Effect of different additives on bacterial cellulose production

Effect of different additives on bacterial cellulose production

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Effect of different additives on bacterial cellulose production by Acetobacter xylinum and analysis of material property

Kuan-Chen Cheng Æ Jeffrey M. Catchmark Æ Ali Demirci

Received: 19 December 2008/Accepted: 15 July 2009/Published online: 9 August 2009 Springer Science+Business Media B.V. 2009

Abstract Bacterial cellulose (BC) demonstrates unique properties including high mechanical strength, high crystallinity, and high water retention ability, which make it an useful material in many industries, such as food, paper manufacturing, and pharmaceutical application. In this study, different additives including agar, carboxymethylcellulose (CMC), microcrystalline cellulose, and sodium alginate were added into fermentation medium in agitated culture to enhance BC production by Acetobacter xylinum. The optimal additive was chosen based on the amount of BC produced. The produced BC was analyzed by using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and dynamic mechanical analysis (DMA). Among the evaluated additives, CMC yielded highest BC production (8.2 g/L) compared to the control (1.3 g/L). The results also indicated that CMC-altered BC production increased with CMC addition and reached saturation around

1%. The variation between replicates for all analysis was \5%. From XRD analysis, however, the crystallinity and crystal size decreased as CMC addition increased. FESEM results showed CMC-altered BC produced from agitated culture retained its interweaving property. TGA results demonstrated that CMC-altered BC had about 98% water retention ability, which is higher than BC pellicle produced with static culture. CMC-altered BC also exhibited higher Tmax compared to control. Finally, DMA results showed that BC from agitated culture loses its mechanical strength in both stress at break and Young’s modulus when compared to BC pellicle. This study clearly demonstrated that addition of CMC enhanced BC production and slightly changed its structure.

Keywords Bacterial cellulose Acetobacter xylinum Cellulose crystallinity, cellulose yield


Cellulose is the most abundant macromolecule on earth (Brown 2004) and most cellulose is produced by vascular plants. However, the ever-increasing demand of industrialization has imposed extreme negative pressure on the delicate ecological balance

K.-C. Cheng J. M. Catchmark (&) A. Demirci Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA e-mail: jcatchmark@engr.psu.edu

A. Demirci The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA

Cellulose (2009) 16:1033–1045 DOI 10.1007/s10570-009-9346-5 of the plant world. Cellulose obtained from wood stock requires the removal of impurities such as lignin, hemicellulose, and pectin. The industrial process of removal of lignin, a major component of the plant cell wall, to obtain cellulose accounts for *50% of the total energy consumed in the liberation of cellulose from wood, and is often accomplished via environmentally hazardous chemicals.

One approach to reduce the demand from plants is the production of cellulose using a microbial system (Lynd et al. 2002; Brown 2004). The Gram-negative Acetobacter xylinum has been the subject of the most intensive inquiry, which permits a detailed biochemical description of the bacterial cellulose (BC) biogenesis in this organism (Cook and Colvin 1980; Delmer 1987). A. xylinum produces BC at the air/ liquid interface. Traditional static culture has been used for BC production, which produces pellicles on the surface of fermentation broth. Several cultivation improvements had been presented later; Yoshino et al. (1996) developed a silicone membrane vessel which provided oxygen from the bottom; the rate of BC production was doubled using the cylindrical vessel. Serafica et al. (2002) made bacterial cellulose in a rotating disk bioreactor that consists of a cylindrical trough with inoculated medium into which are dipped flat, circular disks mounted on a rotating central shaft. Rotating disk bioreactor is more efficient and reduces the time of a run to about 3.5 days instead of the usual 12–20 days. Hornung et al. (2007) developed a novel reactor which both glucose and oxygen were fed directly to the BC- producing cells. However, the production is still a function of surface area to volume ratio.

An alternative method for BC production is using submerged fermentation, for which several strains of BC producing bacteria had been screened for aerated and agitated systems (Ishikawa et al. 1995; Toyosaki et al. 1995). Instead of cellulose pellicle, small pellets of BC were produced in these submerged/agitated fermentations. The BC produced in these agitated systems exhibits a lower degree of polymerization (DP), crystallinity, and Young’s modulus than that produced under static cultivation. The less-organized form of BC may result from shear stress during agitation (Watanabe et al. 1994).

