Morphological Investigation of Bacterial Cellulose/Cassava Starch

Nanocomposites Produced by In-situ Process in Agitated Culture

C. F. Zuhra

, Yugia Muis

, S. Gea

, S. A. Amaturrahim

, K. M. Pasaribu

, and S. U. Rahayu

Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara,

Jl. Bioteknologi No. 1 Kampus Padang Bulan USU Medan 20155, Indonesia

Departement of Physics, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara,

Jl. Bioteknologi No. 1 Kampus Padang Bulan USU Medan 20155, Indonesia

Keywords: Bacterial cellulose, Cassava starch, Nanocomposites, In-situ process, Agitated culture

Abstract: The existence of starch in the formation of bacterial cellulose was found to have the capability to enhance

the viscosity of culture medium, thus it can affect the agitation process. The aim of this study was to

investigate the morphological structure of bacterial cellulose/cassava starch (BC/CS) nanocomposites

produced by in-situ process in agitated culture. The fabrication of BC/CS was carried out at 28°C for 7 days

under agitated condition at 100 rpm with five different masses of CS. The crystallinity of BC/CS

nanocomposites was studied by using X-ray Difraction (XRD) pattern, whereas the morphological structure

was discovered from the digital photograph, optical microscope (OM) picture and scanning electron

microscope (SEM) pictures. From the SEM analysis, it was observed that the cassava starch layer had

occupied the pores of the fibre network of bacterial cellulose and increased the average size of the fibres.

Also, it was well dispersed in the network of bacterial cellulose. The high content of starch on the culture

caused the changes in orientation of the nanocomposites’ surface morphology and reduced the shaking

effect of the culture. The study found that the BC/CS nanocomposites with variation of 5 g CS showed the

best morphological properties.

1 INTRODUCTION

Bacterial cellulose (BC), which produced by

Acetobacter xylinum, was one of environmentally

friendly products that has grown the researchers’

attention nowadays due to its outstanding features,

such as its higher tensile strength, crystallinity, and

capacity of water absorption; besides, it has ultra-

fine fibre network structure, good transparency,

good chemical stability, considerable fibre binding

ability, appropriate biocompatibility,

biodegradability, and moldability [Ishihara et al.

2002; Klemm et al. 2001; Shezad et al. 2009;

Vandamme et al. 1998] . One of its applications is as

a material to solve the problem of the dependence on

the petroleum products and the environmental

damage [Averous, 2004; Lu et al. 2006]. As a

biodegradable material, BC can be employed as a

matrix of nanocomposites material.

Regarding to the above explanation, starch,

which has a proper biodegradability, low cost

production, and a wide availability, could be a good

filler for the matrix of bacterial cellulose to form

BC/cassava starch nanocomposites [Angles and

Dufresne, 2000]. The integration of those two

biodegradable materials was employed since the

starch itself has some drawbacks, namely, lower

mechanical properties, high hygroscopicity, and high

permeability to gases [Vandamme et al. 1998].

Therefore, by combining these two materials, the

new biodegradable materials with enhanced

properties could be yielded.

As reported by Haigler et al. (1982) the starch

which is added to the culture medium of

Acetobacter xylinum does not affect the reaction of

cellulose formation even though different carbon

sources, such as glucose, fructose, and gluconate,

can be used to synthesize the cellulose. In other

word, the starch is not consumed by the bacteria. It

can be proved by the appearance of blue color on the

nanocomposites after being added by iodine, which

shows the existence of starch on the nanocomposites

[Zhang et al. 2006; Kuipers et al.1994]. The

existence of starch on the nanocomposites of

1042

Zuhra, C., Muis, Y., Gea, S., Amaturrahim, S., Pasaribu, K. and Rahayu, S.

Morphological Investigation of Bacterial Cellulose/Cassava Starch Nanocomposites Produced by In-situ Process in Agitated Culture.

