Marine Phages As Tracers: Effects of Size, Morphology, and Physico

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MARINE PHAGES AS TRACERS: EFFECTS OF SIZE, MORPHOLOGY AND PHYSICO-CHEMICAL SURFACE PROPERTIES ON TRANSPORT IN A POROUS MEDIUM Nawras Ghanem, Bärbel Kiesel, Rene Kallies, Hauke Harms, Antonis Chatzinotas, and Lukas Y. Wick Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04236 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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Environmental Science & Technology

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MARINE PHAGES AS TRACERS: EFFECTS OF SIZE, MORPHOLOGY AND PHYSICO-CHEMICAL

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SURFACE PROPERTIES ON TRANSPORT IN A POROUS MEDIUM

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Nawras Ghanem1, Bärbel Kiesel1, René Kallies1, Hauke Harms1,2, Antonis Chatzinotas1,2 and Lukas Y. Wick1*

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Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstraße 15, 04318 Leipzig, Germany.

German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany

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Running title: Marine phages as microbial tracers

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Intended for: Environmental Science and Technology

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Corresponding author: Helmholtz Centre for Environmental Research - UFZ. Department of Environmental Microbiology; Permoserstrasse 15; 04318 Leipzig, Germany. phone: +49 341 235 1316, fax: +49 341 235 45 1316, e-mail: [email protected].

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ACS Paragon Plus Environment

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Abstract

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Although several studies examined the transport of viruses in the terrestrial systems only few studies

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exist on marine phages (i.e. non-terrestrial viruses infecting marine host bacteria) as sensitively

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detectable microbial tracers for subsurface colloid transport and water flow. Here, we systematically

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quantified and compared for the first time the effects of size, morphology and physico-chemical

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surface properties of six marine phages and two coliphages (MS2, T4) during transport in sand-filled

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percolated columns. Phage-sand interactions were described by colloidal filtration theory and the

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extended Derjaguin-Landau-Verwey-Overbeek approach (XDLVO), respectively. The phages

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belonged to different families and comprised four phages never used in transport studies (i.e. PSA-

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HM1, PSA-HP1, PSA-HS2 and H3/49). Phage transport was influenced by size, morphology and

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hydrophobicity in an approximate order of size > hydrophobicity ≥ morphology. Two phages PSA-

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HP1, PSA-HS2 (Podoviridae and Siphoviridae) exhibited similar mass recovery as commonly used

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coliphage MS2 and were sevenfold better transported than known marine phage vB_PSPS-H40/1.

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Differing properties of the marine phages may be used to trace transport of indigenous viruses,

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natural colloids or anthropogenic nanomaterials and, hence, contribute to better risk analysis. Our

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results underpin the potential role of marine phages as microbial tracer for transport of colloidal

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particles and water flow.

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One sentence brief. Differences in the properties of marine phages influence their transport in

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porous media and underpin their potential as microbial tracers for reactive transport of colloidal

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particles and water flow.

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KEYWORDS: Marine phage, transport, colloid, bacteria, microbial tracer, XDLVO.


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TOC / Abstract Art

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INTRODUCTION

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Bacteriophages (or short: phages) are viruses that infect bacteria. In contrast to human- and animal

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health-related microorganisms, viruses infecting environmental microbes in particular in terrestrial

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system are less studied.1,2,3 Next to their impact on their hosts and on manifold ecosystem

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functions,4,5,6 their suitability as markers for water transport in the subsurface has been considered.7,8

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In 1980 Keswick and Gerba suggested to use bacteriophages as tracers because of similar movement

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and survival as animal viruses.9 Many phages (especially coli-phages such as MS2) have been tested

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since then either as potential surrogates for viral pathogens10,11 or as a tool to trace the flow of water

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in hydrology.12,13 An awkwardness of this usage was their differing adsorption behavior from the

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human adenoviruses (hAdVs)14 or the Norwalk virus15 and the isolation of both, coli-phages and

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their hosts from groundwater.9,16 Moreover, strains of E. coli are listed as pathogenic bacteria of

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concern.17 Despite a large number of studies examining the transport of phages in the terrestrial

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subsurface, only very few studies evaluated marine phages, most often phage H40/1,18,19,20,21 as

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biological tracers.22 Marine phages are highly suitable for transport studies because they are virtually

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absent in the terrestrial ecosystem23 and non-pathogenic. Additionally they allow for highly sensitive

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detection22 due to the possibility to apply as many as 1015 phages (~1 g) in tracer experiments and to

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detect as little as one or two phages per mL of recovered water,22 via its specific interaction with the

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host bacterium.

