Influence of Inorganic Ions on Aggregation and ... - ACS Publications

Article pubs.acs.org/est

Influence of Inorganic Ions on Aggregation and Adsorption Behaviors of Human Adenovirus Kelvin Wong,†,* Biplab Mukherjee,‡ Amy M. Kahler,§ Richard Zepp,† and Marirosa Molina† †

Ecosystems Research Division, United States Environmental Protection Agency, 960 College Station Road, Athens, Georgia 30605, United States ‡ The Dow Chemical Company, Midland, Michigan 48667, United States § Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, 1600 Clifton Road, Atlanta, Georgia 30329, United States S Supporting Information *

ABSTRACT: In this study, we investigated the influence of inorganic ions on the aggregation and deposition (adsorption) behavior of human adenovirus (HAdV). Experiments were conducted to determine the surface charge and size of HAdV and viral adsorption capacity of sand in different salt conditions. The interfacial potential energy was calculated using extended Derjaguin and Landau, Verwey and Overbeek (XDLVO) and steric hindrance theories to interpret the experimental results. Results showed that different compositions of inorganic ions have minimal effect on varying the iso-electric point pH (pHiep) of HAdV (ranging from 3.5 to 4.0). Divalent cations neutralized/shielded virus surface charge much more effectively than monovalent cations at pH above pHiep. Consequently, at neutral pH the presence of divalent cations enhanced the aggregation of HAdV as well as its adsorption to sand. Aggregation and adsorption behaviors generally agreed with XDLVO theory; however, in the case of minimal electrostatic repulsion, steric force by virus’ fibers can increase the energy barrier and distance of secondary minimum, resulting in limited aggregation and deposition. Overall, our results indicated that subsurface water with low hardness residing in sandy soils may have a higher potential of being contaminated by HAdV.

■

INTRODUCTION

Compared to bacteria and protozoa, viruses are smaller and able to travel longer distances in the environment.16 Because of their mobility,17 viruses have been selected as the biological agent to model the transport of waterborne pathogens in subsurface environments.18,19 Solution chemistry of environmental compartments can significantly affect the electro-kinetic and size characteristics of suspended particles.20 These properties significantly influence the mobility, adsorption, and removal of viruses.21 The Derjaguin and Landau and Verwey, and Overbeek (DLVO) theory has been used to understand the transport behavior of colloid particles, and it can also be applied to study virus transport because of the similar size of viruses and colloids.22 However, some studies found DLVO theory was not able to predict deposition of viruses,23−25 and suggested that steric hindrance arising from fibers or longer loops can significantly reduce virus deposition. Because of the potential public health risk caused by HAdV and the lack of understanding of its transport behavior, the objectives of this study were to investigate the influence of

Human adenoviruses (HAdVs) are a common cause of gastroenteritis,1,2 upper and lower respiratory system infections, and conjunctivitis.3 Other diseases associated with HAdVs include acute and chronic appendicitis, cystitis, exanthematous disease, and nervous system diseases.3 HAdVs are also considered to be important opportunistic pathogens in immunocompromised patients.4 High prevalence of HAdV has been found in different water environments including marine, river, ground, drinking, recreational, and wastewaters.5−15 Wastewater effluent samples from different sources tested positive for HAdV with a mean concentration of 3.7 × 103 genome copies (GC)/L.12 Infectious HAdV was also found in approximately 70% of biosolid samples,13 and the mean genome concentration of HAdVs in digested sludge was as high as fecal indicators such as Escherichia coli and enterococci.14 HAdV was found in river and lake water samples with a mean concentration of 101−104 GC/L.5,15 Considering its widespread presence and propensity to cause adverse health impacts, HAdV has been included on the U.S. Environmental Protection Agency’s contaminant candidate list one, two, and three in the year 1998, 2005, and 2007, respectively. © 2012 American Chemical Society

Received: Revised: Accepted: Published: 11145

July 16, 2012 September 1, 2012 September 5, 2012 September 5, 2012 dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

directly added to qPCR reaction mix without nucleic acid extraction. We found that 95 °C for 15 min during the enzyme activation step in the qPCR assay was enough to burst the virus capsid and release the DNA for analysis. Standard curves were generated by serial dilution of HAdV in liquid matrix with suspended sand particles so that inhibition effects of the standards and experimental samples were the same, and any differences in threshold value caused by inhibition would not affect viral quantification accuracy. A standard curve was generated for each salt condition since inorganic ions also can affect the threshold value in the qPCR reaction. The standard curves for each condition are illustrated in SI Figure S2. The R2 values for all standard curves were above 0.99 and slopes ranged from 3.41 to 3.48 (SI Figure S2). All viral concentrations in the isotherm study were within the concentration range of the standard curves. The qPCR assay was adopted from Heim et al.27 and details of the reaction mix and amplification conditions are described in the SI. Extended DLVO and Steric Interaction. The virus−virus attachment (aggregation) and virus−sand attachment (deposition) are governed by interfacial potential energy. According to extended DLVO theory (XDLVO), the surface potential energy (ΦXDLVO) is calculated by the sum of potential energy contributed by van der Waals (ΦvdW), electrostatic double layer (ΦEdI), and hydrophobic interactions (Φhydrophobic).28 In addition, previous studies have suggested that steric interactions (ΦSteric) could have a significant influence on the attachment behavior of viruses or bacteria;29,30 therefore, steric energy is added to the XDLVO energies to calculate the total interaction energy:

solution chemistryinorganic ion concentrations and composition, and pHon the colloidal properties and adsorption characteristics of HAdV. In addition, the interfacial potential energies of virus−virus and virus−sand were calculated using extended Derjaguin and Landau, Verwey, and Overbeek (XDLVO) and steric hindrance theories to interpret the aggregation and adsorption results.