DOI: 10.5220/0010096710421046

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

1042-1046

ISBN: 978-989-758-449-7

BC/starch, furthermore, could strengthen the

interaction between cellulose membrane and the

wall inside the culture vessel during the inoculation

process because of the high viscosity of the medium

after gelatinization process of starch. That

interaction, which was called as wall effect, can

compress the production of cellulose by limiting the

increase of cellulose membrane thickness [Homung

et al. 2006]. Besides, the decrease of fluidity and the

increase of viscosity due to the existence of

gelatinized starch could limit the diffusion process

of the glucose substrate on the cellulose matrix and

the motion of Acetobacter xylinum in the culture

medium [Yang et al. 2014].

There are two methods that are commonly used

to produce the BC, namely static method and

agitation method. The use of the static method on

the production of BC has been proved to have some

disadvantages over the agitation method. The

agitation method can provide enough oxygen for the

bacteria, while the static method failed to do so;

thus, the agitation method can increase the

production of cellulose. Also, the agitation method

can reduce the crystal size and form the more stable

crystal. However, based on several studies, the

agitation methods can cause the stretching among

the woven of cellulose fibre, thus forming the larger

pores. This makes the layers of formed cellulose

separated each other, so that the degree of

crystallinity reduced [13–1Watanabe et al. 1998;

Yamanaka et al. 2000; Yamamoto et al. 1996].

Based on the above issues, the production of

BC/starch nanocomposites using agitation method

should be investigated. The agitation method was

chosen because it can provide enough oxygen, while

the existence of starch can reduce the shaking effect

of agitation process. To date, there is no study

discussed about the production of BC/starch

nanocomposites using agitation method. Although

there have been several research studied about the

production of BC/starch nanocomposites [Yang et

al. 2014; Grande et al. 2009; Martin et al. 2009;

Woehl et al. 2010], those studies still used the

conventional static method. This study aimed to

investigate the morphological properties of

BC/cassava starch (BC/CS) nanocomposites

produced by in-situ process in agitated culture

because it can present the effects of starch on BC

visually. Thus, it can provide better understanding in

the effects of starch on the formation of bacterial

cellulose nanocomposites.

2 MATERIALS AND METHODS

2.1 Materials

The materials used in this study were glucose, bacto-

peptone, urea, NaOH, NaOCl, CH

COOH, and

distilled water, which were purchased from Merck,

without having further treatments. Coconut water

was supplied from traditional markets in Medan,

Indonesia and the bacteria, Acetobacter xylinum,

was supplied by the Microbiology Laboratory of

Universitas Sumatera Utara.

2.2 Isolation of Cassava Starch

The isolation of cassava (Manihot esculanta) starch

was done by using conventional method. Briefly, the

cassava was peeled. After that, it was washed using

water, then shredded. The resulted cassava was

added with enough water, then blended. The result

was then precipitated after filtered using the gauze.

The precipitation was washed frequently until the

washed waste was transparent. This precipitation

was called as starch. After that, it was dried in the

oven at 45

C for 24 hours. The dried starch was

ground and sifted in order to obtain the final starch.

2.3 Preparation of BC/CS

Nanocomposite Film

BC/CS nanocomposites were produced by the

Acetobacter xylinum bacterial strain in culture

medium that containing 100 ml coconut water, 0.5%

(w/v) urea, 1.0 % (w/v) glucose, 1.5% (w/v) bacto-

peptone. The pH of culture medium was adjusted to

4.5 by acetic acid. CS with variations of 1 g, 2 g, 3

g, 4 g, and 5 g was added to culture medium,

followed by autoclave for 30 min at 121°C. The

solutions were magnetically stirred for 15 minutes.

Main cultivation were carried out at 28°C for 7 days

under agitated condition at 100 rpm. The BC/CS

nanocomposites were washed under running tap

water and it was immersed overnight in 2.5% NaOH

and also in 2.5% NaOCL. Then, it was rinsed again

under running tap water to remove any solvent until

it reached neutral pH. The BC/CS nanocomposites

were finally pressed using hot-press with wire-mesh

at 115°C for 10 min.

