Nonleaching antimicrobial cotton fibers for hyaluronic acid adsorption

Biochemical Engineering Journal 53 (2010) 44–51

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Nonleaching antimicrobial cotton fibers for hyaluronic acid adsorption David Wibowo, Cheng-Kang Lee ∗ Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Rd. Sec. 4, Taipei 106, Taiwan

a r t i c l e

i n f o

Article history: Received 6 April 2010 Received in revised form 1 September 2010 Accepted 5 September 2010

Keywords: Quaternary ammonium Choline Deep eutectic solvent Hyaluronic acid Adsorption Hydrophobic interaction

a b s t r a c t Quaternary ammonium containing compounds (QACs) such as cetylpyridinium chloride (CPC) is commonly employed in hyaluronic acid (HA) production process as an HA precipitating agent. 3(Trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, a Si containing QAC (Si-QAC) generally used to modify the surface of cotton fibers for the preparation of nonleaching antibacterial textiles, has a chemical structure very similar to CPC. Choline, a natural QAC, can form a deep eutectic solvent with urea and be grafted onto cotton surface when incubating cotton in the deep eutectic solvent. Both of the QAC-modified cottons demonstrated antibacterial activity against Gram (−) Escherichia coli and Gram (+) Bacillus subtilis along with HA adsorption capacity. The HA adsorption isotherm could be well-fitted with Langmuir model with maximum adsorption capacities of 184 mg/g and 351 mg/g for Si-QAC and choline surface-functionalized cotton fibers, respectively. In the presence of high concentration of contaminants, HA could be directly recovered from a B. subtilis culture with a capacity of 15 mg/g by using Si-QAC modified antimicrobial cotton fiber. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Hyaluronic acid (HA) is a linear polysaccharide consists of alternating d-glucuronic acid and N-acetyl-d-glucosamine subunits. The chain lengths of HA can reach up to ∼30,000 repeating disaccharide units, corresponding to a molecular weight of ∼106 Da. HA is highly hydrophilic, and in aqueous solutions shows viscoelastic behavior and water binding capacity due to the high molecular weight and the high number of charged groups. HA has many significant structural, rheological, physiological, and biological functions in the body, such as providing a cushion effect in human joints, stimulating the immune system and maintaining a smooth, elastic skin [1]. These important functions have led to a wide range of applications, such as in cosmetics, ophthalmic surgery, arthritis treatment, postsurgical adhesions, tissue engineering scaffolds for wound healing, and drug delivery devices [2]. Commercially, HA is produced through extraction from rooster combs or via bacterial fermentation but the latter is gradually replacing extraction as the preferred source of HA [3], because the bacterial process presents the opportunity to optimize the product yield and quality through metabolic engineering and control of culture conditions. The separation and purification of HA from bacterial fermentation broth generally involves the precipitation of HA with quaternary ammonium compound (QAC) which is a cationic surface-active agent with antibacterial activity [4–6]. Cetylpyri-

∗ Corresponding author. Tel.: +886 2 27376629, fax: +886 2 27376644. E-mail address: [email protected] (C.-K. Lee). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.09.002

dinium chloride (CPC), an antiseptic agent commonly used in oral or throat care, is often used QAC for precipitating HA. Instead of isolating HA by selective precipitation, adsorption of HA on a selective adsorbent will be a better alternative for HA isolation because the energy-intensive centrifugation step for recovering the precipitate can be avoided. However, CPC as a well-known affinity precipitation ligand does not have a functional group available for easy activation and coupling to a solid matrix for the preparation of selective adsorbent. Quaternary ammonium containing organosilicon salt, 3-(trimethoxysilyl)-propyldimethyl octadecyl ammonium chloride (Si-QAC) has a structure very similar to CPC which consists of a quaternary ammonium group with a long aliphatic tail but with an additional alkoxysilane group, which functions as an anchor can be easily bonded to a solid bearing surface hydroxyls. The antimicrobial properties of Si-QAC immobilized on solid surfaces are known for decades and utilized by the textile industry [7–12]. The major mode of action was identified as the positively charged nitrogen that attracts the negatively charged microorganisms which comes to contact with the long hydrophobic tail which pierces the cell membrane and leads to cytolytic damage. The interaction behavior between the surface modified Si-QAC and HA is rarely studied except Yang et al. [13] have reported that the Si-QAC modified magnetic particles can be used for HA adsorption but with a low capacity. Because of its rich surface hydroxyls, cotton fibers are usually surface functionalized for the preparation of functional textiles. For example, cationic functionalized cotton fibers can be used for the preparation of nonleaching antimicrobial fabrics [14,15]. Recently, an ionic liquid analogue or a deep eutectic solvent based on a

