Bacterial Adhesion to Ultrafiltration Membranes: Role of Hydrophilicity

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Bacterial Adhesion to Ultrafiltration Membranes: Role of Hydrophilicity, Natural Organic Matter, and Cell-Surface Macromolecules Sara BinAhmed, Anissa Hasane, Zhaoxing Wang, Aslan Mansurov, and Santiago Romero-Vargas Castrillón Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03682 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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Environmental Science & Technology

Bacterial Adhesion to Ultrafiltration Membranes: Role of Hydrophilicity, Natural Organic Matter, and Cell-Surface Macromolecules Sara BinAhmed1, Anissa Hasane2, Zhaoxing Wang1, Aslan Mansurov1, Santiago Romero-Vargas Castrillón* 1

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Abstract

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Insight into the mechanisms underlying bacterial adhesion is critical to the formulation of

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membrane biofouling control strategies. Using AFM-based single-cell force spectroscopy, we

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investigated the interaction between Pseudomonas fluorescens, a biofilm-forming bacterium,

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and polysulfone (PSF) ultrafiltration (UF) membranes to unravel the mechanisms underlying

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early-stage membrane biofouling. We show that hydrophilic polydopamine (PDA) coatings

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decrease bacterial adhesion forces at short bacterium-membrane contact times. Further, we find

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that adhesion forces are weakened by the presence of natural organic matter (NOM)

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conditioning films, owing to the hydrophilicity of NOM. Investigation of the effect of adhesion

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contact time revealed that PDA coatings are less effective at preventing bioadhesion when the

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contact time is prolonged to 2 – 5 s, or when the membranes are exposed to bacterial

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suspensions under stirring. These results therefore challenge the notion that simple hydrophilic

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surface coatings are effective as a biofouling control strategy. Finally, we present evidence that

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adhesion to the UF membrane surface is mediated by cell-surface macromolecules (likely to

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be outer membrane proteins and pili) which, upon contacting the membrane, undergo surface-

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induced unfolding.

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Keywords. adhesion, biofouling, polydopamine, NOM, Pseudomonas fluorescens, single-cell

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force spectroscopy, ultrafiltration.

Department of Civil, Environmental, and Geo- Engineering, University of Minnesota, 500 Pillsbury Dr SE, Minneapolis, MN 55455, United States 2 Department of Civil and Environmental Engineering, Norwegian University of Science and Technology, 7491 Trondheim, Norway

1 ACS Paragon Plus Environment


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1. Introduction

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The presence of bacteria near interfaces is ubiquitous in natural and engineered aqueous

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environments. Near most surfaces, adhered bacteria will aggregate in a hydrated matrix of

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proteins and polysaccharides, known as biofilms1. These sessile communities are notoriously

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resistant to removal by biocides and disinfectants due to the protection provided by a matrix of

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predominantly self-produced extracellular polymeric substances (EPS)2–5. Consequently,

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biofilm formation and biofouling constitute a major technical problem in membrane-based

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water treatment, leading to decreased permeance, selectivity, and membrane useful life6–11. The

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first step in biofilm formation and biofouling, termed bioadhesion, is the complex process of

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bacterial attachment to a substrate12–14. A variety of nonspecific interactions drive

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bioadhesion12,15,16 including long-range electrostatic forces14,17,18, and shorter ranged

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hydrophobic14,van der Waals14,17, and hydrogen bond interactions17. These interactions come

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into play through a host of bacterial cell membrane macromolecules (e.g., lipopolysaccharides,

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proteins19), as well as cell-surface adhesins (e.g., pili and flagella) that often determine bacterial

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adhesion to the substrate20–23. Cell appendages such as flagella and pili play a major role in

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overcoming repulsive double layer surface forces, initiating bacterial attachment4,13,14, often at

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several m from the substrate15.

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Given that bioadhesion interactions are reversible24, insight into the mechanisms underlying

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early-stage bacterial attachment is essential to the formulation of biofouling-resistant

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membranes and biofouling prevention strategies. Nonetheless, given the myriad physical,

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chemical, and biological factors intervening, bioadhesion remains a poorly understood

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phenomenon, particularly for chemically heterogeneous interfaces such as membranes for

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water separations, which exist in contact with complex solution matrices25. Earlier studies on

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bioadhesion onto model polymeric (e.g., Teflon) and inorganic surfaces (glass, mica) have


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shown that attachment is enhanced by substrate hydrophobicity15,26. Several other interfacial

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properties influence bacterial adhesion, including nanoscale roughness and surface charge (zeta

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potential), as well as feed water composition (pH, ionic strength, presence of divalent cations,

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organic matter properties), all of which are known to contribute to membrane fouling5,6,11–

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14,27,28

.

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In addition, transport and deposition of bacterial polymers, natural organic matter (NOM), and

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other macromolecules at the substrate-water interface is thought to lead to the formation of a

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conditioning film mainly composed of organic matter29 that may hinder29–31, or promote30,32,33

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bacterial adhesion by providing support and nutrients for the foundation layer of a

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biofilm13,14,20,34. The role of NOM in bacterial and biocolloid adhesion is poorly understood,

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with previous studies reporting contradictory results31,35–39. Suwannee River (SR) NOM

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conditioning films decrease the attachment efficiency of MS2 virus particles to silica surfaces,

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possibly due to electrostatic repulsive forces that dominate at low ionic strength31. Conversely,

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at low ionic strengths, SRNOM was shown to accelerate the deposition kinetics of C. parvum

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oocysts on conditioned silica surfaces, compared to bare silica collectors35. Other studies

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focused on the effect of humic acid (HA), the main hydrophobic36 constituent of NOM37 in

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surface water38. HA had a minor influence on the attachment efficiency of E. coli cells to silica

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particles36, while Suwannee River humic acid (SRHA) resulted in lower deposition of E. coli

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on quartz sand when present in suspension or as a coating film37. On the other hand, the effect

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of HA conditioning on Pseudomonas aeruginosa adhesion was found to be dependent on

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lipopolysaccarride (LPS) structure. Adhesion of P. aeruginosa AK 1401, having an A-band

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antigen in its LPS, exhibited similar adhesion forces to HA-coated silica particles as the bare

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silica surfaces. Conversely, P. aeruginosa PAO1 possesses an additional long antigen (B-band)

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that increased its adhesion to HA-coated silica39.



