Antimicrobial properties of polysulfone membranes modified with carbon nanofibers and silver nanoparticles

Accepted Manuscript Antimicrobial properties of polysulfone membranes modified with carbon nanofibers and silver nanoparticles Alexandra Mocanu, Edina Rusen, Aurel Diacon, Gabriela Isopencu, Gabriel Mustă�ea, Raluca Şomoghi, Adrian Dinescu PII:

S0254-0584(18)30848-4

DOI:

10.1016/j.matchemphys.2018.10.002

Reference:

MAC 21017

To appear in:

Materials Chemistry and Physics

Received Date: 4 June 2018 Revised Date:

9 August 2018

Accepted Date: 2 October 2018

Please cite this article as: A. Mocanu, E. Rusen, A. Diacon, G. Isopencu, G. Mustă�ea, R. Şomoghi, A. Dinescu, Antimicrobial properties of polysulfone membranes modified with carbon nanofibers and silver nanoparticles, Materials Chemistry and Physics (2018), doi: https://doi.org/10.1016/ j.matchemphys.2018.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Antimicrobial properties of polysulfone membranes modified with carbon nanofibers and silver nanoparticles

RI PT

Alexandra Mocanua, Edina Rusena*, Aurel Diacona, Gabriela Isopencua, Gabriel Mustățeab, Raluca Şomoghic, Adrian Dinescud a

Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania

National Institute of Research and Development for Food Bioresources − IBA Bucharest, 5 Ancuta Baneasa,

SC

b

020323 Bucharest 2, Romania c

National Research and Development Institute for Chemistry and Petrochemistry – ICECHIM, 202 Splaiul

M AN U

Independenţei, 060021 Bucharest, Romania d

National Institute for Research and Development in Microtechnologies - IMT-Bucharest, 126 A, Erou Iancu Nicolae Street, 077190, PO-BOX 38-160, 023573 Bucharest, Romania e-mail: [email protected]

Abstract

The aim of this study was the fabrication of polysulfone membranes with antimicrobial properties. The strategy employed was based on the use of carbon nanofibers (CNF) presenting carboxyl functional groups (CNF-COOH)

TE D

and silver nanoparticles (AgNPs). The materials were investigated by XPS, SEM, TEM, EDX, and ICP-MS. The antibacterial activity was ascertained towards Escherichia coli and Bacillus subtilis both in solid and liquid media employing different synthesis routes for the AgNPs. In solid phase the Gram-positive bacteria showed higher sensitivity for PSF-CNF-Ag membranes, while in liquid phase the antimicrobial activity of the hybrid membrane is

EP

more pronounced towards Gram-negative species. Furthermore, in the case of E. coli, the growth inhibition in liquid medium is probably due to the synergetic action of the modified CNF and AgNPs.

AC C

Keywords: carbon nanofibers, silver nanoparticles, polysulfone membranes, antimicrobial properties

1. Introduction

Improvement of water quality involves the development of several technologies related to

filtration, centrifugation, sedimentation, coagulation and/or flocculation, aerobic and anaerobic treatments, distillation, crystallization, evaporation, solvent extraction, precipitation, ion exchange, which are generally expensive, time-consuming and sometimes unproductive procedures in terms of pollutants retention[1]. Thus, serious efforts in terms of constant development of innovative, energy-efficient, environmentally friendly and low-cost technologies 1

ACCEPTED MANUSCRIPT

for water treatment are directed towards the design of polymer membranes for reverse osmosis (RO), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF) procedures[2, 3]. In this context, polymer chemistry presents an important synthesis leverage for the membranes used in dialysis and electrodialysis for water purification at ionic levels[4].

RI PT

The presence of inorganic nanoparticles such as copper, zinc, zinc oxide, silver, zirconia, silica, titania, etc. embedded in the polymer matrix of membranes improved in many cases the gas permeability, selectivity, hydrophilicity, and magnetic properties[5-7], while the presence of carbon-based materials contributed to the enhancement of thermal and mechanical strength of the

SC

final composite membranes[8, 9].

Furthermore, composite membranes based on multiwall carbon nanotubes (MWCNT)

M AN U

and single wall carbon nanotubes (SWCNT) exhibited antimicrobial properties due to several mechanisms such as: i) cell membrane disruption by direct contact of CNTs with the microorganism cells, ii) direct oxidation of cellular components, and iii) secondary oxidation of cellular lipid bilayer[10].

The antimicrobial activity of carbon nanotubes (CNTs) is different in solid media compared to liquid media. Also, the length of the CNT is essential in the antibacterial activity.

TE D

For instance, the longer tube was found to exert higher bactericidal performance than shorter tubes[10, 11]. In case of SWCNT the shorter length may increase the chances for interaction between open ends of nanotubes and the microorganism, leading to extra cell membrane damage. If the length of MWCNTs reaches up to 50 µm, the tube wrapped around the surface of

EP

microbial cell consequently promotes osmotic lysis of the microorganism[12]. In a liquid medium, longer nanotubes have exhibited higher antibacterial performance than shorter ones.

AC C

Due to van der Waals interactions, it is expected for CNTs to form aggregates that can involve a variable number of pathogen cells depending on the size of the agglomerates. Thus, in a liquid system, the shorter CNTs self-aggregate without involving a large number of microbial cells, while longer agglomerated CNTs affect a larger number of cells caught inside the aggregates[13, 14].

