2827 © IWA Publishing 2011 Water Science & Technology | 63.12 | 2011

Synthesis and structure-activity study of quaternary

ammonium functionalized β-cyclodextrin-

carboxymethylcellulose polymers

Danielle Bonenfant, François-René Bourgeois, Murielle Mimeault,

Frédéric Monette, Patrick Niquette and Robert Hausler

ABSTRACT

Carboxymethylcellulose (CMC) and β-cyclodextrin (β-CD)-based polymers functionalized with two

types of quaternary ammonium compounds (QACs), the alkaquat DMB-451 (N-alkyl (50% C14, 40%

C12, 10% C10) dimethylbenzylammonium chloride) (DMD-451) named polymer DMB-451, and FMB

1210-8 (a blend of 32 w% N-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammonium chloride and

48 w% of didecyldimethylammonium chloride) named polymer FMB 1210-8, were synthethized and

characterized by Fourier transform infrared spectroscopy. The antimicrobial activities of these

polymers against Eschericia coli were also evaluated at 25 WC in wastewater. The results have

indicated that the polymer FMB 1210-8 possesses a high-affinity binding with bacterial cells that

induces a rapid disinfection process. Moreover, in the same experimental conditions of disinfection

(mixture of 1.0 g of polymer and 100 mL of wastewater), the polymer FMB 1210-8 has a higher

antimicrobial efficiency (99.90%) than polymer DMB-451 (92.8%). This phenomenon might be

associated to a stronger interaction with bacterial cells due to stronger binding affinity for E. coli

cells and greater killing efficiency of the C10 alkyl chains QAC of polymer FMB 1210-8 to

disrupt the bacterial cell membrane as compared to N-alkyl (50% C14, 40% C12, 10% C10)

dimethylbenzylammonium chloride. Together, these results suggest that the polymer FMB 1210-8

could constitute a good disinfectant against Escherichia coli, which could be advantageously used in

wastewater treatments due to the low toxicity of β-CD and CMC, and moderated toxicity of FMB

1210-8 to human and environment.

doi: 10.2166/wst.2011.630

Danielle BonenfantFrançois-René BourgeoisFrédéric MonetteRobert Hausler (corresponding author)Département de génie de la construction,STEPPE-École de technologie supérieure,1100, Notre-Dame Ouest,Montréal, QC,Canada H3C 1K3E-mail: Robert.Hausler@etsmtl.ca

Murielle MimeaultDepartment of Biochemistry and Molecular

Biology,College of Medicine, Eppley Cancer Institute,7052 DRC,University of Nebraska Medical Center,985870 Nebraska Medical Center,Omaha, NE 68198-5870,USA

Patrick NiquetteIndependant consultant,245, du Manoir Ave.,St Elzear, QC,Canada G0S 2J0

Key words | alkaquat DMB-451, antimicrobial activity, antimicrobial polymers, Escherichia coli,

FMB 1210-8, wastewater

INTRODUCTION

Escherichia coli (E. coli) is the most common member ofcoliform bacteria. E. coli is a Gram-negative rod-shapedand facultative anaerobic non-spore-forming bacterium,

commonly found in the intestines of humans and animalsand which can contaminate the wastewater through fecalmaterials (Brenner ; De la Rosa-Gómez et al. ).

Most E. coli strains are harmless, but the presence of someone in water supply, such as enterotoxigenic, enteropatho-genic, enteroinvasive, and enterohemorrhagic E. coliO157:H7, might be problematic because they can causeserious diarrheal deseases (Robson ). The disinfectionof municipal wastewater is generally achieved by using

chemical and physical agents such as ozone, chlorine andits derivatives, and UV radiations (Tansel ). These treat-ments represent a problem because they might cause the

formation of toxic compounds such as aldehydes, ketones,phenols, aromatic acids, organochloride compounds,chloramines, nitrosamines, etc. (Boorman ; Liberti

et al. ).The disinfection of wastewater with antimicrobial poly-

mers containing quaternary ammonium compounds (QACs)

commonly designed as cationic polymers, can considerablyreduce the environmental contamination caused by tra-ditional treatments. Amphiphilic QACs are surface-active

