Water purification using functionalized cellulosic fibers with non

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Water purification using functionalized cellulosic fibers with non-leaching bacteria adsorbing properties Anna Elizabeth Ottenhall, Josefin Illergård, and Monica Kerstin Ek Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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Environmental Science & Technology

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Graphic for TOC/Abstract art

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Water purification using functionalized cellulosic fibers with non-leaching bacteria adsorbing properties Anna Ottenhall, Josefin Illergård and Monica Ek* Department of Fibre and Polymer Technology, School of Chemical Science and Engineering, KTH Royal Institute of Technology, Teknikringen 56-58, 114 28 Stockholm, Sweden Key words: Emergency treatment, contact-active material, Layer-by-Layer, polyelectrolyte

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multilayer

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ABSTRACT: Portable purification systems are easy ways to obtain clean drinking water when there

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is no large-scale water treatment available. In this study, the potential to purify water using bacteria

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adsorbing cellulosic fibers, functionalized with polyelectrolytes according to the Layer-by-Layer

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method, is investigated. The adsorbed polyelectrolytes create a positive charge on the fiber surface

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that physically attracts and bonds with bacteria. Three types of cellulosic materials have been

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modified and tested for the bacterial removal capacity in water. The time, material-water ratio and

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bacterial concentration dependence, as well as the bacterial removal capacity in water from natural

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sources, have been evaluated. Freely dispersed bacteria adsorbing cellulosic fibers can remove

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greater than 99.9 % of Escherichia coli from non-turbid water, with the most notable reduction

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occurring within the first hour. A filtering approach using modified cellulosic fibers is desirable for

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purification of natural water. An initial filtration test showed that polyelectrolyte multilayer

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modified cellulosic fibers can remove greater than 99 % of bacteria from natural water. The bacteria

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adsorbing cellulosic fibers do not leach any biocides, and it is an environmentally sustainable and

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cheap option for disposable water purification devices.

ACS Paragon Plus Environment

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INTRODUCTION Clean drinking water is one of the basic needs of survival, and the lack of safe drinking water is a

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large global health issue. One of the ten most common causes of death globally is diarrheal diseases,

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which are often caused by fecal bacteria such as Escherichia coli or Vibrio cholerae that are spread via

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contaminated, non-treated drinking water.1 Effective sanitation systems and sterilization of drinking

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water are essential for preventing water-borne diseases from spreading, especially because even

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small doses of pathogen bacteria can lead to fatal diarrheal diseases.2

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Point-of-use (POU) water treatments, i.e., portable water purification devices, are a way to obtain

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safe drinking water when there is no large-scale water treatment infrastructure available, e.g., during

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an emergency response or in remote areas.3, 4 There are a few different POU sterilization methods

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utilized today, such as chlorination with or without a flocculation agent, ceramic membrane filters

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and sterilization by sunlight. 5, 6 Although most methods are efficient at removing bacteria, they also

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have drawbacks. In particular, disinfection by chlorine, which is the most common POU method, is

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less efficient when water is turbid. In addition, chlorination of water with organic matter can result

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in hazardous chlorinated carbon compounds that can have long term health effects.7 Another

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important issue with chlorination is the taste of chlorine in the treated water, which makes it

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unpleasant to drink.6 Ceramic membrane filters and sterilization by sunlight is time consuming and

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can take several hours to obtain sufficient volumes of drinking water. The efficiency of sterilization

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by sunlight can also be reduced if the water is too turbid.5 New methods and new approaches are

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needed for easy, cheap and safe POU sterilization to complement the existing techniques.

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Bacteria adsorbing cellulosic fibers are an interesting alternative to the currently available POU

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methods since cellulose is a relatively cheap, lightweight and biodegradable material, which can be

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incinerated or possibly composted after use to remove pathogenic bacteria. These are all important

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properties when developing disposable devises for POU purification of water emergency situations.

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Cellulose is the most abundant polymer on Earth and is mainly found in plant fibers, e.g., in wood,


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and is used for making many of the products used on a daily basis, such as paper and textiles. A new

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POU product has recently been launched on the market, Safe Water Book (Folia Water, Pittsburg,

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PA, USA), which sterilizes drinking water by filtering it through a cellulosic filter infused with silver

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nanoparticles that kill bacteria.8, 9 Similar to most commercially available antibacterial materials,

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Safe Water Book utilizes the release of biocides to achieve the antibacterial effect, which is often

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referred to as controlled-release surfaces.8-10 Although silver is an effective bactericide and sterilizes

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water, it also has considerable drawbacks. Silver nanoparticles are dangerous for organisms that live

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in water, and research has shown that fecal bacteria that are exposed to silver can develop a