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First studies by Gerba and Goyal proposed that viruses can be grouped by their adhesion behavior as

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mimic for their ability to interact with soil.10 Follow-up studies however rather focused on the effect

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of environmental rather than viral factors on virus transport in soil (e.g. heterogeneity of the soil24,25;

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soil textures and conditions26,27,28,29 or chemical factors).30,31,32,33,34,35 Data on the effect of virus

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properties on their transport, however, are still scarce.36 A few studies aimed at predicting transport

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in relation to physical viral properties such as the isoelectric point,37 a combination of isoelectric


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point and size,38 or the different surface charge, polarity and topography of viral protein structures.39

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Yet no such study on marine phages is known. In the frame of the Collaborative Research Centre

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AquaDiva (http://www.aquadiva.uni-jena.de/) our study aimed at selecting and characterizing

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different marine phages and at studying their transport in porous media.40 Although inherently not

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being as inert and conservative as ideal chemical tracers may be, we here consider marine phages as

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sensitively detectable microbial tracers17 for colloidal transport and advective water flow. We

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hypothesized that size, morphology and physico-chemical surface properties control their transport

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and, hence, their appropriateness as surrogates for transport of colloidal particles. For this purpose,

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we systematically investigated the transport of six marine phages in addition with two commonly

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applied coli-phages in transport studies (T4 and MS2) using sand-filled laboratory columns. The

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selected phages belonged to four different virus families (Myoviridae, Siphoviridae, Podoviridae and

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Leviviridae) and thus differed in their size, morphology and physicochemical properties. The set of

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phages included four marine phages that never had been examined for their suitability as tracers

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(PSA-HM1, PSA-HP1, PSA-HS2 and H3/49). The phages’ physico-chemical properties, were

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assessed by dynamic light scattering and water contact angle analysis, size and morphology

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information were obtained from literature data, while phage transport and phage-sand interactions

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were described by colloidal filtration theory and the extended Derjaguin-Landau-Verwey-Overbeek

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approach (XDLVO), respectively.

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EXPERIMENTAL PROCEDURES

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Material and Methods

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Phages and phage assay: Six lytic marine phages were selected (Table 1) and obtained together

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with their host strains from different sources: Phages PSA-HP1, PSA-HS2 and PSA-HM1 were

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kindly provided by Dr. B. M. Duhaime (University of Michigan, USA); phage H3/49 by Dr. E.


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Roine (University of Helsinki, Finland); phage VB_PSPS-H40/1 and VB_PSPS-H6/1 by Dr. J. Zopfi

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(University of Basel, Switzerland). In addition to marine phages two well-characterized, non-marine

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phages (phage T4 and MS2) were used as controls.41,42 Phages T4 and MS2 and their host E. coli

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were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ,

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Germany). Except for Schewanella baltica that grew in 100% nutrient broth at 10°C, all marine

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bacterial strains were grown at room temperature using dilute (50%) 2216E medium.43Phage titration

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was performed using a modified spotting plaque assay technique. Deviating from Adams44 the top

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layer of the counting agar plates contained solely a bacterial suspension, to which phage lysates (5

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µL) were spotted in triplicate. No significant differences to results obtained with the technique by

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Adams were found in the range of 7 – 22 (±3) plaque forming units (PFU) 5 µL-1. All plates

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containing the phage host pairs were incubated overnight at room temperature (except 10°C for

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H3/49 and 37°C for phages MS2 and T4).