CNF with diameters around 100 nm and length between several tens to hundreds of microns, gained much attention as energy conversion and storage materials, reinforcement agents of composites, sensing, electrical devices, and even catalysts for chemical reduction to obtain

2

ACCEPTED MANUSCRIPT

pharmaceutical compounds[15-19] due to their amazing properties that combine high surface area with flexibility, high mechanical strength and good chemical stability[9, 20]. In terms of biological systems for water treatment, materials modified with CNF registered higher biocompatibility and lower toxicity compared with their counterparts multiwall

RI PT

or single wall carbon nanotubes[10, 21].

Silver has been used for centuries as an antimicrobial agent[22] to fight in infections and it is well known that silver ions and silver-based compounds are highly toxic to Gram-negative and Gram-positive microorganisms[23, 24]. It is generally accepted that free silver ions, present

potential and causing proton leakage[25, 26].

SC

or released from the nanomaterials, can bind cell membrane structures disrupting the membrane

M AN U

One of the major drawbacks of the polymer membranes modified with inorganic nanoparticles compounds consists in the lack of a uniform dispersion/distribution of the nanoparticles in the polymer matrix due to the agglomeration process of the colloidal particles (diameter less than 100 nm)[27, 28].

Thus, the objective of this study was to investigate the antimicrobial activity of polysulfone membranes modified with CNF-COOH and silver nanoparticles (AgNPs) for water

TE D

treatment. Therefore, the antimicrobial polysulfone (PSF) composites membranes were obtained by employing two methods: a) the chemical reduction of a silver salt directly on CNF-COOH surface followed by dispersion in PSF solution (referred as PSF-CNF-Ag in situ); b) mixing of previously synthesized AgNPs dispersion with CNF-COOH dispersion in PSF solution (referred

EP

as PSF-CNF-Ag ex situ). The structured materials (as described in Scheme 1) were investigated by XPS, SEM, TEM, EDX and ICP-MS, while their antibacterial activity to Escherichia coli and

AC C

Bacillus subtilis bacterial cells was monitored both in solid and liquid media.

Scheme 1: Illustration of the membrane structure 3

ACCEPTED MANUSCRIPT

2. Materials and methods 2.1.

Materials

200

µm)

(CNF)

(Aldrich),

polysulfone

beads

RI PT

CNF pyrolytically stripped platelets (conical), (> 98% carbon basis, D × L 100 nm × 20(average

Mn∼22,000)

(PSF)

(Aldrich), acrylic acid (AA) (Merck), dimethyl formamide (DMF) (Merck), 1-N-methyl-2pyrrolidinone (NMP) (Aldrich), silver nitrate (AgNO3) (Aldrich), sodium benzoate (Aldrich),

2.2.

M AN U

97%) (Merck) were used without further purification.

SC

sodium hydroxide (NaOH) (Aldrich), gallic acid (GA) and lauroyl peroxide (LP) (Luperox LP,

Methods

2.2.1. Synthesis of CNF-COOH

The functionalization of CNF was performed by employing a radical solution polymerization reaction in DMF as solvent and AA monomer. Thus, 300 mg of pristine CNF were dispersed in a solution of 8 mL AA and 30 mL DMF. The reaction was kept at 75 °C for 3

TE D

hours under continuous stirring after the addition of 100 mg of LP. The CNF modified with carboxylic groups (CNF-COOH) were recovered after filtration and several washing procedures with DMF and distilled water. The black powder was used after complete drying.

EP

2.2.2. Ex-situ synthesis of AgNPs

Silver benzoate was previously obtained by mixing a solution of 0.4 g AgNO3 in 10 mL

AC C

of water with a solution of 0.34 g sodium benzoate (1:1 molar ratio) in 10 mL of water. The white powder obtained after filtration was dried until constant mass and further used to generate AgNPs in NMP. Thus, gallic acid (0.05 g dispersed in 1 ml NMP) was slowly added to an NMP solution of silver benzoate (0.09 g in 9 mL of NMP) and NaOH for a pH value of 11. After 30 min of continuous stirring 0.125 g of CNF-COOH were introduced and dispersed by sonication. This solution was the used directly for the dissolution of PSF.

4

ACCEPTED MANUSCRIPT

2.2.3. In-situ synthesis of AgNPs In a round bottom flask, 0.125 g of CNF-COOH and 0.09 g of silver benzoate were sonically dispersed in 9 mL of NMP. The solution of gallic acid (0.05 g in 1 mL NMP) was

2.2.4. Synthesis of PSF-CNF-Ag membranes

RI PT

added to generate the AgNPs and the reaction was stirred for 30 min.

To the previous aliquots (described in section 2.2.1 and 2.2.2), 1.25 g of PSF was added in both cases and the mixtures were kept overnight. The membranes were obtained by the wet-

SC

phase inversion method using the method of V. Kaiser et.al. [29]. The casting of the PSF-CNFAg hybrid membranes was performed on glass plates followed by coagulation in deionized water

M AN U

bath. After coagulation, the membranes were kept for 12 hours in distilled water to completely remove the solvent, followed by drying until constant mass was reached.

3. Characterization

FT-IR analysis was performed on pristine and modified CNF using Nicolet 6700 FTIR spectrometer in the range of 4000−400 cm−1.

