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Biochemical Engineering Journal 53 (2010) 4451

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Biochemical Engineering Journal


journal homepage: www.elsevier.com/locate/bej

Nonleaching antimicrobial cotton bers 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 bers 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-modied cottons demonstrated antibacterial activity against Gram () Escherichia coli and Gram
(+) Bacillus subtilis along with HA adsorption capacity. The HA adsorption isotherm could be well-tted
with Langmuir model with maximum adsorption capacities of 184 mg/g and 351 mg/g for Si-QAC and
choline surface-functionalized cotton bers, 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 modied antimicrobial cotton ber.
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 signicant
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 purication 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 [46]. Cetylpyri-

Corresponding author. Tel.: +886 2 27376629, fax: +886 2 27376644.


E-mail address: cklee@mail.ntust.edu.tw (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 afnity 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
[712]. The major mode of action was identied 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 modied Si-QAC and HA is
rarely studied except Yang et al. [13] have reported that the Si-QAC
modied magnetic particles can be used for HA adsorption but with
a low capacity.
Because of its rich surface hydroxyls, cotton bers are usually
surface functionalized for the preparation of functional textiles. For
example, cationic functionalized cotton bers 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) 4451

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 bers 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 bers. In this work, Si-QAC and
choline were grafted onto the surface of cotton bers, respectively.
The antimicrobial activities against Gram () Escherichia coli and
Gram (+) Bacillus subtilis of the modied cotton bers were evaluated. The adsorption of HA at various temperatures and pHs as well
as the reusability of the antimicrobial cotton bers 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 modied bers 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 bers
To prepare Si-QAC modied cotton bers (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 modied cotton
bers (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
bers 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

46

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 4451

Table 1
Elemental analysis and grafting ratio of modied cotton bers.
Samples

C (%)

H (%)

N (%)

Grafting ratio (%)

Unmodied
Si-QAC modied (SMC)
Choline modied (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 modied cotton bers was calculated using the following
formula as described by Roy et al. [15]

where Wg is the dry weight of each grafted cotton bers and Wo is


the initial weight of each cotton bers. 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 glycineHCl buffer (for CMC), at room temperature and
shaken at 200 rpm for 24 h. The cotton bers 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 modied cotton bers was evaluated against E. coli and B. subtilis by the viable cell counting method
[16]. Prior to the antibacterial tests, all cotton bers 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 unmodied cellulose bers (UMC), SMC, and CMC,
respectively and incubated at 37 C for 3 h with 200 rpm shaking. A
control culture without cotton bers was also treated in the similar
way. At the predetermined time, 1 mL of bacteria culture was taken
and serially diluted down to 107 . 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 bers samples
for 15 h. The cell density was measured by spectrophotometer at
wavelength 600 nm.

The HA concentration was estimated by carbazole method [17].


Briey, 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 rst 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 bers was carried
out by mixing 1 mL of HA solution of various concentrations with
5 mg of dry cotton bers. Prior to the addition of cotton bers, the
HA solutions were placed in a water bath for 30 min to achieve the
specic adsorption temperature. HA concentration in the supernatant was analysed by carbazole method [17]. The effect of pH on
HA adsorption onto cotton bers was studied at pH in the range of
38 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 bers
before and after modication. The higher nitrogen and carbon contents detected after modication suggested that both Si-QAC and
choline have been grafted onto the bers. 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) 4451

(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 signicant reduction of cells number


observed for SMC and CMC.
In order to clarify whether the modied cottons indeed have
the antimicrobial activity, the growth curves of bacteria cultured
in the presence of cotton bers were studied. As shown in Fig. 3,
both E. coli and B. subtilis cells show a slower growth rates and a
lower nal 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

The adsorption kinetic of HA onto modied cotton bers was


rst 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 rst

7
6

5
5

(a)

3
3

2
0

20

40

60

80

100

120

140

Time (min)

OD600

Log (viable cell numbers) (CFU/mL)

47

2
Fig. 2. Viable cell number of (a) E. coli and (b) B. subtilis vs. time incubated with modied cotton bers 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 bers addition,
0.02 g of () unmodied bers, () choline modied bers, () Si-QAC modied
bers.