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Advanced surface analytical techniques, notably AFM-based single cell force spectroscopy

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(SCFS), have emerged in recent years to investigate the mechanisms underlying bacterial

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adhesion. In SCFS, an AFM cantilever is functionalized with a single bacterial cell, enabling

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measurement of the nanoscale forces between individual cells and a substrate in native aqueous

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environments16,40. In addition, SCFS can provide information on the mechanical response of

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cell adhesins as they contact a substrate41,42.

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In this study, we use SCFS to unravel the interactions between Pseudomonas fluorescens, a

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biofilm-forming bacterium21,43, and polymeric ultrafiltration (UF) membranes. P. fluorescens

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is an aerobic gram negative bacterium found in soil, drinking and river water, plants, in addition

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to biomedical and industrial equipment25,44–46. We show that hydrophilic polydopamine (PDA)

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surface coatings significantly weaken the adhesion forces of P. fluorescens. While this

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observation underscores the importance of hydrophilicity in mitigating microbial adhesion, we

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also show that the antifouling properties of hydrophilic coatings are less effective for prolonged

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contact between the cell and the membrane surface. Further, we show that NOM conditioning

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films weaken cell-substrate adhesion forces. Finally, a significant fraction of the force

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measurements suggests that adhesion of P. fluorescens is mediated by cell surface appendages

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whose elastic response can be modeled via the worm-like chain (WLC) model. Our WLC

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analysis of adhesion forces suggests that unfolding of adhesins (pili and outer membrane

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proteins) is a determinant of cell attachment to membrane surfaces.

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2. Materials and Methods

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2.1. Membrane Fabrication and Characterization

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We investigate bioadhesion on polysulfone UF membranes fabricated by non-solvent induced

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phase inversion47. Hydrophilic polydopamine48 (PDA) surface coatings, which have been


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shown to be effective in the control of membrane organic fouling49, are explored in this work

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as a surface modification strategy to mitigate bioadhesion27. Polydopamine is formed by the

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deposition of 3,4-dihydroxyphenylalanine (DOPA) from alkaline solution48. Hereinafter, we

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refer to PDA-coated polysulfone UF membranes as PSF-PDA, while the unmodified

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polysulfone control membranes are designated PSF membranes. In addition, the effect of NOM

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conditioning films is investigated by depositing Upper Mississippi River NOM (International

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Humic Substances Society, St. Paul, MN) on the membrane surfaces via pressure-driven

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filtration of 10 ppm NOM solution.

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The interfacial properties (wettability, surface charge and nanoscale roughness) and water

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permeability coefficient of the PSF and PSF-PDA membranes were characterized. Detailed

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experimental procedures and characterization data are presented in the Supporting Information

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(SI).

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2.2. Single-Cell Force Spectroscopy (SCFS) Experiments

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2.2.1. Bacterial Strain and Growth Conditions

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The strain of P. fluorescens used in our experiments, ATCC 13525, originates from pre-filter

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tanks50. As with other bacteria in the Pseudomonas genus, P. fluorescens has a high

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adaptation51 and biofilm formation potential43,52, possessing a variety of proteinaceous

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adhesins53, such as flagella and pili, which are also fundamental to bacterial motility44,54. The

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small radius of pili (1-5 nm55) is believed to help P. fluorescens overcome electrostatic

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repulsive forces with the substrate13,55. Pili also promote P. fluorescens bioadhesion via

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hydrophobic interactions with hydrophobic substrates55, due to the high content of hydrophobic

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amino acids in the constituent pilin proteins56. LPS23,57,58 and outer membrane proteins

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(OMP)14,39, such as LapA20,21, also contribute to adhesion of P. fluorescens. Experimental

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details on bacterial culture preparation and growth conditions are provided in the SI.



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2.2.2. Membrane sample and cantilever preparation

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A UF membrane coupon (~3×3 mm2) was glued using epoxy (3M Quick Set Epoxy Adhesive)

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to a piranha-cleaned and UV/O3-treated glass disc, and allowed to set for 10 minutes.

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Subsequently, a 20-µL droplet of bacterial suspension was deposited directly on the glass,

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allowing the droplet to stand for 30 minutes to permit bacterial attachment to the glass surface.

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The glass disk was rinsed with 2.5 mL PBS to remove unattached bacteria, avoiding contact

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between the membrane surface and the bacterial suspension. Finally, the glass disk was inserted

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in the fluid cell (Fluid Cell Lite, Asylum Research), and the cell was filled with 1.4 mL of test

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solution. We conducted experiments in PBS or in 10 ppm NOM solution containing 156.4 mM

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KCl and 0.5 mM CaCl2 (I = 158 mM, pH 7.2-7.4), i.e., conditions approximating those of PBS,

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to perform experiments under comparable conditions.

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Tipless SiN cantilevers (nominal k = 0.01 N/m, MLCT-O10 probe “C”, Bruker) were used.

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Wet polydopamine (wPDA) was used as a wet adhesive to immobilize a single bacterial cell

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on the cantilever, as done by others15,19,59,60. The adhesive properties of wPDA, which contrast

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the antifouling properties of PDA explored in this work (and previously by others49,61,62), are

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thought to depend on the degree of pH-induced oxidation of DOPA catechol groups to

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quinones63–65, and the extent of crosslinking of DOPA monomers48,66. In our work, we observed

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that longer deposition times on the membranes (~45 min) resulted in hydrophilic anti-fouling

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coatings, whereas shorter (15 minutes) deposition times rendered adhesive cantilevers on

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which bacterial cells could be robustly immobilized. We further observed that wPDA coatings

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completely lost their adhesiveness after ~3 hours, possibly due to conversion to non-adhesive

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(oxidized) PDA. Determination of the structures of adhesive wPDA and non-adhesive PDA,

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as well as their formation mechanisms, are active areas of research67–69 that fall outside of the

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scope of this study.