TE D

The pristine and modified CNF were investigated by XPS analysis performed on a K Alpha instrument from Thermo Scientific, using a monochromated Al Kα source (1486.6 eV), at bass pressure of 2×10−9 mbar. Charging effects have been compensated by a flood gun and binding energy has been calibrated by placing the C 1 s peak at 285 eV as internal standard.

EP

The inorganic particles generated in the presence of modified CNF were analyzed by FEI Tecnai F20 G2 TWIN TEM equipped with EDX X-MaxN 80T detector from Oxford

AC C

Instruments. The samples were dispersed in absolute ethanol and deposited on copper grids. The gun is connected to a high voltage source and the micrographs were obtained at 200 kV. The morphology of CNF and the porous structures of the hybrid membranes have been

investigated by SEM (scanning electron microscopy) using FEGSEM-Nova NanoSEM 630 (FEI).

The experiments for silver migration were carried out using a NexION 300Q ICPMS (Perkin-Elmer Inc., USA) equipped with cross-flow nebulizer and a Quartz torch. ICP-MS NexION instrument software was used to control all instrument operations including tuning, data acquisition and data analysis. Prior to analysis, the ICP-MS was allowed a sufficient period of 5

ACCEPTED MANUSCRIPT

time to stabilize before optimization procedures being carried out. The operating conditions are: Nebulizer Gas flow rates: 0.93 L/min; Auxiliary Gas Flow: 1.2 L/min; Plasma Gas Flow: 16 L/min; Lens Voltage: 7.25 V; CeO/Ce=0.021; Ce++/Ce = 0.020. Silver content from solid samples (initial and final) were digested by microwave heating

the following digestion parameters:

RI PT

in a MWS-2 (Berghoff) microwave oven, using HNO3 65% (4 mL) and 30% H2O2 (1 mL) and Temperature,

Time,

T (°C)

t (min.)

1

160

5

2

220

40

90

3

cooling

20

0

Power, P (%)

SC

Step

80

M AN U

After completing digestion, the residue was filtered and transferred into 25 mL volumetric flask and filled with deionized water (resistivity 18.2 MΩ m and conductivity 0.055µS). The antimicrobial assays were performed against Bacillus subtilis (ATCC 6051a) a Gram-positive bacteria and Escherichia Coli (K-12 MG1655) Gram-negative bacteria. Two methods were used to determine the antimicrobial activity of the composite PSF-CNF-Ag membranes: a. Agar plates were used to determine the inhibition zone of PSF-CNF-Ag membranes against

TE D

B. subtilis and E. coli bacteria. Samples were cut into discs and then placed on LB/NA plates previously inoculated with 100 µL of inoculum containing approximately 107– 108CFU mL-1of cultured bacteria. The plates were incubated at 37°C for 4 days and then

EP

the diameters of the inhibition zones around the PSF-CNF-Ag discs were measured. b. The antibacterial activity of the PSF-CNF-Ag membranes was evaluated against studied

bacteria in liquid media. Test tubes containing 6 mL LB broth were inoculated 100 µL of

AC C

each type of bacteria followed by the immersion of PSF-CNF-Ag discs. All samples were

incubated with untreated control (without membrane disc). After incubation under shaking conditions (150 rpm) at 37°C the optical density (OD) of the samples was measured after each hour at 600 nm length using UV/VIS LLG Spectrophotometer uniSPEC 4.

4. Results and discussion FT-IR analysis was performed on pristine and CNF-COOH. The signals from 3200 cm-1 for the O–H stretch, respectively for C=O of a carboxylic acid appears as an intense band from 6

ACCEPTED MANUSCRIPT

1730 cm-1 were obtained, thus confirming the presence of the carboxylic group on the CNF surface. Further investigation of the pristine and carboxylic CNF consist in the XPS analysis as shown in Fig. 1. The C1s deconvolution sustains the change of C-C sp2 percentage and increase

RI PT

in C-C sp3, thus confirming an increase of the oxygen content to 12.28 % (atomic %) due to the functionalization with poly(acrylic acid) (PAA) (Fig. 1-b)[30]. The functionalization with carboxylic groups is necessary for an improved dispersion in polar media, in order to prevent the relatively strong aggregation of CNF. Signals corresponding to C–C bonds (284.5 eV), π–

SC

π interaction (291.5 eV) and those of oxygen-contained groups such as C–O (287.4 eV) were

M AN U

noticed.

CNF pristine

80000

25000

Atomic % C1s 95.18 O1s 4.82

C1s

C1s

40000

TE D

Counts/s

60000

O1s

20000

total (Counts / s)

20000

15000

10000

5000

0

EP

1200 1000 800 600 400 200

0 0

300

295

290

285

280

Binding energy (eV)

Binding energy (eV)

AC C

(a)

7

ACCEPTED MANUSCRIPT

CNF-COOH 25000

Atomic % C1s 87.72 O1s 12.28

80000

O1s 40000

total (Counts / s)

20000

60000

Counts/s

C1s

C1s

15000

RI PT

100000

10000

20000

0

12001000 800 600 400 200

0

0 300

SC

5000

295

290

285

280

Binding energy (eV)

M AN U

Binding energy (eV)

(b)

Fig. 1. XPS analysis for pristine CNF (a) and CNF modified with carboxylic groups (b)

The morphology of pristine carbon fibers is presented in Fig. 2 revealing not only fibrous structure, but also the presence of large carbon spheres resulted probably from the synthesis of

TE D

the carbonaceous material. The detailed image (Fig. 2-b) reveals nanofibers with a medium

AC C

EP

diameter of around 100 nm.

