain the relative speed of MS2 adsorption to KAO and FG as well as the strong tendency to generate MS2 aggregates. XDLVO models should be revised to include interactions between MS2 aggregates and colloid surfaces and future studies should employ these research techniques to further elucidate the intricacies of the viral adsorption to colloids.
[1] Q. Wang, P. Wang, Q. Yang, Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci. Total Environ. 621 (15) (2018) 990–999. [2] R. Roffey, K. Lantorp, A. Tegnell, F. Elgh, Biological weapons and bioterrorism preparedness: importance of public-health awareness and international cooperation, Clin. Microbiol. Infect. 8 (8) (2002) 522–528. [3] EPA, Progress on Water Sector Decontamination Recommendation and Proposed Strategic Plan, EPA Office of Water, Washington, D.C, 2015 Report Number 817-R15-001. [4] WERF, Collaborative Workshop on Handling, Management, and Treatment of Biocontaminated Wastewater by Water Resource Recovery Facilities, Final Report No.WERF7W15 Water Environmental Research Foundation, Alexandria, VA, 2016. [5] C. Zoli, L. Steinbuerg, M. Grabowski, M. Hermann, Terrorist critical infrastructures, organizational capacity and security risk, Saf. Sci. 110 (Part C) (2018) 121–130. [6] A. Sakoda, Y. Sakai, K. Hayakawa, M. Suzuki, Adsorption of viruses in water environment onto solid surfaces, Water Sci. Technol. 35 (7) (1997) 107–114. [7] Rickert and Hunter, Rapid fractionation and materials balance of solids fractions in wastewater and wastewater effluent, Journal of Water Pollution Control Federation 39 (9) (1967) 1475–1486. [8] Y. Huang, W. li, X. Xie, B. Gao, A. Li, Distribution of endocrine-disrupting chemicals in colloidal and soluble phases in municipal secondary effluents and their removal by different advanced treatment processes, Chemosphere 219 (2019) 730–739. [9] M. Nasser, F. Twaiq, S. Onaizi, Effect of polyelectrolytes on the degree of flocculation of papermaking suspensions, Sep. Purif. Technol. 103 (2013) 43–52. [10] C. Dykstra, H. Giles, S. Banerjee, S. Pavlostathis, Fate and biotransformation of phytosterols during treatment of pulp and paper wastewater in a simulated aerated stabilization basin, Water Res. 68 (2015) 589–600. [11] L. Bennear, S. Olmstead, The impacts of the “right to know”: information disclosure and the violation of drinking water standards, J. Environ. Econ. Manage. 56 (2) (2008) 117–130. [12] EPA, Drinking Water: EPA Needs to Take Additional Steps to Ensure Small Community Water Systems Designated As Serious Violators Achieve Compliance, EPA Office of Inspector General, Washington, D.C, 2016 Report Number 16-P-0108. [13] C.H. Stagg, C. Wallis, C.H. Ward, Inactivation of clay-associated bacteriophage MS2 by chlorine, Appl. Environ. Microbiol. 33 (2) (1977) 385–391. [14] M. Templeton, R. Andrews, R. Hofmann, Inactivation of particle-associated viral surrogates by ultraviolet light, Water Res. 39 (15) (2005) 3487–3500. [15] C. Gerba, Applied and theoretical aspects of virus adsorption to surfaces, Advanced in applied microbiology 30 (1984) 133–168. [16] E. Skripkin, M. Adhin, M. de Smit, J. van Duin, Secondary structure of the central region of bacteriophage MS2 RNA: conservation and biological significance, J. Mol. Biol. 211 (2) (1990) 447–463. [17] K. Toropova, G. Basnak, P. Twarock Stockley, N. Ranson, The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly, J. Mol. Biol. 375 (3) (2008) 824–836. [18] M. Shishovs, J. Rumnieks, C. Diebolder, K. Jauzems, L. Andreas, J. Stanek, A. Kazaks, S. Kotelovica, I. Akopjana, G. Pintacuda, R. Koning, K. Tars, Structure of AP205 coat protein reveals circular permutation in ssRNA bacteriophages, J. Mol. Biol. 428 (21) (2016) 4267–4279. [19] B. McMinn, N. Ashbolt, A. Korajkic, Bacteriophages as indicators of faecal pollution and enteric virus removal, Lett. Appl. Microbiol. 65 (2017) 11–26. [20] M.F. Pouet, N. Azema, E. Touraud, O. Thomas, Physical and aggregate properties, Chapter 6, in: O. Thomas, C. Burgess (Eds.), UV-Visible Spectrophotometry of Water and Wastewater, Elsevier Publishers, London, UK, 2007, pp. 145–161. [21] G. Carlson, F. Woodard, D. Wentworth, O. Sproul, Virus inactivation on clay particle in natural waters, Journal of the Water Pollution Control Federation 40 (2) (1968) R89–R106. [22] B. Moore, B. Sagik, J.F. Malina, Viral association with suspended solids, Water Res.
