Indian Journal of Fibre & Textile Research Vol. 39, March 2014, pp. 93-96

Production and characterisation of microbial cellulosic fibre from Acetobacter xylinum

G Gayathrya & G Gopalaswamy

Department of Agricultural Microbiology, TamilNadu Agricultural University, Coimbatore 641 003, India

Received 26 November 2012; revised received and

accepted 8 February 2013

Bacterial cellulose (BC) has been produced by the Gram negative bacterium Acetobacter xylinum at the air liquid interface of sugary rich medium. The BC has been produced from Hestrin Shramn (HS) medium using the efficient cellulose-producing culture isolated from sugarcane juice under static batch fermentation condition. Results show that A. xylinum (sju-1) produces 11g/L BC after 14 days of fermentation period. The water holding capacity of bacterial cellulose is found to be 84.4 %. The tensile strength, Young’s modulus, viscosity and degree of polymerisation of bacterial cellulosic fibre are found to be 120 MPa, 4.9 GPa, 127.4 cP and 2074 respectively. The FTIR results reveal the presence of hydroxyl and CH2 stretching behaviour at the absorption wavelength of 3229 cm-1 and 2911cm-1.

The banding patterns of bacterial cellulose closely resemble the structure of pure celluloses. A. xylinum (sju-1) isolate produces bacterial cellulose of type Iα in quantities of commercial interest. The findings of the research reveal that bacterial cellulose can be used in making absorbent pads and nonwoven textiles.

Keywords: Absorbent pad, Acetobacter xylinum, Bacterial cellulose, Cellulosic fibre, Fermentation, Fibre, Nonwoven

Cellulose is of great importance in fundamental research and industrial applications because of its widespread occurrence, uniform high molecular structure and unique properties such as polyfunctionality, multichirality, hydrophilicity, and biocompatibility. Cellulose is produced by plants and microorganisms such as fungi, bacteria and algae. Bacterial cellulose (BC) is preferred over the plant cellulose as it can be obtained in higher purity with a higher degree of polymerization (DP) and crystallinity index (CI). It is a pure cellulose aggregate which does not include any impurities, such as hemicellulose, pectin and lignin1. Amongst all the cellulose, BC produced by an advanced type of α-proteobacteria Gluconacetobacter xylinum (Acetobacter xylinum) is

of great commercial interest. It has a microscopic reticular microfibrillar structure with the fibre thickness of about 0.1 µm and a higher degree of crystallinity than wood pulp cellulose. It is generated as a never-dried membrane in a nearly pure form that contains 99% water, of which 0.3% is bound and 98% is free water2. Fibrils of bacterial cellulose are about 100 times thinner than that of plant cellulose, making it a highly porous material which allows transfer of antibiotics or other medicines into the wound, while at the same time serving as an efficient physical barrier against any external infection. The properties are similar to the hydrogels produced from synthetic polymers; for example, it displays good sorption of liquids, is non-allergenic and can be safely sterilised without any change to its characteristics. Being similar to human skin, bacterial cellulose can be applied as skin substitute in treating extensive burns and nonwoven dressing material for chronic wounds. It is therefore used extensively in wound healing. Many potential high value markets exist for thin film of bacterial cellulose, including acoustic diaphragms, artificial skin, pulp and high value paper products, artificial blood vessels, liquid loaded medical pads, super-sorbers and specialty membranes3.

Production of cellulose from A. xylinum was first reported in 1886 by AJ Brown. A. xylinum is a Gram-negative, aerobic bacterium that produces cellulose in the form of interwoven extracellular ribbons as part of primary metabolism. This bacterium grows and produces cellulose from a wide variety of substrates and is devoid of cellulase activity. It has long served as a model organism for the study of bacterial cellulose synthesis, primarily because of the large quantities it produces. The bacteria secrete a structurally homogeneous slimy substance within which the cellulose fibres are formed. This microfibrillar structure of bacterial cellulose was first described by Muhlethaler in 1949. Electron microscopic observations showed that the cellulose produced by A. xylinum occurs in the form of fibres with two forms of cellulose, namely (i) Cellulose I — the ribbon-like polymer and (ii) Cellulose II — the thermodynamically more stable amorphous polymer. A single A. xylinum cell is capable of polymerizing

_______ aCorresponding author. E-mail: gayasaro@yahoo.co.in

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200000 glucose molecules per second into β-1,4-glucan chains which are then excreted into the surrounding medium forming ribbon-like bundles of microfibrils. The fibres are formed in the membrane by cellulose synthase and consequently secreted from a row of 50-80 pore like synthetic sites along the longitudinal axis of the cell and gives a three dimensional cellulosic fibre layer4. A. xylinum previously characterized for cellulose production have been isolated from fruit sources and isolates from sugarcane juice have not been extensively characterized. Previously studied strains from fruit have failed in long-term subculturing under static conditions. The scale of bacterial cellulose production, processing and use is relatively small because of the problems associated with the selection of sufficiently efficient producers. The isolate from sugarcane juice namely Acetobacter xylinum (sju-1) produces bacterial cellulose in quantities of commercial interest. Further the production of BC is receiving great attention because of its benefit for environmental requirements and possess wide application possibilities. Hence, an attempt has been made to produce and characterise bacterial cellulose from Acetobacter xylinum isolated from fermented sugarcane juice.