0
0

lower nitrogen content was detected in Si-QAC modied cotton,


a higher grafting ratio (25.05%) was obtained as compared with
choline modied 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).

10

12

14

16

10

12

14

16

Time (h)
5

(b)
4

3.2. Antibacterial activity


3

OD600

Antibacterial ability of the modied 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 unmodied 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 unmodied cotton. This decrease might be merely due to
the entrapment of the bacterial cells into the interwoven cotton
bers 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 bers

0
0

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

48

D. Wibowo, C.-K. Lee / Biochemical Engineering Journal 53 (2010) 4451

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 () unmodied bers,
() choline modied bers, () Si-QAC modied bers. [HA]initial = 1 mg/mL;
ionic strength = 0.1 M; T = 37 C; pHinitial = 4; shaker speed = 200 rpm; cotton bers
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 signicant decrease
of HA concentration was detected for the unmodied 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 signicant
adsorption was detected at pH 3 for CMC. But at pH higher than 3,
the adsorption efciency 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 efciency 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 efciency 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 efciency 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 ber is known to be around 2.5
[20] which means that the surface of cotton ber will become negatively charged as pH increases above 2.5. Presumably, the PZC of
SMC will be increased to 6.0 with surface modied 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 efciency 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
modied 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
modied cotton bers 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

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

Fig. 6. Schematic representations of (a) hydrophobic attraction (dashed line) on SiQAC modied cotton bers, and (b) electrostatic interaction (dotted line) on choline
modied bers 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) 4451

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

200

(a)

HA adsorbed (mg/g)

180

R2

160

Samples

T (K)

140

Si-QAC modied
(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 modied
(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 modied cotton
bers (pH = 4), and (b) choline modied bers (pH = 7) at different temperatures.
Ionic strength = 0.1 M; incubation time = 3 h; shaker speed = 200 rpm; cotton bers
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-tted 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 modied cotton bers loaded and b is the Langmuir equilibrium
constant. The tting 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-QACmagnetite
(38 mg/g magnetite) reported by Yang et al. [13], QAC modied
cotton demonstrated at least 5 fold higher adsorption capacity. The
higher capacity is probably resulted from the lighter density and
the higher specic surface area of cotton bers available for the
interaction with the high molecular weight HA as compared with
magnetite particles. In addition, the cotton bers 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 modied cellulose bers 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 afnity between adsorbent
and adsorbate, thus, resulting in a higher adsorption capacity at

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

Si-QAC modied (SMC)


Choline modied (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) 4451

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

Number of cycle
400

30

200

20

100

10

Desorption efficiency (%)

HA adsorbed (mg/g)

HA adsorbed
Desorption efficiency

300

0
0

30
in culture broth

60

Cellulose dose (mg/mL)

40

(b)

0
5 mg/mL
in buffer system

Number of cycle
Fig. 8. Repeated use of (a) Si-QAC modied cotton bers at pH 4, and (b) choline
modied bers at pH 7 incubation time = 3 h; shaker speed = 200 rpm; bers
dose = 5 mg/mL. One cycle of operation is dened as the bers incubated with HA
solution, washing, incubated with 1 N NaCl, pH 7 phosphate buffer for SMC and 1 N
NaCl, pH 3 glycineHCl 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 reects 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 reect the slight increase of system randomness as
HA is adsorbed onto the modied cotton bers.
3.5. Recovery of HA by adsorption
In order to recover HA by adsorption process using the antibacterial cotton bers, 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 efciency
of hyaluronic acid adsorbed onto silane modied cellulose bers.

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 efciency 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 efciency 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 efciency. From Fig. 9, it is showed that the high
HA adsorption efciency 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 efciency.
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 modied cotton ber.
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) 4451

SMC. Table 4 shows the HA purication 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
Modication of cotton bers by the antibacterial quaternary
ammonium compound (QAC) was addressed in this work for isolating HA from B. subtilis culture. Both Si-QAC and choline modied
cotton bers 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 bers 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.
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