bacterial cellulose nanocomposite beads prepared using Gluconacetobacter xylinus and their application in lipase immobilization

Carbohydrate Polymers 157 (2017) 137–145

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Alginate/bacterial cellulose nanocomposite beads prepared using Gluconacetobacter xylinus and their application in lipase immobilization Ji Hyun Kim a,1 , Saerom Park a,1 , Hyungsup Kim b , Hyung Joo Kim a , Yung-Hun Yang a , Yong Hwan Kim c , Sang-Kyu Jung d , Eunsung Kan e,f , Sang Hyun Lee a,∗ a

Department of Microbial Engineering, Konkuk University, Seoul 143-701, South Korea Department of Organic and Nano System Engineering, Konkuk University, Seoul 143-701, South Korea c School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 44919, South Korea d Department of Bio & Chemical Engineering, Hongik University, Sejong 339-701, South Korea e Texas A&M AGRILIFE Research & Extension Center, Texas A&M University, Stephenville, TX 76401, USA f Office of Sponsored projects, Tarleton State University, Stephenville, TX 76401, USA b

a r t i c l e

i n f o

Article history: Received 19 April 2016 Received in revised form 29 August 2016 Accepted 23 September 2016 Available online 26 September 2016 Keywords: Alginate Bacterial cellulose Nanocomposite Bead Lipase Immobilization

a b s t r a c t Alginate/bacterial cellulose nanocomposite beads, with well-controlled size and regular spherical shapes, were prepared in a simple manner by entrapping Gluconacetobacter xylinus in barium alginate hydrogel beads, followed by cultivation of the entrapped cells in culture media with a low sodium ion concentration. The entire surface of the alginate hydrogel beads containing the cells was covered with cellulose fibers (∼30 nm) after 36 h of cultivation. The cellulose crystallinity index of the alginate/bacterial cellulose beads was 0.7, which was slightly lower than that of bacterial cellulose prepared by cultivating dispersed cells. The water vapor sorption capacity of the alginate/bacterial cellulose beads increased significantly from 0.07 to 38.00 (g/g dry bead) as cultivation time increased. These results clearly indicate that alginate/bacterial cellulose beads have a much higher surface area, crystallinity, and water-holding capacity than alginate beads. The immobilization of lipase on the surface of the nanocomposite beads was also investigated as a potential application of this system. The activity and specific activity of lipase immobilized on alginate/bacterial cellulose beads were 2.6- and 3.8-fold higher, respectively, than that of lipase immobilized on cellulose beads. The alginate/bacterial cellulose nanocomposite beads prepared in this study have several potential applications in the biocatalytic, biomedical, and pharmaceutical fields because of their biocompatibility, biodegradability, high crystallinity, and large surface area. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Cellulose is the most abundant biopolymer on Earth and is frequently obtained from plant sources. Some bacterial strains such as Gluconacetobacter, Agrobacterium, Rhizobium, Rhodobacter, and Sarcina can produce cellulose extracellularly (Ullah, Wahid, Santos, & Khan, 2016; Iguchi, Yamanaka, & Budhiono, 2000). Because of its inherent biocompatibility and biodegradability, bacterial cellulose (BC) has several potential applications in various biomedical fields, including artificial blood vessel manufacturing, wound dressing, dialysis membranes, and biosensors (Kim, Park, Won, Kim, and Lee,

∗ Corresponding author. E-mail address: [email protected] (S.H. Lee). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.carbpol.2016.09.074 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

2013). The structural features of BC are superior to those of plant cellulose, as BC has unique properties such as high purity, high crystallinity, ultrafine networks, and remarkable mechanical strength (Shah, Ul-Islam, Khattak, & Park, 2013). Under static conditions, cellulose-producing cells produce BC, which has a thick and gelatinous membrane, while pellet BC forms are produced under agitated culture conditions (Lin et al., 2013). To date, most studies of BC focused on the preparation and application of membranous BC. Although cellulose beads have several potential applications in fields such as drug delivery, chromatography, and protein immobilization, the pellet or bead BC forms have not been widely examined. Chao, Ishida, Sugano, and Shoda (2000) reported the large-scale production of pellet BC using a 50-L internal-loop airlift reactor and Cheng, Wang, Chen, and Wu (2002) described the production of a unique elliptical pellet with a 10 mm diameter in a modified airlift reactor. Wu and Lia (2008) used BC pellets to