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To ensure adhesiveness of the wPDA, the AFM probes were coated immediately before each

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experiment as follows: the UV/O3-cleaned AFM probe was immersed in 10 mL of 4 g/L PDA

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solution (pH 8.5) prepared as described in the SI. After 15 minutes under shaking at 60 RPM,

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the probe was rinsed with deionized (DI) water (18.2 M-cm, Barnstead) and dried under a

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gentle nitrogen flow before mounting the probe on the AFM probe holder. The head was then

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lowered into the fluid cell, immersing the probe in the solution (PBS or NOM) and allowing

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the cantilever deflection to reach a stable deflection signal (which was typically observed

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within 30-60 min).

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2.2.3. Force Measurements

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Single-cell force spectroscopy (SCFS) was performed at room temperature using an MFP-3D-

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Bio atomic force microscope (Asylum Research, Santa Barbara, CA) integrated into a Zeiss

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Axio Observer A.1 inverted optical microscope (IOM). The cantilever spring constant was

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obtained using the thermal noise method70, and was always found to be within range specified

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by the manufacturer. The optical lever sensitivity of the cantilever was determined from the

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slope of the contact region of deflection signal (V) vs. piezo extension (nm) curves collected

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over the bare glass. To immobilize a single bacterial cell on the cantilever, we adapted

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previously reported experimental procedures15,19. Using the 60× objective of the IOM, the

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wPDA-coated cantilever was directed towards a cell aligned parallel to the front edge of the

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cantilever. The cantilever was engaged in contact mode over the cell for 5 minutes with a 1 nN

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loading force15, after which the cantilever (functionalized with the bacterial cell) was

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withdrawn.

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To begin force measurements, the cantilever was translated towards the membrane surface

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using the AFM top view digital camera. Approach-retraction force cycles were recorded at a

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400 nm/s cantilever speed and 4 µm ramp (8-µm ramps were used when long range interactions

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were observed), with an approach (target loading) force of 600 pN. A dwell or contact time (0

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≤ tContact ≤ 5 s), defined as the time in which the bacterial cell can remain in contact with the

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membrane surface under a constant loading force, was investigated in a subset of the

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experiments. Experiments in NOM solution and/or conditioning film were carried out at tContact

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= 5 s only. Force curves were collected at multiple randomly chosen locations of the

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membranes, measuring 3 force curves over each point, to minimize the deposition of cell-

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surface macromolecules on the UF membrane (no systematic changes in adhesion force were

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observed in sequential measurements on the same membrane location, or between locations).

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A total of  100 force curves (minimum: 93, maximum: 129) were collected for each

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combination of membrane type and experimental conditions, using at least 3 different bacterial

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cells (i.e., at least 3 separate single-cell experiments). Both approach and pull-off force curves

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were measured, but only pull-off force curves were considered to quantify adhesion. Raw data

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in terms of cantilever deflection as a function of piezo Z position were first converted to force

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versus cell-substrate separation curves using the method described by Senden71. The maximum

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adhesion force and rupture separation (i.e., the separation at which cell-substrate forces vanish)

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were extracted from each pull-off force curve using a Matlab code. A representative

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approach/pull-off force cycle, showing the maximum adhesion force and rupture separation, is

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shown in Figure S1.

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Cell viability was assessed after each experiment using a live/dead assay (Baclight,

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Thermofisher Scientific) consisting of SYTO 9 and propidium iodide (PI) fluorescent nucleic

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acid stains. If the cell was dead (red fluorescence) or dislocated (i.e., if the cell position on the


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cantilever changed during the experiment), the collected data were discarded. Only data

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collected with a live cell (green fluorescence) that remained at its initial location throughout

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the experiment are reported herein.

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3. Results and Discussion

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3.1. Membrane Characterization

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3.1.1. Contact Angle

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Contact angle provides information about the hydrophilicity of the membranes, a well-known

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attribute of fouling-resistant surfaces3,33,72,73. Contact angle measurements of an ultrapure water

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droplet on various membrane surfaces are shown in Figure 1. The contact angle of PSF

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membranes was θ = 62.7° ± 7.4°. A more hydrophilic surface results from PDA deposition,

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which exhibits a contact angle θ = 51.5° ± 5.7°; the observed increase in hydrophilic character

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of the PSF-PDA membrane is consistent with previous results of PSF-PDA-modified

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membranes73 and other organic and inorganic63,66,74–76 substrates. Figure 1 also shows that both

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the PSF and PSF-PDA membranes are rendered more hydrophilic after deposition of an NOM

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conditioning film, when compared to their NOM-free counterparts. This can be attributed to

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the hydrophilic components of NOM, which have been shown to render membrane surfaces

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more hydrophilic after fouling experiments77. The hydrophilic components of NOM are

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believed to be non-humic substances77 such as polysaccharides78,79 and proteins79. The

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presence of polysaccharides on NOM-conditioned PSF and PSF-PDA membranes is supported

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by FTIR data (Figures S2 and S3), showing an increase in the ~1078 cm-1 and 1105 cm-1 bands,

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corresponding to polysaccharides and polysaccharide-like molecules78,80.

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Contact Angle (deg.)

80 70

62.7 51.8 51.5

60 50

35.5

40 30 20 10 0

PSF

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PSF -PDA

PSF -NOM

PSF-PDA -NOM

Figure 1: Sessile water drop contact angle measurements on PSF and PSF-PDA membranes, both pristine and coated with an NOM film (as described in the Supporting Text). For each membrane type, the reported contact angle is an average of at least 18 measurements, collected over 2 separately cast membrane samples of the same type; error bars indicate one standard deviation (p < 0.05 for all pairwise comparisons, except for PSF-PDA compared to PSF-NOM). 3.1.2. Zeta Potential

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The charging behavior of a solid-water interface, as revealed by streaming potential

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measurements, provides information about the functional groups present on the membrane

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surface, as well as surface charges that influence cell deposition. The zeta potential () of PSF,

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PSF-PDA, and NOM-conditioned membranes is shown in Figure S4 as a function of pH. PSF

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membranes exhibited a negative  over the entire pH range (4 to 10), due to OH- adsorption on

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the PSF membrane surface49. In contrast, PSF-PDA membranes attained a positive zeta

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potential at pH < 4.5, due to protonation of –NH– to –NH2+– in PDA. At basic pH,