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resistance to silver and even antibiotics. 11-13,14, 15 Still, new concepts for POU sterilization methods

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based on releasing silver ions or silver particles to kill bacteria in water are continuously developed.4,

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6, 8, 16

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mechanism, i.e., where the antibacterial functionality is permanently attached to the material

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surface, which prevents leaching of the antibacterial agent into the water.10 Several studies have been

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performed on contact-active bacteria adsorbing cellulosic fibers where the fibers have been

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equipped with antibacterial agents.10, 17-19 One of the interesting modification techniques used to

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achieve the bacteria adsorbing effect is to introduce a highly positive charged fiber surface via Layer-

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by-Layer (LbL) modification. In this technique, charged polymers, polyelectrolytes are continually

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absorbed to form polyelectrolyte multilayers on any charged surfaces to provide the desired

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properties. 10, 20-22 The process is usually carried out in water and at room temperature, making it

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more environmentally friendly than other cellulose modifications that covalently link polymers to

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the fibers in organic solvents during harsh conditions.17 The bacteria adsorbing effect is commonly

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achieved by having a highly positive charged polyelectrolyte in the final layer of the multilayer

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modification. This creates a net positive surface charge that will attract and bond with bacteria upon

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physical contact, as the cell envelope of both Gram-positive and Gram-negative bacteria has a

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negative net charge. This makes the contact-active bacteria adsorbing cellulosic fibers efficient for

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many different pathogens and prevents the bacteria from easily developing resistance to the contact-

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active material.10, 23 A polymer that had previously been shown to possess excellent antibacterial

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properties when applied on cellulosic fibers is polyvinylamine (PVAm). Illergård et al. showed it was

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possible to achieve an antibacterial effect greater than 99.9 % on E. coli after 4 hours of incubation

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by the use of treated cellulosic fibers while not leaching any polyelectrolytes.10, 17 Contact-active

A better alternative than releasing biocides would be to use materials with a contact-active


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bacteria adsorbing cellulosic fibers, generated by the LbL technique, could therefore be a better

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alternative for water purification than using materials that leach biocides, both from an

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environmental and a toxicological perspective.

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This article investigates the possibility of a novel method to purify bacterial contaminated water by

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combining a non-leaching bacteria adsorbing modification, using polyelectrolyte multilayers, with

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disposable cellulosic material and it is the first one to evaluate the bacterial removal efficiency of this

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multilayer modification on natural water samples. These are the first steps towards developing a

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disposable POU method for sustainable water purification in emergency situations.

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EXPERIMENTAL METHODS

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Materials and chemicals

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Bleached chemical softwood pulp fibers (hereinafter referred to as cellulose fibers) were supplied by

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SCA Hygiene Products (Mölndal, Sweden), and commercially available coffee filter paper

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(hereinafter referred to as filter paper) made of bleached cellulosic fibers was obtained from

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Klimabolaget AB (Staffanstorp, Sweden). Cationic polyvinylamine Lupamin 9095 was supplied by

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BASF SE (Ludwigshafen, Germany) with a molecular weight of 340 kDa, and anionic polyacrylic acid

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was obtained from Sigma-Aldrich (Stockholm, Sweden) with a reported molecular weight of 240

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kDa. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) was obtained from Sigma-Aldrich (Stockholm,

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Sweden). Filter paper from the POU purification device Safe Water Book (Folia Waters, Pittsburgh,

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PA, USA) was used as a commercial reference.

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Methods

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Pretreatment with TEMPO-oxidation

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TEMPO-oxidation, which occurs when the primary hydroxyl group in the glucose chain is oxidized

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into carboxyl groups, was performed on a fraction of the fibers to increase the negative net charges

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and thereby increase the polymer adsorption.24, 25 An scaled up version of Saito et al. TEMPO-

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oxidation was used, in which 20.0 g of cellulose fibers were purified during constant stirring for 1

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hour at 60°C in an acetate buffer solution at a pH of 4.6 and a NaClO2 concentration of 3.00 g/L.25

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The cellulose fibers were rinsed in deionized water (dH2O) and thereafter oxidized over 4 hours at


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60°C in a phosphate buffer solution at a pH of 6.8, containing 10.50 g/L NaClO2, 8.33 g/L NaClO and

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0.17 g/L TEMPO. The oxidation reaction was terminated by rinsing the cellulose fibers in dH2O.

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Polymer modification

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Cellulose fibers, TEMPO-oxidized cellulose fibers (hereinafter referred to as TEMPO cellulose) and

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filter paper were modified with three layers of polyelectrolytes using the LbL adsorption method.