■

MATERIALS AND METHODS Preparation of HAdV. HAdV serotype 2 (strain 6) was obtained from the Centers for Disease Control (Atlanta, GA) and propagated in A549 cells; viruses were then purified according to the procedure described in Tollefson et al.26 The concentration of purified virus determined by plaque assay was about 107 PFU/mL. Detailed procedures for virus propagation and plaque assay are described in the Supporting Information (SI). Transmission Electron Microscopy (TEM). TEM images were taken to determine the size of HAdV used in this study. Approximately 10−20 drops of the viral suspension were droppered, one at a time, onto a carbon-coated Formvar grid of 400-mesh and later stained with 3% aqueous phosphotungistic acid, pH 7.0. The grid was then dried in a desiccator and TEM images were acquired using JEM-1210 (JEOL, Tokyo, Japan), coupled with a XR41C Bottom-Mount CCD Camera (AMT, Danvers, MA). Zeta Potential Measurement of Adenovirus in Different pH and Salt Conditions. Effect of inorganic salt composition and concentration on the zeta potential (ZP) of HAdV at different pH values was investigated. The ZP was measured under a range of pH (∼3 to ∼10) in the following five conditions: 1 mM NaCl; 10 mM NaCl; 7 mM NaCl and 1 mM CaCl2; 1 mM NaCl and 3 mM CaCl2; 1 mM NaCl and 3 mM Na2SO4. All five matrices have an ionic strength (IS) of 10 mM except for 1 mM NaCl. NaOH and H2SO4 were used to adjust the pH. Each ZP value is the average of four repeated measurements. Virus Aggregation Studies. Effects of pH, salt concentration and inorganic ion composition on the aggregation of HAdV were investigated using dynamic light scattering (DLS) to measure hydrodynamic diameter (DH) changes. The following solution chemistries were used for the aggregation study: 1 and 100 mM of NaCl at three different pH (4, 7, and 10); 0.1, 0.25, 0.5, 0.75, 1.0, 5.0, and 50 mM of CaCl2 and of MgCl2 at ∼pH 7.5. The ZP measurements were also made under these conditions. Detailed procedures for ZP and DLS measurements are described in the SI. Each DH value is the average of 10 repeated measurements. Isotherm Experiment. Batch adsorption isotherm experiments were conducted to evaluate adsorption of the virus to sand particles. Experiments were conducted in glass tubes with three different inorganic ion compositions but the same IS: 10 mM NaCl; 7 mM NaCl and 1 mM CaCl2; 1 mM NaCl and 3 mM CaCl2. Acid-washed Ottawa sand (EMD, SX0070-3) with a particle size of 30−40 mesh was used as the adsorbent. The experiment was performed in duplicate. Detailed procedures for isotherm experiments are described in the SI. Quantitative PCR (qPCR) Analysis. qPCR was used to quantify viral particles during the isotherm experiments. High variations in DNA extraction recovery can potentially create significant errors in calculating virus distribution between liquid and solid phase, thus affecting the accuracy of the isotherm curve. Therefore, liquid phase samples and standards were

ΦXDLVO + steric = ΦvdW + ΦEdI + Φ hydrophobic + Φsteric

(1)

The van der Waals and electrostatic double layer interaction energies are calculated using the following equations: ΦvdW =

AH ·r

(

6·x 1 +

14·x λw

)

(2)

⎛ ⎛ 1 + e−κx ⎞ ⎟ + (U12·U2 2) · ΦEdI = πεw ε0r ⎜2·U1·U2·In⎜ ⎝ 1 − e−κx ⎠ ⎝ ⎞ In(1 − e−κx)⎟ ⎠

⎛ 2·e 2 ·n ·I ⎞1/2 − i ⎟ κ=⎜ ⎝ ε0 ·εw ·K ·T ⎠

(3)

(4)

where AH is the Hamaker constant, r is the radius of HAdV; x is the separation distance between the surfaces of viruses or the surfaces of virus and sand particle; λW is the Dielectric wavelength for water; ε0 and εW are the dielectric permittivity of vacuum and water, respectively; U1 and U2 are the surface zeta potentials of the virus and sand particle, respectively, but for virus−virus interaction, both U1 and U2 are the same value, which is the surface zeta potential of the virus; k is the inverse of Debye length calculated by eq 4; e2‑ is the charge of electron; ni is bulk concentration of the ion; I is the valence charge; K is the Boltzmann constant; T is the temperature. The hydrophobic interaction energy between virus and sand particle is calculated using the empirical approach by Yoon et al.31 11146

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Φ hydrophob = −

K1W2·r x

Article

hydration layer thickness can be observed during DLS measurement, it fails to show up in TEM analysis because of low electron-density in that region. Additionally, the Stokes− Einstein equation is used in DLS measurement to determine the particle size and it assumes that all the particles are spheres. Therefore, fibers (∼31 nm) attached to the HAdV capsid can be regarded as virus surface and increase the DLS measurement. Finally, since DLS depends on light scattering for size estimation and light scattering varies with the sixth power of the particle diameter,20 the presence of a few virus aggregates in the suspension can skew overall virus size distribution toward the larger-size. Nevertheless, the mean polydispersivity index (PDI) for the DLS measurements was found to be ≤0.25, indicating that the viruses in the stock suspension were nearly monodispersed.23 Surface Charge and Isoelectric Point pH. Figure 1 illustrates the effect of IS, inorganic ion composition, and pH

(5)

where K1W2 is the hydrophobicity constant for the interaction between virus and sand particle, and is calculated as follows: ⎛ cos θ1 + cos θ2 ⎞ ⎟+b K1W2 = a⎜ ⎝ ⎠ 2