2.4 Characterization

The BC/CS nanocomposites were characterized by

X-ray diffraction (XRD), scanning electron

Morphological Investigation of Bacterial Cellulose/Cassava Starch Nanocomposites Produced by In-situ Process in Agitated Culture

1043

microscope (SEM), and optical microscope (OM).

The XRD pattern were taken by Shimadzu XRD-

6100 diffractometer using Cu-Kα radiation (λ =

0.154 nm) at scanning rate of 2°/min, a voltage of

40kV and a current of 200mA. The diffraction angle

(2θ) range from 5° to 30° with a step size of 0.02°.

The degree of crystallinity (Crystallinity Index, CrI)

was calculated from diffracted intensity data using

the method described by previous researcher (Seagel

et al., 1959) as shown on the formula (1)

CrI (%) = (1-(I

-I

200

)) x 100% (1)

Where the I

and I

200

represented the intensity of

diffraction in the same units at approximately 2θ =

18° and maximum intensity of (002) lattice

diffraction at approximately 2θ = 22.7°, respectively.

The scanning electron microscope was done

using SEM EDX EVO MA 10 Carl Zeiss Bruker.

All samples were sputter coated with gold-palladium

and observed using an accelerating voltage 20 kV.

Samples were viewed at magnification between

1000 and 10000 times from their original sizes.

The optical microscope pictures were taken using

American Optical Microscope with the

magnification of 100 times

3 RESULT AND DISCUSSION

3.1 XRD Analysis

As shown in Figure 1, the diffraction peak of starch

was found at 2θ of 14,780°, 16,924°, and 22,040°.

This is in agreement with the results reported by

Grande et al. [Grande et al. 2009]. The starch

showed lower crystallinity value. This was caused

by two conditions from the gelatinization process

that occurred during the formation of

nanocomposites. As gelatinized CS, the crystal from

starch granules was damaged and the intensity

related to the peak of diffraction will decrease, or

even disappear. The crystallinity of starch was equal

to zero because there are no crystal peaks observed

on the spectra.

The diffraction peaks for BC was discovered at

2θ of 14.208°, 16.930°, and 22.924°, which showed

the diffraction lattice of (1ȋ0), (110), and (002),

respectively, at the polymorph of cellulose I. This

result is in accordance with the result reported by

Table 1. Diffraction peaks, d-spacing, and degree of

crystalinity of CS, BC, and CS/BC nanocomposites.

Sample 2θ (°) d (nm) Degree of

crystallinity

(%)

CS 15.030° 5.889 ~0%

17.880° 4.956

23.173° 3.835

BC 14.827° 5.969 92%

17.362° 5.103

23.168° 3.836

BC/CS 14.208° 6.228 53%

16.930° 5.233

22.924° 3.876

Yang et al. 2014. As also can be seen from

figure 1, the BC has sharp peaks because of the high

crystallinity degree of it (92%, shown in table 1). It

can be explained as a result of the intermolecular

hydrogen bonding in the cellulose structure.

10 20 30

2θ (degree)

Intensity (-)

BC/CS

Figure 1. X-ray diffraction spectra of CS, BC, and CS/BC

nanocomposites.

For the BC/CS nanocomposites, the diffraction

peaks was placed at 2θ of 14.827°, 17.362°, and

23.168°. This result is also similar to the results

obtained by Yang et al. (2014). As previously

mentioned that the BC itself has a high degree of

crystallinity, the existence of cassava starch,

furthermore, reduce its degree of crystallinity to

53% as shown in table 1. The decrease in degree of

crystallinity of BC/CS nanocomposites occurred due

to two possible reasons: 1.The migration of

Acetobacter xylinum was blocked by the poor

fluidity of medium due to the existence of

gelatinized starch. In other word, the motion of

bacteria was limited; 2. There was a high steric

obstruction in amylopectin branching on that prevent

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

1044

the formation of cellulose bands since the

amylopectin stick in the cellulose microfibrils [Yang

et al. 2014]