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The first and third final layers of polyelectrolytes were cationic PVAm, and anionic PAA was

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adsorbed as the second middle layer. The polyelectrolyte adsorptions were performed at a 1 w/w %

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material consistency in dH2O. The polyelectrolytes were adsorbed over 10 minutes in a 0.1 g/L

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polymer solution with 100 mM NaCl added. The PVAm was adsorbed at a pH of 9.5 and the PAA was

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adsorbed at a pH of 3.5. The material was thoroughly rinsed in dH2O between the adsorption steps.

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To increase the available mobile charges of the final material, the pH of the modified material was

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rinsed with dH2O set to a pH of 3.5.26 The cellulose fibers and TEMPO cellulose were thereafter

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freeze-dried, while the filter paper was dried at ambient temperature.

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Fiber size measurement

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The size of the cellulose pulp fibers was determined to get an approximation of the average fiber

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surface area. The length (l) and width (w) of the fibers were measured using L&W Fiber Tester Plus

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(ABB Lorentzen &Wettre Products, Kista, Sweden) and the results were weighted against the fiber

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area (A) according to Eq. 1 and 2.

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=

∑ × ∑

Eq. 1

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=

∑ × ∑

Eq. 2

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Scanning electron microscopy

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The morphology of the cellulosic materials was visualized by scanning electron microscope (SEM)

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micrographs, using a S-4800 field emission scanning electron microscope (Hitachi, Tokyo, Japan).

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The specimens were dried in a desiccator over night and sputtered with a platinum/palladium


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coating using a 208HR high-resolution sputter coater (Cressington, Watford, United Kingdom) prior

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to the SEM analysis to reduce specimen charging.

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Nitrogen analysis

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Nitrogen analysis was performed using ANTEK 700 nitrogen analyzer (Antek Instruments, Houston,

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TX, USA) using 6 mg of material per measurement, and 6 measurements were performed for each

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material. The amount of PVAm adsorbed to the materials can be estimated by measuring the

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nitrogen content in comparison with the reference material, as the positively charged polymer is the

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only nitrogen containing substance added in the modification process.

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Bacterial reduction

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Microbiology tests were performed with the non-pathogenic bacterial strain Escherichia coli ATCC

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11775, obtained from SIK (Gothenburg, Sweden), as a model bacteria and the number of viable

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bacteria in water was determined as colony-forming units (CFU) per mL. The bacterial reduction

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capacity of the material was tested and duplicated by shaking 0.10 g of material in dH2O or in a

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saline ¼-strength Ringer’s solution with an E. coli concentration of 106 CFU/mL for 4 hours at

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ambient temperature and estimating the remaining bacteria in suspension by cultivation thereafter.

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The number of bacteria removed from the suspension was calculated by subtracting the cultivation

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results from the initial bacterial concentration. The bacteria suspensions were cultivated in duplicate

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on Petrifilm (3M, Sweden), and the CFU were calculated using the ImageJ image-analysis tool.27

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Dependence of material-water ratio

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The dependence of the material-water ratio was evaluated using the same method as the bacterial

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reduction test, but the volume of the bacterial suspension was varied while the bacterial

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concentration was kept constant at 106 CFU/mL or 105 CFU/mL. The amount of TEMPO-oxidized

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cellulose fibers were kept constant at 0.10 g and 10 mL, 25 mL, 50 mL and 100 mL of bacterial

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suspension were used for the bacterial reduction test.

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Dependence of bacterial concentration

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Different initial bacterial concentrations were tested while the volume was kept constant at 10 mL,

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and the amount of fibers was kept constant at 0.10 g. The procedure was similar to the bacterial


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reduction test except that the initial bacterial concentrations tested were 104, 105, 106 and 107

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CFU/mL.

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Dependence of time

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As described in the bacterial reduction test, 0.10 g of material was added to 10 mL of dH2O with a

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bacterial concentration of approximately 106 CFU/mL. The bacteria were incubated with the material

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on a shaking table at room temperature, and samples were collected in 1-hour increments over a 4-

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hour period and cultivated on Petrifilm.

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Water samples from nature

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A total of three different water samples were collected in nature: a water sample from the Mississippi

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River (New Orleans, LA, USA); a water sample from Nybroviken (Stockholm, Sweden); and a turbid

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water sample from a ditch (Vallentuna, Sweden). The initial bacterial concentration in the samples

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was determined through cultivation. The bacterial reduction capacities of the modified materials

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were tested with the three natural water samples using the bacterial reduction test. The turbid ditch

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water sample, which contained a significant amount of clay, was filtered through a qualitative

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Munktell filter paper, grade 3 (Ahlstrom Falun AB, Falun, Sweden) to remove coarse particles before

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it was used in the bacterial reduction test.