(6)

where θ1 and θ2 are the water contact angle of virus and sand, respectively, and a and b are system-specific constants. Due to a lack of instrument and information in the literature, the contact angle of HAdV is not available for this study. Constants and contact angles to calculate the hydrophobicity interaction between HAdV and sand were adopted from a previous study on the interaction between MS2 and sand.32 MS2 was selected because it has a higher hydrophobicity than other two viruses: ΦX174 and Aichivirus. Song et al.33 described steric interaction as the sum of osmotic and elastic repulsive forces from adsorbed polymers. Fibers on the surface of the virus capsid can behave like polymers and create steric repulsion that hinders attachment between virus and sand particles. Osmotic and elastic interaction energies are calculated as follows: Φosm

2·π ·r ·Φp2 ·NA ⎛ 1 ⎞ ⎜ = − χ ⎟(d − hx)2 ⎝ ⎠ 2 V̅

Φelas

3 2·π ·r ·NA ·Φp ·d 2·ρp ⎛ 2 ⎛ x ⎞ 1⎛x⎞ ⎜ − ⎜ ⎟ −⎜ ⎟ = ⎝ ⎠ ⎝ 2d ⎠ 6 d Mw ⎝3

+

⎛ x ⎞ ⎛ x ⎞⎞ ⎜ ⎟In⎜ ⎟⎟ ⎝ d ⎠ ⎝ d ⎠⎠

(7)

(8)

Figure 1. Zeta potential values of human adenoviruses (HAdV) in different pH values. Error bars represent standard deviation of four repeated measurements.

where V̅ is the molar volume of water; χ is the Flory−Huggins solvency parameter; d is the length of the fiber; MW is the molecular weight of the fiber; and ρp is the density of the fiber. ΦP is the volume fraction of the fiber and calculated as follows: Φp = 3

Γmax ·r

on the ZP of HAdV. As expected, ZP became more positive as pH decreased, due to protonation of the negative functional groups on the virus surface. Ionic strength is a combined effect of counter- and co-ions and is responsible for compressing the electrostatic double layer (EDL) around colloids (in our case, HAdV). However, only the counterions (in our case, Na+ and Ca2+) shielded the negative charge on the surface and caused a decrease in the absolute ZP (|ZP|). As observed in other studies,20,36 charge shielding is more effective in the presence of higher valence counterions and higher concentration of counterions, which explains the observed variation trends in ZP shown in Figure 1. At pH > 4, the presence of CaCl2 decreased the |ZP| more than other conditions with the same IS but no Ca2+. The decrease of |ZP| was higher at 10 mM than 1 mM NaCl (pH > 6), and at 3 mM than 1 mM CaCl2 (pH > 4). Like counterions, co-ions can play a significant role in affecting the surface charges of colloids. Because of their smaller hydrated radius, co-ions can approach and get adsorbed to similar-charged surfaces37 and cause an overall increase in |ZP|. Such preferential adsorption of co-ions is also possible on virus surfaces and may affect surface charge. At pH > 8, the ZP of HAdV was found to be more negative in the presence of SO42‑ than when just Cl− was present (Figure 1); this is because each SO42‑ carries two negative charges while each Cl− carries only one negative charge. Preferential adsorption of co-ions has been reported by another study of nanoparticles in aqueous systems.20

2

ρp ·[(d + r )3 − r 3]

(9)

where Γmax is the maximum surface concentration. The values of zeta potential, Debye length and other constants are shown in SI Tables S1, S2, and S3, respectively. Statistical Analysis. To determine whether different conditions can cause significant changes in the ZP and DH of HAdV, analysis of variance (ANOVA) single tests were performed using Sigma Plot 11.0; a p-value of ≤0.05 indicates a significant difference.

■

RESULTS AND DISCUSSION Size Characterization of Adenovirus. SI Figure S3 is the TEM image of purified HAdV; the size of HAdV determined by TEM was about 70 nm, which is similar to previously reported values.34 The image showed that the virus structure remained intact with no apparent damage during the purification and TEM preparation procedure. The DH of HAdV in the storage buffer (pH 5.9, 1 mM NaCl), determined by DLS, was ∼120 nm. Size variation with different measurement methods has been reported previously, and TEM measurement values have always been found to be lower than DLS results.23,35 One possible explanation might be the formation of the hydration layer at the virus surface water interface.23 Although the 11147

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

and DH of MS2 decreased from ∼1500 to ∼1100 nm when the concentration of NaNO3 increased from 1 mM to 100 mM at pH 4, near the pHiep of MS2.52 One explanation could be that fibers at both pHiep and high salt conditions can roll back and lie in parallel conformation to the virus capsid reducing the overall free energy of the virus particle, the same mechanism causing micelle and hemimicelle formation.53 We believe that DH measurements can be reduced by rolling back fibers, but more in-depth investigation is needed to identify and confirm the specific mechanism causing lower DH in the presence of higher salt concentration at pHiep. Our results indicate that adjusting the solution pH to pHiep has a stronger effect on virus aggregation than increasing the monovalent cation concentration. As noted in Figure 2, the DH increased significantly (p ≤ 0.05) as the pH approached pH 4. By lowering pH value from 10 to 4, the DH increased from 90 to 300 nm at 1 mM NaCl, and from 125 to 200 nm at 100 mM NaCl. Colloids tend to aggregate as the pH approaches pHiep because the energy barrier preventing aggregation disappears (see discussion of XDLVO Theory and Steric Effect). Conversely, the further from pHiep, greater stability of the suspended colloids is expected. Thus, like other colloids, the pHiep of HAdV plays a significant role in determining its ability to interact with other surfaces in its vicinity. Effect of Divalent Cation on Aggregation. The effects of divalent cations (Ca2+ and Mg2+) on HAdV aggregation were determined under low (0.1−1.0 mM) and high (5−50 mM) salt concentrations around neutral pH (7.5). At the low salt concentration, the |ZP| of HAdV decreased from ∼25 to ∼18 mV (Figure 3a) as salt concentration increased from 0.1 to 1.0 mM. No aggregation was observed and the DH did not vary significantly with salt type (p ≥ 0.05) (Figure 3a). However, the DH of HAdV significantly increased (p ≤ 0.05) when the