Fig. 2. SEM images of modified pristine CNF at: a) scale – 1 µm; b) scale – 200 nm

The composite membranes were obtained by employing a phase-inversion method followed by immersion precipitation procedure. In both of our first experiment the main purpose consisted in using the same organic solvent for generating the AgNPs as for dissolving PSF. 8

ACCEPTED MANUSCRIPT

Thus, in the first experiment, the inorganic nanoparticles were generated directly on the surface of modified CNF in NMP solvent assisted by ultrasounds. The second procedure involved mixing of preformed AgNPs and CNF dispersion in NMP. Subsequently, PSF beads were added to the CNF-AgNPs in situ or ex situ dispersions and

RI PT

kept under continuous stirring overnight. The aliquots of PSF with CNF-AgNPs were casted in a non-solvent (distilled water) leaving porous composite membrane films.

SEM analysis performed on the two types of membranes revealed porous structures in

M AN U

SC

both cases (Fig.3 and Fig. 4).

TE D

Fig. 3. SEM analysis for composite membrane of PSF-CNF-Ag-in situ a) cross-section; b) detail image of AgNPs attached on CNF surface

In Fig. 3a the cross-section SEM image of the composite membrane PSF-CNF-Ag-in situ presents a channel-like structure specific to asymmetric filtration membranes[31] filled with

EP

modified CNF (marked with white circles). The AgNPs are strongly attached to the surface of CNF as a result of the synthesis procedure (Fig. 3b). Furthermore, the CNF decorated with inorganic nanoparticles protrude through the walls of the polymer channels without blocking the

AC C

pores confirming a good distribution of the filler. In the case of PSF-CNF-Ag-ex situ the AgNPs are distributed in the whole mass of the

composite membrane. The surface of the membrane is covered by inorganic nanoparticles that are uniformly dispersed between the pores or alongside with CNF (Fig. 4a). As for the case of our previous membrane, PSF-CNF-Ag-ex situ presents also a porous channeled structure with CNF that penetrate the walls of the macropores, but with AgNPs unattached to the fibers (Fig. 4b, respectively Fig. S2 - d, e, f - Supplementary data).

9

RI PT

ACCEPTED MANUSCRIPT

SC

Fig. 4. SEM image of composite membrane PSF-CNF-Ag-ex situ a) top of the membrane; b) porous channels in cross-section

M AN U

The purpose of this study was to obtain polymer ultrafiltration membranes for water treatment with antimicrobial properties. For this reason, our first attempt was concentrated on the investigation of the antimicrobial activity of the membranes by disk diffusion procedure for Gram-positive (B. subtilis) and Gram-negative (E. coli) bacterial strains. The inhibition zone assay on the agar plates was performed towards E. coli and B. subtilis

TE D

bacteria using PSF-CNF membrane (as control) and PSF-CNF-Ag-in situ hybrid membrane. PSF-CNF control PSF-CNF-Ag in situ - E. coli PSF-CNF-Ag in situ - B. subtilis

3.0

2.0 1.5

EP

IZ, (mm)

2.5

1.0

AC C

0.5 0.0

0

20

40

60

80

Time (h)

Fig. 5. The inhibition zones for PSF-CNF control membrane and PFS-CNF-Ag-in situ membrane, against E. coli and B. subtilis

In Fig. 5 the evolution of inhibitory activity of the AgNPs reinforced in the PFS-CNF membrane is presented. It is obvious that AgNPs generated in situ directly on the CNF-COOH surface and embedded in PSF matrix have a higher effect on the growth of E. coli compared to B. 10

ACCEPTED MANUSCRIPT

subtilis development (Fig. 5). In the Petri plate with B. subtilis strain (Fig.S1 – Supplementary data) the surrounding areas of the samples present the halo of Ag diffusion in culture media, phenomenon which limits but not expels the cell growth. Some studies based on metallic inorganic particles and their antibacterial activity

RI PT

evidenced that certain proteins bind specifically to AgNPs with an average diameter of around 30 nm. As a consequence, the enzymatic activity is permanently lost leading to an inhibition growth of Gram-negative bacteria [32]. For instance, tryptophanase (TNase), an enzyme present in E. coli metabolism, was noticed to have an increased affinity for binding to the AgNPs surface, but

AgNPs embedded in composite structures[26].

SC

loses completely its activity upon this association. These results highlight the potential effect of

M AN U

Antimicrobial membranes were also obtained by Zodrow et. al. [33] that dispersed AgNPs ranging from 1 to 70 nm in polysulfone as polymer matrix in order to create a material that inhibits the growth of E. coli cells on the materials surface up to 94%. Compared with their study, in our case, for samples in which AgNPs were generated ex situ (Fig. 6), the antimicrobial activity against E. coli remained constant for up to 80 hours, while for B. subtilis, the antimicrobial activity of the hybrid membrane increased significantly. These results could be

TE D

explained on one hand by the size of the inorganic particles obtained in situ and ex-situ and on the other by the binding of AgNPs to the CNF-COOH. As shown in Fig. S2 (from Supplementary data) the AgNPs generated in situ are strongly attached to the CNF-COOH as confirmed by the TEM images (Fig S2-a,b,c), while the ex situ generated particles can be found

EP

both on CNF-COOH and the carbon grid support (Fig. S2-d,e,f). In both cases, our AgNPs have less than 70 nm, which is in good accordance with previous data that correlate the antimicrobial

AC C

properties with the size and size distribution of the silver nanoparticles [33-37]. The formation of AgNPs was also confirmed by EDX data (Fig. S3 – Supplementary data).