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deprotonation of hydroxyl groups in PDA results in negative zeta potential49. Figure S4 further

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shows that the NOM conditioning film results in negative  over the entire pH range

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investigated for both PSF and PSF-PDA membranes. This is consistent with the presence of

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polysaccharides and polysaccharide-like substances that increase the number of negatively

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charged functional groups on the membrane surface78. The deposition of negatively charged

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humic acid (HA), alginate (a polysaccharide), and a combination of HA and alginate has been

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shown to decrease (i.e., render more negative) the zeta potential of polyethersulfone (PES) UF

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membranes79. Comparing the four membrane types, Figure S4 shows that at low pH (pH < 5)


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the zeta potential of NOM-conditioned membranes is lower (more negative) than that of

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pristine PSF and PSF-PDA membranes. On the other hand, all membranes exhibit similar

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surface charge at pH 7.2-7.4, the pH of our SCFS experiments (Section 3.2), irrespective of

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NOM-conditioning or PDA surface modification.

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We also measured the zeta potential of P. fluorescens cells (Figure S5, details in the SI).

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Bacterial cells attain a negative surface charge at pH values corresponding to their normal

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living environment2,81. As shown in Figure S5, bacteria exhibit a negative zeta potential at pH

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7.2-7.4, becoming less negative with increasing ionic strength82–84 due to electrical double layer

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compression82,83. Figure S5 also shows that the zeta potential of P. fluorescens was the same

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when suspended in PBS or NOM solution, suggesting that both solutions yield similar

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screening of bacterial surface charge at comparable ionic strength. This observation agrees with

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the work of Yang et al.37, who found that the zeta potential of motile and nonmotile E. coli was

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the same in the presence and absence of SRHA. Interestingly, the zeta potential of P.

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fluorescens was approximately the same as that of the cell-free NOM solution, possibly due to

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the presence of similar functional groups (e.g., carboxylates) in NOM and the surface of

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bacteria.

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In summary, Figures S4 and S5 show that both the bacterial and membrane surfaces possess

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negative zeta potentials under the conditions of the SCFS experiments reported below,

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irrespective of the presence of NOM or (for the membranes) PDA surface coating.

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3.1.3. Roughness and Permeability

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Figure S6 presents AFM images of PSF and PSF-PDA membranes. The root-mean-squared

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(RMS) roughness, defined as the standard deviation of height z-values measured over the



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scanned area85,86, was calculated from the AFM images. The RMS roughness of PSF and PSF-

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PDA membranes was 2.45 ± 0.32 nm and 3.74 ± 1.44 nm, respectively. The slight increase in

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roughness of PSF-PDA is presumed to be due to PDA nanoscale aggregates formed during

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deposition49. The permeability coefficients of the membranes presented in Figure S7, are within

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the range expected for UF membranes87,88. The slightly higher permeability of PSF-PDA

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membranes can be attributed to the increased hydrophilicity of the PDA layer covering both

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the membrane surface and the inner walls of the pores62.

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3.2. Single-Cell Force Spectroscopy

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In this section, we systematically investigate the effects of UF membrane hydrophilicity, cell-

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substrate contact time, and NOM conditioning on the adhesion of P. fluorescens to polysulfone

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UF membranes. Given the stochastic nature of microbial adhesion19,59, raw data (maximum

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adhesion force and rupture separation, cf. Figure S1 for definition) are reported as histograms

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(see SI).

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3.2.1. Effect of Membrane Hydrophilicity and Contact Time

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The adhesion of single P. fluorescens cells on polysulfone (PSF) and polydopamine-coated

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polysulfone (PSF-PDA) membrane surfaces is investigated at contact times (tContact) of 0, 2 and

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5 s in Figure 2 (A) and (B), which report the average maximum adhesion force and rupture

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separation, respectively. All experiments were performed in PBS buffer, pH 7.4, at room

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temperature. The histograms of the maximum adhesion force (Figure S8) and rupture

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separation (Figure S9), from which Figure 2 was obtained, show broad distributions, derived

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from complex interactions between the membrane and biopolymers present on the cell surface

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(see Section 3.2.3). By contrast, cell-free control measurements (Figures S10-S15) between a

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wPDA-coated cantilever and the membrane surface showed significantly higher average


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adhesion forces (~1 nN, compared to forces < 1 nN with bacterial probes, cf. Figure S12) and

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much shorter rupture distances (~100 nm, compared to rupture at > 1 m with bacterial probes,

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cf. Figure S15), demonstrating that the forces presented in Figures 2 (A) and S8 are due to

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bacterium-membrane interactions.

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Figures 2 (A) and S8 show that the distribution of adhesion force measurements became

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broader, i.e., the average adhesion force increased, as tContact increased from 0 to 5 s. Similarly,

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Figure S8 shows that the fraction of predominantly repulsive forces (corresponding to the

307

column labeled “NO”) decreased with increasing contact time over both PSF and PSF-PDA

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membranes. In these instances, attractive forces were too weak (on the order of 40 pN or less),

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and could not be discriminated from the random fluctuations in the deflection of the cantilever

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about zero force. Data on Figure 2 (A) thus show that adhesion of bacteria to membrane

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surfaces is a time-dependent phenomenon15, irrespective of the membrane chemistry, with

312

bacterial cells adhering more strongly to a surface when a sufficiently prolonged contact time

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is allowed. The higher mean adhesion force at longer tContact suggests that adhesins undergo

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structural changes that strengthen attachment on contacting the membrane surface. The range

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of tContact explored in Figure 2 (0 – 5 s) is shorter than the particle-substrate contact times

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encountered in membrane filtration, which would exceed the hydraulic residence time of the

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feed channel (> 10 s for typical module sizes and crossflow velocities of 10 cm s-1 89,90), but

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nonetheless suffices to observe surface-induced conformational changes in bacterial adhesins,

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which occur over times scales of ~100 – 700 ms 91. An important observation in Figure 2 (A)

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is that average adhesion forces over PDA-coated membranes were always weaker than those

321

measured on PSF membranes at the same tContact. This is due to the interfacial properties of the

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pristine and PDA-coated polysulfone membranes. The surface charge of both PSF and PSF-

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PDA membranes is similar ( -25 mV at pH 7.4, Figure S4), and the bacterial zeta potential is



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also weakly negative ( -5 mV, extrapolating data in Figure S5 to the ionic strength of PBS).

