Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis
- First Online:
- 21k Downloads
Bacterial cellulose has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated foods. It has also been used in the paper manufacturing industry to enhance paper strength, the electronics industry in acoustic diaphragms for audio speakers, the pharmaceutical industry as filtration membranes, and in the medical field as wound dressing and artificial skin material. In this study, different types of plastic composite support (PCS) were implemented separately within a fermentation medium in order to enhance bacterial cellulose (BC) production by Acetobacter xylinum. The optimal composition of nutritious compounds in PCS was chosen based on the amount of BC produced. The selected PCS was implemented within a bioreactor to examine the effects on BC production in a batch fermentation. The produced BC was analyzed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and dynamic mechanical analysis (DMA). Among thirteen types of PCS, the type SFYR+ was selected as solid support for BC production by A. xylinum in a batch biofilm reactor due to its high nitrogen content, moderate nitrogen leaching rate, and sufficient biomass attached on PCS. The PCS biofilm reactor yielded BC production (7.05 g/L) that was 2.5-fold greater than the control (2.82 g/L). The XRD results indicated that the PCS-grown BC exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control. FESEM results showed the attachment of A. xylinum on PCS, producing an interweaving BC product. TGA results demonstrated that PCS-grown BC had about 95% water retention ability, which was lower than BC produced within suspended-cell reactor. PCS-grown BC also exhibited higher Tmax compared to the control. Finally, DMA results showed that BC from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young's modulus when compared to the control BC. The results clearly demonstrated that implementation of PCS within agitated fermentation enhanced BC production and improved its mechanical properties and thermal stability.
Cellulose is the most abundant macromolecule on earth  and most cellulose is produced by vascular plants. A substitute to reduce the demand from plants is the production of cellulose using a microbial system [1, 2]. Bacterial cellulose (BC) has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated food, in the paper manufacturing industry to enhance paper strength, in acoustic diaphragms for audio speakers, and in the pharmaceutical industry as a filtration membrane, wound dressing and artificial skin [3, 4].
BC produced by Acetobacter xylinum in static cultures is initially extruded from the cell surface as microfibers and entangle together to form ribbons, which then intertwine to form a dense, gelatinous pellicle at the air/liquid interface. Traditional static culture has been used for BC production, which produces pellicles on the surface of fermentation broth. The pellicle grows downward since cells that are entrapped into the pellicle become inactive or die from lack of oxygen .
Several cultivation improvements have been presented to enhance BC production; Yoshino et al.  developed a cylindrical silicone membrane vessel that provided oxygen from the bottom, resulting in a two-fold improvement in BC production. Serafica et al.  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. A 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.  developed a novel reactor where both glucose and oxygen were fed directly to the BC-producing cells. These modified production processes were explored to increase the oxygen rich surface area to reactor volume ratio, which improves BC production.
An alternative method for BC production is submerged fermentation. Several strains of BC-producing bacteria had been screened for aerated and agitated cultivation [9, 10]. Instead of a cellulose pellicle, small pellets of BC were produced. These BC pellets exhibit a lower degree of polymerization, crystallinity, and Young's modulus than that produced under static cultivation. The less-organized form of BC may have resulted from shear stress during agitation .
High biomass density also proved to be beneficial for BC production in many cases [12, 13]. High density of biomass can be achieved by several ways such as cell immobilization, cell-recycle reactor, and hollow-fiber reactors. Cell-recycle reactors and hollow-fiber reactors have their limitations due to the high capital and operational cost, as well as the potential risk of membrane fouling and/or contamination during fermentation. Biofilm reactors, on the other hand, provide a substitute of high-biomass density systems with lower capital cost. Biofilm reactors have demonstrated a very high volumetric productivity of submerged fermentation, especially the continuous cultures when compared with suspended-cell cultures due to the high cell density maintained in the reactor .
Biofilms grow on the solid support when microorganisms attach, and are a natural form of cell immobilization . Plastic composite support (PCS) is an extrusion product of a mixture between polyprolylene and nutritious compounds . Polypropylene acts as a matrix and integrates agricultural mixtures, such as ground soybean hulls, soybean flour, and microbial nutrients such as bovine albumin, red blood cells, yeast extract, and peptone, as well as mineral salts. As a result, PCS not only provides an ideal physical structure for biofilm formation, but also releases nutrients slowly during fermentation.