2828 D. Bonenfant et al. | Synthesis and characterization of QAC functionalized polymers Water Science & Technology | 63.12 | 2011

substances constituted of a cationic moiety containing a

nitrogen atom of valency five bonded to four alkyl or hetero-cyclic radicals, which is associated with a counteranion(chloride, bromide, iodide anions) (Roy et al. ). Thesecompounds are moderately toxic (FMB 1210-8 and DMB-451 LD50 oral, rat of about 500 mg/kg), not carcinogenicand non-corrosive, and exist as stable salts at normal pHof wastewater (Bore et al. ; Rhodia Inc. ; Lonza

Inc. ). Moreover, they display a rapid antimicrobialactivity against several types of microorganisms, includingGram-positive and Gram-negative bacteria, virus, and

fungi (Ahlström et al. ). The following mechanism hasbeen proposed to describe the mode of action of QACs :(1) absorption of the cationic salt onto the bacterial cell sur-

face through an ionic interaction; (2) diffusion through thecell wall; (3) binding to the cytoplasmic membrane follow-ing by its disruption; (4) release of electrolytes; (5)degradation of proteins and nucleic materials (DNA,

RNA) follows by (6) the wall lysis and the death of the cell(Chen et al. ; Tashiro ; Gilbert & Moore ).The antimicrobial activity of QACs is dependent of their

intrinsic properties, including the charge density, hydropho-bicity, molecular size, and counteranion (Chen et al. ;Kügler et al. ; Majumdar et al. ). More particularly,

the antimicrobial activity of QACs enhances with theircharge density and alkyl chain length attached to the nitro-gen atom, with an optimum chain length of 16–18 carbon

atoms and a highest activity achieved for C10. The inte-gration of QACs into polymers can be advantageous todisinfect wastewater. In addition to the great efficiency ofQACs as disinfectants, these cationic polymers have a

higher charge density than monomeric cations that mayenhance their adsorption onto the negatively charged bac-terial cell surfaces, and thereby facilitate their binding to

the cytoplasmic membrane of the bacterium (Roy et al.; Majumdar et al. ). Moreover, these polymers arealso insoluble in water, and consequently they can be recov-

ered and re-used after the treatment.The present study was undertaken to evaluate the anti-

microbial properties against E. coli, of two cationic

polymers, which differ by their substituent radicals. Thestudied polymers were synthesized through a reaction con-sisting of the condensation of β-cyclodextrin (β-CD) withcarboxymethylcellulose (CMC), epichlorohydrin (Epi), and

two commercial QACs, DMB-451 80% and FMB 1210-8.The cationic polymers, DMB-451 and FMB 1210-8, werecharacterized by Fourier transform infrared (FTIR) spec-

troscopy and their antimicrobial activity against E. coliwas measured by culturing a disinfected wastewater with

them. The structure-activity relationship is discussed in func-

tion of common structure of their alkyl chains and thepresence of didecyldimethylammonium chloride into FMB1210-8 structure.

MATERIAL AND METHODS

Materials

β-cyclodextrin (98%), epichlorohydrin (99%), sodium boro-hydride (98%) and sodium hydroxide beads werepurchased from Sigma-Aldrich Canada ltd (Oakville,

Ontario), sodium CMC from Univar Canada Ltd (Dorval,Quebec), hydrochloride acid (37%) from Fisher ScientificCo. (Nepear, Ontario), Dico™ rosolic acid from Becton,

Dickinson and company (Sparks, MD), alkaquat DMB-45180% from Rhodia Inc (Cranbury, NJ), FMB 1210-8 fromLonza Inc (Allendale, NJ), and m-FC Broth (9 g enzymatic

digest of casein, 2.5 g enzymatic digest of animal tissue,6.5 g yeast extract, 5 g sodium chloride, 12.5 g lactose, 1.5 gbile salts, and 0.1 g aniline blue per liter) from Acumedia

Manufacturers inc (Lansing, Michigan). All these productswere used without additional purification. The wastewatersamples were collected from affluent of the La Pinièrewastewater treatment plant (Laval, Quebec), and were not

disinfected before sampling.

Preparation and characterization of the polymerscontaining QACs

The antimicrobial polymers were prepared as following: 20 gof β-CD was mixed with 5 mg of NaBH4, 8.534 g of CMC,3 g of NaOH and 50 mL of deionized water in a reactor

at 25± 1 WC during 24 h. After, 2.46 g of DMB-451 80%(5.52 mmol DMB-451) or FMB 1210-8 (6.84 mmol FMB1210) was added to this mixture under agitation. Then,

14 mL of Epi was added to mixture dropwise at 60 WCunder vigorous stirring. At the end of the Epi addition, thepolymers were neutralized with 3N HCl and washed in deio-

nized water during 24 h at 25± 1 WC under stirring. Finally,the polymers were collected by filtration, dried at 60 WC for24 h, and crushed.