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immobilize glucoamylase, and Park, Kim, Kwon, Hong, and Jin (2009) prepared BC/multiwalled carbon nanotube pellets. Furthermore, Zhu, Li, He, and Duan (2015) prepared BC/graphene oxide pellets and investigated their cytotoxicity. A major limitation associated with the preparation of bead BC forms is the inability to control their size and shape. In general, preparing BC beads with a regular spherical shape is very difficult because these parameters are highly dependent on the culture conditions such as aeration rate, shaking speed, and cell type. Therefore, BC beads with well-controlled size and shape should be prepared to expand the applications of BC beads. Alginate is an anionic polysaccharide widely present in the cell wall of brown algae. Alginate is a linear copolymer of ␤d-mannuronic acid and ␣-l-guluronic acid, and can be gelated using divalent cations such as Ca2+ and Ba2+ (Bajpai & Sharma, 2004). Alginate has been used to entrap proteins, enzymes, and cells in the food and pharmaceutical industries because of its low cost, non-toxicity, biocompatibility, and biodegradability. Recently, hybrid nanocomposites of BC and alginate were developed for use as scaffolds for tissue engineering, antibacterial films for wound dressing, yeast cell carriers for ethanol production, membranes for the separation of ethanol-water mixtures, and supports for drug delivery (Kirdpompattara & Phisalaphong, 2013; Kirdponpattara, Khamkeaw, Sanchavanakit, Pavasant, & Phisalaphong, 2015; Shao et al., 2015; Shi et al., 2015; Suratago et al., 2015). Because of their inherent biocompatibility and the high surface area of their nanofibrous structures, nanocomposites of BC and alginate have many potential applications in the biocatalytic, biomedical, and pharmaceutical fields. In this study, alginate was used as a template to prepare BC beads. Gluconacetobacter xylinus entrapped in alginate hydrogel beads led to the formation of alginate/BC nanocomposite beads with well-controlled size. This is the first report of the production of BC beads with regular spherical shape prepared by simple cultivation of G. xylinus. The methods associated with gelation of alginate hydrogel beads, medium composition for cultivation, and bead purification were optimized to prepare rigid alginate/BC nanocomposite hydrogel beads. Several characteristics of the alginate/BC beads, including surface morphology, interaction between alginate and BC, and cellulose crystallinity, were investigated using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and x-ray diffraction (XRD). In order to confirm the continuous production of BC by G. xylinus, the water vapor sorption capacity of the alginate/BC beads was determined at various cultivation times. As a potential application of the alginate/BC nanocomposite beads, lipase from Candida rugosa was immobilized on the bead surface. In addition, the biodegradability of alginate/BC beads was investigated by cellulase-catalyzed hydrolysis.

2. Experimental 2.1. Materials Sodium alginate, barium chloride, barium hydroxide, butyrate, p-nitrophenol, 1-ethyl-3p-nitrophenyl methylimidazolium acetate ([Emim][Ac]), lipase from C. rugosa, cellulase from Trichoderma reesei, and the glucose assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Yeast extract and peptone were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Citric acid, glucose, mannitol, ethanol, and acetonitrile were purchased from Samchun Pure Chemical (Gyeonggi-do, South Korea). Gluconacetobacter xylinus (ATCC 11142) was purchased from the Korean Culture Center of Microorganisms (Seoul, South Korea). Cellulose was obtained from Hyosung Co. (Seoul, South Korea).