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Phage propagation and purification: Phages were propagated on their hosts using the double agar-

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layer technique and high phage concentrations to reach completely lysis plates. Phage particles were

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gathered after confluent lysis of bacteria by adding 5 mL of SM buffer (100 mM NaCl, 8 mM

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MgSO4 x 7H2O, 50 mM Tris-HCl; pH = 7) and a few drops of chloroform with mild shaking for 2 h

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at room temperature. The supernatant was recovered and cell debris was removed by centrifugation

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at 10,000g for 15 min. The supernatant was mixed with an equal volume of chloroform and

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centrifuged at 5,000g for 5 min to further purify the phage particles. The phage particle-containing

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supernatant was filtrated through 0.22 µm polyvinylidene fluoride (PVDF) CHROMAFIL®

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membrane filters45 and the phage suspension was stored at 4°C.

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Genome sequencing of phages: Genomes of all marine phages were sequenced in order to either

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confirm characteristics of phages described recently i.e. PSA-HM1, PSA-HP1, PSA-HS2 and H3/49

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(in particular to exclude nucleotide changes in the genomes that may occur during phage

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propagation) or to characterize phages whose genome was still unknown i.e. vB_PSPS-H40/1 and


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vB_PSPS-H6/1. DNA was extracted using the protocol of Thurber et al.46 and shotgun genome

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sequencing was performed on an Illumina MiSeq system using standard Illumina protocols for

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generation of paired 150-bp reads. Resulting sequencing reads were de novo assembled using

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Geneious assembler (Geneious R9, Biomatters, Auckland, New zealand) and alignments were

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performed with ClustalW.47 Genome sequences of PSA-HM1, PSA-HP1, PSA-HS2 and H3/49 were

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100% identical with the corresponding sequences available at GenBank (KF302034, KF302037,

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KF302036 and KJ018214). Sequences of vB_PSPS-H40/1 and vB_PSPS-H6/1 (GenBank acc. Nos.

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KU747973 and KX257490) confirmed these two phages belong to the Siphoviridae family.

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Characterization of surface properties. The zeta-potentials (ζ) were approximated from the

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electrophoretic mobility measured by Doppler electrophoretic light scattering analysis (Zetamaster,

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Malvern Instruments, Malvern, UK) of phage suspension in 100 mM phosphate buffer (PB; 0.87 g L-

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1

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contact angles of water θw, formamide θf and methylene iodide θmi were measured using a DSA 100

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drop-shape analysis system (Krüss GmbH, Hamburg, Germany) as described earlier48. Phage lawns

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were prepared by deposition of phage lysate of high concentration (i.e. 109 - 1010 PFU mL-1) onto

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Whatmann inorganic AnoDisc filter (0.02 µm, diameter: 25 mm). The filters were then dried for 2 h

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and contact angles were measured by applying 3–5 droplets on a filter using at least two filters per

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liquid tested (i.e. n ≥ 6 measurements).

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Column deposition experiments: All experiments were performed in triplicates at 25°C in heat-

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sterilized vertical percolation columns (i.d.: 1 cm; L.: 10 cm). These were made of borosilicate glass

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and confined at the bottom by a glass frit (pore size: 100 – 160 µm). The columns were wet packed

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in 100 mM PB with clean, sterile commercial quartz sand (Euroquarz-group) with a d50 median

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particle size of 0.31 mm, a porosity of ≈ 0.4 (estimated gravimetrically), and a total pore volume

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(PV) of 2.86 mL. Prior to the filling, the sand was washed with deionized water, heated at 400°C for

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3 h and then allowed to cool down under sterile conditions. Unfilled columns showed no retention of

K2HPO4, 0.68 g L-1 KH2PO4; pH = 7). In order to calculate the surface free energies of the phages,


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any of the phages. Sand-filled columns were allowed to equilibrate by flushing at least 8 PV of clean

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PB. Then homogenized phage suspensions (106-107 PFU mL-1 in 100 mM PB) were circulated with a

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peristaltic pump at a hydraulic flow rate of 0.7 × 10-4 m s-1 (19.8 mL h-1) from the top to the bottom

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of the columns, i.e. at flow conditions lower than in coarse gravel aquifers (23 - 44 m day-1).32 To

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characterize the flow conditions prevailing in the columns and as reference to evaluate the arrival

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time of studied phages. Bromide tracer tests were conducted in triplicate using 0.05 g L-1 KBr.