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

45

Fig. 1. Reaction schemes for grafting cotton surface with (a) 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium; (b) choline in deep eutectic solvent.

mixture of inexpensive choline chloride and urea has been reported to be able to directly cationize cellulose fibers surface by grafting choline molecule [14]. The choline molecule consists of a quaternary ammonium group but with no aliphatic tail like Si-QAC. Therefore, it is interesting to know the effect of the long aliphatic tail of the quaternary ammonium containing molecules grafted surface on its antimicrobial activity and HA adsorption behavior. In addition, the antimicrobial cotton fabrics can be used as a wounddressing biomaterials if the HA could be strongly adsorbed onto the surface of the antimicrobial cotton fibers. In this work, Si-QAC and choline were grafted onto the surface of cotton fibers, respectively. The antimicrobial activities against Gram (−) Escherichia coli and Gram (+) Bacillus subtilis of the modified cotton fibers were evaluated. The adsorption of HA at various temperatures and pHs as well as the reusability of the antimicrobial cotton fibers were studied. Equilibrium isotherm and thermodynamic analysis was utilized to examine the effect of temperature and the mechanism of adsorption. The feasibility of applying the modified fibers for adsorbing HA directly from bacterial culture was also investigated. 2. Materials and methods 2.1. Materials Defatted cotton wool was obtained from a local drug store. Si-QAC, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium

chloride (AEM 5700) from Dow Corning was provided as a 42% solution in methanol. 2-Chloroethyltrimethyl ammonium chloride also known as chlorocholine chloride with 98% purity was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Sodium hyaluronate was obtained from Fine Chemical Division of Q.P. Co. (Tokyo, Japan). E. coli Top10 and B. subtilis BCRC 51921 were obtained from Invitrogen and Bioresource Collection and Research Center (Hsinchu, Taiwan), respectively. All other chemicals were purchased from either Acros or Sigma unless otherwise stated. 2.2. Preparation of antimicrobial cotton fibers To prepare Si-QAC modified cotton fibers (SMC) (Fig. 1a), defatted cotton as much as 0.1 g was mixed with 4 mL of 95:5%, (v/v) methanol:water solution and 0.4 mL of Si-QAC for 24 h at room temperature followed by heating to 90 ◦ C for 12 h. The recovered cotton was washed thoroughly with methanol to remove the nonhydrolyzed and physically absorbed Si-QAC. The washed SMC was dried at 60 ◦ C and stored a 4 ◦ C until use. Choline modified cotton fibers (CMC) were prepared by using a chlorocholine chloridebased deep eutectic solvent as shown in Fig. 1b [14]. The solvent was made by mixing 12.96 g of chlorocholine chloride and 9.78 g of urea, heated to 80 ◦ C and stirred occasionally for 30 min. Cotton fibers as much as 0.1 g was added to 5 mL of deep eutectic solvent followed by 0.372 g of sodium hydroxide. The mixture was heated

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D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

Table 1 Elemental analysis and grafting ratio of modified cotton fibers. Samples

C (%)

H (%)

N (%)

Grafting ratio (%)

Unmodified Si-QAC modified (SMC) Choline modified (CMC)

41.02 ± 0.16 46.14 ± 1.53 47.97 ± 0.63

6.62 ± 0.02 7.94 ± 0.48 8.29 ± 0.28

0.08 ± 0.01 0.59 ± 0.08 1.16 ± 0.28

0 25.05 ± 2.57 14.61 ± 1.34

with stirring at 90 ◦ C for 15 h. After washing with copious amount of deionized water, the CMC was then dried at 60 ◦ C.

CMC). Each experiment was carried out in duplicate and the results are shown with error bars.