The amount of β-CD incorporated into the antimicro-

bial polymers was evaluated using an internal standard(Monnier et al. ). This method has consisted to comparethe absorbance of the FTIR bands of β-CD at 1028.1 cm�1 of

the dried antimicrobial polymers (Apolymer) and a mixturecontaining 2 g of dried antimicrobial polymer and 430 mg

2829 D. Bonenfant et al. | Synthesis and characterization of QAC functionalized polymers Water Science & Technology | 63.12 | 2011

of β-CD (Amixture) (Bonenfant et al. , ). The percen-

tage (%) of β-CD incorporated was calculated as described inEquation (1).

%β - CD incorporated ¼ ðApolymer=Amixture � ApolymerÞ× ðCtCa=CpÞ × 100 ð1Þ

In Equation (1), Ct, Cp, and Ca are the values of theamount of β-CDþEpiþCMC added into the preparationof the antimicrobial polymers, and the amounts of polymer

(2g) and β-CD added (430 mg) in the mixture, respectively.The amounts of DMB-451 and FMB 1210-8 incorporated

into the polymers were obtained from their FTIR bands at

1,597.1 and 1,595.1 cm�1 respectively, and standard curvesestablished for aqueous DMB-451 and FMB 1210-8 solutions.

Disinfection testing

Firstly, the disinfection testing has been effected in mixturescontaining 0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.5, and 2.0 gof polymers DMB-451 and FMB 1210-8, and 100 mL of

wastewater in stopped flasks during 24 h at 25± 1 WCunder agitation. After, disinfection tests were made in a mix-ture containing 1.0 g of these polymers and 100 mL ofwastewater during 5, 10, 15, 30, 60, 120, 180, 240, 360,

480, 720, 960, 1,200, and 1,440 min at 25± 1 WC under agita-tion. These assays were effected with three samples ofwastewater containing 1.59 × 106, 2.05 × 106, and 2.39 ×

106 CFU/100 mL. All these assays were made in triplicate.After disinfection, coliforms (E. coli) were enumerated bya technique approved in drinking water filtration and waste-

water treatment plants. This method consisted of filtering10, 100, and 500 μL aliquots of supernatants of all these mix-tures through 47 mm diameter and 0.45 μm pore-sizedsterile cellulose nitrate membrane filters. These membranes

were transferred into m-FC nutrient plates. The nutrient waspreviously prepared by mixing 7.8 g of m-FC Broth, 150 mLof deionized water and 1.5 mL of 1% aqueous rosolic acid

solution (0.1 g rosolic acidþ 10 mL of 0.2N NaOH), andthis mixture was heated at boiling. The medium plateswere cultured in an incubator LabTech (Daihan Labtech

Co. Ltd, Kyungki-Do, Korea) at 44.5 WC, and the blue colo-nies (E. coli) were counted at 24 h.

Characterization by FTIR spectroscopy

Infrared spectra of polymers DMB-451 and FMB 1210-8,mixtures containing 2 g of antimicrobial polymers and

430 mg of β-CD, and aqueous DMB-451 and FMB 1210-8

solutions (11.262 g QACþ 50 mL deionized water) wereobtained using a Varian Excalibur 3100 FTIR spectrometerequipped with a reflexion ATR module, and continuously

purged with dry air. For each spectrum, covering the 500–7,900 cm�1 region, 200 interferograms were coadded, apo-dized and Fourier transformed to give a resolution of4 cm�1. After FTIR data acquisition, spectra of aqueous

DMB-451 and FMB 1210-8 solutions were corrected for thespectral contributions of water bands and water vapour.The amounts of β-CD contained in the polymers were evalu-

ated by the method described at section 2.2, whereasthe amounts of DMB-451 and FMB 1210-8 were obtainedfrom standard curves established for aqueous DMB-451

(y¼ 0.0022xþ 0.0018; r2¼ 0.9851) and FMB 1210-8 (y¼0.006x � 0.0058; r2¼ 0.9502) solution at 1,652.99 and1,654.9 cm�1, respectively.