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Water filters were created by adding 0.7 g of cellulose fiber, unmodified and LbL modified, to 12 mL

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plastic syringes with a diameter of 15 mm. A water sample from Nybroviken (Stockholm, Sweden)

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was used for the filtration test where 10 mL of the natural water sample was filtered through the

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cellulose fiber filter and the filtrate was cultivated on Petrifilm to determine the efficiency of the

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filtration. Filter paper from the Safe Water Book was used as a commercial reference for the

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filtration test. All tests were performed using duplicate samples.

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RESULT

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A new method for POU water purification was investigated using cellulosic pulp fibers and filter

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paper modified with PVAm/PAA/PVAm multilayers to obtain bacteria adsorbing properties. This

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modification method yields a non-leaching, contact-active material that had previously been found

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to attract large amounts of bacteria when applied to cellulosic pulp fibers.17 TEMPO oxidized pulp


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fibers were included in the study, as this pretreatment has previously been shown to give an

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increased bacteria adsorbing effect.10 To evaluate the suitability of these materials for a POU water

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treatment, characterization for polymer content (nitrogen analysis) and bacterial assays were

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conducted, using E. coli as a model bacteria as well as natural water samples.

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Fiber size measurement

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The size of the cellulose pulp fibers was determined to get an approximation of the surface area on

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fibers. The average fiber length was 2.209 mm and the average fiber width was 32.1 μm, both

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weighted towards the fiber area. The average surface area of one fiber is then approximately 0.14

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mm2.

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The length of E. coli varies between 1.6 to 3.1 μm and the average width is between 0.7 to 1.1 μm, but

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the bacteria size varies depending on bacteria strain and cultivation conditions.28 If the surface area

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occupied by one E. coli on the cellulose fiber is roughly estimated to 3 μm2, then approximately 46

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000 bacteria could theoretically fit in an optimized monolayer on an average cellulosic pulp fiber.

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The number of fibers per gram of pulp is usually somewhere between 2.3 to 5.2 million fibers per g

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for softwood pulp, as the one used in this study.29 If 46 000 bacteria could fit on one fiber, then 1 g of

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pulp fibers could adsorb approximately 1011 E. coli. That imply that 1 g of modified pulp could

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theoretically remove all bacteria from 100 L of water, if the bacterial concentration of the water was

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106 CFU/mL. These are theoretical estimations, based on optimal bacteria attachment, but it could

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be interesting when estimating the potential of using freely dispersed fibers in a POU water

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purification process.

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Nitrogen analysis

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Nitrogen analysis was performed to estimate the amount of adsorbed cationic PVAm on the

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modified materials, as PVAm is the only nitrogen-containing compound added in the LbL process.

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The modified TEMPO oxidized cellulose fibers were expected to have a higher nitrogen content than

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the modified cellulose fibers because the TEMPO oxidation increases the net negative charge of the

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fiber and thereby increases the possibility to adsorb cationic PVAm.10, 25 Taking large variations into


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account, no significant difference in the nitrogen content was observed for the three different

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modified materials, i.e., the amount of adsorbed PVAm was approximately the same for all materials

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(Figure 1). However, the nitrogen content in the TEMPO cellulose fibers was much lower than the

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results presented in the study by Illergård et al., which indicated the potential to adsorb more

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cationic PVAm onto the oxidized fibers and enhance the bacteria adsorbing effect.10 The unmodified

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reference cellulose fibers and unmodified TEMPO oxidized cellulose fibers did not contain any

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nitrogen, while the unmodified reference filter paper did contain a small amount of nitrogen, which

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is probably because it is treated to increase the wet strength of the filter.10 The results of the nitrogen

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content in the modified materials have large variations, as shown by the error bars in Figure 1. This

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behavior has been observed in previous studies for multilayer modification with PVAm and could be

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caused by an uneven distribution of PVAm on the cellulose fibers.10

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Figure 1. Result from the nitrogen analysis for modified and reference material. The error bars represent a 95 % confidence interval.

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Scanning electron microscopy

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The macrostructure of the cellulose fibers and the filter paper can be seen in the SEM micrographs

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at x150 magnification (Figure 2). It can be seen that the fibers in the filter paper are tightly packed

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together, resulting in less surface area available for bacteria adsorption compared to the free

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cellulose fibers. It is debated how the morphology of a surface affects bacterial adhesion, some

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researcher claim that bacteria adhesion is promoted with an increased roughness of the surface as it

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will increase the specific surface area available for attachment, other studies have shown that a

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change in the surface roughness do not affect the bacteria adsorption.30 The morphology of the


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adsorbed polyelectrolyte multilayer is however difficult to visualize since the thickness of the

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polyelectrolyte multilayer is in the nanometer range and the cellulose fibers have an average width

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of 32.1 μm.31 A previous study by Lingström et al. has shown that adsorbed multilayers of

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polyelectrolytes on cellulose fibers can be visualized in environmental SEM as a smoothening of the

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surface.32 They used 11 polyelectrolyte layers and concluded that the roughness of the fiber surface

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decreased when the polyelectrolytes were adsorbed. A similar smoothening effect of the fiber surface

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could possibly be detected in the high magnification SEM micrographs of the cellulose fiber. No

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difference on the fiber surface could be seen on fibers in the filter paper, before and after the

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polyelectrolyte adsorption.