2.3. Characterization methods

2.6. Repeated use of the antimicrobial cottons

The content of carbon, nitrogen and hydrogen in the original and treated samples was determined by combustion followed by chromatographic separation and thermal conductivity detection using a Heraeus Vario ELIII elemental analyzer. The grafting ratio (wt.%) of the modified cotton fibers was calculated using the following formula as described by Roy et al. [15]

where Wg is the dry weight of each grafted cotton fibers and Wo is the initial weight of each cotton fibers. FTIR spectra were obtained from discs containing 2 mg of samples in approximately 120 mg of KBr on a Bio-Rad FTS 3500.

One milliliter of 1 mg/mL HA solution was incubated with 5 mg (dry weight) of either SMC (at 37 ◦ C, pH 4) or CMC (at 4 ◦ C, pH 7) with constant shaking (200 rpm) for 3 h. After thorough washing with 0.1 M of buffer used for adsorption, the samples were collected by centrifugation and dried until constant weight. Desorption experiments were performed by mixing the collected samples after adsorption with 1 N NaCl, pH 7 phosphate buffer (for SMC) or 1 N NaCl, pH 3 glycine–HCl buffer (for CMC), at room temperature and shaken at 200 rpm for 24 h. The cotton fibers were then undertaken for next cycle of HA adsorption and desorption after thorough washing with 0.1 M buffer and drying until constant weight. The supernatant of each adsorption and desorption step were analysed for HA concentration by carbazole method.

2.4. Antibacterial assessments

2.7. HA concentration determination

The antibacterial activity of modified cotton fibers was evaluated against E. coli and B. subtilis by the viable cell counting method [16]. Prior to the antibacterial tests, all cotton fibers samples were sterilized with 70% ethanol solution and kept under UV-light for overnight. One-loop full of the bacteria from a colony on agar plate was inoculated into 5 mL liquid culture of Luria Bertani (LB) medium and incubated for 15 h at 37 ◦ C. The bacteria-containing broth was centrifuged (5000 rpm, 2 min) and the obtained cells pellets were washed twice with Ringer saline solution (8.6 g/L NaCl, 0.3 g/L KCl, 0.33 g/L CaCl2 ). The bacteria stock solution was diluted with saline solution to give cell number of approximately 107 –108 CFU/mL for antimicrobial activity test. Five milliliters of bacteria suspension was then added to the each test tube containing 20 mg of unmodified cellulose fibers (UMC), SMC, and CMC, respectively and incubated at 37 ◦ C for 3 h with 200 rpm shaking. A control culture without cotton fibers was also treated in the similar way. At the predetermined time, 1 mL of bacteria culture was taken and serially diluted down to 10−7 . Aliquots (100 ␮L) of the diluted samples were then spread, in duplicate, onto nutrient agar plate. After incubation at 37 ◦ C for 12 h, the number of colony formed was counted. In order to study the effect of antibacterial activity on the cell growth, the bacteria were grown in 50 mL of LB at 37 ◦ C and shaken at 200 rpm along with 0.2 g of cotton fibers samples for 15 h. The cell density was measured by spectrophotometer at wavelength 600 nm.

The HA concentration was estimated by carbazole method [17]. Briefly, a serial dilution of HA standard or sample of 150 ␮L was added into 900 ␮L of a cooled solution of 25 mM sodium tetraborate in 98% sulfuric acid placed in test tube. The test tube was closed and shaken gently at first then vigorously for 10 s and heated for 10 min at 100 ◦ C in an aluminous heating block. After cooling in an ice bath for 5 min, 30 ␮L of 0.125% carbazole in ethanol were carefully added. After heating at 100 ◦ C for 10 min and cooling in an ice bath for 5 min, the absorbance of the reaction solution was measured by means of Jasco V-530 visible spectrophotometer at 525 nm wavelength. The HA concentration was determined from the established calibration curve.

Grafting ratio (wt.%) =

Wg − Wo × 100% Wo

(1)

2.8. HA recovery from B. subtilis culture B. subtilis was grown at 37 ◦ C in 50 mL of LB broth for 15 h. The pH of B. subtilis culture was then adjusted to 4. HA powder was also added so that the culture broth contained as much as 1 mg/mL of HA. Three grams of SMC were mixed with the culture broth in order to study the feasibility of directly adsorbing HA from the culture. After thorough washing with pH 4, 0.1 M acetate buffer, the recovered SMC was incubated with 50 mL of 0.1 M, pH 7 phosphate buffer containing 1 N NaCl for 24 h to desorb the adsorbed HA. The supernatant of each recovery step were analysed for HA and protein concentrations by carbazole method and Bradford method [18], respectively.

