Influence of Cellulose Charge on Bacteria Adhesion and Viability to

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Influence of Cellulose Charge on Bacteria Adhesion and Viability to PVAm/CNF/PVAm Modified Cellulose Model Surfaces Chao Chen, Torbjörn Pettersson, Josefin Illergård, Monica Ek, and Lars Wågberg Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00297 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 24, 2019

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Biomacromolecules

1

Influence of Cellulose Charge on Bacteria

2

Adhesion and Viability to PVAm/CNF/PVAm

3

Modified Cellulose Model Surfaces

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Chao Chen, Torbjörn Petterson, Josefin Illergård, *Monica Ek and *Lars Wågberg

5

Department of Fibre and Polymer Technology, School of Engineering Science in Chemistry,

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Biotechnology and Health (CBH), KTH Royal Institute of Technology. Teknikringen 56-58,

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100 44 Stockholm, Sweden

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9

ABSTRACT

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A contact-active antibacterial approach based on the physical adsorption of a cationic

11

polyelectrolyte onto the surface of cellulose material is today regarded as an environment-

12

friendly way of creating antibacterial surfaces and materials. In this approach, the electrostatic

13

charge of the treated surfaces is considered to be an important factor for the level of bacteria

14

adsorption and deactivation/killing of the bacteria. In order to clarify the influence of surface

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charge density of the cellulose on bacteria adsorption as well as on their viability, bacteria were

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adsorbed onto cellulose model surfaces which were modified by physically adsorbed cationic

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polyelectrolytes to create surfaces with different positive charge densities. The surface charge

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was altered by layer-by-layer (LbL) assembly of cationic polyvinylamine (PVAm)/anionic

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cellulose nanofibrils (CNF)/PVAm onto the initially differently charged cellulose model

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surfaces. After exposing the LbL treated surfaces to Esherichia coli in aqueous media, a

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positive correlation was found between the adsorption of bacteria as well as the ratio of non-

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viable/viable bacteria and the surface charge of the LbL-modified cellulose. By careful colloidal

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probe AFM measurements, it was estimated, due to the difference in surface charges, that

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interaction forces at least 50 nN between the treated surfaces and a bacterium could be achieved

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for the surfaces with the highest surface charge, and it is suggested that these considerable

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interaction forces are sufficient to disrupt the bacterial cell-wall, and hence kill the bacteria.

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KEYWORDS: Antibacterial mechanism, Cellulose model surface, Colloidal probe, Layer-by-

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Layer, Surface charge, Surface potential

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INTRODUCTION

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Cellulose and its modification and functionalization have attracted considerable attention in

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recent years to meet the increasing demand for environmentally friendly and biocompatible

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products 1. One important field is the development of antimicrobial properties of cellulose

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surfaces due to the huge potential of these materials in applications such as water purification,

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medical biomaterials and wound-healing membranes. Several approaches have been used to

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fabricate antibacterial cellulosic substrates. One approach is a leaching antibacterial technique,

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where cellulose materials are impregnated with antibiotic metal ions or nanoparticles, which

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are subsequently released and thereby able to reach the bacteria. 2-3 Non-leaching approaches,

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on the other hand, have no release of antimicrobial substances that to make a surface deactivate

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bacteria upon contact 4-6. They have been developed via graft polymerization/copolymerization

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from cellulose 7-8, or via self-assembly of antibiotic polymers. The antibiotic polymers used in

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the non-leaching approach are usually cationic polymers, whose antibacterial properties are


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Biomacromolecules

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believed to target the bacterial cell membrane by physical interaction and are thus active

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without leaching substances to the surroundings 9-10. These modifications are also considered

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to be more sustainable and able to suppress the bacteria resistance, because the negatively

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charged bacteria are adsorbed by a charge-induced interaction, which subsequently weakened

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the membrane of the adsorbed bacteria, and make them more vulnerable on the surfaces 9.

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However, the reasons behind these interactions are still not settled 11. The importance of the

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charge density of a contact-active antimicrobial surfaces was studied by Kugler et. al

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discovered a charge-density threshold for achieving an optimum bactericidal effect, and

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suggested an ion-exchange mechanism to explain the existence of this threshold. Murata et. al

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9

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methacrylate) (polyDMAEMA) polymer by surface-initiated atom transfer radical

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polymerization and subsequent quaternization to tertiary amino groups, and demonstrated that

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the charge density might be more important than the chain length of the polyelectrolyte.

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Nevertheless, little has been done in recent years to clarify the importance of the surface charge

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for the adsorption of bacteria and how bacteria are inactivated/killed by the surface-treated

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materials.

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Our previous works have presented a three-layer polyelectrolyte system of PVAm/PAA/PVAm

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was therefore adsorbed onto pulp fibres through a layer-by-layer (LbL) deposition and showed

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excellent antibacterial properties which were attributed to a re-charging of fibre surface by the

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adsorbed polyelectrolytes 13-14. Further investigations showed that more bacteria were adsorbed

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onto PVAm/PAA/PVAm modified cellulose fibers when using the initial cellulose charge

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density was higher as a result of a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)

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oxidation of the fibers 15. It was demonstrated that the adsorption of bacteria increased due to

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the higher amount of adsorbed PVAm on the fibers, which was considered as a result of a higher

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positive charge. However, it still lacks direct evidence of how surface charge density influences

12

who

were able to precisely control the molecular weight of a poly(dimethylaminoethyl



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the bacteria viability, because cellulose-rich wood based fibres are heterogeneous, porous and

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rough, and it is hence difficult to visualize the viability of bacteria and perform a quantitative

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analysis of bacterial interaction on fibres using the commonly used high resolution

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microscopy/spectroscopy. This problem can be circumvented by using cellulose model surfaces.