Many studies showed that using PCS biofilm reactors can enhance production of ethanol, organic acid, and bacteriocin [14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Therefore, a PCS biofilm reactor system was evaluated for BC production by A. xylinum, which is suitable for submerged fermentation .
The goal of this study was to evaluate effects of PCS implementation on bacterial cellulose production by A. xylinum, which was divided into two specific objectives: (1) to evaluate BC production in a PCS biofilm reactor and compare to one produced from the conventional suspended-cell culture, and (2) to analyze the material properties of the produced BC, including degree of crystallinity and crystal size by X-ray diffraction (XRD), thermogravimetric analysis (TGA) to determine its water content and thermal decomposition behavior, scanning electron microscopy analysis (SEM) for determining the structure of BC, and dynamic mechanical analysis (DMA) for determining tensile strength of BC.
Materials and methods
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 CSL-Fru medium with agitation 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 10,000 rpm for 1 min and filtered through a sterile gauze to remove BC. Ten ml of the filtrate containing the cell suspension was added to 90 ml of CSL-Fru medium and cultivated at 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 previously described , and contained the following constituents per liter: 50 g of fructose, 20 ml of CSL (corn steep liquor, Nihon Starch Industry, Kagoshima, Japan), 1.0 g of KH2PO4, 0.25 g of MgSO4. 7H2O, 3.3 g of (NH)2SO4, 3.6 mg of FeSO4. 7H2O, 1.5 mg of CaC12. 2H2O, 2.4 mg of Na2MoO2. 2H2O, 1.7 mg of ZnSO2. 7H2O, 1.4 mg of MnSO4. 5H2O, 0.05 mg of CuSO4. 5H2O, 2.0 mg of inositol, 0.4 mg of nicotinic acid, 0.4 mg of pyridoxine. HCl, 0.4 mg of thiamine. HCl, 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. Agar (Becton, Dickinson Co., Sparks, MD), carboxymethylcellulose (Fluka Co., Buchs SG, Switzerland), microcrystalline cellulose (FMC Co., Newark, DE), and sodium alginate (Sigma, St. Louis, MO) were used as additives at various concentrations.
Plastic composite support
List of PCS ingredients
% dry weight
Flask Fermentations for Selection of PCS
Thirteen types of PCS with different compositions were evaluated for both biofilm formation and BC production using flask fermentation with three replicates. For each replicate, 3 g of PCS disks was sterilized dry in 150 mL flask for 1 h at 121°C. Fifty mL of sterilized medium with 5% (w/v) fructose as a carbon source was added aseptically to the sterile PCS before being incubated at 30°C overnight in order to hydrate the PCS. The medium was then aseptically decanted, and fresh sterile medium was added with 1% (v/v) inoculum of 24-h A. xylinum before incubating at 30°C for 120 h. Each replicate was determined the biomass on the PCS, BC production, and residual fructose. The best PCS type was selected according to the biofilm formation on PCS (g-biomass/g-PCS), BC production (g/mL), nitrogen content (% w/w), and nitrogen leaching rate from PCS (% w/w of total nitrogen after the 120-h fermentation). A control experiment without PCS rings was also performed simultaneously. The results on nitrogen content and nitrogen leaching rate were obtained from the study of Ho et al. .
BC Fermentation in the Bioreactor
Measurement of biomass and bacterial cellulose
At the end of the 5-day incubation, BC on the PCS shaft was detached using a knife and combined with culture broth and then centrifuged at 3300 × g for 20 min (Super T-21, Sorvall Co., Norwich, CT). For biomass, the precipitated pellicle was added to 90 ml of buffer (0.1 M potassium acetate-acetate buffer (pH 5.0) and 10 ml of 20% cellulase (Celluclast, Novo Nordisk A/S, Denmark) and incubated at 50°C with shaking at 100 rpm for 1 h to hydrolyze BC . Then, the solution was centrifuged at 3,300 × g for 20 min. The precipitate was dried in an oven at 80°C overnight and then weighed to determine biomass. For BC determination, the precipitated BC pellicle was treated with 0.1 N NaOH solution at 80°C for 30 min to remove the bacterial cells and medium components . This NaOH treatment was repeated three times and then, the solution was centrifuged at 3,300 × g for 20 min. The obtained cellulose was dried in an oven at 80°C overnight and then weighed.