2.2. Cultivation of G. xylinus Gluconacetobacter xylinus was cultured on a solid nutrient medium containing 5 g/L yeast extract, 3 g/L peptone, 25 g/L mannitol, and 15 g/L agar at 26 ◦ C for 48 h. The colonies were picked from agar plates and suspended in modified Hestrin-Schramm (HS) medium containing 20 g/L glucose, 2 g/L yeast extract, 5 g/L peptone, 1.15 g/L citric acid, and 1% ethanol (Hestrin & Schramm, 1954; Park, Jung, & Park, 2003). All culture media were sterilized at 121 ◦ C for 15 min. Ethanol was added to the sterilized media to enhance cellulose productivity (Yunoki, Osada, Kono, & Takai, 2004), and the pH was adjusted to 5.5 by adding NaOH. 2.3. Preparation of alginate/BC nanocomposite beads Sodium alginate (3% w/w) was dissolved in distilled water at 60 ◦ C for 1 h. A total of 4 mL alginate solution was mixed with 1 mL of G. xylinus suspension in modified HS medium. The optical density of the cell suspension was approximately 80 at 600 nm (about 80 mg dry cell weight). The resulting alginate and cell mixture (1 mL) was then added drop-wise into 1 L of 0.2 M BaCl2 at a rate of 50 ␮L/min, and the mixture was subjected to vigorous stirring using a 1-mL plastic syringe with a 26-gauge needle and syringe pump (LSP012A, Longer Pump, Hebei, China). Barium alginate hydrogel beads containing G. xylinus were cured in BaCl2 solution for 1 h, and were then washed with sterilized water for 20 min. Approximately 100 beads were prepared using a mixture of 1 mL alginate and cells. Fifteen barium alginate hydrogel beads containing G. xylinus cells were incubated in 25 mL of modified HS medium at 26 ◦ C in a shaking incubator at 70 rpm. After cultivation for various periods, the alginate/BC hydrogel beads were incubated in 30 mM Ba(OH)2 solution for 24 h at room temperature to remove the cells, followed by repeated washing with distilled water for neutralization. The purified alginate/BC nanocomposite beads were stored in distilled water at 4 ◦ C until use. In order to prepare alginate-free BC beads for lipase immobilization, alginate/BC nanocomposite beads were incubated in 5% (w/v) NaCl solution at 40 ◦ C for 2 h with vigorous shaking and then the beads were incubated in the same solution at 60 ◦ C for 3 days under static conditions. NaCl-treated BC beads were washed with distilled water and stored at 4 ◦ C until use. 2.4. Characterization of alginate/BC nanocomposite beads The sizes of the alginate/BC nanocomposite hydrogel beads were measured using a digital caliper (Fuso, Japan). The diameter of each bead was measured at three different angles and the average was calculated. Three beads were used to calculate the average bead size. The dry weights of the alginate/BC hydrogel beads were measured after drying at 60 ◦ C for 24 h. To analyze the bead surface, alginate/BC hydrogel beads were frozen overnight at −80 ◦ C and then freeze-dried under a vacuum for 12 h. All freeze-dried samples were sputter-coated with platinum prior to observation. The surfaces of the samples were examined by SEM (SUPRA 55VP, Carl Zeiss, Jena, Germany) and fiber diameters were measured using the SEM images and image analysis software (EyeViewAnalyzer, Digiplus, Inc.). In addition, the freeze-dried beads were used for FTIR analysis (Nico-let 6700, ThermoElectron, Waltham, MA, USA). Scanning was conducted from 4000 cm−1 to 650 cm−1 , and 64 repetitious scans were averaged for each spectrum. The resolution was 4 cm−1 and the scanning interval was 2 cm−1 . 2.5. Cellulose crystallinity measurement Freeze-dried alginate/BC nanocomposite beads were ground using a homogenizer and analyzed by XRD. The samples were scanned on a D8 Advance Diffractometer (Bruker, Billerica, MA,

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Table 1 Characteristics of various alginate/BC nanocomposite beads. Cultivation time (h)

Average diameter of hydrogel bead (mm)

Average diameter of cellulose fiber on the surface of bead (nm)

Water vapor sorption capacity of bead (g/g dry bead)

0 12 18 24 36 48 72

2.5 ± 0.1 3.0 ± 0.1 3.0 ± 0.1 3.0 ± 0.1 3.2 ± 0.2 3.5 ± 0.1 3.6 ± 0.2

– a ND ND ND 30.2 31.1 33.4

0.07 ± 0.00 6.06 ± 0.42 15.25 ± 0.62 26.26 ± 1.50 37.01 ± 1.48 38.88 ± 1.26 35.19 ± 2.83

a

ND: not determined because no fiber was detected on the surface of the bead.