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Further, both membranes have similarly low (< 4 nm) RMS surface roughness (Figure S6).

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These results suggest that the weaker adhesion on PDA-coatings does not stem from surface

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electrostatic interactions or nanoscale roughness, but rather from the increased hydrophilicity

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of PSF-PDA membranes (= 51.5º ± 5.7º, compared to = 62.7º ± 7.4º for PSF, p < 0.05, cf.

329

Figure 1). Stronger bioadhesion on more hydrophobic substrates (PSF in this case) has been

330

reported by different studies using various bacterial strains13–15,26,59. Our results therefore

331

indicate that P. fluorescens uses adhesins (probably of proteinaceous nature, such as pili and

332

outer membrane proteins53) that depend on surface hydrophobicity for interaction.

333 334

Figure 2 (B) presents the mean rupture separation (defined as the distance at which the adhesion

335

force vanishes, cf. Figure S1), obtained from the distributions shown in Figure S9. Long rupture

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separations, reaching up to 9 µm, were observed in some measurements (Figure S9), which can

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be explained by the presence of long flagella and pili in P. fluorescens15,54,56,92,93. For both PSF

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and PSF-PDA, longer rupture separations were recorded as tContact was increased from 0 to 2 s,

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but remained unchanged (or increased slightly for PSF-PDA) on increasing tContact to 5 s. The

340

increase in average rupture separation as tContact was varied from 0 to 2 s indicates that at longer

341

tContact, longer adhesins (possibly pili) mediated attachment or more contact points along a

342

single pili were established. Beyond 2 seconds, the rupture separation did not vary significantly

343

but the adhesion became stronger (Figure 2 (A)), suggesting that more adhesins of similar

344

length were involved15. Additionally, we note that average rupture separation was always

345

smaller over PSF-PDA compared to PSF membranes for the same tContact (Figure 2 (B)). This

346

observation is analogous to the stronger adhesion observed on PSF compared to PSF-PDA

347

surfaces (Figure 2 (A)), suggesting that adhesins can engage more binding sites on the (more

348

hydrophobic) PSF membrane surface, resulting in longer rupture separation distances.


Page 15 of 35


|Mean Adhesion Force| (nN)

349

1.0

tContact = 0 s tContact = 2 s tContact = 5 s

A

0.8

0.6

0.4

0.2

0.0

PSF-PDA

PSF

Mean Rupture Separation (m)

350

4.0

B

tContact = 0 s tContact = 2 s tContact = 5 s

3.5 3.0 2.5 2.0

*

*

1.5

*

1.0

*

0.5 0.0

PSF

PSF-PDA

351 352 353 354 355 356

Figure 2: Average maximum adhesion force (A) and mean rupture separation (B) of single P. fluorescens cells on PSF and PSF-PDA membranes investigated for different contact times (tContact) denoted in the inset. The histograms from which the reported means were computed are given in Figures S8 and S9 (p < 0.05, except for pairwise comparisons indicated by *).

357

3.2.2. Effect of NOM on Microbial Adhesion

358

To investigate the effect of NOM on microbial adhesion, we performed two types of

359

experiments at tContact = 5 seconds. In the first set, we investigated the effect of dissolved NOM

360

on bioadhesion by conducting SCFS in a 10 mg/L Upper Mississippi River NOM solution at

361

ionic strength (158 mM) and pH (7.2-7.4) that approximate those of PBS. The selected

362

concentration of NOM is a typical upper limit of the NOM concentration in surface waters (2-

363

10 mg/L)94. In the second set of experiments, NOM was deposited on the surface of the



Page 16 of 35

membranes as a conditioning film (see SI). SCFS experiments with NOM-conditioned

365

membranes were also performed in 10 mg/L NOM solution at the ionic strength and pH

366

indicated above. The NOM conditioning film resulted in brown coloration of the membrane,

367

and its presence was verified by FTIR as explained in Section 3.1.1 (see also Figures S2 and

368

S3). Figure 3 summarizes the mean of the maximum adhesion force and mean rupture

369

separation computed from histograms presented in Figures S16 and S17 of the SI. Data for

370

experiments performed in PBS (tContact = 5 s) are also included in Figure 3 for comparison.

|Mean Adhesion Force| (nN)

364

PBS A NOM solution NOM solution & film

1.0

0.8

0.6

0.4

* *

* *

0.2

0.0

PSF-PDA

PSF

Mean Rupture Separation (m)

371

4.0

PBS B NOM solution NOM solution & film

3.5 3.0 2.5 2.0 1.5 1.0

*

0.5

*

0.0

PSF

PSF-PDA

372 373 374 375 376 377 378

Figure 3: Mean of the maximum adhesion force (A) and mean rupture separation (B) of single P. fluorescens cells on PSF and PSF-PDA membranes investigated in different solution chemistries, denoted in the inset: PBS, 10 ppm NOM solution, and NOM-conditioned membranes in 10 ppm NOM solution. The histograms from which the reported means were computed are given in Figures S16 and S17 (p < 0.05, except for pairwise comparisons indicated by *).


Page 17 of 35


379 380

Figure 3 (A) shows that the presence of NOM simultaneously in solution and as a conditioning

381

film results in weaker adhesion, compared to experiments in NOM solution (without surface

382

conditioning) and PBS. Also, weaker adhesion forces are observed on the PSF membrane when

383

NOM is present only in solution compared to experiments performed in PBS. On the other

384

hand, the experiment performed on PSF-PDA membranes in NOM solution resulted in stronger

385

adhesion forces compared to PBS (-0.39 nN vs. -0.30 nN, respectively). Experiments with 3

386

different bacterial cell probes were conducted with the PSF-PDA membrane tested in NOM

387

solution, yielding mean adhesion forces (-0.48 nN, -0.65 nN and -0.08 nN) that varied by one

388

order of magnitude. We speculate that the large variation in adhesion – which may be due to

389

variations in bacterial surface hydrophobicity across different cells95 – may explain the stronger

390

adhesion observed in NOM solution with the PSF-PDA membrane. The effect of NOM on the

391

mean rupture separation is described in Figure 3 (B), showing smaller rupture separations in

392

NOM solution, which become shorter still for adhesion on NOM-conditioned membranes.