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The smooth cellulose II surface introduced by Gunnars et.al 16, makes it possible to study the

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bacteria adsorption on the surfaces and to quantify bacteria/cellulose interactions, since no

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lignin is present.

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oxidation can be controlled by the addition of sodium hypochlorite

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charge of the cellulose fibres, and thus the final positive charge density of the LbL-modified

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surface.

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Therefore, in the present work, a method was developed to study the influence of charge density

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on bacterial interaction on a smooth cellulose surface modified by a PVAm/CNF/PVAm LbL

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treatment. This newer three-layer polyelectrolyte system of PVAm/CNF/PVAm was chosen

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because it is more bio-based, and has also shown even better antibacterial properties when

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adsorbed on pulp fibres than ones modified by PVAm/PAA/PVAm

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therefore deposited on cellulose model surfaces with different charge densities, which were

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tuned by TEMPO oxidation, and the surface charge of the surfaces was calculated based on

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precise surface potential determinations from AFM colloidal probe measurements. From these

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measurements of the surface charge it was possible to quantify the importance of the interaction

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force between the charged cellulose surface and bacteria with regard both to bacterial

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adsorption and to the viability of the adsorbed bacteria.

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The model surfaces can be TEMPO oxidized and the extent of cellulose

90 91 92

MATERIALS AND METHODS


18

19

to alter the negative

. The multilayers were

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Biomacromolecules

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Materials

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The PVAm with the trade-name Xelorex RS 1300 was supplied by BASF AG (Ludwigshafen,

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Germany). Xelorex RS 1300 contains 20 wt% of PVAm with an average molecular weight of

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340 kDa according to the supplier. The polymer was dialysed against deionized water and then

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freeze-dried prior to use. Carboxymethylated cellulose nanofibrils (CNFs) were supplied by

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RISE Bioeconomy, formerly Innventia AB, (Stockholm, Sweden) as a gel-like dispersion with

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a solids content of 2.5% by weight in MilliQ water (MQ) (Millipore, Solna, Sweden). This CNF

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stock was dispersed to lower concentrations and colloidally stable dispersions according to an

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earlier described procedure

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Oxidation of Cellulose Fibres for the Preparation of Cellulose Model Surfaces with

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Different Charge Densities

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A disintegrated bleached chemical softwood fluff pulp, supplied by Essity, formerly SCA

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Hygiene Products (Mölndal, Sweden), was washed according to a standard procedure

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remove unwanted contaminants and to convert the carboxyl groups of the fibres into their

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sodium form. The original pulp had a carboxylic acid content of 40 μmol/g and the pulp was

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oxidized further to four different charge levels corresponding to 90 μmol/g, 500 μmol/g, 800

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μmol/g and 1300 μmol/g carboxylic acid contents respectively. The oxidation was performed

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on the original fibres using 2,2,6,6-Tetramethylpiperiding 1-oxyl (TEMPO)-mediated

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oxidation by adding different amounts of sodium hypochlorite at pH 10 following the procedure

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developed by Saito et al.

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oxidation, the oxidized pulps were reduced in a dibasic sodium phosphate dihydrate (0.01 M,

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1.42 g, Sigma Aldrich) and sodium borohydride (0.053 M, 2 g, Sigma Aldrich) for 1 hour at

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room temperature. The carboxylic acid contents were determined by conductometric titration

116

23

20

.

18, 22

21

to

. In order to remove aldehydes or ketones created during the

. In this procedure about 0.5 g of each pulp was used, and the reported values are the averages



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of triplicate measurements with a maximum of 3% deviation. The different fiber samples and

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their resulting total charges are listed in Table 1.

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Table 1. Total charge densities of cellulose pulp as determined by conductometric titration

120 121 122

No. cellulose pulp

1

2

Total charge density (μmol/g)

40

90

3

4

5

500 800 1300

123 124

Fourier Transform Infrared Spectroscopy (FTIR)

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Infrared spectroscopy was utilized to determine the change in concentration of carboxylic

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groups during the preparation of the model cellulose surfaces. The fibers of different charge

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were kept in their sodium form. A small amount of dried fibers was taken from each of five

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different charged un-dissolved pulp samples and analyzed with the aid of FTIR (Perkin-Elmer

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Spectrum 2000). For the dissolved pulps, 2 ml of the dissolved pulp solution was taken and

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precipitated (regenerated) in 96 % ethanol for 1 hour, the ethanol being exchanged 3 times, and

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then left overnight together with ethanol at 4 C sealed by parafilm. The resulting gel-like

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precipitated cellulose was dried in an oven at 60 C for 2 hours until it was completely

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dehydrated. Each dried and regenerated dissolved cellulose sample was disintegrated into small

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pieces and subjected to FTIR analysis.