Experimental

Bacterial cellulose production and recovery

The starter culture of Acetobacter xylinum (sju-1) was inoculated in 1.0 L sterilised Hestrin Shramn medium at 30˚C for 14 days. After the incubation period, bacterial cellulose (BC) was formed as a white pellicle floating at the top of the medium. The pellicle was lifted from the medium and washed thoroughly in running tap water. Pellicle was treated with 4 per cent (w/v) sodium hydroxide solution in boiling water bath for 30 min to remove microbial cells attached to the pellicle. After that, it was washed with excess water to remove the alkali completely. Then it was pressed with a 10 ton press unit to squeeze out water and to get the BC wet sheets. The BC wet sheets were bleached by soaking in 5 L of 1.5 per cent H2O2, which was adjusted to pH 11 with 0.5 per cent NaOH, in a sealed plastic bag. The plastic bag with the BC was then shaken for 10 min and put into boiling water for 30 min or until the BC colour turned white. After bleaching, the white BC fibres were washed with tap water and pressed again with a 10 ton press unit; the wet white BC was dried on an electrical press unit at a high temperature of 140˚C for 30s. The dried white BC were tested for physical properties further5.

Physicochemical characteristics of bacterial cellulose

The fresh cellulosic pellicle formed was lifted and after several washing, the excess water was dripped off completely and weighed for measuring the fresh weight (g). The moisture content (%) was determined based on the weight loss of the cut cubes when dried under vacuum for 8 h at 25 bar pressure at 75˚C and the dry weight was calculated. Thickness (mm) was tested by using a micrometer type calliper. Cellulosic mat after cutting into perfect cubes of equal dimensions was wrapped in filter paper and centrifuged at 5000g for 10 min. During centrifugation the water released was absorbed by the filter paper. The per cent ratio of the moisture in the centrifuged cellulose to the original moisture content yielded the water holding capacity of the BC. A suspension containing disintegrated bacterial cellulose (BC) was cast in a plastic petri dish and completely dried at 50˚C overnight. The tensile strength of the sheet was measured with an ASTM Standard D 638. The sheet was cut into ribbons (5 × 30 mm) for measurement of tensile strength. The thickness of the ribbons was 100 µm, and the elongation rate was 1.0 mm/min. Young’s modulus was determined from the ratio of the stress exerted on the sample to that exerted on the deformed sample, as measured by a tensile tester (SGA-100, Shin Gang Co. Ltd., Korea)6.

Viscosity and degree of polymerisation measurement

The viscosity of finely divided BC sample was measured as centipoises (cP) using cupriethylendiamine (CED) as a solvent and a capillary viscometer7. From viscosity values, the average degrees of polymerization (DP) of the BC sample were calculated using the following equation:

P0:905 = 0:75(954 log(X) - 325, where X =TAPPI viscosity in cP

Air resistance of cellulose (s/50 ml) was the time measurement in seconds on the air resistance of approximately 1 inch2 of a circular area of cellulosic layer using a pressure differential of 1.22 kPa. Castor oil penetration (min/0.05 mL) was the measurement of the time required for a drop of castor oil to produce a uniform translucent spot on the underside of the test specimen by permeating through the bacterial cellulose8. FTIR and SEM studies

The cellulose samples obtained from HS medium was analysed to study conformational characteristics by FTIR spectrometer (Perkin-Elmer S2000) using

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KBr plate method. 1.0 mg of dried bacterial cellulose samples were mixed with KBr powder and pressed into a small tablet. Then FTIR spectrum was measured in the transmittance mode with the resolution of 1.00 cm-1 at wavenumbers ranging from 4000 cm-1 to 400 cm-1. The morphological investigations of the bacterial cells on the cellulose and the cellulose fibrils were characterized using scanning electron microscope (SEM) (Model: S-3400 HITACH Co., Japan). Thin layers of freeze dried cellulose were gold coated using ion sputter (Fisons Instruments, UK). The gold coated sample was viewed and photographed.

Results and Discussion

Production of cellulosic fibre

BC has been obtained as a thick layer of mat on the surface of the HS medium and the yield of cellulose by Acetobacter xylinum (sju-1) is found to be 11.00 g/L (Fig. 1). The thickness of wet cellulosic pellicle is 13.00 mm and the thickness of dry sheets is 0.23 mm. The water content is recorded as 92.3% and water holding capacity as 84.4% which indicates that bacterial cellulose could be used as absorbent pads.