2.5. HA adsorption 3. Results and discussion Adsorption of HA by the antimicrobial cotton fibers was carried out by mixing 1 mL of HA solution of various concentrations with 5 mg of dry cotton fibers. Prior to the addition of cotton fibers, the HA solutions were placed in a water bath for 30 min to achieve the specific adsorption temperature. HA concentration in the supernatant was analysed by carbazole method [17]. The effect of pH on HA adsorption onto cotton fibers was studied at pH in the range of 3–8 at 37 ◦ C and ionic strength 0.1 M. The effect of temperature was investigated at 4, 18, and 37 ◦ C at pH 4 (for SMC) and at pH 7 (for

3.1. Characterization Table 1 lists the results of elementary analysis of cotton fibers before and after modification. The higher nitrogen and carbon contents detected after modification suggested that both Si-QAC and choline have been grafted onto the fibers. The grafting either SiQAC or choline to the hydroxyl groups of cotton causes an increase in weight as represented by grafting ratio in Table 1. Although a

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

(a) 8 7 6 5 4 3 2 0

20

40

60

80

100

120

140

Time (min)

Log (viable cell numbers) (CFU/mL)

10

might also contribute to the significant reduction of cells number observed for SMC and CMC. In order to clarify whether the modified cottons indeed have the antimicrobial activity, the growth curves of bacteria cultured in the presence of cotton fibers were studied. As shown in Fig. 3, both E. coli and B. subtilis cells show a slower growth rates and a lower final cell density when cultured either with SMC or CMC. The highest cell density achieved in the SMC culture is approximately 1 OD lower than that of CMC culture. This indicates both Si-QAC and choline molecules grafted onto cotton surface indeed have the antimicrobial activity and SMC is more effective for inhibiting the growth of bacterial cells than CMC. Evidently, the higher antimicrobial activity is mainly resulted from the long aliphatic tail of Si-QAC. Presumably, the long aliphatic tail of Si-QAC displays on the surface of SMC will interact and disrupt the cell membrane of bacteria that leads the bacteria to death. On the other hand, CMC does not have an aliphatic tail long enough to pierce into the cell membrane and bacteria are possibly bound by electrostatic interaction but still alive.

(b) 9

3.3. HA adsorption

8

The adsorption kinetic of HA onto modified cotton fibers was first studied at 37 ◦ C in pH 4, 0.1 M acetate buffer. As shown in Fig. 4, a very steep HA concentration decline during the first

7 6

5 5

(a)

4

4

3 3

2 0

20

40

60

80

100

120

140

Time (min)

OD600

Log (viable cell numbers) (CFU/mL)

9

47

2 Fig. 2. Viable cell number of (a) E. coli and (b) B. subtilis vs. time incubated with modified cotton fibers as estimated by spread plate method. The bacteria were incubated in 5 mL of salt solution and shaken at 200 rpm at 37 ◦ C with () no fibers addition, 0.02 g of (䊉) unmodified fibers, () choline modified fibers, () Si-QAC modified fibers.

1

0 0

lower nitrogen content was detected in Si-QAC modified cotton, a higher grafting ratio (25.05%) was obtained as compared with choline modified cotton (14.61%). Evidently, this is due to the fact that the nitrogen content in Si-QAC molecule itself (ca. 3%, w/w) is much lower than that in choline (ca. 10%, w/w).

2

4

6

8

10

12

14

16

10

12

14

16

Time (h) 5

(b) 4

3.2. Antibacterial activity 3

OD600

Antibacterial ability of the modified cottons was explored by estimating the number of E. coli and B. subtilis cells survived after being incubated with cottons for various time. Two control experiments were run with unmodified cotton and without cotton addition, respectively. As shown in Fig. 2, the number of viable cells decreases with respect to incubation time for both SMC and CMC. After 2 h incubation with SMC, the decreases for E. coli and B. subtilis were 62% and 34%, respectively. In the case of CMC, the viable cell reduced to 51% and 33% for E. coli and B. subtilis, respectively. In contrast, only less than 10% decreases were observed for the unmodified cotton. This decrease might be merely due to the entrapment of the bacterial cells into the interwoven cotton fibers mat formed during shaking thus reduces the cells number in the supernatant. In other words, the electrostatic adsorption and entrapment of bacterial cells between the interwoven cotton fibers

2

1

0 0

2

4

6

8

Time (h) Fig. 3. Growth curves of (a) E. coli and (b) B. subtilis in 50 mL of LB media () without fibers, (䊉) 0.2 g unmodified fibers, () 0.2 g choline modified fibers, () 0.2 g Si-QAC modified fibers.