Addition of different chemicals in BC production medium was found to enhance BC production both static and submerged cultivation. Addition of

Calcofluor White ST, a stilbene derivative used as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose, but disrupted the assembly crystalline cellulose I when the concentration is above 0.1 mM (Benziman et al. 1980). The negatively charged water-soluble cellulose derivative, carboxymethylcellulose (CMC), was widely used to enhance BC production in static culture (Haigler et al. 1982; Hirai et al. 1998; Seifert et al. 2003). The improvement of BC production with the presence CMC is varied from its DP and/or (DS). Water content of CMC-altered BC pellicle also increased from 73 to 96% (w/w). Addition of other water soluble polysaccharides such as agar, sodium alginate was also reported to enhance BC production in agitated cultivation (Ishida et al. 2003; Bae et al. 2004; Zhou et al. 2007).

Moreover, the mechanical properties of BC pellicle from static cultivation were investigated (Yamanaka et al. 1989; Backdahl et al. 2006). Yamanaka found that sheets of bacterial cellulose gave Young’s modulus around 15 GPa and future improved to 30 GPa by alkaline and oxidation solution treatment (Nishi et al. 1990). Backdahl reported that bacterial cellulose exhibited similar stress-strain response of the carotid artery as a potential scaffold for tissue engineered blood vessels (TEBV). Hsieh et al. (2008) studied on a single filament of bacterial cellulose and Young’s modulus reached 114 GPa. Mechanical properties of cellulose composite such as cellulose/ pectin or xyloglucan, and cellulose/CAB (cellulose acetate butyrate) have also been studied (Astley et al. 2003; Gindl and Keckes 2004). This collective research on enhanced production of BC using agitation cultivation and different additives has provided a wealth of information, but comprehensive analyses on the material properties of these BC products are sparse.

Therefore, the goal of this study was to evaluate effects of different additives on bacterial cellulose production by Acetobacter xylinum and analyze material properties. This work consisted of the following objectives: (1) to evaluate the yield of BC production in agitation culture with various additives; (2) to analyze the degree of crystallinity and crystal size by x-ray diffraction (XRD); (3) TGA to determine its water content and thermal decomposition behavior; (4) scanning electron microscopy analysis (SEM) for determining the structure of BC; and (5) dynamic mechanical analysis (DMA) for determining tensile strength of BC.

Materials and methods Bacterial strains

The bacterial strain used in this study was Acetobacter xylinum (ATCC 700178) obtained from the American Type Culture Collection (Rockville, MD), which was grown in an agitated culture as a BC producer. The cell suspension of A. xylinum was stored at -80 C in a 20% glycerol solution. One milliliter of cell suspension stored at -80 C was added to 100 mL of CSL-Fru medium in a 500 mL flask and statically cultivated at 30 C for 3 days. The cellulose pellicle formed on the surface of the broth was homogenized using a homogenizer (model 7011S, Waring Co., Torrington, CT) at 10,0 rpm for 1 min and filtered through a sterile gauze to remove BC. Ten milliliter of the filtrate containing the cell suspension was added to 90 mL of CSL-Fru medium and cultivated 30 C and 200 rpm for 24 h and used as an inoculum.


All the chemicals used were of analytical grade and commercially available unless specified description. For BC production, corn steep liquor with fructose (CSL-Fru) medium was slightly modified as described previously (Kouda et al. 1997), and contains the following constituents per liter: 50 g of fructose, 20 mL of CSL (corn steep liquor, Nihon Starch

of MnSO4 5H2O, 0.05 mg of CuSO4 5H2O, 2.0 mg of inositol, 0.4 mg of nicotinic acid, 0.4 mg of pyridoxineHCl, 0.4 mg of thiamineHCl, 0.2 mg of pantothenic acid Ca salt, 0.2 mg of riboflavin, 0.2 mg of pamino-benzoic acid, 0.002 mg of folic acid, and 0.002 mg of biotin. The final pH was adjusted to 5.0. Agar (Cat # 214010, Lot # 6080253, Becton, Dickinson Co., Sparks, MD), carboxymethylcellulose (CMC, 0.60–0.95 substitution; Cat # 21900, Lot # 434516, Fluka Co., Buchs SG, Switzerland), microcrystalline cellulose (Cat # 9004346, Lot # P206879308, FMC Co., Newark, DE), and sodium alginate (Cat # A-2033, Lot # 39H0154, Sigma, St. Louis, MO) were used as additives at various concentrations.

Flask fermentation

Ten milliliter of the prepared inoculum was added to 90 mL of fresh CSL-Fru medium supplemented with various additives including agar, carboxymethylcellulose (CMC), microcrystalline cellulose, and sodium alginate at different concentrations (0.2 and 0.5%) in a 250 mL flask and incubated at 30 C and 200 rpm for 5 days. A control experiment without addition of additives was also performed simultaneously.