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LbL Modification of Cellulose Model Surfaces

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The preparation of cellulose model surfaces for the modification were described in detail in the

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supporting information (SI), and their preparation method can be found in elsewhere.24 The

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LbL modification of the cellulose surface was achieved with a dipping sequence of PVAm-


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Biomacromolecules

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rinse-CNF-rinse-PVAm-rinse-dry. The surfaces were dipped in 0.1 g L-1 PVAm containing 100

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mM NaCl for 30 mins at pH 9.5, rinsed with MilliQ for one minute and then dipped in 0.1 g L-

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1

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PVAm as in the first step (30 minutes). After rinsing in MilliQ, dried under a flow of N2 gas,

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the LbL-modified surfaces were kept under vacuum in a desiccator until use.

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Colloidal Probe Measurements

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AFM Colloidal probe measurements were made with a MultiMode IIIa (Veeco Instruments Inc.

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Santa Barbara, CA) with a PicoForce extension, using tipless rectangular cantilevers (CLFC-

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NOCAL, Bruker) with a normal spring constant of approximately 0.18 N/m.

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calibrated 26-27 in air under ambient conditions using the AFM Tune IT 2.5 (Force IT, Sweden)

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software and were then used after silica particles (Lot No: 31443, Dry Borosilicate Glass

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Microspheres, Duke Scientific Corporation) with a diameter of 9.6  1.0 μm had been glued to

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the cantilevers with thermo setting glue (Epikote 1001, Shell Chemical Co.) using a manual

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micromanipulator (HS 6 Manuell, Marzhauser Wetzlar GmbH & Co. KG) and a reflection

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microscope (Olympus). Before gluing, these particles were dispersed for 15 min in 1 M NaOH

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followed by rinsing with MilliQ water until the dispersion had a neutral pH. A small volume of

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the dilute particle suspension was then applied to a clean microscope slide and allowed to dry.

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The force measurements were performed in a liquid cell on the different cellulose sample

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surfaces in 0.1 and 10 mM NaCl solutions at pH 6.5. Measurements in a particle vs particle

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geometry were also made to assess the interaction between two particles of the same type (i.e.

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a silica particle was glued onto flat silica wafer in the same way the particles were attached to

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the cantilever) in order to make it possible to estimate the surface potential of the silica particle

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subsequently used to determine the surface charge of the cellulose surfaces. AFM Force IT

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version 3.0 (ForceIT, Sweden) software with the plug-in dlvoIT (ForceIT, Sweden) was used

CNF solution containing 10 mM NaCl for 30 mins at pH 7.5, rinsed and again dipped in


25

They were


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to convert the raw data and to compare the force profiles to both symmetric and asymmetric

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DLVO models 28. For the asymmetric surface set-up, the program Asymm_pc_v2_2 by Johan

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C. Fröberg was used, based on models created by both Bell and Peterson 29 and Devereux and

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De Bruyn 30. In the asymmetric model, the value obtained by a symmetrical fitting of the silica

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particle/particle data was used as the value for the silica particle.

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Fluorescence Microscopy

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A fluorescence microscope Nikon Eclipse Ti-U (Bergman Labora AB, Danderyd, Sweden) was

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used to quantify the adsorption and distribution of the bacteria, E. coli K-12 (Biorad

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Laboratories AB, Solna, Sweden) on the cellulose model surfaces. The bacteria were

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transformed with a PGreen plasmid (Biorad, Solna, Sweden) to express green fluorescent

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protein (GFP) according to a calcium-chloride-heat-shock-transformation protocol

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visualize the bacteria adsorption, unmodified and modified cellulose model surfaces were

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submerged in 2.5108 CFU ml-1 E. coli PGreen in 10 mM NaCl solution for 4 and 18 hours

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with continuous shaking at 37 C. This was followed by a gentle dip-rinsing with ¼ Ringer’s

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solution (Sigma Alderich, Stockhom, Sweden) after which the surfaces were observed at 100x

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magnification. The bacteria adsorbed on the surfaces was calculated by subtraction of the

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bacterial populations left in the solution after incubation with modified surfaces for 4 and 18

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hours from a blank reference (bacterial solution with no specimen added), and the number of

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bacteria left in the solution was determined by optical density (OD) at  = 620 nm using a

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MultiSkan FC microplate spectrophotometer (Thermo Scientific, Stockholm, Sweden).

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In order to determine the viability of the bacteria, the E. coli K-12 was stained for 15 minutes

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in the dark by a LIVE/DEAD BacLightTM Bacterial Viability Kit, L13012 containing SYTO 9

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and propidium iodide (PI) fluorescent dyes (Molecular Probes, Invitrogen Grand Island, NY,


31

. To

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Biomacromolecules

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USA). In this procedure, the SYTO 9 shows green fluorescence, whereas PI shows red only in

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damaged bacteria membrane. The green fluorescence was measured at 520 nm and the red

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fluorescence above 630 nm at 400x magnification. The images were captured using an

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INFINITY 2-3 digital CCD camera with 3.3-megapixel resolution (Lumenera, Ontario, Canada)

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with the same exposure time. The numbers of live/dead bacteria were determined by ImageJ

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from each sample after 18 hours incubation with bacteria solution. Percentage of live and dead

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bacteria was the average obtained by analyzing 5 images taken from different areas on the same

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charged surfaces.

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Scanning Electron Microscopy (SEM)

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Prior to the SEM observation, the bacterial cells were fixed according to the procedure

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described in SI. Thereafter, dried surfaces with fixed bacteria were coated with 10 nm of Pt in

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a 208HR high-resolution sputter coater (Cressington, Watford, UK), after which, the samples

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were subjected to SEM imaging using a S-4800 field emission scanning electron microscope

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(Hitachi, Tokyo, Japan). The images were captured under magnifications of 18000x and 15000x

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at 5.0 kV. The bacterial population was quantified by counting on 50  50 μm2 areas at different

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positions in the SEM images. The counting was done by the particle analyzing function in an

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image analysis software ImageJ (NIH, USA).