11

ACCEPTED MANUSCRIPT

PSF-CNF control PSF-CNF-Ag in situ - E.coli PSF- CNF-Ag ex situ - E.coli PSF-CNF-Ag in situ - B. subtilis PSF-CNF-Ag ex situ - B. subtilis

9 8 6 5 4 3 2 1 0 0

20

40

60

80

Time (h)

RI PT

IZ, (mm)

7

SC

Fig. 6. The inhibition zones for the composite membranes PSF-CNF- Ag (in situ and ex situ)

M AN U

against B. subtilis and E. coli.

The effects of carbon-based nanomaterials on the living cells are different, concerning the concentration, the length and the orientation of the fibers in the hybrid compounds, causing over 80% cell inactivation at only 2-3 wt% of carbon nanomaterial [12, 14, 38]. Their antimicrobial potential towards both Gram-positive and Gram-negative bacteria can be increased to 99.5 % if metallic nanoparticles are generated on their surface[12]. In our case, the results obtained for the

TE D

PSF-CNF-Ag-in situ membranes exhibit a lower antimicrobial activity compared to PSF-CNFAg-ex situ probably due to a lower diffusion rate of the AgNPs in the solid media. This can be attributed to a rigid binding between CNF-COOH and AgNPs that were generated directly on the surface of the modified CNF. Furthermore, the complex created by CNF-COOH with AgNPs-in

EP

situ, exhibits lower antimicrobial activity probably due to the larger conformation of the aggregates formed by CNF which are more predisposed to entanglement and folding than their MWCNT or SWCNT counterparts[12]. Consequently, the inhibition zones are smaller (Fig. S4 –

AC C

right side - Supplementary data). On the other hand, the PSF-CNF-Ag-ex situ membranes, in which AgNPs are unattached

to the CNF-COOH surface, allow an individual diffusion of AgNPs and CNF respectively (by penetrating the base polymer PSF) and synergetic antimicrobial action leading to larger inhibition zones (Fig. S4 – left side - Supplementary data). To validate the observation regarding the antimicrobial action determined by the different obtained structures, experiments were also carried out in the liquid phase, following the variation of the optical density (OD). Also, the diffusion of AgNPs from the hybrid membranes was 12

ACCEPTED MANUSCRIPT

determined by ICP-MS in order to correlate the concentration of inorganic nanoparticles released in the medium with the concentration of possible viable cells of Gram-positive and Gramnegative bacteria (Fig. 7, respectively Fig. 8). Thus, in liquid phase PSF-CNF-Ag membranes exhibit higher antimicrobial activity

RI PT

against Gram-negative bacteria compared to Gram-positive bacteria. The wall of B. subtilis bacteria may be penetrated by the AgNPs depending on the size of the nanoparticles[39]. Consequently, the OD obtained for B. subtilis growth illustrates a higher development of the biofilm and free cells in the liquid medium and on the surface of the membrane regardless of the

0.25

0.20

0.20

0.15

Concentration of Ag (µg/L)

0.25

35

0.30

M AN U

OD, B. subtilis-PSF-CNF control OD, B. subtilis-PSF-CNF-Ag in situ Release of Ag in situ (µg/L)

0.30

OD

SC

synthesis method applied for AgNPs (Fig. 7).

30 25

0.15

0.10

0.10

0.05

0.05

0.00

20 15

0.00

0

2

4

6

8

10

TE D

Time, h

(a)

EP

0.30

0.30

OD

0.25

AC C

0.35

0.25

0.20

0.20

0.15

0.15

0.10

0.10

0.05

0.05

0.00 0

0.00 2

4

6

24

Concentration of Ag (µg/L)

OD, B.subtilis-PSF-CNF control OD, B.subtilis-PSF-CNF-Ag ex situ Release of Ag ex situ (µg/L)

23

22

21

20

8

Time, h

(b) Fig 7. The OD variation in time, for B. subtilis in the presence of PSF-CNF-Ag membranes a) in situ, b) ex situ

13

ACCEPTED MANUSCRIPT

The concentration of Ag ions released from PSF-CNF-Ag in situ membrane varies from 12.09 to 30.63 µg/L as a quasi-linear increasing function (Fig. 7-a – blue line), while for ex situ membranes the liberation of AgNPs is oscillatory between releasing and adsorption (Fig. 7-b blue line). In the case of PSF-CNF-Ag-in situ membranes the growth of the microorganism is not

RI PT

suppressed by the presence of CNF-COOH and AgNPs (Fig. 7-a- red line) which are deactivated by the exo - biopolymers excretion, while the biofilm formation for the control sample and PSFCNF-Ag-ex situ membranes are similar (Fig. 7-b, black and red line).