USA) at 40 kV and 40 mA, and the 2 theta (␪) was scanned from 2◦ to 40◦ with a 0.1◦ width. The cellulose crystallinity index (CrI) was determined using the XRD peak height method and the following equation (Park, Baker, Himmel, Parilla, & Johnson, 2010). CrI =

I002 − IAM I002

I002 is the height of the 002 peak and IAM is the height of the minimum between the 002 and 101 peaks. 2.6. Water vapor sorption capacity of alginate/BC nanocomposite beads To determine the water vapor sorption capacity of alginate/BC nanocomposite beads, 60 beads were equilibrated in distilled water in closed vessels for 5 days at 25 ◦ C until no further water uptake was observed. The total weights of the hydrogel beads (Wt ) were measured after equilibrium was reached. The beads were then dried at 60 ◦ C for 24 h, and the dry weights of the beads (W0 ) were measured. The water vapor sorption capacity was calculated using the following equation: Water vapour sorption capacity(g/gbead) =

(Wt − W0 ) W0

2.7. Adsorption of lipase on alginate/BC nanocomposite beads Alginate/BC nanocomposite hydrogel beads cultivated for 48 h and NaCl-treated alginate-free BC hydrogel beads were used to immobilize lipase. To prepare the lipase solution, 10 mg of C. rugosa lipase was added to 1 mL of 50 mM MES buffer (pH 7.0) and the sample was shaken for 1 min. After centrifugation, the supernatant was diluted with MES buffer and used for the immobilization experiments. To physically adsorb lipase onto the alginate/BC hydrogel beads or alginate-free BC hydrogel beads, 10 beads were added to 2 mL of lipase solution (approximately 60 ␮g protein/mL), followed by incubation with vigorous shaking at 120 rpm and 25 ◦ C. After incubating the beads and lipase for various periods, the beads were filtered to remove unbound enzymes, were then washed three times with 50 mM MES buffer (pH 7.0) for 5 min at 25 ◦ C to remove weakly adsorbed lipase. The filtrate and wash solutions were collected for protein measurement. The amount of protein adsorbed on the beads was indirectly estimated by calculating the difference between the initial protein content in the enzyme solution and remaining protein content in the filtrate and wash solutions. Protein content in the solutions was measured using a Micro BCATM protein Assay Kit (Thermo Scientific, Waltham, MA, USA) following a standard protocol. In order to compare the immobilization efficiency of alginate/BC beads with those of various beads, two types of alginate beads and cellulose beads were prepared. To prepare alginate beads with sizes similar to those of alginate/BC beads (ALG1), 2.4% sodium alginate was dissolved in 4 mL of distilled water at 60 ◦ C for 1 h. The alginate