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Figure 2. SEM micrographs showing the macrostructure and fiber surface of the cellulosic materials. a) Unmodified cellulose fibers at x150 magnification, b) surface on unmodified cellulose fiber at x5000 magnification, c) surface on 3L modified cellulose fiber at x5000 magnification, d) unmodified filter paper at x150 magnification, e) surface on fiber in unmodified filter paper at x5000 magnification and f) surface on fiber in 3L modified filter paper at x5000 magnification.

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The bacterial removal capacities of the modified materials were tested in dH2O and ¼-Ringer’s

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solution. Filter paper from Safe Water Book, containing silver was used as a commercial reference.

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The initial bacterial concentration in this study was set to 106 CFU/mL, and all materials were

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compared with non-modified material, which showed no bacterial reduction. The bacterial

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reduction test showed that more than 99.9 % of the initial bacteria are removed after 4 hours of

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incubation with the polyelectrolyte modified materials (Figure 3). The highest bacterial removal

Bacterial reduction


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capacity, which was 99.98 % of the initial bacteria after 4 hours, was noted for the cellulose fibers in

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dH2O and TEMPO cellulose in ¼-Ringer’s solution. The commercial reference reduced the viable

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bacteria with 100 % in dH2O and 99.996 % in ¼-Ringer’s solution.

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Figure 3. The percentage of removed bacteria from suspension after 4 hours of incubation with modified materials, in dH2O and ¼-Ringer’s solution. SWB is the commercial reference Safe Water Book, 3 L Cellulose is the modified cellulosic fiber, 3 L TEMPO is the modified TEMPO-oxidized cellulosic fiber and 3 L Filter is the modified filter paper. The initial bacterial concentration was 106 CFU/mL and the error bars represent the standard deviation.

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Previous investigations of the bacteria adsorbing effect of multilayer-treated fibers have mainly been

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conducted using ¼-strength Ringer’s solution to provide a more beneficial environment for the

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bacteria.17, 33 The ¼-strength Ringer’s solution used in this study had a pH of 6.45 and the dH2O had

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a pH of 5.10. It is still unclear how the ion strength affects the bacterial adhesion.34 Some studies

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report that increasing the ionic strength will increase the bacterial adhesion, while others report the

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opposite.35-37 The bacterial reduction test shows a difference in the bacterial reduction for the

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cellulose fibers when using ¼-Ringer’s solution compared to using dH2O, but no notable difference

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was observed for the TEMPO cellulose and for the filter paper (Figure 3). Illergård et al. reported

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that no difference in bacterial removal was observed when analyzing the bacterial removal efficiency

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of the multilayer modified cellulosic fibers in different salt concentrations.24 The ions in water will

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interact with the charged polyelectrolytes, but bacterial adhesion to surfaces is complex and is

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affected by many different parameters, e.g., salt concentration, pH, type of bacteria, etc.

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Time dependence

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The time it takes to remove the bacteria is an important parameter when treating drinking water.

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The number of remaining bacteria in suspension after 1, 2, 3, and 4 hours of contact-time were

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therefore evaluated. The test shows that all modified materials greatly decreased the bacterial

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concentration after only one hour of contact-time (Figure 4). The TEMPO cellulose removed

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99.89 % of the bacteria from the suspension within the first hour while the cellulose fibers and the

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filter paper removed 99.65 % and 98.03 % of the bacteria during the first hour, respectively. The

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filter paper needed more time to remove the same number of bacteria as the cellulose fibers and the

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TEMPO cellulose. The reason for this is most likely attributed to the cellulose and TEMPO cellulose

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fibers being freely dispersed in the water compared to the fixed fibers in the filter paper. This

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difference will increase the probability for the material to attract and bind to the bacteria. The

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results from the time dependence test are promising, as many techniques used to purify water

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require several hours to obtain safe drinking water. 5

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Figure 4. The results from the time dependence test showing the percentage of bacteria remaining in suspension in hourly increments after incubation with modified materials. The initial bacterial concentration was 106 CFU/mL. The error bars represent the standard deviation.