393

Figure 3 thus shows that NOM, when present simultaneously as a dissolved foulant and as an

394

adsorbed film, weakens, and shortens the range of, adhesive interactions between the microbe

395

and the membrane. A further observation from Figure 3 is that, for the same NOM solution

396

chemistry, adhesive forces are weaker and shorter-ranged over PSF-PDA membranes

397

compared to PSF membranes; this observation is consistent with findings in PBS, as depicted

398

in Figure 2, and are attributable to the more hydrophilic PSF-PDA surface.

399 400

The weaker adhesion of P. fluorescens in the presence of simultaneously dissolved and

401

adsorbed NOM is at odds with the notion that macromolecular adsorbed films promote

402

bacterial attachment20,30,96. Nevertheless, we note that observations of lower deposition of

403

biocolloidal particles on NOM-coated surfaces are not unprecedented; lower deposition rates



Page 18 of 35

404

of oocysts on NOM-coated silica collectors have been reported35, while NOM was found to

405

decrease adhesion of Gram negative E. coli and Gram positive S. suis bacteria to soil colloidal

406

particles, possibly due to electrosteric repulsive forces83. In our experiments, the surface charge

407

of control and NOM-conditioned membranes was similar (  -25 mV, cf. Figure S4), while

408

bacteria suspended in NOM solution and PBS also exhibited similar  values ( -5 mV,

409

extrapolating data in Figure S5 to the ionic strength of PBS). These measurements indicate that

410

NOM does not significantly modify the surface charge of the bacteria or the membranes.

411

Nonetheless, we note that the NOM-conditioned PSF membranes are as hydrophilic as the non-

412

conditioned PSF-PDA membranes, and that the NOM-coated PSF-PDA membrane exhibits

413

the lowest contact angle of all the systems measured (see Figure 1). Consequently, NOM

414

creates a hydrophilic coating on the membrane that weakens bacterial attachment, and reduces

415

the rupture separation through a reduction of binding sites available to adhesins. The

416

hydrophilic character of NOM films is expected given that NOM contains functional groups,

417

such as carboxylic acid and amine groups, that can form hydrogen bonds with water36. Further,

418

our FTIR data (Figures S2 and S3) show that NOM contains polysaccharides and

419

polysaccharide-like components, both of which are hydrophilic constituents of NOM. The

420

hydrophilicity of the NOM film can also be explained in terms of the high water content of the

421

NOM conditioning layer97. Steric repulsion, which acts at short distances (< 15 nm)26,83, may

422

also weaken adhesion on NOM-coated surfaces: cell-surface structures, such as

423

lipopolysaccharide chains, can introduce steric repulsive forces26,39 against macromolecular

424

polysaccharide chains that protrude from the NOM adsorbed on the membrane.

425 426

The weaker adhesion observed in PSF membranes when NOM is present only in solution can

427

be attributed to Ca2+-promoted complexation of NOM with sulfonyl functional groups on the

428

membrane surface98. Ca2+ ions, present in NOM solution, can adsorb to the UF membrane


Page 19 of 35


429

surface99 reducing the electrostatic repulsion between NOM and the membrane79 and forming

430

a bridging complex with carboxyl groups in NOM100. The aromatic and aliphatic content of

431

NOM could also lead to the adsorption of NOM to hydrophobic PSF membrane surfaces via

432

hydrophobic interactions99,101. Since the NOM employed in this work is predominantly

433

hydrophilic (cf. Figure 1), its adsorption to PSF membranes decreases the magnitude of

434

bacterial adhesion forces and the rupture separation (cf. Figure 3 (A) and (B)). At the same

435

time, it is possible that NOM also adsorbs on the bacterial cell surface39,102, possibly due to

436

mediation of Ca2+ ions in solution. This would result in a more hydrophilic bacterial cell

437

surface, leading to weaker adhesion forces. Steric hindrance arising when a bacterial cell and

438

substrate are both coated with a macromolecule (e.g., NOM)102 may also be responsible for

439

weaker adhesion forces.

440 441

3.2.3. Molecular Basis of Microbial Adhesion to UF Membranes

442

A significant fraction (~30% at tContact = 5 s in PBS) of the pull-off force-separation

443

measurements collected over PSF and PSF-PDA membranes exhibited a series of consecutive

444

spring instabilities, resembling the “sawtooth” pattern characteristic of force unfolding of

445

biomolecule domains103. The representative force curve in Figure S1 shows that following

446

strong, unspecific adhesion at short separations, consecutive sawtooth peaks are observed,

447

which are usually ascribed to progressive unwinding of biomolecule domains42,104,105. In

448

agreement with previous work on protein unfolding106, we find that each peak is well fitted (R2

449

≥ 0.98) to the worm-like chain (WLC) model of polymer elasticity107 (cf. Figure S1). The WLC

450

model describes the polymer (in this case, the bacterial surface macromolecule) as a semi-

451

flexible chain that is randomly oriented at any point41,107,108.

452 453

The WLC fit to the force (F) – separation (z) profile along the sawtooth-like peaks (Figure S1)



454

Page 20 of 35

is given by 𝐹(𝑧) =

455

𝑘𝐵 𝑇 1 𝐿𝑐 −𝑧 −2 𝐿𝑝

[4 (

𝐿𝑐

)

𝑧

1

+ 𝐿 − 4] 𝑐

(1)

456

where kB is Boltzmann’s constant, and T = 298 K is the absolute temperature41,106. Two fitting

457

parameters characterize a semi-flexible chain as described by the WLC model: the persistence

458

length (Lp) and the contour length (Lc). Lp is a property that reflects the flexibility of the chain

459

bonds109, and is thus a measure of the chain rigidity23; while Lc is the total length of the extended

460

polymer without stretching its backbone41,109,110. The WLC model conceptualizes the polymer

461

as an entropic chain: when undeformed, macromolecular chains are in a high entropy, random

462

coil configuration that resists stretching; an external force F must be applied to elongate the

463

polymer chain, i.e., to unfold the macromolecule107. Stretching results in a lower

464

configurational entropy due to alignment of the constituent segments along the direction of

465

load111.