solution was then added drop-wise into 1 L of 0.2 M BaCl2 at a rate of 50 ␮L/min by using an 18-gauge needle. To prepare beads with similar alginate contents to those of alginate/BC beads (ALG2), 4 mL of 3% sodium alginate solution was mixed with 1 mL of modified HS medium. The resultant mixture was then added drop-wise into 1 L of 0.2 M BaCl2 at a rate of 50 ␮L/min by using a 26-gauge needle. To prepare cellulose beads with sizes similar to those of alginate/BC beads (CEL), 2.4% cellulose was dissolved in 4 mL of [Emim][Ac] at 60 ◦ C for 1 h. The cellulose solution was then added drop-wise into 1 L of distilled water at a rate of 50 ␮L/min by using a 18-gauge needle. The cellulose beads were washed five more times with distilled water. The absence of [Emim][Ac] in the beads was confirmed by measuring the optical density of the washing solution at 211 nm (Park et al., 2015). The procedure for lipase immobilization on alginate/BC beads described above was also used to immobilize lipase on alginate beads (ALG1 and ALG2) and cellulose beads (CEL). 2.8. Determination of lipase activity A spectrophotometric assay was used to determine the hydrolytic activity of immobilized lipase. Immobilized lipase was placed in a 50 mL Falcon tube containing 9.5 mL of 50 mM MES buffer (pH 7.0). The reaction was initiated by adding 0.5 mL of substrate solution prepared by dissolving 10 mM p-nitrophenyl butyrate in isopropanol, and the solution was maintained at 25 ◦ C in a shaking water bath (120 rpm). Periodically, 300 ␮L aliquots were removed, diluted with 300 ␮L of acetonitrile, and centrifuged to obtain the supernatant. The activity was expressed as the initial rate and was determined by measuring the increase in absorbance at 400 nm (10 mm path length cuvette with 1 mL volume). Absorbance increased when p-nitrophenol was produced during the lipasecatalyzed hydrolysis of p-nitrophenyl butyrate. The initial rate measurements were conducted in triplicate (Park et al., 2015). 2.9. Cellulase-catalyzed degradation of cellulose in alginate/BC nanocomposite beads The cellulase-catalyzed hydrolysis reaction was performed in 50 mL vials on a rotary shaker at 120 rpm and 37 ◦ C in 10 mL with three alginate/BC beads and cellulase (5 mg/mL) in 50 mM citrate buffer (pH 5.0). Samples (50 ␮L) were periodically removed and boiled for 3 min to quench the enzymatic reaction. After centrifugation of the boiled samples, glucose concentration was measured using a hexokinase-based glucose assay kit. All reactions were performed in triplicate (Lee, Doherty, Linhardt, & Dordick, 2009). 3. Results and discussion 3.1. Preparation of alginate/BC nanocomposite beads The preparation of uniform BC beads that retain a nanofibrous structure is challenging because tight control of the bacterial growth conditions is required. Therefore, alginate was used as a

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Fig. 1. SEM images of alginate/BC nanocomposite beads prepared using various cultivation times. (a–c): 0 h; (d–f): 24 h; (g–i): 36 h; (j–l): 48 h; (m–o): 72 h.

template to produce BC beads in this study. Gluconacetobacter xylinus entrapped in alginate hydrogel beads successfully produced BC with a narrow distribution of fiber diameters. The prepared alginate/BC nanocomposite beads showed consistent sizes and regular spherical shapes. The barium alginate hydrogel beads could produce more rigid alginate/BC nanocomposite hydrogel beads than calcium alginate hydrogel beads. Calcium alginate beads containing G. xylinus quickly swelled and became weak during cell cultivation, while barium alginate beads containing cells remained rigid for a longer period. However, barium alginate beads also became weaker after an incubation period of one day because of the high sodium ion content in HS medium, and thus sodium content in the HS medium was reduced. NaHPO4 was removed and the yeast extract content was decreased in the modified HS medium. As a

result, barium alginate beads containing G. xylinus remained rigid in the modified HS medium for more than three days. During the purification step, barium hydroxide was used rather than sodium hydroxide to remove cells without decreasing the rigidity of the alginate/BC nanocomposite hydrogel beads.

3.2. Characteristics of alginate/BC nanocomposite beads The average diameter of alginate/BC hydrogel beads increased from 2.5 to 3.6 mm with increasing cultivation time (Table 1). Alginate/BC hydrogel beads prepared for the same cultivation time deviated by less than 6%, indicating that consistently sized alginate/BC beads can be prepared by controlling the cultivation time. The average diameter and volume of alginate/BC hydrogel beads

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141

Fig. 2. Frequency distribution of cellulose fibers on the surface of alginate/BC nanocomposite beads. (a): 36 h cultivation (n = 38); (b): 48 h cultivation (n = 49); (c): 72 h cultivation (n = 36).

increased by 144% and 300%, respectively, after 72 h cultivation. The sizes of alginate/BC hydrogel beads after cultivation may have increased because of swelling of alginate and the enhanced waterholding capacity of alginate/BC beads from the produced BC. Fig. 1 shows SEM images of alginate/BC beads prepared for various cultivation times. Although cellulose fibers were not detected on the surface of the alginate/BC beads until 24 h, the entire alginate/BC bead surface was covered with cellulose fibers after 36 h of cultivation. The average diameter of alginate/BC hydrogel beads initially increased from 2.5 to 3.0 mm after 12 h, but the diameter did not change until 24 h. However, the diameter increased again between 36 and 72 h of cultivation. These results indicate that the BC produced by G. xylinus was present inside the alginate beads until 24 h, and then the cellulose fibers were released to the bead surface. With increasing cultivation times from 36 h to 72 h, the number of cellulose fibers and number of cellulose mats layered on the bead surface increased. The BC fiber size on the surface of alginate/BC nanocomposite beads was measured using 100,000-fold enlarged SEM images. Fig. 2 shows the frequency distribution of cellulose fibers on the surface of alginate/BC beads. The average cellulose fiber diameters were 30.2, 31.1, and 33.4 nm for alginate/BC beads cultivated for 36, 48, and 72 h, respectively (Table 1). The average cellulose fiber diameter was slightly enhanced by increasing the cultivation time