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Concentration dependence

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The concentration dependence test shows that the number of bacteria remaining in the suspension

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depends on the initial bacterial concentration (Figure 5). The number of remaining bacteria in the

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water increases significantly when the initial bacterial concentration increases from 106 CFU/mL to


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107 CFU/mL, but the percentage of bacteria removed from the suspension also increases when the

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initial bacterial concentration increases. This is an effect of a large increase initial bacterial load

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compared to the increase of remaining bacteria. The amount of remaining bacteria is approximately

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6 times larger for the initial concentration of 107 CFU/mL compare to the concentration of

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105 CFU/mL. The increase in initial bacterial load is however 100 times larger when increasing the

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bacterial concentration from 107 CFU/mL to 105 CFU/mL, resulting in higher bacterial removal

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efficiency for 107 CFU/mL. A similar effect of increase in removal efficiency has been seen in a

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previous study when the bacterial load was increased for contact active materials, where it has been

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suggested that the amount of bacteria remaining in suspension at lower concentrations could be an

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equilibrium effect.24 This might be avoided if using the materials in cross flow filtration mode. The

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increase in remaining bacteria in suspension when the initial concentration increase to 107 CFU/mL

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could be a result of increased saturation of the bacteria adsorbing surface of the fibers.

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Figure 5. Result from the concentration test showing the number of remaining bacteria in suspension and the percentage of removed bacteria after 4 hours of incubation with 0.1 g modified TEMPO cellulose. The initial bacterial load ranges from 104-107 CFU/mL. The error bars represent the standard deviation.

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Material-water ratio dependence

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The material-water ratio dependence test shows that the bacterial reduction efficiency increases

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when the material-water ratio increases (Figure 6). However, the results show that it is possible to

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use 1/5 of the amount of modified materials compared to previous tests, i.e., 0.2 w/w % material-

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water ratio, and still obtain a bacterial reduction greater than 99.9 %. The difference in bacterial

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reduction for the different ratios is small compared to the initial bacterial concentration.


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Specifically, only 0.16 percentage points of bacterial reduction efficiency differs between the smallest

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and largest water volume tested. These results indicate that it may be possible to use less material

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and scale-up the water treatment method.

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Figure 6. Result of the material-water ratio dependence, showing the percentage of removed bacteria vs. the material-water ratio after 4 hours of incubation with modified TEMPO cellulose. Ci is the initial bacterial concentration, the light grey bars are Ci =105 CFU/mL and the dark gray bars are Ci =106 CFU/mL. The error bars represent the standard deviation.

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Water samples from nature

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To get a more realistic lab set-up, the modified materials bacterial removal capacities were tested

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with three different water samples from nature. It is known that chlorine is less efficient for

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sterilization of turbid water with a significant amount of particles; thus, it is important to test water

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treatment techniques with water samples containing different turbidities.38 The water sample from

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the ditch water contained approximately 8 x 103 CFU/mL, the Mississippi River contained

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approximately 1.5 x 105 CFU/mL, and the water sample from Nybroviken contained approximately

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2 x 104 CFU/mL and (Table 1). Natural water samples can contain a large number of different

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microorganisms and the colony growth on the cultivation on Petrifilm indicates that the water

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samples contained different colony forming bacteria.

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When tested in the bacterial reduction assay, the bacterial concentration of the Mississippi River

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water sample was reduced by 97 % with the cellulose fibers, 95 % with the TEMPO cellulose and

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94 % with the filter paper (Table 1). The bacterial content in the sample from Nybroviken was

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reduced by 93 % with modified the cellulose fibers and 91 % with the TEMPO cellulose, while the

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bacterial reduction was only 68 % of the initial concentration for the water sample treated with filter

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paper after 4 hours of incubation. No significant reduction in bacterial concentration was observed

386

for the ditch water sample treated with the modified materials, the bacterial reduction was below

387

60 % of the initial concentration.

388 389 390 391

Table 1. Result from the bacterial reduction test with natural water samples, shown as bacterial removal efficiency after 4 hours of incubation with the modified materials. Water sample

Initial bacterial conc. [CFU/mL]

Cellulose fibers

TEMPO cellulose

Filter paper

Ditch water

8 x 103

< 60 %

< 60 %

< 60 %

97 %

95 %

94 %

93 %

91 %

68 %

Mississippi River

1.5 x 10

5

water Nybroviken water

2 x 104

392 393

Water from Nybroviken was collected at a later occasion to be used for the filtration test. This time

394

the water sample had a bacterial concentration of approximately 130 CFU/mL, which was possible to

395

detect through cultivation. The cellulose fiber filter, containing 0.7 g fiber, had a thickness of 20 mm

396

and a density of approximately 0.20 g/cm3. The three layer modified filter removed greater than 99

397

% of the bacteria compared to the unmodified filter that removed 43 % of bacteria from the water

398

sample (Figure 7). The commercial reference removed greater than 99 % of the bacteria in the

399

natural water sample.