RESULTS AND DISCUSSION

Characterization of the antimicrobial polymers

The results obtained from the FTIR measurements have indi-

cated that 100 w% of β-CD, 100 w% of DMB-451 (14.68mmol DMB-451/100 g polymer) and 100 w% of FMB1210-8 (16.96 mmol FMB 1210) added during the prep-

arations, were incorporated into polymers DMB-451 andFMB 1210-8. These results are in agreement with the datafrom the previous studies that indicated that the incorpor-ation of β-CD, MCT-β-CD, and Quat-188 into crosslinked

β-CD-CMC polymer and MCT-β-CD-starch polymer withQuat-188 (Hebeish et al. ; Bonenfant et al. ).

The infrared spectra obtained for the aqueous DMB-451

and FMB 1210-8 solutions, and polymers DMB-451 andFMB 1210-8 measured in the 2,700–3,100 and 1,500–1,800 cm�1 regions are showed in Figure 1. The FTIR bands

detected in the 2,700–3,100 cm�1 region are assigned to asym-metric and symmetric CH2 stretching vibrations (νCH2),whereas bands located in the 1,500–1,800 cm�1 can be associ-

ated with a combination of C–H and C–N stretching vibrationsof amino group (νHCN) (aqueous DMB-451 and FMB 1210-8solutions); and a combination of asymmetric and symmetricstretching of COO� (νC55O) associated to CMC, and C–H

and C–N vibrations (dried antimicrobial polymers) (Table 1)(Mimeault & Bonenfant ; Bocanegra-Diaz et al. ;Shi et al. ; Tomas et al. ; Prabaharan & Gong ;

Kowalczyk ). Figure 1(a) shows that the presence ofDMB-451 and FMB 1210-8 into polymers causes a decrease

Figure 1 | FTIR spectra of the CH2 stretching region of (a) aqueous DMB-451 solution; (b)

aqueous FMB 1210-8 solution; (c) dried polymer DMB-451; (d) dried polymer

FMB 1210-8, and the 1,500–1,800 cm�1 region of (e) aqueous DMB-451 sol-

ution; (f) aqueous FMB 1210-8 solution; (g) dried polymer DMB-451; (h) dried

polymer FMB 1210-8, measured at 25± 1W

C.

Table 1 | Maximum wavenumber of FTIR bands of aqueous DMB-451 and FMB 1210-8

solutions, and dried antimicrobial polymers, in CH2 stretching and 1,500–

1,800 cm�1 regions

Wavenumber (cm�1)

Mode DMB-451 FMB 1210-8 Polymer DMB-451 Polymer FMB 1210-8

νCH2 2,956.9 2,956.9 2,951.1a 2,953.0a

2,924.1 2,926.0 2,922.1 2,924.1

2,854.6 2,854.6 2,854.6 2,856.6

νHCN 1,653.0 1,654.9 1,597.1 1,595.1

νC55O Overlapped by νHCN

aShoulder.

2830 D. Bonenfant et al. | Synthesis and characterization of QAC functionalized polymers Water Science & Technology | 63.12 | 2011

of νCH2 of 2.0 and 1.9 cm�1 as compared with those observed

for aqueous DMB-451 and FMB 1210-8 solutions, respectively.These shifts of νCH2 can be due to the strengthening of theinteractions in the alkyl chains following the binding of

QACs with β-CD or CMC. Figure 1(b) shows also a significantdecrease of νHCN for the polymer DMB-451 (55.9 cm�1) andpolymer FMB 1210-8 (59.8 cm�1) as compared with those of

aqueous DMB-451 and FMB 1210-8 solutions, which areaccompanied by an increase of the bands intensity. Thesechanges may be associated with the binding of β-CD with

alkyl chains of DMB-451 and FMB 1210-8 that could haveas a consequence to increase the sterical hindrance aroundthe amino group due to the special arrangement of β-CD mol-

ecules. However, the greater decrease between νHCN ofaqueous FMB 1210-8 solution and polymer FMB 1210-8 canbe due to smaller didecyldimethylammonium chloride that

may be easier to incorporate into the polymer than N-alkyl(50% C14, 40% C12, 10% C10) dimethylbenzylammoniumchloride and can adopt a special arrangement to restrict theamino group vibrations. All together, these results are in agree-

ment with the polymerization model of MCT-β-CD and Quat-188 with starch that was suggested by Hebeish et al. ().According to this model, when reactions of MCT-β-CD and

Quat-188 with starch occur in one-step, there is a competitionwhere the alkyl chain of Quat-188 binds to OH groups ofstarch, whereas MCT-β-CD binds to OH groups of starch

and Quat-188.