from 36 to 72 h. The BC fiber size ranged from 20 to 45 nm and was narrowly distributed with a bell-shaped graph. The sizes of BC fibers produced by G. xylinus in the barium alginate bead were similar to those produced by G. xylinus in suspended culture (Guhados, Wan, & Hutter, 2005). FTIR spectroscopy was used to investigate the potential interactions between alginate and BC in the alginate/BC nanocomposite bead (Fig. 3). The FTIR spectra of the alginate, alginate/BC bead, and BC were measured at wavelengths of 4000–650 cm−1 . Pure alginate showed characteristic peaks centered at 1586 and 1408 cm−1 , which are commonly assumed to be asymmetric and symmetric carboxyl stretching bands, respectively. Additionally, a peak centered at 1028 cm−1 was observed and assigned to the characteristic vibration of a sugar ring. In contrast, the BC film and alginate/BC nanocomposite bead showed peaks centered at 3341 and 2897 cm−1 , which were assigned to O H and C H stretching, respectively. A peak centered at 1032 cm−1 was also observed in the spectra of the BC and alginate/BC (Table 2). The patterns of the FTIR spectra for alginate/BC nanocomposite beads were very similar to those of the FTIR spectra for the BC/alginate membrane (Cacicedo et al., 2016). The band for alginate at 1586 cm−1 indicated the presence of a carboxyl group. The FTIR spectra of the BC showed a band at 1652 cm−1 , which was attributed to the glucose carbonyl of cellulose. The carboxyl group band for the alginate/BC bead was shifted from 1586 to 1595 cm−1 . These results indicate that

(a) 3276

(b)

2988 1586

(002)

1408 1028

(c)

1595 1422 3342 1652

2897

1412 3341

1031

Intensity (a.u.)

2898

(101) (10 I )

(040)

(a)

(b) (c) (d)

1032

4000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm ) Fig. 3. FTIR spectra of alginate (a), alginate/BC nanocomposite bead prepared with 48 h cultivation (b), and BC film (c).

10

15

20

25

30

35

40

2-theta Fig. 4. X-ray diffraction patterns of BC film (a) and alginate/BC nanocomposite beads prepared using various cultivation times. (b): 72 h cultivation; (c): 48 h cultivation; (d): 36 h cultivation.

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Table 2 FTIR analysis of alginate, alginate/BC nanocomposite bead, and BC. Alginate (Wavenumbers, cm−1 )

Alginate/BC

BC

Assignments

3276 2988 1586 1408 1028

3342 2898 1595 1422 1031

3341 2897 1652 1412 1032

OH stretching C H stretching Asymmetric COO stretching Symmertic COO stretching C O C and C O H stretching vibration of sugar ring

specific interactions occur between the hydroxyl group of cellulose and carboxyl group of alginate. Similar observations were previously reported for the preparation of BC/alginate blend membranes (Phisalaphong, Suwanmajo, & Tammarate, 2007). 3.3. Cellulose crystallinity of alginate/BC nanocomposite beads Fig. 4 shows the XRD patterns of BC film and alginate/BC nanocomposite beads prepared for various cultivation times. The BC film displayed the expected four peaks at 2␪ = 14.3◦ , 16.7◦ , 22.6◦ , and 34.5◦ , which corresponded to the (101), (10¯ı), (002), and (040) crystalline planes of cellulose (Park et al., 2010). Alginate/BC nanocomposite beads also displayed these four peaks and alginate/BC beads exhibited an unreported peak at 2␪ = 24.0◦ . The intensity of this unknown peak increased with increasing cultivation time. This peak may represent the formation of a new crystalline structure, but additional studies are needed to verify this. The CrI values of alginate/BC beads, calculated using the peak height method, were approximately 0.70, and only slight changes were observed with varied cultivation times (Table 3). Compared to the CrI value of BC film produced in static culture, the CrI values of the alginate/BC beads were reduced by 19%. Zhou, Sun, Hu, Li, and Yang (2007) also reported that compared to the CrI values of