The tensile strength and Young’s modulus are recorded to be 120 MPa and 4.9 GPa respectively. The viscosity and degree of polymerisation are 127.4 cp and 2074 respectively (Table 1). According to Keshk and Shameshima9 viscosity and degree of polymerisation of bacterial cellulose produced by A. xylinum (ATCC 10245) are found to be 129 cP and 2681 respectively. The results of the present study using A. xylinum (sju-1) are in close proximity with that of bacterial cellulose produced by A. xylinum (ATCC 10245). Castor oil penetration test shows that it completely offers resistance to oil penetration and air could not penetrate into it as confirmed by the air penetration test. Yamamoto and Horri10 has reported that the native Acetobacter pellicle has mechanical properties including shape retention and tear resistance that are superior to many synthetic fibres. FTIR and SEM analysis

Figure 2 shows the IR spectrum of bacterial cellulose. IR spectrum obtained for BC produced from HS medium shows strong absorption peak at 3229 cm-1 and 2911 cm-1 representing OH and CH2

grouping. Broader bands of 3229 cm-1 indicate the presence of more hydrogen bonding patterns. The results of the present study correlate with the findings of Sluraska et al.11 who has indicated that Acetobacter

xylinum grown in HS medium produces cellulose showing IR spectrum in the region of 3400 cm-1. The strong absorption peak at 1644 cm-1

confirms the presence of carboxylic acid groups (COOH) in cellulose structure. The band at 1428 cm-1

is attributed to the occurrence of carbonyl group in BC. The bands at 1163 cm-1 and 1068 cm-1 show the possibilities of C-O-C functionalities present in the BC. According to Jung et al.12 IR spectrum of BC produced from molasses medium is in the region of 3240 cm-1,

Fig.1 — Fresh cellulosic fibre formed by A. xylinum (sju-1)

Table 1— Physical property of bacterial cellulosic fibre Parameter Value

Dry weight, g/L 11.0 Moisture content, % 92.3 Water holding capacity, % 84.4 Thickness, mm 0.2 Tensile strength, MPa 120 Young’s modulus, GPa 4.9 Viscosity, cP 127.4 Degree of polymerisation (DP) 2074 Castor oil penetration Nil Air penetration test, s/50 mL Nil

Fig. 2— FTIR spectrum of bacterial cellulose

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attributing to the presence of more quantities of cellulose Iα. In this investigation also, the transmittance peak near to 3240 cm-1 is achieved, indicating that cellulose Iα is abundantly present in BC from HS medium. The present study indicates an appropriate coincidence with earlier studies relating to IR spectrum of pure cellulose and bacterial cellulose and authentically proves that the component produced by the sugarcane juice isolate of A. xylinum (sju-1) is cellulose and the structural components obtained for the BC from HS medium show higher content of cellulose type Iα. Figure 3 shows the SEM micrographs of thread like cellulosic microfibrils and the bacterial cells entangled in it. The thickness of the fibrils varies from 128nm to 207nm at ×8000 magnification. The fibrils are tightly packed and conferred morphological features similar to that of pure microcrystalline cellulose.

Cellulose obtained from A. xylinum (sju-1) has opened new avenues in the field of fibre and textiles. The production of bacterial cellulose is more convenient and a relatively pure form of cellulose can

be obtained with simple alkali treatment. Based on the FTIR studies it is concluded that the properties of bacterial cellulose are closer to that of the pure microcrystalline cellulose. With a very high water retention capacity and tensile strength bacterial cellulose appears to be a specific substitute for plant cellulose in selected applications such as absorbent pads and in production of nonwoven textiles.

Acknowledgement

One of the authors (G. Gayathry) thanks TamilNadu Agricultural University for granting Research Assistantship and Seshasayee Paper boards, Pallipalayam, Tamilnadu for technical assistance and to Central Instrumentation Facility (CIF) at Pondicherry University, Puducherry (U.T) for IR analysis and SEM micrographs of BC.

References 1 Orts W J, Shey J, Imam S H, Glenn G M, Guttman M E &

Revol J F, J Polym Environ, 13 (2005) 301. 2 Kersters K, Lisdiyanti P, Komagata K & Swings J, in The

Prokaryotes (Springer, New York) 2006, 163. 3 Bielecki S, Krystynowicz A, Turkiewicz M & Kalinowska H,

in Bacterial Cellulose: Polysaccharides and Polyamides in

the Food Industry (Wiley-VCH Verlag, Weinheim, Germany) 2005, 31.

4 Delmerl D P & Amor Y, The Plant Cell, 7 (1995) 987. 5 Thompson D & Hamilton M, Appl Biochem Biotechnol, 91

(2001) 503. 6 Annual Book of ASTM Standards, Section 8, Plastics, 540

(ASTM, Pennsylvania), 1993. 7 Viscosity of Pulp (Capillary Viscometer Method), T230 om-89

(TAPPI, Atlanta) 2005. 8 TAPPI Test Method, T460 om-96, T462 om-93 (TAPPI,

Atlanta), 1998. 9 Keshk S & Sameshima K, Appl Microbiol Biotechnol, 72(2)

(2006) 291. 10 Yamamoto H & Horri F, Cellulose, 1 (1994) 57. 11 Slusarska B S, Presler S & Danielewicz D, Fibres Text

Eastern Eur, 16(4) (2008) 108. 12 Jung H O, Lee O M, Jeong J H, Jeon Y D, Park K H, Kim H S,

Ann W G & Son H, J Appl Biochem Biotechnol, 162 (2010) 486.

Fig. 3— SEM micrograph of cellulosic fibrils formed by A. xylinum (sju-1)