48

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

HA concentration (mg/mL)

1.2

1.0

0.8

0.6

0.4

0.2

0.0 0

30

60

90

120

150

180

210

240

270

Time (min) Fig. 4. Time courses of hyaluronic acid adsorption onto (䊉) unmodified fibers, () choline modified fibers, () Si-QAC modified fibers. [HA]initial = 1 mg/mL; ionic strength = 0.1 M; T = 37 ◦ C; pHinitial = 4; shaker speed = 200 rpm; cotton fibers dose = 5 mg/mL.

15 min adsorption and level-off after 60 min were noticed for SMC. Whereas a gradual decrease of HA concentration lasts for 120 min was observed for CMC. In contrast, no significant decrease of HA concentration was detected for the unmodified cotton. After 3 h incubation, the remaining HA concentrations were 0.34 and 0.76 mg/mL for SMC and CMC, respectively. Apparently, the adsorption performance of SMC is superior to that of CMC with an initial adsorption rate and equilibrium adsorption capacity approximately 6 and 3 fold higher, respectively. Since HA is consisted of glucuronic acid and N-acetylglucosamine, its net charge will be dependent on environment pH. As shown in Fig. 5, no significant adsorption was detected at pH 3 for CMC. But at pH higher than 3, the adsorption efficiency increased steadily until pH 7. This adsorption behavior is mainly attributed to the electrostatic interaction between the strong cationic choline molecules of CMC and polyanionic character of HA at pH above 2.5 [19] due to its glucuronic acid subunits. In contrast, an appreciable adsorption of HA was observed for SMC at pH 3. The adsorption efficiency increased 4 fold to about 80% as pH increased from 3 to 4 and 5. Apparently, this substantial adsorption increase is mainly due to the fact that HA becomes more negatively charged as pH increases. However, contradictory

results were obtained that adsorption efficiency deceased from 80% to 35% as pH increased further above 6. Apparently, interactions other than electrostatic may contribute to the adsorption of HA onto SMC. The decrease of HA adsorption efficiency is probably because SMC becomes negatively charged at pH above 6. The negatively charged surface will repulse the anionic HA. The point of zero charge (PZC) of native cotton fiber is known to be around 2.5 [20] which means that the surface of cotton fiber will become negatively charged as pH increases above 2.5. Presumably, the PZC of SMC will be increased to 6.0 with surface modified by the positively charged QAC. In other words, SMC is positively charged at pH lower than 6.0 which is favorable for HA adsorption via electrostatic interaction. The HA adsorption efficiency observed at pH higher than 6.0 may result from the hydrophobic interaction between HA and SMC. HA is generally considered as an anionic hydrophilic polysaccharide but it also has extensive hydrophobic patches, about 8 CH units are in its secondary structure [21]. On the other hand, Si-QAC modified SMC also carries hydrophobic long aliphatic tails (18 CH2 units). Therefore, the hydrophobic interaction between HA and SiQAC grafted on SMC (Fig. 6a) can explain the appreciable amount of HA adsorbed at pH 3 that could not be achieved by CMC which does not have a long hydrophobic chain. In addition to the electrostatic interaction, the hydrophobic interaction should also contribute to the substantial increase of HA adsorption observed at pH 4 and 5 for SMC. 3.4. Adsorption isotherm The effect of temperature on equilibrium adsorption of HA to the modified cotton fibers was studied at its optimum pH. As shown in Fig. 7, temperature has a pronounced effect on the adsorption capacity. The amount of HA adsorbed onto SMC increased

100

HA adsorbed (%)

80

60

40

20

0 2

3

4

5

6

7

8

9

pH Fig. 5. Effect of pH on the equilibrium adsorption of hyaluronic acid onto () unmodified fibers, () choline modified fibers, () Si-QAC modified fibers. [HA]initial = 1 mg/mL; ionic strength = 0.1 M; T = 37 ◦ C; t = 3 h; pHinitial = 4; shaker speed = 200 rpm; cotton fibers dose = 5 mg/mL.