Table 3 Cellulose crystallinity index values of alginate/BC nanocomposite beads. BC film obtained by static culture

CrI

0.86

Alginate/BC bead based on cultivation time 36 h

48 h

72 h

0.72

0.70

0.70

BC produced without alginate, the CrI values of cellulose produced by Acetobacter xylinum decreased in the presence of alginate. 3.4. Water vapor sorption of alginate/BC nanocomposite beads One of major characteristics of BC is its high water-holding ability. To measure the water-holding ability of alginate/BC nanocomposite beads, the water vapor sorption capacity of alginate/BC beads was determined (Table 1 and Fig. 5). The water vapor sorption capacity of alginate beads was only 0.07 (g/g dry bead), while that of alginate/BC nanocomposite beads prepared for 48 h of cultivation was 38.88 (g/g dry bead). The content of BC in the alginate/BC nanocomposite beads prepared by 48 h cultivation was approximately 25% (w/w). Thus, BC in the nanocomposite beads showed a water sorption capacity of approximately 155 (g/g dried

Fig. 5. Water vapor sorption capacity of alginate/BC nanocomposite beads prepared using various cultivation times. (a): 0 h; (b): 12 h; (c): 18 h; (d): 24 h; (e): 36 h; (f): 48 h; (g): 72 h.

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143

Hydrolyzed cellulose (%)

100

80

60

40

20

0 0

20

40

60

80

100

120

Incubation time (min) Fig. 7. Cellulase-catalyzed hydrolysis of cellulose in alginate/BC nanocomposite beads.

3.5. Immobilization of lipase on alginate/BC nanocomposite beads

Fig. 6. (a) Protein content adsorbed on alginate beads (䊉: ALG1 (alginate beads with sizes similar to those of alginate/BC beads); 䊏: ALG2 (alginate beads with alginate contents similar to that of alginate/BC beads)), cellulose beads (), alginate/BC nanocomposite beads (), and BC nanocomposite beads (). (b) Specific activities of immobilized lipases.

BC). This result is similar to the water sorption by BC produced using G. hansenii (Feng et al., 2015). The water vapor sorption capacity of alginate/BC beads was dramatically enhanced with increasing cultivation time. As shown in Fig. 1, cellulose fibers were not observed on the surface of the alginate/BC composite beads cultivated for 24 h. However, the water-holding power of these beads was 375fold higher than that of alginate beads. This result clearly shows that G. xylinus produced BC continuously inside the alginate bead until 24 h, although the cellulose fiber was not detected on the alginate/BC nanocomposite bead surface.

Lipase from C. rugosa was physically adsorbed on alginate/BC beads cultivated for 48 h and alginate-free BC beads treated with NaCl to remove alginate. Three types of beads were also examined for comparison: alginate beads with sizes that were similar to alginate/BC beads (ALG1), alginate beads with alginate content that was similar to alginate/BC beads (ALG2), and cellulose beads with sizes that were similar to alginate/BC beads (CEL) (Fig. 6). When the adsorption process reached equilibrium, protein content adsorbed on the alginate/BC beads was similar to protein content adsorbed on the ALG1 and CEL beads, which was 120% higher than the protein content adsorbed on ALG2 beads. ALG2 beads can be considered as non-cultivated (0 h) alginate/BC beads. Therefore, the protein content adsorbed on alginate/BC beads was enhanced by the production of cellulose using G. xylinus. The high surface area of cellulose nanofibers on the surface of the alginate/BC beads may aid in the immobilization of additional protein. However, the protein loading capacity of alginate/BC beads was lower than expected. The enhanced protein loading capacity of alginate/BC may have resulted from the increased bead size, as the protein content adsorbed on the alginate/BC bead was similar to that adsorbed on ALG1 beads. In addition, the existence of alginate on the surface of alginate/BC beads may inhibit the interaction between lipase and BC nanofibers. Therefore, alginate on alginate/BC beads was removed by treatment with NaCl solution at high temperature. The protein content adsorbed on the treated BC beads was 1.9-fold higher than the protein content adsorbed on alginate/BC. These results clearly show that the high surface area of BC nanofibrous structures is very useful for protein adsorption. The specific activity of lipase adsorbed on alginate/BC beads was much higher than that adsorbed on alginate (ALG1 and ALG2) beads and cellulose (CEL) beads. When the adsorption process reached equilibrium, the specific activity of lipase adsorbed on alginate/BC beads was 159%, 192%, and 245% higher than that adsorbed on ALG1, ALG2, and CEL beads, respectively. When the specific activity of lipase reached a maximum value, the specific activity of lipase adsorbed on the alginate/cellulose beads was 187%, 245%, and 381% higher than that adsorbed on ALG1, ALG2, and CELL