15


Page 16 of 22

400 401 402 403 404

Figure 7. Result from the filtration test using natural water from Nybroviken. The water filter contained 0.7 g of cellulose fibers, unmodified or modified with the LbL technique. SWB is the commercial reference Safe Water Book. The error bars represent the standard deviation.

405

DISCUSSION

406

The bacteria adsorbing cellulosic materials have capacity to remove more than 99.9 % of bacteria in

407

water with an initial bacterial concentration of 106 CFU/mL. The main reduction of bacteria is

408

performed within one hour, and a 0.2 w/w % material-water ratio can be used to purify water as long

409

as contact between the bacteria adsorbing material and the bacteria is maintained. These results

410

present the initial stages of developing an environmentally friendly and low cost disposable POU

411

treatment. The modified materials used in this study do not release any biocides to water, which is a

412

significant advantage compared to other sterilization methods.7, 8, 10

413 414

Modified TEMPO cellulose was the most efficient material for removing bacteria from water,

415

followed by modified cellulose fibers and then the modified filter paper. The cellulose fibers were

416

slightly better at removing bacteria from water than the filter paper, which is potentially attributed

417

to the cellulose fibers being freely dispersed in the water compared to the fixed fibers of the filter

418

paper. This gives the cellulose fibers a larger active adsorption area and makes it easier for the fiber

419

to attract and bond with the bacteria. The material-water ratio dependence test indicates that it is

420

very important to have good circulation of the fibers to attain an efficient bacterial removal when

421

using larger volumes of water. Contact-active materials need to maintain contact with the bacteria in

422

order to affect the bacteria, e.g. to adsorb them to a contact-active surface. It is more difficult for the

423

fibers to attract and bond with the bacteria when the average distance between the fibers and the


16

Page 17 of 22


424

bacteria increases, e.g., when the fiber concentration decreases with larger volumes of water, which

425

is an issue that could possibly be resolved by filtering water through the modified material. The

426

advantage of having the fibers freely dispersed in the water is that it allows bacteria to adsorb an all

427

parts of the fibers. The cellulose fibers have a very large specific surface area that could adsorb large

428

amount of bacteria, giving an excellent bacterial removal capacity per gram of fibers, if the bacteria

429

adsorption was optimized. The fibers need however to be removed from the water after the

430

purification process, and this implies that a filtration step will probably be required. One way to

431

avoid this two-step process is to create filters from the bacteria adsorbing cellulosic fibers. This will

432

decrease the surface area available for bacterial adsorption but it will make the fibers easier to

433

handle and use for water purification. Filtering will also improve the performance of the bacteria

434

adsorbing materials for natural water.

435 436

The water samples from nature showed that the modified materials are less efficient at removing

437

microorganisms from real-life water samples, when using the materials dispersed in the water,

438

compared to removing bacteria from bacterial suspensions prepared in the lab. The modified

439

cellulose fibers removed 97 % of the bacteria in the Mississippi river water sample and 93 % of the

440

bacteria in the Nybroviken water sample, while no reduction was observed for the turbid ditch water

441

sample. One explanation for the reduced bacterial removal efficiency compared to previous test

442

could be that the natural water samples contain particles that shield the bacteria.38 The particle

443

surface provides a good foundation for biofilms that can host and protect microorganisms and

444

thereby prevent the modified fibers from attracting and bonding with the bacteria.39 This has been

445

shown to be an issue for other sterilization methods as well, e.g., chlorination and UV-light

446

treatment.38, 39 Another possible explanation could be the complex mixture of microorganisms in

447

natural water samples, the antibacterial effect of the LbL modification has previously been

448

successfully tested with the gram-positive bacteria Bacillus subtilis, but research on other types of

449

bacteria and microorganisms present in water, e.g. protozoa, would be valuable for understanding

450

the issue.24

451 452

The presence of particles renders the notion of having modified fibers freely dispersed in water

453

insufficient, and it will most likely be desired to remove the particles before exposing the


17


Page 18 of 22

454

contaminated water to the bacteria adsorbing materials during the decontamination process. Using

455

multilayer modified cellulosic materials in filtration mode will both remove larger particles and

456

promote for an increased contact between the bacteria adsorbing material and bacteria in the water,

457

which is critical for an efficient bacterial removal when using a contact-active method. The filtration

458

test using natural water showed that it is possible to improve the bacterial removal efficiency for

459

natural water samples by filtering it through the modified fibers. The water filter containing

460

modified cellulose fibers had a bacterial removal efficiency greater than 99 %, similar to the

461

commercial reference. It can be compared to the filter with unmodified fibers, which removed 43 %

462

of the bacteria from the natural water sample. This indicates that it should be possible to develop an

463

efficient water treatment method if the modified materials are used in filtration mode to remove

464

both bacteria and particles that impair bacteria removal. Removing particles from water would also

465

decrease the turbidity and make the water more pleasant to drink, which is an important parameter

466

when promoting the use of POU water treatments in developing countries, especially for children. It

467

should be noted that the bacterial concentration, possible to detect through cultivation, was

468

considerably lower for the water sample collected for the filtration test compared the high bacterial

469

concentration determined for the water sample collected for the reduction test, with the material

470

dispersed in the water, even though the water samples were collected at the same spot but at two

471

different occasions.