The iso-electric point pH (pHiep) of HAdV (Figure 1) was in the range of 3.5−4.0 and varied minimally with different solution chemistry and IS; this indicates that none of the cations or anions became specifically associated on the virus surfaces and therefore interacted reversibly.38 These observations are similar to previous studies showing that the pHiep of bacteriophage and pathogenic bacteria varied slightly with different IS and inorganic ion composition.39−41 Only a few studies have reported the pHiep of HAdV: the pHiep of HAdV serotypes 4 and 5 were reported as 2.6 and 4.5, respectively.42,43 Favier et al.44 predicted the pHiep of different serotypes of HAdV, based on external structural protein; however, none of the pHiep values closely match the experimental pHiep values measured by our and other studies.42,43 Based on the review by Michen and Graule,45 the pHiep of most microorganisms is in the range of 3.5−7.0, and our study showed the pHiep of HAdV2 is within this range. Two types of bacteriophage, MS2 and ΦX174, have been widely used as the enteric virus surrogates to study virus transport.46−49 Studies reported the pHiep of MS2 to be around 3.0 to 4.1, while most of the pHiep of ΦX174 are between 6.0 and 7.0.45,50 Considering the similarity in pHiep, MS2 may be a more representative surrogate than ΦX174 to predict transport behavior of HAdV in the environment. Effect of Monovalent Cation (Na+) and pH on Aggregation. Figure 2 shows the effect of NaCl concentration

Figure 2. Hydrodynamic diameters of HAdV in 1 and 100 mM of NaCl at pH 4, 7, and 10. The numbers inside the bars represent the zeta potential values. Error bars represent standard deviation of 10 repeated measurements.

and pH on the size of HAdV. The DH at 100 mM was significantly larger than at 1 mM NaCl at both pH 7 and 10 (p ≤ 0.05). This aggregation was due to the presence of Na+ which caused surface charge neutralization. However, the effect of Na+ on viral aggregation was minimal since the DH increased by only ∼30 nm in the presence of higher Na+ concentrations. Previous studies also suggested that monovalent cations have little or no effect on virus aggregation even though the concentration was equal to and/or greater than 1 M.39,41,51 Over a pH range from 2 to 7, MS2 did not aggregate when the background salt concentration of NaNO3 and NaHCO3 was increased from 1 mM to 100 mM.39,41 No aggregation of norovirus (NoV)-GII was observed with an increase in NaCl concentration from 1 mM to 1000 mM.51 Interestingly, the DH at pH 4 in 100 mM NaCl was lower, compared to 1 mM NaCl (p < 0.05) (Figure 2). Similar results were observed by others:51,52 da Silva et al.51 noted that DH of norovirus was larger at lower salt concentration of some pHs,

Figure 3. Hydrodynamic diameters and zeta potentials of HAdV in (a) 0.1−1.0 mM of CaCl2 and MgCl2 at pH 7.5, and (b) 5 and 50 mM of CaCl2 and MgCl2 at pH 7.5. The numbers inside the bars represent the zeta potential values. Error bars represent standard deviation of 10 repeated measurements. 11148

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

not statistically significant (P > 0.05). Though not statistically significant at 95% confidence interval, the increasing trend of KF within minimal change in Ca2+ indicates that the presence of higher concentration of Ca2+ can enhance adsorption of virus on sand particles. The same explanation of why viruses aggregated with the presence of Ca2+ applies in the case of adsorption behavior of viruses to sand particles, where electrostatic repulsion between the surfaces becomes attenuated as |ZP| approaches zero and divalent cations have stronger influence than monovalent cations on both viral aggregation and deposition on the sand surface. To the best of our knowledge, this is the first study to investigate the adsorption of HAdV to sand particles. Previous studies found that the adsorption capacity of bacteriophages (MS2 and ΦX174) on Ottawa sand particles in phosphate buffered saline (PBS) was very low, with KF ranging from 0 to 0.44.16,47,58 Those results are similar to what we observed with HAdV since PBS is composed of monovalent cation salts, and neither the previous studies nor ours observed significant adsorption of viruses to sand particles when only the monovalent cation is present. In another isotherm study, the adsorption capacity of poliovirus by Ottawa sand in the presence of 1 mM Ca2+ was 505,59 indicating the positive influence of divalent cation on virus adsorption. It should be noted that the HAdV adsorption capacity by silt−clay−loam soils in PBS found in our other study60 was around 103, much higher than the values reported here, suggesting that adsorption of HAdV to pure sand particles can be relatively insignificant compared to silt and clay. Modification of sand surface such as by metal oxide coating,61 however, can significantly increase the adsorption capacity. Interpretation of Aggregation and Adsorption Results by XDLVO Theory and Steric Effect. The virus−virus and virus−sand interaction energy profiles are illustrated in Figure 5. Aggregation and adsorption results generally agree with the XDLVO theory, while smaller DH and lower adsorption capacity are mostly associated with higher energy barriers. The hydrophobic interaction had no significant effect on the energy profile since we did not see any difference in the energy profile of classic and extended DLVO theories (data not shown). For virus−virus interaction, no energy barrier under 50 mM CaCl2 (Figure 5a) and 50 mM MgCl2 and 100 mM NaCl (SI Figure S4) was observed; however, virus aggregation was minimal compared to other nanoparticle studies20 where the aggregation increased several fold when the energy barrier was absent. To interpret further, steric interaction energy contributed by osmotic and elastic repulsive force from the virus’s fibers was added to other XDLVO energies (Figure 5b). Results showed that steric interaction increased the attachment hindrance slightly. Equations 7 and 8 determined only the steric force from the attaching particle, not the attached surface; therefore, the actual maximum energy barrier should be higher than the one shown in Figure 5b. Also, the effect of steric forces was estimated from certain assumptions and values from the literature, which might or might not represent the true values in the case of HAdV. The effects of steric force on the interaction energy between virus and sand particle are illustrated in Figures 5c−e. Steric interaction increased the maximum energy barrier and reduced the distance of secondary minimum. Even though steric interactions contributed the same amount of energy (around 1kBT) to the maximum energy barrier in all three conditions, it had different effects on the distance of secondary minimum,