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RESULTS AND DISCUSSION

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This study was performed to investigate the interaction between bacteria and differently

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charged surfaces prepared by layer-by-layer deposition of PVAm/CNF/PVAm on charged

208

cellulose surfaces in order to elucidate the mechanism behind the antibacterial action of

209

cationically treated cellulose surfaces. To obtain the differently charged cellulose model



210

surfaces, cellulose-rich, wood-based fibers were processed through a series of steps including

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oxidation, dissolution, and regeneration as a coating on smooth silica surfaces. The cellulose-

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rich materials were carefully characterized before and after surface preparation in order to

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ensure that no major changes had taken place during the processing of the cellulose. The

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prepared surfaces were then treated with different LbL-strategies to alter the surface charge and

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the charge properties of these surfaces were then evaluated using colloidal probe AFM and the

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antibacterial properties were assessed using live-dead analysis as well as SEM-investigations.

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FTIR analysis of oxidized fibers and the regenerated cellulose surfaces

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The fibers were analyzed with FTIR both before and after dissolution in order to determine

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possible changes resulting from the dissolution and regeneration process. The FTIR spectra of

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the different samples are summarized in Figure 1(a) Shows spectra of fibers with different

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degrees of oxidation before dissolution. The greatest difference between the spectra is around

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1614 cm-1 corresponding to the carboxylate groups in their sodium form which is in good

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agreement with earlier published data

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with respect to the absorbance at 1320-1310 cm-1, corresponding to the ring (CH2 rocking at

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C6) stretch, the height of the peak at 1614 cm-1 was used to assess the increase in the amount

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of COO- Na+ due to the oxidation of the fibres. The Figure 1 (b) shows spectra of the films after

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dissolution and regeneration. The peaks in the inset in Figure 1b show basically the same trends

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as in Figure 1(a), indicating that there was no significant loss of carboxylic carbonyls as a result

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of the dissolution and regeneration of the modified cellulose in 96% ethanol. Ethanol was used

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instead of water because an earlier investigations had shown that there is a significant loss of

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carboxylic carbonyls loss after regeneration of charged cellulose in water, at charge levels

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around 1200 μmol/g 32. The present results show that by using ethanol it is possible to avoid

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charge loss due to the much lower solubility of cellulose in ethanol.

32

. Since all the spectra, in the inset, were normalized


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Biomacromolecules

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Figure 1: (a) FTIR spectra of cellulose fibers with different total charge densities. (b) FTIR

236

spectra of regenerated cellulose in 96% EtOH after dissolution in NMMO/DMSO 1:3 solutions.

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The insets in both a) and b) show the region representing the COO- Na+ groups and the different

238

spectra have been normalized with respect to the absorbance at 1310 -1320 cm-1.

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Characterization of Cellulose Surfaces by AFM

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The morphology of the cellulose-coated silica surfaces was examined by AFM to evaluate the

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surface roughness as well as the thickness in both dry and wet states (10 mM NaCl), in order to

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see the state of the surface where the bacteria were adsorbed. The results are shown in Table 2.

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Table 2: Cellulose film thickness h on silica substrate and the root mean square roughness Sq

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(10  10 m2) in air and in 10 mM NaCl aqueous media. Fiber total charge hdry (nm)

hwet (nm)

Sqdry (nm)

Sqwet (nm)

40

20.5

28.7

8.6

18.4

90

15.8

25.4

5.6

17.1

500

15.6

35.4

5.0

13.8

(mol g-1)



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800

10.3

32.6

4.2

8.9

1300

10.8

45.5

3.9

11.1

245 246

The dry thickness of the cellulose film decreased with increasing fiber charge, which is

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attributed to a decrease in viscosity of the dissolved cellulose which results in a thinner dried

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spin-coated layer. The viscosity is related to the solubility and molecular mass of the cellulose

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in NMMO/DMSO but the study of the exact reason was beyond the scope of the present work.

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It has been shown 24 that dissolution in NMMO leads to a slight decrease in the molecular mass

251

of the cellulose, but since the decrease was small it was considered not to affect the results of

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the present work. The thickness in the wet state is a combination of the original amount of

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material i.e. dry thickness swelling due to the increased charge. The greatest wet thickness with

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the highest charge implies, in accordance with the FTIR results, that the charge was not lost

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significantly during the dissolution and regeneration and that it was also maintained upon

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reswelling in water. The dry roughness decreased with increasing charge. The surface

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roughness trend in the wet state is not as clear, but there was a decrease when the charge was

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increased up 600 ueq/g and a slight increase with the highest charge. This behavior is probably

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a combined effect of swelling and the dry roughness. The wet surface roughness it is still low

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compared with the dimensions of E. coli bacteria which have dimensions greater than 1 µm.

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The surfaces can therefore be considered flat and homogeneous for studies of bacteria

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adsorption and for the evaluation of the interactions between adsorbed polyelectrolytes and

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bacteria. From an interaction point of view, the surfaces can be considered flat and the

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roughness should not be of major importance for the interaction.