472 473

There is little data in literature about bacteria adsorbing POU methods, most research has been

474

focused on leaching materials that require a slightly different experimental setup. The commercial

475

reference used in this study inactivated all bacteria in dH2O and had a very high bacterial reduction

476

in ¼-strength Ringer’s solution. This was expected since the reference filter contains silver, which is

477

known to be an efficient bactericide and the 4 hour incubation will allow plenty of time for bacterial

478

inactivation, provided that the concentration of silver is high enough.15 Dankovich et al. reported a

479

bacterial log reduction value of 6 for cellulosic filters impregnated with silver particles, similar to the

480

commercial reference, which could be compared to a log reduction value of 3-4 for the bacteria

481

adsorbing cellulosic fibers modified with multilayers.8 However, the study evaluating the silver

482

infused filter paper used a higher initial bacterial concentration, 108 CFU/mL, but the number of

483

remaining bacteria in the effluent water was in the same range as for the contact-active cellulosic


18

Page 19 of 22


484

fibers.8 Zeng et al. reports that they obtained a bacterial reduction over 97 % when filtering water

485

containing E. coli with a concentration of 105 CFU/mL through bactericidal hydrogel filters made

486

from reduced graphene oxide and silver, which is far less efficient than the bacteria adsorbing

487

cellulosic fibers outlined in this study.16

488 489

Cellulose is a natural biomaterial that is relatively cheap and safe to handle, and combined with the

490

non-leaching bacteria adsorbing modification, it presents an environmentally friendly option for

491

current POU treatment techniques based on releasing toxic compounds. Cellulosic fibers are great

492

for making paper sheets and are currently used in a variety of filters ranging from simple coffee

493

filters to advanced fuel and air filters in cars. The functionalized cellulosic fibers can be processed

494

into paper sheets, which could be used for water filtration. The water filter would then rely on

495

physical removal of bacteria through electrostatic interactions and should be able to allow a higher

496

flux compared to filters relying on size exclusion of bacteria. However, the pore size of the filters

497

must still be small enough to obtain sufficient contact between the functionalized fibers and all

498

bacteria in the water, as it is critical for an efficient contact-active POU technique.

499 500

There are potentially more advantages using a contact-active material for water purification than

501

only the removal of bacteria, as most impurities found in natural water are negatively charged.

502

Cationic polyelectrolytes are often used in water treatment as they have been proven to be the most

503

effective flocculent agent.40 The cationic PVAm on the modified cellulosic fibers could possibly

504

remove other impurities from water in addition to removing bacteria. Mayer-Gall et al. successfully

505

used polyester textile functionalized with PVAm to remove toxic chromate ions from contaminated

506

groundwater. The ability to purify water from other harmful compounds could potentially be a great

507

advantage for the contact-active bacteria adsorbing cellulosic fibers compared to other water

508

treatment techniques based on leaching biocides. The bacterial reduction capacity of the

509

functionalized cellulosic fibers is good and the material could with an optimized product design,

510

preferably cross flow filtration, be a promising alternative for a disposable POU water purification

511

device.

512 513

AUTHOR INFORMATION


19


514

Corresponding Author

515

* Tel: +46 8 790 8104, [email protected], Teknikringen 56-58, SE-100 44, Stockholm, Sweden

516

ORCID ORCID

517 518 519 520

Anna Ottenhall: Josefin Illergård: Monica Ek:

521

ABBREVIATIONS

522

CFU

Colony forming units

523

Ci

Initial bacterial concentration

524

dH2O

Deionized water

525

LbL

Layer-by-Layer

526

PAA

Polyacrylic acid

527

POU

Point-of-use

528

PVAm

Polyvinylamine

529

SEM

Scanning electron microscope

530

TEMPO

2,2,6,6-Tetramethyl-1-piperidinyloxy

Page 20 of 22

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2. Cabral, J. P., Water microbiology. Bacterial pathogens and water. Int. J. Environ. Res. Pub. Health 2010, 7, (10), 3657-3703.

538 539

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Water purification using functionalized cellulosic fibers with non

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