concentration of divalent cations increased from 5 to 50 mM (Figure 3b). At neutral pH, the DH of HAdV in 50 mM CaCl2 and MgCl2 was larger than (p ≤ 0.05) and equal to (p ≥ 0.05) the DH in100 mM NaCl, respectively. These results indicate that aggregation kinetics of HAdV are more sensitive to divalent than monovalent counterions because divalent counterions are more effective than monovalent ions in charge shielding and shrinking the EDL;37 additionally, divalent ions are also able to coagulate by bridging colloidal particles via specific interactions and/or complexation.36,51 The DH significantly increased (p ≤ 0.05) when the MgCl2 concentration was increased from 5 mM to 50 mM, but not when CaCl2 was used as the background salt (p ≥ 0.05). However, the DH of HAdV in both 5 and 50 mM of CaCl2 were significantly larger than in either 5 or 50 mM of MgCl2 (p ≤ 0.05). In addition, |ZP| values of HAdV in 5 and 50 mM of CaCl2 were significantly lower than those in the same concentrations of MgCl2 (p ≤ 0.05). Similar results were observed by others, where Ca2+ had a stronger effect than Mg2+ on aggregation of rotavirus23 and MS2.24 Authors of both cited studies suggested that these effects might be due to the ability of Ca2+ to form bridges across the carboxylate groups on the virus capsid more easily than Mg2+ because the ionic radius of Mg2+ is larger. Glutamic acid and aspartic acid are the amino acids containing carboxylate moieties35,54,55 and both are present in the capsid of HAdV.56 Thus, larger aggregates of HAdV in CaCl2 than in MgCl2 solution could be due to stronger cation-bridging by Ca2+. Based on our results and the fact that the concentration of HAdV in the environment is usually lower than the concentrations used in this study and the natural organic matter can stabilize the virus,57 we anticipate that there will be minimal aggregated forms of HAdV in freshwater environments, where the matrix is usually at neutral pH and low IS. Adsorption of HAdV to Sand Particles. Figure 4 illustrates the adsorption isotherm curves of HAdV and Ottawa

Figure 4. Adsorption isotherm curves of HAdV and Ottawa sand (liquid phase pH ∼6.5). Error bars represent standard deviation of replicate measurements.

sand under the same IS but different Na + and Ca 2+ concentrations. No isotherm curve for 10 mM NaCl was reported since the viral concentration in the liquid phase after the isotherm experiments was not significantly different from the initial concentration, indicating that no adsorption of HAdV to sand particles occurred. The Freundlich constant (KF) increased slightly from 18 to 40 by increasing the concentration of Ca2+ from 1 mM to 3 mM, but the increase in the constant is 11149

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

Figure 5. Interface energy profile: (a) virus−virus by XDLVO theory; (b) virus−virus by XDLVO+Steric in 50 mM CaCl2; (c−e) virus−sand by XDLVO+Steric. DH = hydrodynamic diameter. KF = Freundlich constant.

constant) was not found to be statistically significant at the 95% confidence interval. Finally, the removal of viruses could be higher in the column setting since the tumbling and centrifugation processes could obviate the attachment force at secondary minimum force and straining during column filtration could also contribute a significant amount of viral removal.62 Interpretation of the effect of virus aggregation and adsorption mechanism influenced by solution chemistry can be complicated. Based on observations in this study, interpretation that relies solely on XDLVO theory may be adequate when a high maximum energy barrier is present, but steric hindrance and cationic bridging should also be considered in conditions where a maximum energy barrier is absent or extremely low. Environmental Application. In summary, this study provides initial findings on the influence of inorganic ions on human adenovirus surface charge, isoelectric point, and aggregation and deposition behavior. Based on our results,

where weak and reversible attachment takes place. After incorporating the steric interaction, the distance of secondary minimum increased by approximately 2, 5, and 9 nm in the conditions of 10 mM NaCl, 7 mM NaCl/1 mM CaCl2, and 1 mM NaCl/3 mM CaCl2, respectively. Thus, even though the distance of the secondary minimum computed by XDLVO theory in these three solution conditions varied from 18 to 26 nm, their difference by XDLVO + steric was reduced to 1 nm (ranging from 26 to 27 nm). These results indicate that steric effects not only hindered the attachment between virus and sand particles, but can also be a controlling factor in reversible/ weak adsorption. Since the distances of secondary minimum by XDLOV+steric are very similar in all three conditions, higher viral adsorption capacity by sand particles in the presence of higher Ca2+ concentration is likely a result of a lower maximum energy barrier and the bridging effect by divalent cation discussed earlier. Additionally, the small decrease of maximum energy barrier from 1 to 3 mM of CaCl2 could be the reason why the increase in the adsorption capacity (or Freundlich 11150