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Colloidal Probe Measurements for Model Surfaces


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Biomacromolecules

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To evaluate the surface charge, the surface potentials of the cellulose surfaces and of the

267

multilayer-coated cellulose surfaces were determined by fitting the force measurements from

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AFM colloidal probe measurements to the DLVO theory. The electrical double layer repulsion

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between the target surface and the particle probe when the probe has a charge similar to that of

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the targeted surface leads to a repulsive force detectable at a distance from the surface during

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the probe approach until direct contact is made with the surface. The measured force profile (an

272

example is shown in Figure S1 in the Supporting Information) can be divided into four different

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regimes (noted as different stages in the figure) during the approach to the surfaces. At large

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separations (Stage 0) the regime is outside the detectable force limit (not shown in the figure,)

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In Stage 1 a repulsive force can be detected where the electrical double layer force is in action.

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When the separation is short (around 12 nm), it is possible to detect a “kink” in the force profile,

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which is the point where the probe comes into physical contact with the swollen cellulose

278

surface or with the LbL layers on the swollen cellulose. This point is referred to D0 (off-set) in

279

the DLVO fitting procedure. This is the beginning of Stage 2 and at shorter separations the

280

cellulose is compressed until one enters the last regime. Stage 3 is the hard-wall contact, where

281

the cantilever is bent in full agreement with the piezo movement, and no further compression

282

of the surfaces is possible. The D0 values for the different samples in the different salt

283

concentrations are presented in Table S1. D0 is similar for all the cellulose surfaces but is

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dependent on the salt concentration. It is close to 12 nm in the 0.1 mM NaCl and decreases with

285

increasing salt concentration. Similar trends were observed for the LbL-modified surfaces.

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These estimated D0 values were used when the DLVO theory was fitted to the force curves (a

287

typical example is shown in the inset in Figure S1. When using the asymmetric model, which

288

is necessary since the probe and the flat surface are different, the potential of the probe was first

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determined from measurements made between two similar silica particles by fitting the

290

symmetric DLVO model to force curves in a sphere-sphere geometry of similar spheres. The



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surface potentials of the silica particle were -57 mV at 0.1 mM NaCl, and -30 mV at 10 mM

292

NaCl, and the surface potentials of silica particles modified with a monolayer of PVAm were

293

+45 mV at 0.1 mM NaCl, and +15 mV at 10 mM NaCl. These probe values were then used as

294

input to the DLVO fitting process in the asymmetric model. The Hamaker constant used was

295

the silica/water/silica with a value of 4.6 x 10-21 J 33. It can be argued that this value could be

296

different for all the different samples, but the actual value used does not affect the final value

297

obtained for the surface potential, since this value only affect the shape of the curve in the last

298

few nanometers before entering stage 2. The surface potentials were obtained by fitting the

299

collected force data to the DLVO theory in both a symmetric and an asymmetric model. The

300

potential data obtained from fitting of the symmetric model are shown in the supporting

301

information Figure S2. Figure 2 shows the surface potentials of (a) cellulose surfaces and (b)

302

LbL-modified cellulose surfaces, obtained by fitting the DLVO theory to asymmetric models.

303

The absolute value of the surface potential increasing with the charge density of the different

304

fibers initially used to make the cellulose surfaces. In general, the same trend was observed for

305

the LbL-modified (PVAm/CNF/PVAm) cellulose surfaces, suggesting that the increased

306

charge of the cellulose also affect the surfaces after LbL-modification, resulting in a higher

307

final charge of the PVAm-treated surfaces. This recharging is typical of the LbL-process but

308

there are few quantitative data in the literature of the surface potential of the surfaces and the

309

less precise zeta potential is usually given although this is dependent on many factors and does

310

not reflect the true value of the surface potential. The fundamental molecular reasons for the

311

high potentials of the PVAm-treated surfaces are very important for the properties of the treated

312

surfaces but they are beyond the scope of the present investigation. The values of the potentials

313

are however very valuable in the analysis of the bacteria adsorption and bacteria-killing

314

efficiency of the surfaces. It must however be remembered that they are derived from force

315

measurements and from a fitting to the DLVO-theory, and that factors such as swelling and


Page 14 of 31

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

316

deswelling of the surfaces when treated with salt and oppositely charged polyelectrolytes will

317

affect the estimated surface potentials. Nevertheless, the data in Figure 2 show how the fibre

318

charge affects the potential of the model surfaces and how the addition of salt and

319

polyelectrolyte will affect the surface potential. The results show that the addition of salt leads

320

to a decrease in the absolute value of the surface potential in agreement with theoretical

321

predictions. The results also show that a higher charge leads to a higher surface potential, that

322

with 10 mM NaCl the most highly charged surface has an anionic potential of 80 mV, and that

323

PVAm treatment of this surface results in a cationic surface potential of 110 mV. These are

324

very high values, and it has earlier been shown that a high cationic surface potential is important


Biomacromolecules

325

for the killing of adsorbed bacteria9.

326 327

Figure 2: (a) Surface potential of charged cellulose surfaces, and (b) the surface potential of

328

cellulose surfaces modified with PVAm/CNF/PVAm layer-by-layer deposition. The surface

329

potential was obtained by fitting the force curve with DLVO theory in s asymmetric model at

330

0.1 mM and 10 mM NaCl. The error bar shows the standard deviation of three values obtained 0

Surface potential (mV)

-20 -40 -60 -80 -100 -120 -140

Asymmetric model cellulose surfaces 10 mM NaCl Asymmetric model cellulose surfaces 0.1 mM NaCl

-160 40

90 500 800 Pulp charge (μmol/g)

1300

(a)

Surface potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 31

160

Asymmetric model modified surfaces 10 mM NaCl

140

Asymmetric model modified surfaces 0.1 mM NaCl

120 100 80 60 40 20 0 40

331

90 500 800 Pulp charge (μmol/g) (b)

1300

from the force curve of more than 15 cantilever approaches.


