430789 Attachment of Bacteriophage P22 to Nano Structured Iron Oxide Ceramics: Implications for Drinking Water Treatment

Monday, November 9, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
Guillermina Gentile1, Maria V. Gallardo1, Fernando M. Yrazu1, Oscar Oppezzo2, Ramon Pizarro2, Hugo R. Poma3, Veronica B. Rajal3 and Maria M. Fidalgo4, (1)Instituto Tecnologico de Buenos Aires, Buenos Aires, Argentina, (2)Comision Nacional de Energia Atomica, (3)INIQUI, Universidad Nacional de Salta, (4)Department of Civil and Environmental Engineering, University of Missouri, Columbia, MO


Availability of safe water is important for welfare and improvement of human populations, challenged by growth of demography, development, and waste. Researchers have been paying more attention to links between disease outbreaks and viral pathogens in drinking water.

Adsorption processes can remove contaminants, avoiding energy consumption associated with pumping and disposal issues of concentrated streams generated. Bulk porous adsorbents, contrary to suspended colloidal particles and co-precipitation schemes, do not require extra separation stages, rendering the system safer to handle, easier to use and applicable to mobile devices.

In this work, a nanostructured iron oxide ceramic filter was built and tested as adsorbent for bacteriophage P22.


Attachment kinetics experiments were conducted in order to investigate the time and evolution of the attachment process as well as to determine the appropriate equilibration time to be used in the equilibrium isotherm of bacteriophage P22.

The virus and iron oxide were characterized with respect to size and surface charge, as they play a key role in the removal process.

A ceramic filter was fabricated and tested for the continuous treatment of contaminated water.

Since electrostatic interactions are expected to dominate the virus attachment to metal oxide surfaces, DLVO theory was applied to the analysis of the attachment data as well as the virus stability and aggregation conditions.

Materials and methods

All chemicals were of reagent grade, except FeCl2 that was technical grade. Type I water was used. P22 phage stock suspensions had a concentration of approximately 2 x1010PFU/mL.

Salmonella typhimuriumstrain DA1468, was spiked in LB broth Lenox, and then incubated for 24 h at 37°C. Virus was replicated and afterwards the suspension was centrifuged at 15000 rpm for 60 minutes. The supernatant was filtered through a 0.22 μm PVDF membrane (Millipore GVW P02500). Purification included dialysis through a 100 kDa MWCO membrane (SpectraPor Biotech CE, Spectrum Laboratories, USA) twice: first, against Type I water, and secondly, against 15 mM NaCl solution, for 20 hours each. The final suspension was filtered again and kept at 4ºC.

Iron oxide nanoparticles were synthesized in the laboratory following previously published methods, from the reaction of lepidocrocite (obtained by oxidation of FeCl2) with acetic acid (Anedra, Argentina) to yield nanoparticles by the attack of the acid on the hydrogen bonds of the mineral structure. These nanoparticles are precursors to ceramic membranes; they can be either deposited onto support matrices or used as a self-standing material. Finally, the particles were sintered at 450°C and converted to iron oxide ceramic (hematite).Ferroxane was characterized by X-ray diffraction (PW1730-10 diffractometer, Phillips). Specific surface area was measured by BET N2 method and pore size was calculated by BJH N2adsorption/desorption isotherm at 77 K in a surface area analyzer (Coulter SA 3100).

Size of P22 was determined by TEM (Philips EM301) and DLS (Zetasizer Nano ZS, Malvern). Zeta potentials of the bacteriophage and iron oxide particles were measured by Laser Doppler Micro-electrophoresis (Zetasizer Nano ZS, Malvern).

Adsorption experiments were carried out in centrifuge tubes containing iron oxide (0.010 g to 0.150 g) at different solvent chemistries where the virus was inoculated. Control experiments were carried out without iron oxide with and without stirring, to evaluate natural inactivation of the virus and to quantify the effect of shear. The tubes were shaken in horizontal position in an orbital shaker at room temperature for 7 hours. Samples were taken from each batch at the beginning and at the end of each experiment.

The operational set-up for the attachment during filtration consisted of an alumina-supported iron oxide ceramic tube with a dead end. Samples were taken at different times to assess evolution of virus concentration. The potential inactivation effect of the alumina itself was investigated in an experiment using an alumina tube with no iron oxide. To exclude viral inactivation due to stress, another run was performed with no alumina-supported iron oxide ceramic tube.