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

■

HAdV is likely to remain suspended as an individual virus in a fresh water environment with low turbidity and neutral pH. If the environment is composed of mostly sandy soils without metal oxide coating (e.g., AlOH3, FeOH3) and under the water conditions described above, HAdV can potentially be transported longer distances. On the other hand, a high salinity environment such as seawater should neutralize or reverse the surface charge of HAdV and enhance its attachment to solid particulates. Katayama et al.63 developed a viral sampling method using a negatively charged membrane. Since this method is based on electrostatic interaction, not entrapment, a divalent cation such as MgCl2 was added to the water sample before sample filtration to reverse the virus surface charge to positive. This study showed that adjusting the water pH to pHiep aggregates HAdV more significantly than adding the divalent cation; therefore, further studies involving the sampling of HAdV by negatively charged-membranes can consider including an additional step of adjusting water pH to around the pHiep of HAdV since larger viral aggregates can potentially be entrapped more efficiently on the filter, thus increasing recovery. In addition, replacing MgCl2 with CaCl2 may enhance the attachment of virus to the filter membrane. However, stronger attachment to the filter may not guarantee higher recovery since recovery is a combination of viral attachment to and detachment from the filter; an extremely strong attachment may result in a low number of virus particles eluted from the filter.64

■

REFERENCES

(1) Adrian, T.; Wigand, R.; Richter, J. Gastroenteritis in infants associated with genome type of adenovirus 31 and with combined rotavirus and adenovirus 31 infection. Eur. J. Pediatr. 1987, 146 (1), 38−40. (2) Krajden, M.; Brown, M.; Petrasek, A.; Middleton, P. J. Clinical features of adenovirus enteritis: A review of 127 cases. Pediatr. Infect. Dis. J. 1990, 9 (9), 636−641. (3) Jiang, S. C. Human adenoviruses in water: Occurrence and health implications: A critical review. Environ. Sci. Technol. 2006, 40 (23), 7132−7140. (4) Wadell, G. Molecular epidemiology of human adenoviruses. Curr. Top. Microbiol. Immunol. 1984, 110, 191−220. (5) Albinana-Gimenez, N.; Miagostovich, M. P.; Calqua, B.; Huguet, J. M.; Matia, L.; Girones, R. Analysis of adenoviruses and polyomaviruses quantified by qPCR as indicators of water quality in source and drinking-water treatment plants. Water Res. 2009, 43 (7), 2011−2019. (6) Aslan, A.; Xagoraraki, I.; Simmons, F. J.; Rose, J. B.; Dorevitch, S. Occurrence of adenovirus and other enteric viruses in limited-contact freshwater recreational areas and bathing waters. J. Appl. Microbiol. 2011, 111 (5), 1250−1261. (7) Castignolles, N.; Petit, F.; Mendel, I.; Simon, L.; Cattolico, L.; Buffet-Janvresse, C. Detection of adenovirus in the waters of the seine river estuary by nested-PCR. Mol. Cell. Probes 1998, 12 (3), 175−180. (8) Chapron, C. D.; Ballester, N. A.; Fontaine, J. H.; Frades, C. N.; Margolin, A. B. Detection of astroviruses, enteroviruses, and adenovirus types 40 and 41 in surface waters collected and evaluated by the information collection rule and an integrated cell culture-nested PCR procedure. Appl. Environ. Microbiol. 2000, 66 (6), 2520−2525. (9) Fong, T. T.; Mansfield, L. S.; Wilson, D. L.; Schwab, D. J.; Molloy, S. L.; Rose, J. B. Massive microbiological groundwater contamination associated with a waterborne outbreak in Lake Erie, South Bass Island, Ohio. Environ. Health Perspect. 2007, 115 (6), 856− 864. (10) Jiang, S.; Noble, R.; Chui, W. P. Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California. Appl. Environ. Microbiol. 2001, 67 (1), 179−184. (11) Katayama, H.; Haramoto, E.; Oguma, K.; Yamashita, H.; Tajima, A.; Nakajima, H.; Ohgaki, S. One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan. Water Res. 2008, 42 (6−7), 1441−1448. (12) Simmons, F. J.; Kuo, D. H. W.; Xagoraraki, I. Removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing. Water Res. 2011, 45 (9), 2739−2750. (13) Wong, K.; Onan, B. M.; Xagoraraki, I. Quantification of enteric viruses, pathogen indicators, and salmonella bacteria in class B anaerobically digested biosolids by culture and molecular methods. Appl. Environ. Microbiol. 2010, 76 (19), 6441−6448. (14) Wong, K.; Xagoraraki, I. A perspective on the prevalence of DNA enteric virus genomes in anaerobic-digested biological wastes. Environ. Monit. Assess. 2011, 184 (8), 5009−5016. (15) Xagoraraki, I.; Kuo, D. H. W.; Wong, K.; Wong, M.; Rose, J. B. Occurrence of human adenoviruses at two recreational beaches of the great lakes. Appl. Environ. Microbiol. 2007, 73 (24), 7874−7881. (16) Jin, Y.; Flury, M., Fate and transport of viruses in porous media. Adv. Agron., 2002, 77, 39−100. (17) Scheuerman, P. R.; Farrah, S. R.; Bitton, G. Reduction of microbial indicators and viruses in a cypress strand. Water Sci. Technol. 1986, 18 (10), 1−8. (18) Herboldpaschke, K.; Straub, U.; Hahn, T.; Teutsch, G.; Botzenhart, K. Behavior of pathogenic bacteria, phages and viruses in groundwater during transport and adsorption. Water Sci. Technol. 1991, 24 (2), 301−304. (19) Keswick, B. H.; Gerba, C. P. Viruses in groundwater. Environ. Sci. Technol. 1980, 14 (11), 1290−1297. (20) Mukherjee, B.; Weaver, J. W. Aggregation and charge behavior of metallic and nonmetallic nanoparticles in the presence of competing