Measurement of biomass and bacterial cellulose

At the end of 5-day incubation, the samples of culture broth were centrifuged at 3,300g for 20 min (Super T-21, Sorvall Co., Norwich, CT). For biomass, the BC pellets were added to 90 mL 0.1 M potassium acetate-acetate buffer (pH 5.0) and 10 mL of 20% cellulase solution (Cat # C2730, Lot # 074K1156, Sigma, St. Louis, MO) and incubated at 50 C with shaking at 100 rpm for 1 h to hydrolyze BC (Kouda et al. 1997). Then, the solution was centrifuged at 3,300g for 20 min. The precipitate was washed with deionized water twice and centrifuged. Finally, the precipitate was dried in an oven at 80 C overnight and then weighed to determine biomass. For BC determination, the precipitated BC pellets were treated with 0.1 N NaOH solution at 80 C for 30 min to remove the bacterial cells and medium components (Hwang et al. 1999). This NaOH treatment was repeated three times and then, the solution was centrifuged at 3,300g for 20 min. The purified cellulose was dried in an oven at 80 C overnight and then weighed.

Fructose concentration

Fructose concentration was determined using a Waters high-performance liquid chromatography instrument equipped with column heater, autosampler, computer controller, and a refractive index detector (Waters, Franklin, MA). Components were separated on a Bio-Rad Aminex HPX-87H column

(300 m 9 7.8 m; Bio-Rad, Richmond, CA) with 0.012 N sulfuric acid as a mobile phase at a flow rate of 0.8 mL/min with an injection volume of 20 lL and a column temperature of 65 C. Before injection, the samples were centrifuged in a microcentrifuge at 2,000g for 5 min (Model C-1200, National Labnet Co., Woodbridge, NJ) and filtered through 0.2 lm nylon filters (13 m diameter disk filters, Millipore, Bedford, MA) to remove suspended solid particles.

X-ray diffraction

To determine the crystallinity of the BC, the X-ray diffraction (XRD) patterns of the samples were collected on a Scintag PADV theta-2-theta diffractometer (Scintag, Cupertino, CA) using a copper x-ray source. Scans were collected at 2 deg per minute from 5 to 70 degree 2h. Samples of BC were lyophilized first by using a lab-scale freeze dryer (Model FreeZone 2.5L, Labconco co., Kansas, MO) and pressed into a thin and flat layer (*1.0 m) for analysis. MDI Jade 8 software (Materials Data, Inc., Livermore, CA) was used to process diffraction pattern and to calculate the crystallinity of BC. The degree of crystallinity was taken as Cr I = (I200- Iam)/I200, where I200 is the overall intensity of the peak at 2h (about 2.9 ) and Iam is the intensity of the baseline at 2h (about 18 ; Mihranyan et al. 2004).

The crystal size was calculated by the Scherrer equation (Zhang et al. 2003):

bhkl ¼ Kk Lhkl cos hhkl where b is the breadth of the peak of a specific phase (hkl), K is a constant that varies with the method of taking the breadth (0.89\K\1), k is the wavelength of incident x-rays, h is the center angle of the peak, and L is the crystallite length (size).

Field emission scanning electron microscopy

BC samples after removal of cells were lyophilized and then coated with a thin Platinum film around 5 nm. A LEO 1530 field emission scanning electron microscope (Leo Co., Oberkochen, Germany) operating at 2 kV and imaging magnification about 50,0 times was used for examination of BC samples.

Thermogravimetric analysis

The dynamic weight loss tests were conducted on a thermogravimetric analyzer (TGA) machine (model Q500, TA instruments-Water LLC, New Castle, DE). For water content determination of cellulose, all sample tests were conducted in a N2 purge (40 mL/ min) over a temperature range 30–650 Ca ta n increase rate of 10 C/min. BC samples were placed on the absorbent wipers to eliminate excess water. For thermal decomposition behavior test, cellulose samples were dried at 80 C and were then tested in a

N2 purge (40 mL/min) over a temperature range of 80–650 C at an increase rate of 10 C/min. Samples were initially dehydrated by fixing the temperature at 80 C and holding until no decrease in sample weight was observed. The weight became the initial 100% weight value.

Mechanical properties of bacterial cellulose

The mechanical properties of BC were measured using a dynamic mechanic analyzer (DMA; model Q800, TA instrument-Water LLC, New Castle, DE). BC from agitated culture after removal of cells was freeze dried first and pressed into flat piece at 2,500 psi using a press. The BC samples were then cut to make 20 9 5 m pieces for evaluation of tensile modulus. Samples were mounted between upper (fix) and lower (movable) clamps, and two ends were fixed to avoid slip. Force was applied to lower clamp to pull sample in tension. Experiments were run at 0.1 N/min. Tests were done at an environmental temperature of 35 C. Stress (r) was calculated by F/A where A is the area measured as width X thickness of sample and F is force in Newton. Strain

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