3.2 The Morphology of BC/CS

Nanocomposites Analysis

Figure 2 shows the digital photograph of the BC

membrane and the BC/CS membrane with various

mass of CS after 7-day agitation process. It can be

seen that the mass of CS affects the layer yielded,

the more CS added, the more organized the BC/CS

nanocomposites obtained. The BC/CS

nanocomposites with the variation of 5 g CS show

the most organized one. It is similar to the BC/CS

nanocomposites produced by static method. This can

be occurred because the starch can increase the

viscosity of culture medium, thus it can reduce the

shaking effect during the agitation process.

The optical microscope pictures from BC and

BC/CS nanocomposites with the variation of 5

grams of CS in never-dried state were given in

figure 3. As seen in figure 3(a), the surface of BC is

more transparent than that of BC/CS

nanocomposites. The surface of BC reveals the fibre

network of the BC, while the surface of BC/CS

nanocomposites looks solid without having any fibre

(figure 3(b)). This happened because the pores of

BC have been filled by the swollen starch granules.

Figure 2. Digital photos of pure BC and BC/CS

nanocomposites with various mass of starch with

magnitude (a) 100x in never-dried state.

The surface morphology of CS, BC and BC/CS

nanocomposites is also further investigated through

SEM with 10,000 times of magnification and is

shown in Figure 4. Figure 4(a) gives the shape of

starch granules clearly with a perfect oval shape.

Based on our analysis, the average size of them is

around 12.755 µm. The large size of the granules

indicates the high capability of capping the water

during the gelatinization process.

Figure 3. Optical microscope images of BC and BC/CS

nanocomposites with magnitude (a) 100x in never-dried

state

Figure 4. SEM images of (a) CS, (b) BC, and (c) BC/CS

nanocomposites with magnitude 10,000x after hot-

pressing.

Figure 4(b) indicates the irregularity of BC

network obtained by agitation method. This can be

occurred because during the process, the bacteria

move to the oxygen-rich area. The agitation method

will cause the stretching happened among the woven

of cellulose fibre and result in larger pores among

them. Still, this method can reduce the size of fibre

or the size of crystal [Watanabe et al.1998].

The two above results formed another structure

when they combined as BC/CS nanocomposites due

to the interaction between them. This is shown in

Figure 4(c). The gelatinized CS was dispersed

evenly in the matrix of BC. The high content of

starch caused the integration of BC network and

gelatinized starch; the starch did not only stick in the

BC fibre, but also resulted in the changes in the

orientation of nanocomposites’ surface morphology.

Based on the SEM analysis, it was discovered that

the BC has average size of fibre of 67.907 nm, while

BC/CS nanocomposites have the average size of

fibre of 73.470 nm. The increase of fibre size

occurred because the shaking effect decreased after

the addition of starch.

Morphological Investigation of Bacterial Cellulose/Cassava Starch Nanocomposites Produced by In-situ Process in Agitated Culture

1045

4 CONCLUSION

In this study, BC/CS nanocomposites were

successfully obtained by in-situ process in agitated

culture. The XRD pattern shows that the degree of

crystallinity of BC/CS nanocomposites was lower

than that of the BC. From the digital photograph,

OM and SEM pictures, it was proven that the

existence of starch could rectify the irregularity of

cellulose layer produced by agitation method. The

gelatinized CS is well dispersed in the network of

BC and filled the pores of the BC fibre, thus

increases the average size of the nanocomposite

fibres. Also, it causes the changes in the orientation

of nanocomposites’ surface morphology. The more

CS added, the more organized layer yielded. This

can be occurred because the increase gelatinized

starch can raise the viscosity of culture medium and

reduce the shaking effect. Furthermore, it was found

that the BC/CS nanocomposites with the variation of

5 g CS performed the best result.

ACKNOWLEDGEMENT

The authors would like to thank to the Rector of

University of Sumatera Utara for financial support

from the project of PUU-Talenta 2018.

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