Compared to B. subtilis bacteria, the PSF-CNF-Ag membranes determined a higher

SC

growth inhibition of E. coli in liquid medium. After 2 h of exposure the growth of E.coli is limited to a concentration of 4·107 cells/mL, which corresponds to 23 µg/L Ag released in the

M AN U

media. The concentration of cells was estimated based on OD using a correlation method described by Volkmer et. al.[40]. If the exposure time increases the AgNPs no longer influences the bacterial growth. Compared to the control membrane, the PSF-CNF-Ag-in situ membranes exhibit 7% higher bacterial strain inhibition, while the PSF-CNF-Ag-ex situ membranes present 13% increase (Fig. 8 - a,b – black and red lines).

Although the bacterial growth exhibits the same profile of the curve variation, the ICP-

TE D

MS results for Ag diffusion and the OD are increasing up to 6 h, until the threshold concentration of 23 µg/L AgNPs is reached. Subsequently, bacterial development stops and the decline phase is installed.

In the case of PSF-CNF-Ag-in situ, due to the linear releasing of the AgNPs, the

EP

microorganism gains resistance to the toxic components from composite membrane and the curve profile follows the control curve behavior, but at smaller values of OD, with strong

AC C

variation on the decline period of the cellular growth (Fig 8-a). In the case of PSF-CNF-Ag-ex situ, during the exponential period of E. coli growth, the

microorganism inhibition is due to the synergetic action of the CNF-COOH-AgNPs, because in this specific period the AgNPs concentration is small, according with ICP-MS results (Fig.8-b).

14

ACCEPTED MANUSCRIPT

0.030

35

0.028

30

0.030

0.026

0.020

25 20

0.024

0.015

RI PT

OD

0.025

15

0.010

0.022

0.005

0.020

5

0.000

0.018

0

0

2

4

6

8

10

SC

Time, h

Concentration of Ag (µg/L)

OD, E. coli- PSF-CNF control OD, E. coli- PSF-CNF-Ag in situ Release of Ag in situ (µg/L)

(a)

0.032

0.030

24

0.030

0.025

OD

0.028

0.020

23

0.026

0.015

0.024

0.010

22

0.022

0.005

TE D

0.020

0.000

0

2

21

Concentration of Ag (µg/L)

M AN U

OD, E.coli-PSF-CNF-control OD, E.coli-PSF-CNF-Ag ex situ Release of Ag ex situ

0.018 20 4

6

8

Time, h

(b)

EP

Fig 8. The OD variation in time for E.coli in the presence of PSF-CNF-Ag membranes a) in situ, b) ex situ

AC C

As presented in previous studies, a clear differentiation between the Ag ions and AgNPs role in the antimicrobial properties of AgNPs containing materials cannot be clearly made, regardless of the type of bacteria. However, we have shown that a clear important aspect controlling the antimicrobial properties of the materials is the overall Ag ions content in the surrounding media. Considering that the Ag ions release rate is governed by the overall surface area of the particles and by the electrostatic interaction with the close environment, the slower release rate of the in situ AgNPs samples can be explained. Moreover, although the larger particle size of the ex situ AgNPs, their weaker interaction within the composite, leads to higher release rates and a better antimicrobial activity. 15

ACCEPTED MANUSCRIPT

These results represent a good premise to use PSF membranes modified with CNF functionalized with carboxylic groups and AgNPs for wastewater treatment.

5. Conclusion

RI PT

This study presents the synthesis of a PSF membrane modified with carbonaceous nanomaterials and metallic inorganic nanoparticles with antimicrobial properties. The CNF were functionalized with carboxylic groups using poly(acrylic acid) which favors a better dispersion in polar media. FTIR and XPS analyses were used to evidence the chemical modification of pristine

SC

CNF. Silver nanoparticles were generated in situ or ex situ resulting in two types of hybrid membranes, PSF-CNF-Ag in situ membranes, respectively PSF-CNF-Ag ex situ membranes

M AN U

characterized by SEM, TEM and EDX analyses.

The antimicrobial properties of the membranes were studied both in solid and liquid phase. Both types of membranes have their strengths and weaknesses, but for practical application one must balance the antimicrobial properties with the lifetime of the composite membrane.

Thus, in our case, in solid phase the Gram-positive bacteria showed higher sensitivity for

TE D

PSF-CNF-Ag ex situ membrane due to the relatively strong release of unattached AgNPs to the CNF-COOH, determining cell wall damage highlighted through inhibition zones. In liquid phase the antimicrobial activity of PSF-CNF-Ag ex situ membranes was more pronounced towards bacterial Gram-negative species. These results can be also attributed to the presence of CNF-

EP

COOH which determines the constriction and finally the destruction of the cell walls in the form of bacillus in solid phase. On the contrary, in the liquid medium CNF-COOH forms aggregates

AC C

that surround cells-type coco-bacilli and expose it to the toxic effect of AgNPs. Thus, these results represent a good premise to use PSF membranes modified with CNF functionalized with carboxylic groups and AgNPs for wastewater treatment.

Acknowledgements

The authors would like to acknowledge the financial support provided by the National Authority for Scientific Research from the Ministry of Education, Research and Youth of Romania through the PN-III-P2-2.1-PED-2016-0545–FlexMetCut and PN-III-P2-2.1-PTE-0047 projects. 16

ACCEPTED MANUSCRIPT

Appendix A. Supplementary data Supplementary data related to this article can be found at:

RI PT

Conflict of Interest: The authors declare that they have no conflict of interest.