Fig. 6. Schematic representations of (a) hydrophobic attraction (dashed line) on SiQAC modified cotton fibers, and (b) electrostatic interaction (dotted line) on choline modified fibers with hyaluronic acid. Shadowed hexagonal, circles, and squares represent hydrophobic patches, acetamido, and carboxylate groups, respectively, in hyaluronic acid structure.

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

Table 2 Temperature effect on Langmuir model for hyaluronic acid adsorption by modified cotton.

200

(a)

HA adsorbed (mg/g)

180

R2

160

Samples

T (K)

140

Si-QAC modified (SMC) pH 4

277

78.7

5.8

0.980

291 310 277

153.6 183.7 351.3

6.4 7.7 11.3

0.993 0.997 0.997

291 310

225.3 100.2

9.3 7.4

0.980 0.975

120 100

Choline modified (CMC) pH 7

80 60 4 oC

40

18 oC 37 oC Langmuir model

20 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

HA equillibrium concentration (mg/mL) 400

(b) HA adsorbed (mg/g)

49

300

200

100 4 oC o

18 C 37 oC Langmuir model

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

HA equillibrium concentration (mg/mL) Fig. 7. Adsorption isotherm of hyaluronic acid on (a) Si-QAC modified cotton fibers (pH = 4), and (b) choline modified fibers (pH = 7) at different temperatures. Ionic strength = 0.1 M; incubation time = 3 h; shaker speed = 200 rpm; cotton fibers dose = 5 mg/ml.

with temperature, indicating the hydrophobic interaction plays a major role since hydrophobic interaction is generally known to be promoted by temperature. In contrast, the adsorption capacity of CMC decreased with temperature which implies hydrophobic interaction is not crucial in HA adsorption by CMC. The adsorption equilibrium were well-fitted by Langmuir isotherm model as follow bCe Qe = Qm 1 + bCe

(2)

where Ce (mg/mL) and Qe (mg/g solid) are HA concentration in the aqueous solution and the adsorbed HA on the solid at equilibrium, respectively. Qm is the maximum adsorption capacity for the modified cotton fibers loaded and b is the Langmuir equilibrium constant. The fitting results are summarized in Table 2. For SMC, the values of Qm and b increase with temperature while the opposite is observed for CMC. The maximum adsorption capacity for SMC and CMC is 183.7 mg/g (at 37 ◦ C, pH 4) and 351.3 mg/g (at 4 ◦ C, pH 7),

Qe (mg/g solid)

b (L/mg)

respectively. Evidently, the higher adsorption capacity achieved by CMC is due to its high surface quaternary ammonium concentration (Table 1) which in turn gives a higher positive charge density thus resulted in higher driving force of HA adsorption. In comparison with maximal HA adsorption capacity of Si-QAC–magnetite (38 mg/g magnetite) reported by Yang et al. [13], QAC modified cotton demonstrated at least 5 fold higher adsorption capacity. The higher capacity is probably resulted from the lighter density and the higher specific surface area of cotton fibers available for the interaction with the high molecular weight HA as compared with magnetite particles. In addition, the cotton fibers as a matrix for HA adsorption can provides a short diffusion path and less steric hindrance for the high molecular weight HA to be interacted with so that a fast adsorption kinetic can be achieved to shorten the operation time for HA recovery. The thermodynamic parameters, free energy of adsorption (G0 ), enthalpy (H0 ), and entropy (S0 ) changes for the HA adsorption by SMC and CMC were calculated and presented in Table 3. By using the equilibrium constant b obtained for each temperature from the Langmuir model, the Gibbs free energy (G0 ) can be calculated according to Eq. (3) G0 = −RT ln K

(3) G0

where K corresponds to b in the Langmuir equation. (J/mol) is the standard free energy change, R is the universal gas constant, 8.314 J/mol K, and T (K) is absolute temperature. G0 is the function of change in enthalpy of adsorption (H0 ) as well as change in standard entropy (S0 ) G0 = H 0 − TS 0