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

332 333

Based on the surface potentials determined from colloidal probe measurements, the surface

334

charge was calculated according to Gouy-Chapman model (equation 1) :

335

𝜎

336

where 𝜎 is the surface charge in (C/m2) and Φ is the surface potential in (V), for a flat surface

337

based on the Poisson-Boltzmann equation describing the distribution of ions outside a charged

338

surface.

339

concentration in the bulk (m-3), 𝜀 is the vacuum permittivity, 𝜀 is the relative permittivity, 𝑧

340

is the valency of the ion, and 𝑒 is the elementary charge. The surface charges based on the

341

measurements on LbL-modified cellulose in 10 mM NaCl are presented in Table 3. The surface

342

charge is plotted against the surface potential in Figure S3.

343

Table 3: Surface potentials determined based on AFM colloidal probe measurements and

344

calculated surface charges of LbL-modified cellulose surfaces in 10 mM NaCl with different

345

initial total charges.

8𝒌𝑇𝑐 ∗ 𝜀 𝜀

/

sinh

(1)

𝒌

34

, 𝒌 is the Boltzmann constant, 𝑇 is the temperature in Kelvins, 𝑐 ∗ is the ion

Fiber total

Surface potential 𝚽𝟎

Surface charge 𝝈

(mV)

(mC m-2)

40

30

7.29

90

60

17.1

500

65

19.2

800

105

44.5

1300

110

47.2

charge -1

(μmol g )

346



347

Bacterial Adsorption on Different Charged Surfaces

348

As reported previously, the bacterial removal effect is based on the capacity for bacterial

349

adsorption on PVAm/CNF/PVAm modified cellulose surfaces. 19 The adsorption of the bacteria

350

to the surface is a prerequisite for any bactericidal effect. How an increase in surface charge

351

will influence both bacteria adsorption and bacteria viability on the surface and both effects

352

were investigated in the present work.

353 354

The adsorption of bacteria was examined by fluorescence microscopy after adsorption for 4 and

355

18 hours in 10 mM NaCl and the results are shown in Figure 3 (a), it shows the number of

356

bacteria adsorbed (cm-2) as a function the surface charge. The adsorption of bacteria increases

357

with increasing surface charge, and on the two most highly charged surfaces the adsorption

358

increases when the adsorption time is extended from 4 to 18 hours. This suggests that there is

359

a change in the adsorption process between the surfaces with the lower charges and the more

360

highly charged surfaces. Since the adsorption of bacteria is driven by the release of counterions

361

to the charges on the bacteria and the surface 12, a higher charge of the surface will lead to a

362

higher adsorbed amount. From the dimension of the bacteria it can be estimated that the area of

363

one E. coli is 210-12 m2 and that the maximum adsorbed bacterial population would be 5107

364

CFU/cm2 at full surface coverage not taking into account the exact packing of these bacteria.

365

The results in Figure 3 (b) shows that the two most highly charged surfaces, is at a level close

366

to 5107 CFU/cm2 after 4 hours, i.e. that the surfaces are fully covered with bacteria and any

367

remaining charges on the surface will have a very weak interaction with the bacteria, This

368

adsorption continues however and this means that the extra bacteria adsorbed after 4 hours will

369

have a weaker interaction with the treated surface.


Page 18 of 31

Page 19 of 31

4 hours

18 hours

7.3

17

19

47

100 90

4 hours

18 hours

80 70 60 50 40 30 20 10 0

371

45

a

370 Number of E. coli (106 CFU/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

20 40 Surface charge (mC/m2)

60

b

372

Figure 3: (a) Fluorescent microscopy using 100 times magnification of E. coli on differently

373

charged LbL-modified cellulose surfaces after 4 and 18 hours incubation. The surface charge

374

is indicated in mC/m2 below the images. (b) The number of bacteria adsorbed on LbL-modified

375

cellulose surfaces with different initial surface charges after 4 hours and 18 hours.

376

The samples were thereafter fixed and studied under the SEM and the numbers of adsorbed

377

bacteria were counted with the help of ImageJ. This is illustrated in Figure 4 where the number

378

of bacteria remaining in the SEM study is compared with the count of bacteria from the optical

379

images. The difference between the two counts indicates that about half of the bacterial

380

population was not firmly attached, but that the number of firmly attached bacteria was greater


Biomacromolecules

381

with a higher surface charge. It has been reported that when the bacteria were attracted by

382

positively charged surfaces, not all the bacteria were immobile but that some could flip back

383

and forth or rotate in a circle 35. The rotation of the bacteria was generated by flagella, when

384

they still functioned and maintained their viability. It has been suggested at distances greater

385

than 10-20 nm, bacteria (E. coli) were still able to move laterally by flagella movement or by

386

Brownian motion and that at these distances the bacteria can be considered to be in a state of

387

reversible adsorption on the surface. At distances of less than 10 nm from the surface the

388

bacteria are considered to be irreversibly adsorbed 36, due to the strong attractive force between

389

the bacteria and charged surface. It is here suggested that the higher surface charge induces a

390

higher interaction force between the surface and bacteria and that this leads to a strong

391

attachment which prevents the bacteria from being rinsed away during fixation.