466 467

Equation 1 was fitted to all the force curves exhibiting the sawtooth-like peaks. In all cases, we

468

excluded the unspecific, high-adhesion peak (cf. Figure S1), where the force-extension is not

469

well-defined, and excluded peaks resulting in Lp values < 0.15 nm (i.e., smaller than the length

470

of a C-C bond108) and > 1.0 nm107; persistence lengths exceeding 1 nm indicate that, in addition

471

to entropy, hydrophobic interactions which are not accounted for by the WLC model contribute

472

to protein extension112. The distribution of best-fit Lp values is presented in Figure 4 (A),

473

showing that the persistence length is narrowly distributed between 0.15 nm and 0.3 nm, with

474

an average Lp of 0.26 nm and 0.27 nm obtained for bacteria adhering on PSF and PSF-PDA,

475

respectively. The approximately equal values obtained over both membrane types reflect the

476

fact that Lp is an intrinsic property of the biopolymers intervening in bacterial adhesion, and is

477

thus independent of the chemistry of the substrate. A salient observation is that the mean

478

persistence length is consistent with that obtained from protein unfolding experiments (Lp =


Page 21 of 35


479

0.36-0.4 nm21,106,107), i.e., with the persistence length of an unfolded polypeptide106,107. The

480

data in Figure 4 (A) therefore indicate that proteinaceous adhesins play a major role in

481

attachment of P. fluorescens on the UF membrane surfaces.

482 483

Figures 4 (B) and (C) present the distribution of forces measured at the spring instabilities in

484

each sawtooth-like peak (cf. Figure S1), and the number of sawtooth-like peaks per cell pull-

485

off, respectively. We refer to these quantities as the unfolding forces (FUnfolding, Figure 4 (B))

486

and number of unfolding events per cell pull-off (NUnfolding, Figure 4 (C)), respectively. We

487

observed that the distribution of unfolding forces is approximately Gaussian over both

488

membrane types, but a higher average unfolding force is observed over PSF compared to PSF-

489

PDA (-0.35 nN vs. -0.24 nN, respectively, cf. Figure 4 (B)). Moreover, we observed a broader

490

distribution of the number of unfolding events per cell pull-off over PSF membranes than over

491

PSF-PDA membranes (Figure 4 (C)), and, on average, fewer unfolding events over PSF-PDA

492

(5.3 ± 5.2) compared to PSF (6.4 ± 5.0).

493 494

We propose that data in Figures 4 (B) and (C) can be explained in terms of unfolding of cell-

495

surface proteins of P. fluorescens cells during bacterial adhesion. In support of this view, we

496

note the well-established observation that proteins lose conformational stability near

497

hydrophobic interfaces113, given that hydrophobic interactions between the hydrophobic

498

protein core and a hydrophobic substrate stabilize the unfolded state16,111,113,114. Protein

499

unfolding is more significant over PSF surfaces (note the higher average number of unfolding

500

events, cf. Figure 4 (C)) because PSF surfaces are more hydrophobic than PSF-PDA

501

membranes (cf. Figure 1). We surmise that adhesin proteins in P. fluorescens unfold, or

502

partially unfold to a “molten” globule state116 on the surface of the membrane, and that the

503

forces reported in Figure 4 (B) are those necessary to desorb the unfolded (or partially



Page 22 of 35

504

unfolded) adhesin proteins from the membrane surface. On the surface of PSF membranes,

505

adhesin proteins can establish stronger hydrophobic interactions, hence the higher unfolding

506

forces shown in Figure 4 (B). These unfolding forces are in fair agreement with values (100 -

507

400 pN) observed during force-unfolding of proteins20,96,106,117,118. The observations made in

508

Figure 4 (B) are consistent with reported stronger adhesion forces of lysozyme on PSF

509

membranes compared to adhesion on the more hydrophilic 2-hydroxyethyl methacrylate

510

(HEMA)-grafted polysulfone114. Similarly, using AFM, adhesion forces of three different

511

proteins (bovine serum albumin (BSA), fibrinogen, and human FXII) were weaker over more

512

hydrophilic surfaces (contact angle less than 60°) than on more hydrophobic surfaces119. The

513

tendency of proteins to adhere more readily to hydrophobic surfaces was also shown using

514

lysozyme, fibrinogen, and BSA120.

515 516

We also obtained best-fit values of the contour length, finding that it ranged from 286 nm for

517

sawtooth-like peaks at short separations to 9950 nm at long separations. Further, we calculated

518

the increase in the contour length Lc between two consecutive unfolding peaks (work not

519

shown), as done by others20,110,121, finding that Lc was not constant, suggesting that the

520

adhesin proteins are partially unfolded upon contact with the surface20. In P. fluorescens,

521

proteins such as LapA are known to mediate adhesion 20,21,52. However, Lc for LapA does not

522

extend beyond 1875 nm20,21, whereas we observe contour lengths of several microns. It is

523

therefore likely that membrane proteins such as LapA are responsible for bacterial adhesion

524

when the observed rupture separation is 1 - 2 m. For longer rupture separations, adhesion is

525

likely due to pili, which are several-micrometer-long structures comprising pilin proteins122,123.

526

Finally, we examined the force-separation data collected on NOM-coated membranes (Figure

527

S18), finding that a significant fraction (39% for PSF, 24% for PSF-PDA) of the measurements

528

exhibit the signatures of force unfolding. The distribution of best-fit Lp values revealed mean


Page 23 of 35


529

values (Figure S18 (A)) in close agreement with those obtained with NOM-free membranes

530

(Figure 4 (A)), suggesting that the sawtooth patterns are due to the elasticity of bacterial

531

adhesins, rather than stretching of adsorbed NOM. We also note that a smaller number of

532

unfolding events per pull-off was observed on NOM-coated membranes (Figure S18 (C))

533

compared to NOM-free membranes (Figure 4 (C)). It is possible that the NOM-conditioning

534

film, through a combination of hydrophilicity and steric repulsion, results in a smaller number

535

of adhesin proteins contacting and attaching to the substrate.