6. References

AC C

EP

TE D

M AN U

SC

[1] V.K. Gupta, I. Ali, T.A. Saleh, A. Nayak, S. Agarwal, Chemical treatment technologies for waste-water recycling-an overview, RSC Adv., 2 (2012) 6380-6388. [2] G.M. Geise, H.-S. Lee, D.J. Miller, B.D. Freeman, J.E. McGrath, D.R. Paul, Water purification by membranes: The role of polymer science, J. Polym. Sci., Part B: Polym. Phys., 48 (2010) 1685-1718. [3] E.J. Vriezekolk, T. Kudernac, W.M. de Vos, K. Nijmeijer, Composite ultrafiltration membranes with tunable properties based on a self-assembling block copolymer/homopolymer system, J. Polym. Sci., Part B: Polym. Phys., 53 (2015) 1546-1558. [4] M. Ulbricht, Advanced functional polymer membranes, Polymer, 47 (2006) 2217-2262. [5] L.Y. Ng, A.W. Mohammad, C.P. Leo, N. Hilal, Polymeric membranes incorporated with metal/metal oxide nanoparticles: A comprehensive review, Desalination, 308 (2013) 15-33. [6] N. Ghaemi, S.S. Madaeni, P. Daraei, H. Rajabi, S. Zinadini, A. Alizadeh, R. Heydari, M. Beygzadeh, S. Ghouzivand, Polyethersulfone membrane enhanced with iron oxide nanoparticles for copper removal from water: Application of new functionalized Fe3O4 nanoparticles, Chem. Eng. J., 263 (2015) 101-112. [7] Y.T. Chung, M.M. Ba-Abbad, A.W. Mohammad, A. Benamor, Functionalization of zinc oxide (ZnO) nanoparticles and its effects on polysulfone-ZnO membranes, Desalin. Water Treat., 57 (2016) 78017811. [8] R. Das, M.E. Ali, S.B.A. Hamid, S. Ramakrishna, Z.Z. Chowdhury, Carbon nanotube membranes for water purification: A bright future in water desalination, Desalination, 336 (2014) 97-109. [9] L. Feng, N. Xie, J. Zhong, Carbon Nanofibers and Their Composites: A Review of Synthesizing, Properties and Applications, Materials (Basel), 7 (2014) 3919-3945. [10] C. Yang, J. Mamouni, Y. Tang, L. Yang, Antimicrobial activity of single-walled carbon nanotubes: length effect, Langmuir, 26 (2010) 16013-16019. [11] L. Dong, A. Henderson, C. Field, Antimicrobial Activity of Single-Walled Carbon Nanotubes Suspended in Different Surfactants, J. Nanotechnol., 2012 (2012) 1-7. [12] S.C. Smith, D.F. Rodrigues, Carbon-based nanomaterials for removal of chemical and biological contaminants from water: A review of mechanisms and applications, Carbon, 91 (2015) 122-143. [13] A. Al-Jumaili, S. Alancherry, K. Bazaka, M. Jacob, Review on the Antimicrobial Properties of Carbon Nanostructures, Materials, 10 (2017) 1066. [14] H.J. Johnston, G.R. Hutchison, F.M. Christensen, S. Peters, S. Hankin, K. Aschberger, V. Stone, A critical review of the biological mechanisms underlying the in vivo and in vitro toxicity of carbon nanotubes: The contribution of physico-chemical characteristics, Nanotoxicology, 4 (2010) 207-246. [15] L. Zhang, A. Aboagye, A. Kelkar, C. Lai, H. Fong, A review: carbon nanofibers from electrospun polyacrylonitrile and their applications, J. Mater. Sci., 49 (2013) 463-480. [16] M. Ashfaq, N. Verma, S. Khan, Copper/zinc bimetal nanoparticles-dispersed carbon nanofibers: A novel potential antibiotic material, Mater. Sci. Eng. C., 59 (2016) 938-947. [17] S. Chen, Q. Chen, D. Cai, H. Zhan, Defect-mediated synthesis of Pt nanoparticles uniformly anchored on partially-unzipped carbon nanofibers for electrochemical biosensing, J. Alloys Compd., 709 (2017) 304-312. 17