(4)

Eq. (3) can be inserted into Eq. (4) to obtain ln K = −

H 0 S 0 + RT R

(5)

The standard enthalpy change (H0 ) and standard entropy change (S0 ) of the adsorption process can be obtained from the slope and intercept of the ln K versus 1/T plot. As presented in Table 3, the negative values of G0 show that the adsorption of HA onto modified cellulose fibers is spontaneous. For SMC, as the temperature increased from 4 to 37 ◦ C, G0 became more negative, suggesting that adsorption was more favorable at high temperature. Additionally, a more negative G0 implies a greater driving force of adsorption, means a stronger affinity between adsorbent and adsorbate, thus, resulting in a higher adsorption capacity at

Table 3 Thermodynamic constants for the equilibrium adsorption of HA onto modified cottons. Samples

Si-QAC modified (SMC) Choline modified (CMC)

G0 (kJ/mol) 277 K

291 K

310 K

−4.0 −5.6

−4.5 −5.4

−5.2 −5.2

H0 (kJ/mol)

S0 (kJ/mol K)

6.1 −9.0

0.011 0.012

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

200

100

150 60 100 40 50 20

Adsorption capacity (mg/g)

HA adsorbed (mg/g)

80

Desorption efficiency (%)

(a)

100

160

HA adsorbed Desorption efficiency

Adsorption capacity amount HA adsorbed

140

80 120 100

60

80 40

60 40

20

Adsorption efficiency (%)

50

20 0

0 0

1

2

3

0

4

Number of cycle 400

30

200

20

100

10

0

Desorption efficiency (%)

HA adsorbed (mg/g)

HA adsorbed Desorption efficiency

300

0 0

1

2

3

5

30 in culture broth

60

Cellulose dose (mg/mL)

40

(b)

0 5 mg/mL in buffer system

4

Number of cycle Fig. 8. Repeated use of (a) Si-QAC modified cotton fibers at pH 4, and (b) choline modified fibers at pH 7 incubation time = 3 h; shaker speed = 200 rpm; fibers dose = 5 mg/mL. One cycle of operation is defined as the fibers incubated with HA solution, washing, incubated with 1 N NaCl, pH 7 phosphate buffer for SMC and 1 N NaCl, pH 3 glycine–HCl buffer for CMC to desorb HA, and washed again with the buffer used for adsorption.

37 ◦ C (Table 2). Whereas for CMC, G0 became more negative as the temperature decrease and therefore, the adsorption capacity at 4 ◦ C shows the highest value (Table 2). The positive H0 values of HA adsorption onto SMC reflects its endothermic nature whereas the negative H0 values for CMC implies its exothermic process. In general, the enthalpy change due to chemical adsorption (>20 kJ/mol) is considerably larger than due to physical adsorption (<20 kJ/mol). Hence, the adsorption of HA onto SMC and CMC which have H0 equal to 6.1 kJ/mol and −9.0 kJ/mol, respectively, is attributed to the physical adsorption process. The small positive values of S0 reflect the slight increase of system randomness as HA is adsorbed onto the modified cotton fibers. 3.5. Recovery of HA by adsorption In order to recover HA by adsorption process using the antibacterial cotton fibers, desorptions of the HA adsorbed at their optimum conditions (37 ◦ C, pH 4 for SMC and 4 ◦ C, pH 7 for CMC) were studied. As much as 31.28% and 40.60% of HA can be effec-

Fig. 9. Effect of culture broth to the adsorption capacity and adsorption efficiency of hyaluronic acid adsorbed onto silane modified cellulose fibers.