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beads, respectively. These results clearly show that the surfaces of the alginate/BC beads were more favorable than those of alginate beads and cellulose beads for lipase immobilization. Immobilization efficiency (%) determined by the relative specific activity of immobilized lipase compared with free lipase (1.80 ␮mol/min/mg protein) for the lipase adsorbed on alginate/BC ranged from 25% to 47%. Immobilization efficiency of lipases adsorbed on barium alginate beads in this study ranged from 13% to 24%, which was slightly lower than the immobilization yield of lipases entrapped in calcium alginate beads (Won, Kim, Kim, Park, & Moon, 2005). The specific activity of lipase adsorbed on NaCl-treated BC beads was similar to that of lipase adsorbed on CEL beads, although the protein content adsorbed on the BC beads was much higher than that on CEL beads. The low specific activity of lipase adsorbed on NaCl-treated BC beads may have resulted from steric hindrance of the lipase resulting from the higher density of protein loading on the BC beads. 3.6. Cellulase-catalyzed degradation of cellulose in alginate/BC nanocomposite beads In order to investigate the biodegradability of alginate/BC nanocomposite beads, cellulase from Trichoderma reesei was used to hydrolyze the cellulose in alginate/BC beads. Approximately 85% of cellulose in alginate/BC beads was hydrolyzed into glucose in citrate buffer at pH 5.0, when the hydrolysis reaction reached equilibrium (Fig. 7). This result indicates that the cellulose of alginate/BC nanocomposite beads can be degraded by cellulaseproducing microorganisms when the beads are exposed to the environment. Therefore, biodegradable alginate/BC nanocomposite beads have many potential applications in environmental fields. 4. Conclusions In this study, alginate/BC nanocomposite beads of wellcontrolled size and regular spherical shape were simply prepared by entrapment of G. xylinus into barium alginate hydrogel beads, followed by cultivation of the entrapped cells in modified HS medium. This process can be simply applied to the classical fermentation industry because only classical techniques such as cell entrapment and cell cultivation are involved. The surfaces of alginate/BC beads were fully covered with approximately 30 nm BC fibers with a crystallinity index of 0.7. The water vapor sorption capacity of alginate/BC beads was 542-fold higher than that of alginate beads. As a result, alginate/BC nanocomposite beads showed high surface area, high crystallinity, and high water-holding power, and they maintained the inherent biocompatibility and biodegradability of alginate and BC. In addition, the size of the alginate/BC nanocomposite beads can be easily controlled by altering the size of the barium alginate bead. The ratio of alginate to cellulose in the alginate/BC bead can also be controlled by changing the cultivation conditions such as temperature, pH, and shaking speed. Therefore, alginate/BC nanocomposite beads have many potential applications in the biocatalytic, biomedical, and pharmaceutical fields. In this study, lipase was successfully immobilized to the surface of alginate/BC beads as a potential application of the beads. Therefore, alginate/BC beads can be used as enzyme supports for the immobilization of various enzymes. Acknowledgements This work was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education (2015R1D1A1A01060206). This work was also supported by a Korea CCS R&D Center (KCRC)

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