Determination of antimicrobial activity of polymerFMB 1210-8

The results of the antimicrobial efficiency of polymer FMB1210-8 are shown in Figure 2. The curves presented inFigure 2(a) have showed a rapid killing to reach a maximum

at 1.0–2.0 g of polymer/100 mL of wastewater where 99.90–99.96% E. coli was killed with 24 h of disinfection. More-over, the polymer FMB 1210-8 has a great initial killingactivity, suggesting a high-affinity binding where the

majority of the polymer was bound to bacterial cells.These results are in agreement with Ioannou et al. ()who showed that didecyldimethylammonium chloride pos-

sesses a very rapid and strong initial interaction withStaphylococcus aureus cells. In parallel, the results

Figure 2 | Percentage of survival E. coli colonies after disinfection of wastewater using (a)

polymer FMB 1210-8 as a function of the concentration of polymer used; and

(b) 1.0 g of polymer FMB 1210-8/100 mL of wastewater as a function of the

contact time. The assay was made at 25± 1W

C under stirring.

2831 D. Bonenfant et al. | Synthesis and characterization of QAC functionalized polymers Water Science & Technology | 63.12 | 2011

presented in Figure 2(b) have indicated that in a mixture

containing 1.0 g of polymer and 100 mL of wastewater, thepolymer FMB 1210-8 has a high antimicrobial efficiency inthe first 15 min of contact, where 56% E. coli was killed,

suggesting a strong and rapid interaction with E. coli cells.Moreover, these data suggest an immediate interactionbetween the polymer FMB 1210-8 and E. coli membrane,rapidly causes the death of the bacterial cell following the

leakage of its intracellular constituents (Ioannou et al.). This uptake was followed by a diminution of the anti-microbial activity to reach a maximum of disinfection after

16–24 h. This diminution of killing activity may be due to

a reduction of the interaction between the polymer FMB1210-8 and E. coli cells which be associated to a decreaseof unbound polymer and/or to accumulation of cell debris

on the surface of polymer (Shi et al. ).

Comparison of antimicrobial characteristics of polymersDMB-451 and FMB 1210-8

The E. coli killing efficiencies of 92.80 and 99.90% have been

measured with the polymers DMB-451 and FMB 1210-8 in amixture containing 1.0 g of polymer/100 mL of wastewater,respectively. These results indicate that the polymer FMB1210-8 has a greater killing efficiency than polymer DMB-

451 in the same experimental conditions. This is presumablydue to different compositions of these QACs. It has beenshowed that the didecyldimethylammonium chloride pos-

sesses a stronger binding affinity for bacterial cells than N-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammo-nium chloride (Ioannou et al. ). It has been also

reported that the cationic biocides with C10 alkyl chains aremore effective to disrupt the cell membrane, followed byC12, C14, and C16 chains (Chen et al. ; Hu et al. ).Then, the presence of a large amount of didecyldimethylam-

monium chloride into FMB 1210-8 can contribute toenhance its antimicrobial activity against E. coli and explainthe higher killing efficiency of the polymer FMB 1210-8 as

compared to polymer DMB-451.

CONCLUSIONS

The results from the present study have indicated that the

polymers DMB-451 and FMB 1210-8 possess high antimi-crobial activities against E. coli. The polymer FMB 1210-8has a high-affinity binding and quickly and strongly interacts

with the E. coli membrane, inducing a rapid process of thebacterial cell. However, at the concentrations studied, thepolymer FMB 1210-8 was significantly more potent than

the polymer DMB-451. This phenomenon may be associatedto a stronger interaction of polymer FMB 1210-8 with E. colicells probably due to the stronger binding affinity and thegreater efficiency of didecyldimethylammonium chloride

to disrupt the bacterial cell membrane as compared toN-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammo-nium chloride. Then, the disinfection of wastewater with the

polymer FMB 1210-8 could constitute an advantageoustechnology due to its high antimicrobial activity against

2832 D. Bonenfant et al. | Synthesis and characterization of QAC functionalized polymers Water Science & Technology | 63.12 | 2011

E. coli, low toxicity of β-CD and CMC, and moderated tox-

icity of FMB 1210-8 to human and environment.

ACKNOWLEDGEMENTS

This work was supported by grants from Natural Sciencesand Engineering Research Council of Canada (NSERC).

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First received 16 November 2010; accepted in revised form 28 March 2011