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Bromide concentrations in the outflow of the columns were quantified by ion chromatography

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(Dionex ics-2000; Dionex Corporation, USA). Phage transport and deposition under saturated

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conditions were determined by comparing the PFU concentrations of the influent and effluent as

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described earlier:27,49 about 8 PV of phage suspensions in PB were applied to the columns, the

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columns then rinsed with ≈ 4 PV with phage-free PB to assess the reversibility of phage adhesion

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and, finally, with ≈ 4 PV of pure water (Milli-Q) to study the effect of electrostatic repulsion on

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phage adhesion. The colloidal stability of the phage suspensions over 3 h (i.e. the duration of column

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transport experiments) was quantified in independent batch experiments using identical buffer

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composition and temperature. PFU in samples taken over time was followed assuming that any

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aggregate forms only a single plaque. Colloidal stability was calculated as PFU relative to the initial

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number. Phage lysate was prepared in concentrations of 108 - 1010 PFU mL-1 using PB buffer and

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stored in glass vials at room temperature under static condition (i.e. no agitation). Samples were

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taken at the beginning of the experiment and after 3 h to evaluate the colloidal stability and viability

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of the phages.

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Theory

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Calculation of phage collision efficiency ( ), deposition rate coefficient (Kd) and mass recovery

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(M). Filtration of phages was approximated using the colloidal filtration theory50 in columns


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assuming spherical phages with properties as given in Table 1. For a detailed explanation of the

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calculations, the reader is referred to Tufenkji & Elimelech 2004,51 the filtration equation of

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Rajagopalan and Tien52 and the SI, respectively. Therein, the was defined as the ratio of the

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experimental single-collector removal efficiency ( ; f. eq. S1) to the predicted single-collector

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contact efficiency ( ; eq. S2)51. The is a function of four dimensionless groups: the

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aspect ratio NR, the Peclet number NPe, the van der Waals number NvdW, and the gravitational number

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Ngr. 53,54 The deposition rate coefficient (Kd, h-1 ) relates to the attachment efficiency ( )55,51 by eq. 1

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= ()

(1)

! ɳ#

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where vP is the pore water velocity (cm min-1), L the column length in cm, ac the mean radius of the

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sand (0.29 mm), $ the porosity of the column packing, and ɳ0 the single collector efficiency

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determined as given by Tufenkji and Elimelech, 2004.51 The Kd is calculated by eq. 2

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%& =

!

'

+

ln * +# -

(2)

,

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where C0 is the initial concentration of the phages (PFU mL-1) and Ct is the effluent concentration

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calculated as the average of > 10 steady state data points of the phages’ breakthrough curves (PFU,

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mL-1) as represented in present study.55 The mass recovery (M) was calculated as the ratio of phages

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in the effluent relative to that of the influent as inferred from the difference of inlet and outlet phage

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concentration as described by eq. 3

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∑ + ∆

. = ∑ + , ∆ ∗ 100

(3)

#

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Prediction of the effect of XDLVO phage-sand interaction energy. Phages were assumed to

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follow the principles of colloid chemistry.50 As Lewis acid-base interactions are known to play an

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important role in the total interaction energy between phages and surfaces,56 the extended XDLVO

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interaction energies between phages and sand particles. Therein the total interaction energy 345678


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is the sum of the electrostatic repulsion (3956 ), the Lifshitz-van der Waals (36: ) and the acid-base

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(3;< ) interaction energy. While 3956 and 36: are functions of the separation distance h (nm)

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between two surfaces57,58, 3;< compares the energy status between attached and nonattached

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situations (eq. 4):

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345678 (ℎ) = 3;< +3956 (ℎ) + 36: (ℎ)

(4)

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As the phages studied are far smaller than sand particles we applied a sphere-plate geometry.56 3?@'

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3'A and 3BC were calculated as described by Chrysikopoulos and Syngouna, 2012.56 For 3BC and

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3'A calculations and in contrast to Chrysikopoulos and Syngouna, 2012 we used experimentally

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determined Gibbs free energies and Hamaker constants of all phages, respectively. The Gibbs free