Number of E.coli (106 CFU/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

100 90 80 70 60 50 40 30 20 10 0

After fixation Before fixation

0 392

10

20 30 40 50 2 Surface charge (mC/m )

60

393

Figure 4: Numbers of E. coli on the surfaces before and after fixation. The numbers of bacteria

394

after fixation were determined on SEM images by ImageJ.

395

Viability of bacteria on differently charged surfaces

396

The ability of bacteria to survive under specific conditions can be examined by the


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

397

LIVE/DEAD staining technique, which allows a clear discrimination between live and dead

398

bacteria. A red fluorescence indicates a dead bacterium, due to the propidium iodide (PI) dye,

399

a common DNA-binding dye that only penetrates compromised bacterial membranes and

400

replaces the green nucleic acid stain SYTO 9 that indicates a live bacterium. SYTO 9 can easily

401

penetrate the cell membrane and stain cells green.

402

Figure 5 shows an image of E. coli adsorbed onto PVAm/CNF/PVAm-modified cellulose

403

surfaces for 18 hours observed under fluorescence microscopy after staining with the live/dead

404

kit. The numbers of bacteria were determined by image analysis using ImageJ software. Not

405

only did the total population of adsorbed bacteria increase with increasing surface charge, but

406

the proportion of dead bacteria did also increase to about 29% of the total population. The most

407

highly charged surface, 47 mC/m2, corresponds to 3.11013 charges/cm2 whereas the surface

408

with 17 mC/m2 corresponds to 1.11013 charges/cm2. This is in the range about 50 – 100 times

409

less than the critical charge threshold of 2-3 1015 charges/cm2 found earlier by Murata et. al9.

410

They reported that E. coli K-12 would be efficiently killed, that at least a monolayer of bacteria

411

would be able to eliminate within a short time at a cationic surface has greater than 31015

412

charge/cm2. The results suggested that in the present case an even higher surface charge would

413

be needed for a more efficient killing of the adsorbed bacteria.



414 415

Figure 5: Fluorescence microscopy images of 400 times magnification of E. coli K-12 stained

416

by LIVE/DEAD kit on different PVAm/CNF/PVAm-modified cellulose surfaces with initially

417

different surface charges. Percentage of live and dead bacteria was the average obtained by

418

analyzing 5 images taken from different areas on the same charged surfaces. The cationic

419

charges of the LbL-treated surfaces, estimated by colloidal probe measurements, are shown in

420

mC/cm2.

421

Mechanism of bacteria-surface interaction force for biocidal effect

422

It has shown that the bacterial viability is affected by the surface charge, but this does not

423

explain why the dead/live ratio was greater with a higher surface charge. The bacterial viability

424

was determined based on live/dead assay, which had shown the bacteria were killed due to the

425

disruption of the cell membrane, and for a higher charged surface the cell membrane is easier

426

to compromise. Therefore, the antibacterial action for such cationic surfaces was always

427

proposed to be due to the cell membrane disruption. It has been suggested that the killing of the


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

428

bacteria is due to an ion-exchange effect, and that the polycation can destabilize the bacterial

429

membrane through an exchange of divalent cations, Ca2+ and Mg2+ with adsorbed cationic

430

polyelectrolyte 12. Later a new “phospholipid sponge model” was proposed 37, suggesting that

431

the strong electrostatic attraction forces from the adsorbed polyelectrolyte can tear off the lipid

432

molecules from the cell membrane. Asri et al. pointed out the bacterial-killing effect on highly

433

charged surface was a result of physio-chemical interaction 38, since the immobilized cationic

434

polymer on the surface has a very limited contact with the bacterial cell instead of covering the

435

entire cell membrane as they were in the polymer solution. Although numerous studies have

436

demonstrated the dissolved cationic polymers in solution yield membrane-damage and cell

437

death,39-41 strong adhesion force against bacteria generated from the surface with immobilized

438

cationic polymer would also slowly tear bacterial cell apart. In order to clarify the hypothesis

439

based on our observation, the force between the adsorbed polyelectrolyte and the adsorbed E.

440

coli bacterium cell was calculated to estimate whether the forces are high enough to cause a

441

physical disruption of the bacterial cell wall. The forces (F) or actually the pressure (F/area),

442

between the charged surfaces and adsorbed bacteria, were estimated using the following

443

equation. 42 64𝒌𝑇𝑐 ∗ Γ Γ exp

444

tanh

𝜅ℎ

(2)

445

Γ

446

where Γ and Γ are the Gouy-Chapman coefficients of the positively charged modified

447

cellulose surface and negatively charged bacterial surface, calculated from their surface

448

potentials using equation (3), the surface potential of E. coli is -0.03 V at pH 7.0 and 10 mM

449

salt concentration 43. 𝜅

450

ℎ is the separation distance between the charged surfaces. Figure 6 shows that the attraction

𝒌

(3)

is the Debye- screening length, which is around 3 nm at 10 mM NaCl,



451

force presented as pressure (F/area, kPa) increases exponentially with decreasing distance

452

between surface and bacteria and that the higher the surface charge, the greater is the adhesive

453

force. Assuming the bacteria are attached on the charged surface h = 0, the pressures between