ASSOCIATED CONTENT

S Supporting Information *

More details about virus preparation, analytical and isotherm experiment procedure, virus−virus interface energy profile, and inputs for XDLVO and steric force calculations are provided in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Article

AUTHOR INFORMATION

Corresponding Author

*Phone: 1-706-355-8133; fax: 1- 706-355-8104; e-mail: wong. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr. Vincent Hill from the Centers for Disease Control and Prevention (Atlanta, GA) for providing the adenovirus stock, Dr. Maricarmen Garcia and Sylvia S. Riblet from the Poultry Diagnostic Research Center at the University of Georgia (Athens, GA) for their assistance in viral purification, and Dr. Dermont Bouchard of EPA for valuable review comments. This report has been subjected to the U.S. EPA’s peer and administrative review and has been approved for publication. The findings and conclusions in this presentation are those of the authors and do not necessarily represent those of the Centers for Disease Control and Prevention. The mention of trade names or commercial products in this report does not constitute endorsement or recommendation for use. 11151

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

similarly-charged inorganic ions. Environ. Sci. Technol. 2010, 44 (9), 3332−3338. (21) Schijven, J. F.; Hassanizadeh, S. M. Removal of viruses by soil passage: Overview of modeling, processes, and parameters. Crit. Rev. Environ. Sci. Technol. 2000, 30 (1), 49−127. (22) Gerba, C. P. Applied and theoretical aspects of virus adsorption to surfaces. Adv. Appl. Microbiol. 1984, 30, 133−168. (23) Gutierrez, L.; Mylon, S. E.; Nash, B.; Nguyen, T. H. Deposition and aggregation kinetics of rotavirus in divalent cation solutions. Environ. Sci. Technol. 2010, 44 (12), 4552−4557. (24) Pham, M.; Mintz, E. A.; Nguyen, T. H. Deposition kinetics of bacteriophage MS2 to natural organic matter: Role of divalent cations. J. Colloid Interface Sci. 2009, 338 (1), 1−9. (25) Syngouna, V. I.; Chrysikopoulos, C. V. Interaction between viruses and clays in static and dynamic batch systems. Environ. Sci. Technol. 2010, 44 (12), 4539−4544. (26) Tollefson, A. E.; Kuppuswamy, M.; Shashkova, E. V.; Doronin, K.; Wold, W. S. Preparation and titration of CsCl-banded adenovirus stocks. Methods Mol. Med. 2007, 130, 223−236. (27) Heim, A.; Carmen, Ebnet; Gabi, Harste; Pring-Åkerblom, P. Rapid and quantitative detection of human adenovirus DNA by realtime PCR. J. Med. Virol. 2003, 70 (2), 228−239. (28) Michen, B.; Meder, F.; Rust, A.; Fritsch, J.; Aneziris, C.; Graule, T. Virus removal in ceramic depth filters based on diatomaceous earth. Environ. Sci. Technol. 2012, 46 (2), 1170−1177. (29) Jucker, B. A.; Zehnder, A. J. B.; Harms, H. Quantification of polymer interactions in bacterial adhesion. Environ. Sci. Technol. 1998, 32 (19), 2909−2915. (30) Penrod, S. L.; Olson, T. M.; Grant, S. B. Deposition kinetics of two viruses in packed beds of quartz granular media. Langmuir 1996, 12 (23), 5576−5587. (31) Yoon, R. H.; Flinn, D. H.; Rabinovich, Y. I. Hydrophobic interactions between dissimilar surfaces. J. Colloid Interface Sci. 1997, 185 (2), 363−370. (32) Attinti, R.; Wei, J.; Kniel, K.; Sims, J. T.; Jin, Y. Virus’ (MS2, phi X174, and Aichi) attachment on sand measured by atomic force microscopy and their transport through sand columns. Environ. Sci. Technol. 2010, 44 (7), 2426−2432. (33) Song, J. E.; Phenrat, T.; Marinakos, S.; Xiao, Y.; Liu, J.; Wiesner, M. R.; Tilton, R. D.; Lowry, G. V. Hydrophobic Interactions increase attachment of gum arabic- and PVP-coated Ag nanoparticles to hydrophobic surfaces. Environ. Sci. Technol. 2011, 45 (14), 5988− 5995. (34) Maier, R. M.; Pepper, I. L.; Gerba, C. P. Environmental Microbiology; Academic Press: New York, 2008. (35) Gutierrez, L.; Li, X.; Wang, J. W.; Nangmenyi, G.; Economy, J.; Kuhlenschmidt, T. B.; Kuhlenschmidt, M. S.; Nguyen, T. H. Adsorption of rotavirus and bacteriophage MS2 using glass fiber coated with hematite nanoparticles. Water Res. 2009, 43 (20), 5198− 5208. (36) Bouchard, D.; Zhang, W.; Powell, T.; Rattanaudompol, U. S. Aggregation kinetics and transport of single-walled carbon nanotubes at low surfactant concentrations. Environ. Sci. Technol. 2012, 46 (8), 4458−4465. (37) Elimelech, M.; Omelia, C. R. Kinetics of deposition of colloidal particles in porous media. Environ. Sci. Technol. 1990, 24 (10), 1528− 1536. (38) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: San Francisco, CA, 1981. (39) Langlet, J.; Gaboriaud, F.; Duval, J. F. L.; Gantzer, C. Aggregation and surface properties of F-specific RNA phages: Implication for membrane filtration processes. Water Res. 2008, 42 (10−11), 2769−2777. (40) Schinner, T.; Letzner, A.; Liedtke, S.; Castro, F. D.; Eydelnant, I. A.; Tufenkji, N. Transport of selected bacterial pathogens in agricultural soil and quartz sand. Water Res. 2010, 44 (4), 1182−1192. (41) Yuan, B. L.; Pham, M.; Nguyen, T. H. Deposition kinetics of bacteriophage MS2 on a silica surface coated with natural organic