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energies (Table S1) and Hamaker constants were calculated using the surface free energies of studied

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phages applying eqs. S8 and S4, whereas surface free energy calculations were based on measured

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contact angles (θ) of phages using water, formamide and methylene iodide as liquids and the Young

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equation.59

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RESULTS

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Physico-chemical surface properties, size and morphology of phages

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Surface charge and hydrophobicity of the six marine phages and the two coli-phages were

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determined by their zeta-potentials (ζ) and water contact angles, respectively (Fig. S1A). A broad

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contact angle range from 38o to 95o was observed (Table 1). The phages could be clustered into three

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groups: (i) poorly hydrophobic phages (≈ 40o: PSA-HM1, PSA-HP1 and PSA-HS2), (ii) moderately

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hydrophobic phages (≈ 53-61o: vB_PSPS-H40/1, vB_PspS-H6/1 and H3/49), and (iii) hydrophobic

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phages (≈ 84-95o: T4 and MS2). In the transport buffer (pH = 7) all phages had slightly negative

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surface charges as inferred from similar zeta potentials (ζ = -11 – -18 mV). Phage morphology and


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size varied according to previous own work60,61 and literature data (cf. Table 1). Belonging to

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different families, the phages represented four different morphologies: contractile tail (Myoviridae),

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noncontractile long tail (Siphoviridae), noncontractile short tail (Podoviridae), and icosahedral

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without tail (Leviviridae). Phage sizes ranged from 25 to 200 nm (Table 1).

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Phage transport in saturated percolated columns

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In order to determine transport and deposition characteristics of the phages, experiments were

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conducted using sand-packed columns under continuous, saturated flow conditions. Each experiment

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was divided in three phases: after flushing of 7 PV of dilute phage suspensions in PB, 4 PV of phage-

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free PB followed by a switch in the column inflow to deionized water were applied. The last two

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phases served to assess the reversibility of phage adhesion and to study if electrostatic repulsion

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would lead to phage detachment, respectively. Plots of relative phage densities in the outflow (C/C0)

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against time (normalized to the number of PV exchanged) showed about 0.5 of the C0 at 1 PV and

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were similar to the behavior of KBr tracer (Fig. S2). One exception was for the phage H3/49 which

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exhibited rapid attachment to sand and reached quasi-equilibrium (Fig. S2). After about 2 PV a

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leveling off of the breakthrough curves at plateaus of different heights were observed (Fig. S2).

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Calculated maximal coverages of the sand surface of < 0.2% were calculated and excluding

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substantial changes of the collector surface properties. Subsequent flushing with phage-free PB

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resulted in poor tailing pointing at irreversible attachment of the phages. Changing the inflow from

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PB to deionized water led 0 - 6% of the attached phages to flush out (Table 1); i.e. only partially

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reverted the deposition of the phages despite of a clearly increased electrostatic repulsion between

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the sand and the phage surfaces.

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efficiencies (eq. 1) and deposition rate coefficients (eq. 2) of all phages. It further shows the

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following trend of the different phages’ mass recoveries: MS2 > PSA-HP1 > PSA-HS2 > PSA-HM1

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> vB_PspS-H6/1 > vB_PspS-H40/1 > T4 > H3/49. Conspicuously, the smallest phages MS2 and

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PSA-HP1 exhibited the highest mass recovery regardless of their hydrophilicity or hydrophobicity.

62,63

Table 1 summarizes the mass recoveries (eq. 3), collision


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Deposition rate coefficients and calculated collision efficiencies ranged from 0.21 to 0.02 h-1 and

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0.34 to 0.01, respectively. Phages with fast deposition rates and high collision affinities (i.e.

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vB_PspS-H40/1, T4 and H3/49) thereby exhibited low mass recoveries (Table 1). Hydrophobic and

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moderately hydrophobic phages exhibited lower collision efficiencies (Fig. S2B) and deposition rate

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coefficients (Fig. S2Dand thus a lower mass recovery (Fig. 1) than poorly hydrophobic phages. One

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exception such latter trend was phage MS2 where the small size of MS2 seems to determine its

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transport behavior. The mass recoveries of the poorly hydrophobic phages varied by the order of:

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PSA-HP1 (Podoviridae) > PSA-HS2 (Siphoviridae) > PSA-HM1 (Myoviridae) (Fig. 1). Data from

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control experiments revealed high colloidal stability and no loss of phage viability in the transport

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buffer used (Table 1).