454

the surface and bacteria are summarized in Table 4. The interaction force between the surface

455

and bacteria can be roughly estimated based on the contact area, however, this area depends

456

on the degrees of attraction on differently charged surface, a greater pressure leads to a larger

457

contact area. Since the values of Young’s modulus of E. coli that had been reported in literature

458

have a huge variation among different studies (range from 0.05 – 221 MPa)44, there is not

459

possible to obtain an accurate contact area value in this pressent studie. Nevertheless, it is

460

possible to assume that even a small contact area of 1.510-13 m2 (LW = 1.510-6 m  1.010-

461

7

462

surface with the highest surface charge is up to 50 nN. If the actual contact area reasonably

463

surpass this assumption, the attraction force will be even higher than 50 nN. E. coli is a rod-

464

shape bacterium, whose outer cell wall membrane is approx. 8 nm thick and it consists of

465

lipopolysaccharides and phospholipids 42. These dimensions are hence of the same magnitude

466

as the roughness of the cellulose surfaces, as shown in Table 2. Although the cellulose model

467

surface is relatively smooth with respect to the scale of the bacterial cell, it is still “rough” with

468

respect to the thin bacterial cell membrane, and this effects the pressure on the cell membrane

469

when the surfaces approach each other. Francius et. al 46 investigated the properties of E. coli

470

using the AFM colloidal probe technique and a force of 4 to 5 nN was sufficient to disrupt the

471

membrane of the bacteria cell. They explained this result using the Sneddon model:

472

𝐹

473

where 𝐹 is the loading force, 𝛿 the indentation depth, 𝐸 the Young’s modulus, 𝜈 the Poisson

474

ratio, and 𝜉 the tip geometry (semi-top angle of the tip). Based on the same model, Sen et al. 47

m based on the dimension of E. coli cell

𝛿

45

), the interaction force between E. coli and the

(4)


Page 24 of 31

Page 25 of 31

475

also reported a lysis force for a cell membrane of E. coli of 25 nN, which is close to the cell

476

lysis using pyramidal tips in a force range of 14-27 nN as found by Hategan et al

477

therefore suggested that a part of the population of adsorbed bacteria will lose their viability

478

due to the strong interaction forces created by the adsorbed cationic polyelectrolytes and the

479

negative charges on the bacteria. Besides the strong forces, it would be also reasonable that the

480

bacterial cell membrane are significantly weakened based from previous studies of decreased

481

viability of bacteria in cationic polymer solutions,

482

vulnerable under unexpected stress. In our study, the charge probably needs to be increased

483

further to destroy the cell membrane of the entire population. Another approach would be to

484

investigate is the use of stiff, µm-rough surfaces that would create local stresses on the bacteria

485

that would be sufficient to disrupt the cell membrane.

39-41

48

. It is

making the bacteria even more

400 350 Interaction pressure F/area (kPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

110 mV 300 105 mV 250

65 mV

200

60 mV

150

30 mV

100 50 0 0

5

10

15

20

Separation (nm)

486 487

Figure 6: Calculated attraction force (Pressure, F/area kPa) between PVAm/CNF/PVAm-

488

modified cellulose model surface, as the separation distance (nm).

489

Table 4: Interaction forces between charged, LbL-treated surfaces and single cell E. coli



490

Page 26 of 31

membranes.

Surface

1

2

3

4

5

7.29

17.1

19.2

44.5

47.2

127

236

251

346

355

Surface charge (mC/m2) Pressure F/area (kPa) 491 492 493

CONCLUSIONS

494

This work has shown that the LbL technique depositing a 3-layer of PVAm/CNF/PVAm can

495

be used to study antibacterial effect on differently charged cellulose model surfaces. A higher

496

surface charge of the cellulose induces a higher cationic surface potential on the LbL-treated

497

surfaces and these surfaces were able to adsorb and kill more E. coli. The interaction force

498

between the bacteria and the surface was estimated at least 50 nN at high surface charges,

499

corresponding to surface pressures of up to 355 kPa. These interaction forces are sufficiently

500

high to disrupt the cell membrane of E. coli firmly attached on the surface, and the present

501

results are in accordance with earlier indentation measurements on similar bacterial cells.

502 503 504

ASSOCIATED CONTENT


Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

505

Supporting Information

506

The Supporting Information is available free of charge. A figure illustrating the three stages in

507

a force curve when a silica probe approached the cellulose surface, fitting the DLVO theory at

508

Stage 1 to estimate surface potentials; A table presenting the D0 values, the off-set for the

509

fitting procedure; A figure showing the surface potentials obtained by fitting the DLVO

510

theory in symmetrical model; A figure showing the correlation between the surface charge

511

and surface potential.

512 513

AUTHOR INFORMATION

514

Corresponding Author

515

Lars Wågberg, email: [email protected]

516

Monica Ek, email: [email protected]

517 518

Author Contributions

519

The manuscript was written through contributions of all authors. All authors have given

520

approval to the final version of the manuscript.

521 522

ACKNOWLEDGEMENT

523

We thank the Chinese Scholarship Council for financial support and RISE Bioeconomy for

524

supplying carboxymethylated cellulose nano-fibrils (Generation 2). We also acknowledge the

525

Wallenberg Wood Science Centre at KTH Royal Institute of Technology for financial support

526

and technical support with the CPD instrument.

527 528

REFERENCES



529

530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

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