Analytical methods: Double-agar layer method and qPCR were applied to determine the concentration of P22. The classical double layer method was complemented and enhanced by qPCR. Briefly, DNA was extracted from P22 suspensions using Pure Link Viral RNA/DNA Mini Kit (Invitrogen, USA) and detected with GeneAmp 5700 Sequence Detection System (Applied Biosystems, USA). Amplification was initiated using the hot start method at 95ºC for 10 min; 40 cycles of 95ºC for 15 s, and 60ºC for 1 min. The detection limit was calculated to be 10 bacteriophages per sample.

DLVO modeling: P22 was considered a spherical virus-like-particle and the ceramic membrane an infinite plate. Electrostatic double layer repulsion and van der Waals attraction between two particles of bacteriophage and between bacteriophage and membrane were calculated according to classical works presenting DLVO theory such as J. Gregory’s “Interaction of unequal double layers at constant charge” H.C. Hamaker´s “The London-van der Waals attraction between spherical particles”, Physiology 4 (1937).

Results and discussion

Characterization: XRD showed that the iron oxide was converted to hematite, a-Fe2O3. BET specific surface area was 29.3±1.5 m2/g. Pore size distribution yielded a mean pore size of 62 nm. Zeta potential of P22 was -27.9 mV at pH 7. The iron oxide PZC was 6. Consequently, the virus will be subjected to attractive forces at pHs below 6, while attachment is expected to be hampered by electrostatic repulsion at higher pHs. Virus size was measured by DLS (71±0.8 nm) and by TEM (58±9 nm).

Attachment kinetics: In the first seven hours an average LRV of 1.4 was observed for the samples containing the adsorbent, while no reduction in virus concentration in the control experiments. The decrease in LRV continued, but inactivation in the control samples started at 24 hrs, therefore LRV could not be attributed entirely to attachment to the iron oxide. A rapid decrease in virus concentration is observed in the first seven hours, followed by a marginal increase in removal up to the 48 hour time frame of testing; hinting a two phase process: a rapid removal corresponding to available easier to reach surface sites, and a slower stage in which the adsorbate diffuses into the internal structure of the iron oxide particles finding new sites.

Adsorption isotherms: Data indicated loss of virus activity due to attachment onto iron oxide and increase of the adsorbed concentration with increasing liquid equilibrium concentration. No plateau was reached, but the slope slightly decreased for the highest concentration point. The isotherms were fitted to the linearized form of Langmuir, Freundlich, and Temkin models. The coefficients of determination (R2) were calculated. The equations depict well the experimental data. The ferroxane-derived ceramics showed higher adsorption when ionic strength is increased and this augmented affinity is also reflected in higher h, hinting an electrostatic mechanism for virus attachment.

Filtration experiments: A virus suspension was filtered through ferroxane-coated alumina filters. This breakthrough experiment as originally conceived did not yield a measurable reduction in the viral load.

DLVO modeling: At low values of pH (3-4), van der Waals attraction overweighs electrical double layer repulsion, and thus aggregation between the particles is expected. At pHs close to neutral, an energy barrier develops and aggregation is prevented. At intermediate pHs (4.5-5) a low energy barrier arises; some particles may overcome it due to Brownian motion and aggregate. PFU and qPCR showed equivalent concentrations at pH 7, and relatively lower values given by PFU for pH 4-6. We can explain the difference at the middle points by limited aggregation due to a low energy barrier; however, the predicted instability at pH 4 does not agree with the measurements, that did not revealed a significant difference between both techniques and therefore, not extensive aggregation can be concluded. The results suggested an underestimation of the repulsive forces by the DLVO theory.

The interaction between the bacteriophage and the oxide was analyzed for pH 4-7. The results showed a strong dependence of attachment on pH. For pH 4-6 a primary minimum in the interaction energy is predicted, and strong attachment is expected. As pH gets closer to the adsorbent PZC, repulsive forces arise. A moderate energy barrier is predicted at pH 6.5, while unfavorable conditions for deposition develop at pH 7.


Iron oxide ceramic membranes were successfully applied for the removal of virus from water.  Iron oxide coated alumina filters showed improved removal capacity compared to the stand-alone material tested in batch mode, as inner pore sites that otherwise would be considered inaccessible due to long diffusion times become operational.

The removal mechanism was observed to be electrostatic in nature. DLVO analysis of the attachment predicted it to be effective up to pH 6.5.

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