Frequency (%)

75

Page 24 of 35

A PSF Avg. Lp = 0.26  0.18 nm

50 25 75

PSF-PDA Avg. Lp = 0.27 0.14 nm

50 25 0 0.15

0.30

0.45

0.60

0.75

0.90

1.05

Lp (nm) Frequency (%)

60

B

PSF Avg. Force=-0.35 0.12 nN

40 20 60

PSF-PDA Avg. Force=-0.24  0.08 nN

40

NO

20 0 -0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

FUnfolding (nN) Frequency (%)

40

C

30

PSF Avg. Nunfolding = 6.4 5.0

20 10 40 30

PSF-PDA Avg. Nunfolding = 5.3  5.2

20 10 0

0

2

4

6

8

10

12

14

16

18

NUnfolding

536 537 538 539 540 541 542 543 544 545 546

Figure 4: (A) Distribution of best-fit persistence length values (Lp), obtained from WLC model fits to the pull-off force curve of single P. fluorescens cells. (B) Distribution of the unfolding forces (FUnfolding, the force measured at the sawtooth peak, cf. Figure S1), defined as the force necessary to unwind a macromolecular domain. (C) Distribution of the number of unfolding events (NUnfolding, the number of sawtooth-like peaks per cell pull-off). The mean of each histogram is shown in the inset. Data were collected with single bacterial cells over PSF membranes (upper panel in A, B and C), and PSF-PDA membranes in PBS buffer (pH 7.4) at tContact = 5 s. The histograms reflect 179 WLC fits to sawtooth-like unfolding events, identified in 28 retraction force curves over PSF membranes; and 221 sawtooth-like unfolding events observed in 42 retraction force curves over PSF-PDA membranes.


Page 25 of 35


547

3.3. Environmental Implications

548

This work employed SCFS to understand bacterial adhesion to UF membrane surfaces, the first

549

stage in membrane biofouling. Using P. fluorescens as a model biofilm-forming bacterium, we

550

investigated the effect of PDA deposition on bioadhesion to polysulfone UF membranes.

551

Weaker adhesion forces were observed on the more hydrophilic PSF-PDA UF membranes

552

compared to the unmodified control PSF membranes. Nevertheless, we observed that the

553

adhesion force increased with bacterium-UF membrane contact time, with the PDA membrane

554

exhibiting an average adhesion force at tContact = 5 s that is greater than that of the unmodified

555

(and more hydrophobic) polysulfone at 0 s. When the membranes were challenged with

556

bacterial suspensions in batch deposition experiments (Figure S19, experimental details are

557

given in the SI), we observed that the difference in the number of deposited cells (PSF: 760 ±

558

720 mm-2; PSF-PDA: 340 ± 250 mm-2) was not statistically significant (p = 0.11, two sided,

559

unpaired t-test). The strong bioadhesion forces observed at long tContact, together with the data

560

in Figure S19, call into question the effectiveness of simple hydrophilic coatings in controlling

561

biofouling, considering that bacterium-membrane contact would be frequent and prolonged in

562

membrane filtration. Similar observations have been made by others27,124. On the other hand,

563

the combination of PDA surface modification with biocidal nanoparticles (e.g., Ag)61, appears

564

to be a promising strategy to slow down biofouling rates. Insights into molecular level

565

phenomena underlying bacterial adhesion to membrane surfaces also emerge from this study.

566

In bacteria which rely on hydrophobic adhesins for attachment, results pressented herein

567

suggest that surface-induced protein unfolding on hydrophobic membrane substrates (possibly

568

involving outer membrane proteins, or pilin proteins) promotes bacterial attachment.

569 570 571

Supporting Information



Page 26 of 35

572

Additional materials and methods information (membrane fabrication and surface

573

modification; membrane characterization). Sample force curve (Figure S1); FTIR spectra

574

(Figures S2 and S3); zeta potential of membrane surfaces (Figure S4); zeta potential

575

measurements of P. fluorescens cells (Figure S5); AFM surface roughness (Figure S6); water

576

permeability coefficient (Figure S7); distribution of adhesion forces on PSF and PSF-PDA

577

membranes for 0, 2, and 5 s contact time (Figure S8); distribution of rupture separations on

578

PSF and PSF-PDA membranes for 0, 2, and 5 s contact time (Figure S9); distribution of

579

adhesion forces and rupture separations for control measurements (cell-free cantilever) over

580

PSF and PSF-PDA membranes (Figures S10 to S15); distribution of adhesion forces on PSF

581

and PSF-PDA membranes in the presence of NOM (Figure S16); distribution of rupture

582

separations on PSF and PSF-PDA membranes in the presence of NOM (Figure S17);

583

distribution of Lp, FUnfolding and NUnfolding observed with NOM-coated membranes (Figure S18);

584

results of bacterial deposition experiments (Figure S19). This material is available free of

585

charge via the Internet at http://pubs.acs.org.

586 587

Corresponding Author

588

Santiago Romero-Vargas Castrillón. E-mail: [email protected]

589 590

Acknowledgments

591

We gratefully acknowledge funding from the United States Geological Survey through the

592

Minnesota Water Resources Center (grant no. MN WRC 2015MN362B), and from 3M Co.

593

(Non-Tenured Faculty Award Program). Parts of this work were carried out in the

594

Characterization Facility and Minnesota Nano Center at the University of Minnesota, which

595

receive partial support from NSF through the MRSEC and NNIN programs, respectively. S.

596

BA. acknowledges the support of a Ling Graduate Fellowship in Environmental Engineering.


Page 27 of 35


597

A. H. acknowledges the support of the Norwegian Center for International Cooperation in

598

Education Partnership Program with North America. We also acknowledge Nathan Karp for

599

his contributions to the preliminary stages of this study. We thank the University Imaging

600

Centers at the University of Minnesota for support, and Dr. Guillermo Marqués for technical

601

assistance.



602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651

Page 28 of 35

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652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701


22. 23.

24.

25.

26. 27.

28. 29. 30. 31.

32.

33.

34. 35.

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