tively desorbed from SMC at pH 3 and pH 7, respectively. On the other hand, only about 5.31% of HA was desorbed from CMC at pH 3. In addition to pH shift, NaCl was employed to desorb HA by suppressing the electrostatic interaction. The HA desorbed from CMC could reach 15.80% as NaCl concentration increased to 0.5 N and enhanced further to 30.37% by doubling NaCl concentration to 1 N. For SMC, the desorption efficiency as much as 82.02% and 90.11% can be reached as 1 N NaCl is employed in the pH 3 and 7 desorption buffer, respectively. As shown in Fig. 8, the HA adsorption capacity and desorption efficiency decreased with the number of repeated use. The adsorption capacity of SMC decreased 49% after 3 cycles, whereas the capacity of CMC decreased 89%. The decrease of HA adsorption capacity possibly resulted from the incomplete desorption of the tightly bound HA during the previous cycle since about 10% and 60% of the adsorbed HA could not be desorbed from SMC and CMC, respectively. The bound HA not only decreased the available binding sites but also decreased the positive charge on the surfaces for attracting the incoming HA. The feasibility of recovering HA directly from B. subtilis culture was demonstrated by SMC adsorption at pH 4, 37 ◦ C because of its higher desorption efficiency. From Fig. 9, it is showed that the high HA adsorption efficiency was not satisfactorily achieved as demonstrated in the equilibrium adsorption study using buffer (Fig. 7). Only about 30% of HA was adsorbed from the broth (ca. 63.26 mg HA/g SMC), compared with 80% of HA adsorbed in the buffer system (ca. 145.25 mg HA/g SMC). This results can be explained by the fact that the broth contained various charged components of the rich fermentation medium as well as proteins, thus, prevent the interaction between HA and SMC. When SMC dose was increased to 30 mg/mL, about 49% of HA was adsorbed but the adsorption capacity reduced to 17.22 mg HA/g SMC (Fig. 9). One interesting observation is that the culture broth became transparent after incubation, suggesting that the B. subtilis cells were adsorbed by the SMC as well. The competition from B. subtilis cells for adsorption onto SMC therefore resulted in a lowered HA adsorption efficiency. SMC dose was further increased to 60 mg/mL, 85% of HA could be adsorbed and the adsorption capacity reduced to 14.94 mg HA/g

Table 4 Recovery of hyaluronic acid from Bacillus subtilis culture by using Si-QAC modified cotton fiber. Samples

HA conc. (mg/mL)

Protein conc. (mg/mL)

Yield (%)

Culture broth Supernatant (after adsorption) Eluted (after desorption)

1.054 ± 0.023 0.158 ± 0.032 0.402 ± 0.013

0.194 ± 0.002 0.162 ± 0.015 0

100.00 14.99 38.14

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 44–51

SMC. Table 4 shows the HA purification table using SMC for adsorption. The initial HA and protein concentrations in the culture broth were 1.054 and 0.194 mg/mL, respectively. After adsorption, only 0.158 mg/mL HA left in the supernatant. By incubating with 1 N NaCl, pH 7 phosphate buffer for desorption, HA of 0.4 mg/mL free of contaminant proteins was obtained. As much as 38% of HA in the B. subtilis culture was recovered by using SMC for adsorption. 4. Conclusion Modification of cotton fibers by the antibacterial quaternary ammonium compound (QAC) was addressed in this work for isolating HA from B. subtilis culture. Both Si-QAC and choline modified cotton fibers showed antibacterial activity against E. coli and B. subtilis, with the former had better activity due to the presence of long alkyl chain on the quaternary ammonium groups of SMC. The adsorption of HA onto antibacterial cotton fibers was a function of pH and temperature. Hydrophobic attractions are relatively more important for HA adsorption by SMC at pH 4, while electrostatic interactions are more important for the adsorption of HA by CMC at pH 7. The amount of adsorbed HA on SMC increases with temperature, conversely, the adsorption capacity of CMC decreases with temperature. The equilibrium adsorption isotherm can be well described by Langmuir model. The maximum adsorption capacities for HA were 183.7 mg/g for SMC and 351.3 mg/g for CMC. The adsorbed HA on SMC could be effectively desorbed as much as 90.11% by 1 N NaCl, pH 7 while changing the pH to 3 was effective to desorbed 36.50% HA from CMC. SMC could be used to recover HA directly from the B. subtilis culture about 15 mg of HA was recovered from B. subtilis culture by 1 g of SMC. References [1] H.G. Garg, C.A. Hales, Chemistry and Biology of Hyaluronan, Elsevier, Amsterdam, 2004. [2] L. Lapˇcík Jr., L. Lapˇcík, S.D. Smedt, J. Demeester, P. Chabreˇcek, Hyaluronan:, Preparation, structure, properties, and applications, Chem. Rev. 98 (1998) 2663–2684.

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