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[18] K.P. De Jong, J.W. Geus, Carbon Nanofibers: Catalytic Synthesis and Applications, Catal. Rev., 42 (2000) 481-510. [19] M.J. Fernández-Merino, L. Guardia, J.I. Paredes, S. Villar-Rodil, A. Martínez-Alonso, J.M.D. Tascón, Developing green photochemical approaches towards the synthesis of carbon nanofiber- and graphenesupported silver nanoparticles and their use in the catalytic reduction of 4-nitrophenol, RSC Adv., 3 (2013) 18323. [20] C. Kim, Y.I. Jeong, B.T.N. Ngoc, K.S. Yang, M. Kojima, Y.A. Kim, M. Endo, J.-W. Lee, Synthesis and Characterization of Porous Carbon Nanofibers with Hollow Cores Through the Thermal Treatment of Electrospun Copolymeric Nanofiber Webs, Small, 3 (2007) 91-95. [21] M. Ashfaq, S. Singh, A. Sharma, N. Verma, Cytotoxic Evaluation of the Hierarchical Web of Carbon Micronanofibers, Ind. Eng. Chem. Res., 52 (2013) 4672-4682. [22] S. Silver, L.T. Phung, G. Silver, Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds, J. Ind. Microbiol. Biotechnol., 33 (2006) 627-634. [23] M. López-Heras, I.G. Theodorou, B.F. Leo, M.P. Ryan, A.E. Porter, Towards understanding the antibacterial activity of Ag nanoparticles: electron microscopy in the analysis of the materials-biology interface in the lung, Environ. Sci.: Nano, 2 (2015) 312-326. [24] C. Losasso, S. Belluco, V. Cibin, P. Zavagnin, I. Mičetić, F. Gallocchio, M. Zanella, L. Bregoli, G. Biancotto, A. Ricci, Antibacterial activity of silver nanoparticles: sensitivity of different Salmonella serovars, Front. Microbiol., 5 (2014). [25] J.-Y. Maillard, P. Hartemann, Silver as an antimicrobial: facts and gaps in knowledge, Crit. Rev. Microbiol., 39 (2013) 373-383. [26] B. Reidy, A. Haase, A. Luch, K.A. Dawson, I. Lynch, Mechanisms of Silver Nanoparticle Release, Transformation and Toxicity: A Critical Review of Current Knowledge and Recommendations for Future Studies and Applications, Materials (Basel), 6 (2013) 2295-2350. [27] R.Y. Hong, Q. Chen, Dispersion of Inorganic Nanoparticles in Polymer Matrices: Challenges and Solutions, in: S. Kalia, Y. Haldorai (Eds.) Organic-Inorganic Hybrid Nanomaterials, Springer International Publishing, Cham, 2015, pp. 1-38. [28] V.S. Nguyen, D. Rouxel, B. Vincent, Dispersion of nanoparticles: From organic solvents to polymer solutions, Ultrason. Sonochem., 21 (2014) 149-153. [29] V. Kaiser, C. Stropnik, V. Musil, M. Brumen, Morphology of solidified polysulfone structures obtained by wet phase separation, Eur. Polym. J., 43 (2007) 2515-2524. [30] K.A. Wepasnick, B.A. Smith, J.L. Bitter, D. Howard Fairbrother, Chemical and structural characterization of carbon nanotube surfaces, Anal. Bioanal. Chem., 396 (2010) 1003-1014. [31] H.D. Saier, H. Strathmann, Asymmetric Membranes: Preparation and Applications, Angew. Chem., 14 (1975) 452-459. [32] N.S. Wigginton, A.d. Titta, F. Piccapietra, J. Dobias, V.J. Nesatyy, M.J.F. Suter, R. Bernier-Latmani, Binding of Silver Nanoparticles to Bacterial Proteins Depends on Surface Modifications and Inhibits Enzymatic Activity, Environ. Sci. Technol., 44 (2010) 2163-2168. [33] K. Zodrow, L. Brunet, S. Mahendra, D. Li, A. Zhang, Q. Li, P.J.J. Alvarez, Polysulfone ultrafiltration membranes impregnated with silver nanoparticles show improved biofouling resistance and virus removal, Water Res., 43 (2009) 715-723. [34] E. Rusen, A. Mocanu, L.C. Nistor, A. Dinescu, L. Calinescu, G. Mustatea, S.I. Voicu, C. Andronescu, A. Diacon, Design of Antimicrobial Membrane Based on Polymer Colloids/Multiwall Carbon Nanotubes Hybrid Material with Silver Nanoparticles, ACS Appl. Mater. Interfaces, 6 (2014) 17384-17393. [35] S. Ahmed, M. Ahmad, B.L. Swami, S. Ikram, A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: A green expertise, J. Adv. Res., 7 (2016) 17-28. [36] N.G. Mlalila, H.S. Swai, A. Hilonga, D.M. Kadam, Antimicrobial dependence of silver nanoparticles on surface plasmon resonance bands against Escherichia coli, Nanotechnol., Sci. Appl., 10 (2017) 1-9. 18

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[37] S. Prabhu, E.K. Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects, Int. Nano Lett., 2 (2012) 32. [38] H.Z. Zardini, M. Davarpanah, M. Shanbedi, A. Amiri, M. Maghrebi, L. Ebrahimi, Microbial toxicity of ethanolamines—Multiwalled carbon nanotubes, J. Biomed. Mater. Res. A, 102 (2014) 1774-1781. [39] T.C. Dakal, A. Kumar, R.S. Majumdar, V. Yadav, Mechanistic Basis of Antimicrobial Actions of Silver Nanoparticles, Front. Microbiol., 7 (2016). [40] B. Volkmer, M. Heinemann, Condition-dependent cell volume and concentration of Escherichia coli to facilitate data conversion for systems biology modeling, PLoS One, 6 (2011) e23126.

19

ACCEPTED MANUSCRIPT

HIGHTLIGHTS 1. The fabrication of polysulfone membrane with antimicrobial properties. 2. The strategy employed was based on the use of carbon nanofibers (CNF)

AC C

EP

TE D

M AN U

SC

4. The materials were investigated by XPS, SEM, ICP-MS.

RI PT

3. CNF presenting carboxyl functional groups (CNF-COOH) and silver nanoparticles