Disclaimer The views expressed in this article are those of the authors and do not reflect the official policy or position of the Air Force Institute of Technology, the United States Air Force, the Department of Defense, or the United States government. The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development partially funded and collaborated in the research described here under Interagency Agreement DW-057-92440901-3. It has been subjected to the EPA’s review and has been approved for publication. Note that approval does not signify that the contents necessarily reflect the views of EPA. Any mention of trade names, products, or services does not imply an endorsement by the EPA or the U.S. Government. EPA does not endorse any commercial products, services, or enterprises. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank Dr. Daniel Felker (AFIT) and John Hixenbaugh (AFIT) for assistance handling hazardous material. The authors also thank Brian McMinn (EPA) for providing bacteriophage MS2 stocks. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2019.124099. 6
Colloids and Surfaces A xxx (xxxx) xxxx
Y. Xing, et al. 9 (197) (1975) 203. [23] M. Tong, Y. Shen, H. Yang, H. Kim, Deposition kinetics of MS2 bacteriophages on clay mineral surfaces, Colloids Surf. B Biointerfaces 92 (2012) 340–347. [24] M.I. Bellou, V.I. Syngouna, M.A. Tselepi, P.A. Kokkinos, S.C. Paparrodopoulos, A. Vantarakis, C.V. Chrysikopoulos, Interaction of human adenoviruses and coliphages with kaolinite and bentonite, Sci. Total Environ. 517 (2015) 86–95. [25] APHA, Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA, 2016. [26] S. Douven, C. Paez, C. Gommes, The range of validity of sorption kinetic models, J. Colloid Interface Sci. 448 (2015) 437–450. [27] C.V. Chrysikopoulos, V.I. Syngouna, Attachment of bacteriophages MS2 and ΦX174 onto kaolinite and montmorillonite: Extended-DLVO interactions, Colloids Surf. B Biointerfaces 92 (2012) 74–83. [28] J. Ryan, Effects of ionic strength and flow rate on colloid release: relating kinetics to intersurface potential energy, J. Colloid Interface Sci. 164 (1994) 21–34. [29] H. Schwegmann, J. Ruppert, F. Frimmel, Influence of the pH-value on the photocatalytic disinfection of bacteria with TiO2-explnanation by DLVO and XDLVO theory, Water Res. 47 (2013) 1503–1511.
[30] W. Zhang, X. Zhang, Adsorption of MS2 on oxide nanoparticles affects chlorine disinfection and solar inactivation, Water Res. 69 (2015) 59–67. [31] M. Haerifar, S. Azizian, Fractal-like adsorption kinetics on heterogeneous solid surfaces, J. Phys. Chem. C 118 (2014) 1129–1134. [32] A. Palomino, J. Santamarina, Fabric map for kaolinite: effect of pH and ionic concentration on behavior, Clays Clay Miner. 53 (3) (2005) 209–222. [33] P. Yunker, T. Still, M. Lohr, A. Yodh, Suppression of the coffee-ring effect by shapedependent capillary interactions, Nature 476 (2011) 308–311. [34] V. Dugyala, M. Basavaraj, Control over coffee-ring formation in evaporating liquid drops containing ellipsoids, Langmuir 30 (29) (2014) 8680–8686. [35] S. Vaishanav, K. Chandraker, J. Korram, R. Nagwanshi, K. Ghosh, M. Satnami, Protein nanoparticle interaction: a spectrophotometric approach for adsorption kinetics and binding studies, J. Mol. Struct. 1117 (2016) 300–310. [36] J. Ciejka, K. Wolski, M. Nowakowska, K. Pyrc, K. Szczubiałka, Biopolymeric nano/ microspheres for selective and reversible adsorption of coronaviruses, Mater. Sci. Eng. C 76 (July (1)) (2017) 735–742 2017. [37] A. Julbe, M. Drobek, A. Ayral, About the role of adsorption in inorganic and composite membranes, Curr. Opin. Chem. Eng. 24 (2019) 88–97.
7