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Calculation of the phage-sand interaction energies

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Phage-sand interaction energy profiles were calculated using the extended DLVO (XDLVO) theory

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(Fig. S3, Table 2) based on the sphere-plate model.56 For the experimental conditions applied, the

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interaction energy profiles indicated repulsive interactions for MS2, PSA-HP1, PSA-HS2 and PSA-

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HM1 (Φmax1 = 0.003–0.37 kbT) and attractive interactions for vB_PspS-H6/1, vB_PspS-H40/1, H3/49

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and T4 phages (Φmax1 = -0.046 – -0.011 kbT) (Fig. S3, Table 2). The two phages with the highest

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mass recovery (PSA-HP1, MS) exhibited the highest Φmax1 (53). Although the XDLVO calculations

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indicated the existence of secondary energy minima (Φmin2) at h ≈ 12 nm, their depths for all phages

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were shallow ( hydrophobicity ≥ morphology. Likely due to size exclusion effects69,70 smaller

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phages (≤ 77 nm) were best transported with their transport efficiency being independent of the

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surface hydrophobicity, as seen from similar mass recovery of the poorly hydrophobic PSA-HP1 (M

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= 75%) and the hydrophobic MS2 phage (M = 82%). Good transport arising from low collision

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efficiency of small phages is also in line with comparisons of differently sized phi X 174, MS2, and

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T4 phages showing better retention of large phages in rapid sand filtration42 as well as with a report

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of efficient transport of the MS2 phage.71,42,72 It also agrees with a previous report demonstrating that

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phages of < ≈ 60 nm size are less retained than larger phages37 and that rod shaped particles (such as

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long tailed phages) have higher retention and lower reversibility than spherical particles due to

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orientation effects during transport in saturated porous media.73 Such findings confirm our results

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that the morphology of phages of similar hydrophobicity (PSA-HP1, PSA-HS2 and PSA-HM1 or


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vB_PspS-H6/1 and H3/49 (Fig. 1)) is highly influential. Comparison of PSA-HP1, PSA-HS2 and

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PSA-HM1 shows that the short-tail phage PSA-HP1 is less retained than the noncontractile long tail

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phage PSA-HS2, or the contractile tail phage PSA-HM1. Due to probable interferences of size and

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morphology we observed the following trend of phage mass recovery: Leviviridae (icosahedral, no

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tail) > Podoviridae (noncontractile short tail) > Siphoviridae (noncontractile long tail) ≥ Myoviridae

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(contractile tail). This suggests that morphology controls the transport of phages as also has been

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described for the human adenovirus (HAdV).74 Small-sized MS2 phages excluded, we found a

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negative correlation of water contact angle and mass recovery (Fig. 1) underpinning the effect of

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hydrophobic interactions on the deposition and removal of phages36,75,76,39; in particular at water

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contact angles of > 65o.56

302 303

Phage-sand interaction energies and phage deposition

304

Using the XDLVO approach, we calculated the distance-dependent interaction energies between the

305

phages and the sand surface (Table 2 and Fig. S3). This model estimates the interaction energy as a

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function of surface to surface distance h for a phage approaching a sand grain (eq. 4). It is

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characterized by three distinct interaction energies: the primary minimum (Φmin1) as the deep energy

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at small h from the sorbent surface, the secondary minimum (Φmin2) as the shallow energy at

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relatively large distance, and the maximum energy barrier to attachment and detachment (Φmax1).50

310

Although phages do not have smooth surfaces and are composed of various protein macromolecules

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and hence are far from being ideal colloidal particles, we found good agreement between calculated

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interaction energy profiles and our experimental results which were also shown in other studies

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under similar conditions.56 The XDLVO calculations proposed the existence of secondary energy

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minima (Φmin2) at h ≈ 12 nm; their depths, however, were shallow (

Marine Phages As Tracers: Effects of Size, Morphology, and Physico

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