matter in a radial stagnation point flow cell. Environ. Sci. Technol. 2008, 42 (20), 7628−7633. (42) Salo, R. J.; Mayor, H. D. Isoelectric focusing of parvoviruses. Intervirology 1978, 10 (2), 87−93. (43) Trilisky, E. I.; Lenhoff, A. M. Sorption processes in ion-exchange chromatography of viruses. J. Chromatogr., A 2007, 1142 (1), 2−12. (44) Favier, A. L.; Burmeister, W. P.; Chroboczek, J. Unique physicochemical properties of human enteric Ad41 responsible for its survival and replication in the gastrointestinal tract. Virology 2004, 322 (1), 93−104. (45) Michen, B.; Graule, T. Isoelectric points of viruses. J. Appl. Microbiol. 2010, 109 (2), 388−397. (46) Jin, Y.; Chu, Y. J.; Li, Y. S. Virus removal and transport in saturated and unsaturated sand columns. J. Contam. Hydrol. 2000, 43 (2), 111−128. (47) Jin, Y.; Yates, M. V.; Thompson, S. S.; Jury, W. A. Sorption of viruses during flow through saturated sand columns. Environ. Sci. Technol. 1997, 31 (2), 548−555. (48) Zhuang, J.; Jin, Y. Virus retention and transport as influenced by different forms of soil organic matter. J. Environ. Qual. 2003, 32 (3), 816−823. (49) Zhuang, J.; Jin, Y. Interactions between viruses and goethite during saturated flow: Effects of solution pH, carbonate, and phosphate. J. Contam. Hydrol. 2008, 98 (1−2), 15−21. (50) Chrysikopoulos, C. V.; Syngouna, V. I. Attachment of bacteriophages MS2 and Phi X174 onto kaolinite and montmorillonite: Extended-DLVO interactions. Colloids Surf., B 2012, 92, 74− 83. (51) da Silva, A. K.; Kavanagh, O. V.; Estes, M. K.; Elimelech, M. Adsorption and aggregation properties of norovirus GI and GII viruslike particles demonstrate differing responses to solution chemistry. Environ. Sci. Technol. 2011, 45 (2), 520−526. (52) Dika, C.; Duval, J. F. L.; Ly-Chatain, H. M.; Merlin, C.; Gantzer, C. Impact of internal RNA on aggregation and electrokinetics of viruses: Comparison between MS2 phage and corresponding virus-like particles. Appl. Environ. Microbiol. 2011, 77 (14), 4939−4948. (53) Balnois, E.; Stoll, S.; Wilkinson, K. J.; Buffle, J.; Rinaudo, M.; Milas, M. Conformations of succinoglycan as observed by atomic force microscopy. Macromolecules 2000, 33 (20), 7440−7447. (54) Aoki, S. T.; Settembre, E. C.; Trask, S. D.; Greenberg, H. B.; Harrison, S. C.; Dormitzer, P. R. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing fab. Science 2009, 324 (5933), 1444−1447. (55) Dormitzer, P. R.; Sun, Z. Y. J.; Wagner, G.; Harrison, S. C. The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J. 2002, 21 (5), 885−897. (56) Kern, A.; Schmidt, K.; Leder, C.; Muller, O. J.; Wobus, C. E.; Bettinger, K.; Von der Lieth, C. W.; King, J. A.; Kleinschmidt, J. A. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J. Virol. 2003, 77 (20), 11072−11081. (57) Mylon, S. E.; Rinciog, C. I.; Schmidt, N.; Gutierrez, L.; Wong, G. C. L.; Nguyen, T. H. Influence of salts and natural organic matter on the stability of bacteriophage MS2. Langmuir 2010, 26 (2), 1035− 1042. (58) Thompson, S. S.; Flury, M.; Yates, M. V.; Jury, W. A. Role of the air-water-solid interface in bacteriophage sorption experiments. Appl. Environ. Microbiol. 1998, 64 (1), 304−309. (59) Moore, R. S.; Taylor, D. H.; Sturman, L. S.; Reddy, M. M.; Fuhs, G. W. Poliovirus adsorption by 34 minerals and soils. Appl. Environ. Microbiol. 1981, 42 (6), 963−975. (60) Wong, K.; Voice, T.; Xagoraraki, I. In The effect of organic matter on sorption of human adenovirus to soil particles. 12th International Conference on Environmental Science and Technology, Rhodes, Greece, September 8−10, 2011. (61) Pecson, B. M.; Decrey, L.; Kohn, T. Photoinactivation of virus on iron-oxide coated sand: Enhancing inactivation in sunlit waters. Water Res. 2012, 46 (6), 1763−1770. 11152

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153

Environmental Science & Technology

Article

(62) Tufenkji, N. Modeling microbial transport in porous media: Traditional approaches and recent developments. Adv. Water Resour. 2007, 30 (6−7), 1455−1469. (63) Katayama, H.; Shimasaki, A.; Ohgaki, S. Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Appl. Environ. Microbiol. 2002, 68 (3), 1033−1039. (64) Wong, K.; Fong, T. T.; Bibby, K.; Molina, M. Application of enteric viruses for fecal pollution source tracking in environmental waters. Environ. Int. 2012, 45, 151−164.

11153

dx.doi.org/10.1021/es3028764 | Environ. Sci. Technol. 2012, 46, 11145−11153