Fructose concentration was determined using a Waters high-performance liquid chromatography unit 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 mm × 7.8 mm) (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 μl and a column temperature of 65°C. Before injection, the samples were centrifuged in a microcentrifuge at 2,000 × g for 5 min (Model C-1200, National Labnet Co., Woodbridge, NJ) and filtered through 0.22 μm filters (13 mm diameter disk filters, Millipore, Bedford, MA) to remove suspended solid particles.
where β 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), λ is the wavelength of incident x-rays, θ is the center angle of the peak, and L is the crystallite length (size).
Field emission scanning electron microscopy (FESEM)
Both BC samples before and after removal of cells were lyophilized overnight at 0.133 mbar and then coated with thin Platinum film around 5 nm. A LEO 1530 field emission scanning electron microscope (Leo Co., Oberkochen, German) operating at 2 kV was used for examination of the samples. An imaging magnification of approximately 27,000, 7,000, and 250 times was used for BC, bacteria, and PCS rings, respectively.
Thermogravimetric analysis (TGA)
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°C at an 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 tests were then conducted in a N2 purge (40 ml/min) over a temperature range 80–650°C at an increase rate of 10°C/min. The initial weight at 80°C was set as 100%.
Mechanical properties of bacterial cellulose
The mechanical properties of BC were performed by a dynamic mechanic analyzer (DMA) (model Q800, TA instrument-Water LLC, New Castle, DE). BC from agitation cultivation after removal of cells was lyophilized first and pressed into a flat piece at 2,500 psi using a press. The BC samples were then cut to make 20 × 5 mm plates for testing. Sample were mounted between upper (fix) and lower (movable) clamps, and two ends were fixed to avoid slip. Force was applied to the lower clamp to pull the sample in tension. Experiments were run at 0.1 N/min force. Tests were performed at an environmental temperature of 35°C. Stress (σ) was calculated by F/A where A is the area measured as width × thickness of sample and F is force in Newtons (N). Strain (ε) was calculated by ΔL/Lowhere ΔL is exerted extension from starting point Lo. Young's modulus was calculated by Stress/Strain in the linear region.
All treatments were replicated at least three times. The significant difference of the results was evaluated using the Generalized Linear Model (GLM; with p < 0.05) and Tukey's honestly significant differences (HSD) multiple comparison module of the MINITAB Statistical Software package (Release 13.30; State College, PA, USA)
Results and Discussion
BC production by A. xylinum in the PCS biofilm reactor
A. xylinum was grown in CSL-Fru medium with the selected PCS in a biofilm reactor. Fermentation in the PCS biofilm reactor yielded 7.05 g/l BC, which is 2.5-fold greater than BC production in the suspended-cell reactor (2.82 g/l). This result was probably due to the higher biomass density accumulated on PCS and the cell-density-dependent BC production relation as previously described [9, 12].
BC structure and physical properties
Comparison of crystallinity and crystal size between BC from PCS biofilm and suspended-cell reactor.
Crystallite size of
93 ± 2.1
5.2 ± 0.3
85 ± 1.5
5.2 ± 0.5
Based on the results of this study, BC production by A. xylinum can be enhanced using a PCS biofilm reactor system. The material properties of BC are changed when grown in the presence of the PCS. BC produced in a biofilm reactor grew primarily on the PCS supports and is mainly in cellulose I-β pattern. Among thirteen types of PCS, PCS blended with dried soybean hulls, defatted soybean flour, yeast extract, dried bovine red blood cells, and mineral salts (SFYR+) was selected for biofilm BC fermentation. The PCS biofilm reactor yielded the highest BC (7.05 g/l) which is 2.5 fold compared to control (2.82 g/l). The XRD studies indicated that crystallinity of PCS-grown BC is about 93%, which is higher than control, and a crystal size of 5.2 nm in <200> crystal plane. FESEM image provided the evidence that A. xylinum can grow and produce BC on the PCS. TGA results showed that PCS-grown BC possessed 95% water content and demonstrated higher thermal stability (~30°C) when compared to control BC. BC harvested from the biofilm reactor also exhibited higher mechanical strength than BC produced from agitated culture. The PCS-grown BC exhibited higher stress at break, Young's modulus, and strain at break than one produced from agitated culture.
In conclusion, PCS biofilm reactor system successfully demonstrated several advantages for BC production, such as enhanced production yield, higher thermal stability, and stronger mechanical strength. Based on these results, the development of a semi-continuous and continuous fermentation process for producing BC will be the next challenge. Further study on determining other properties of BC produced using PCS, such as gas-penetration ability, metal-ion chelating capacity, and crystallization characteristics are needed.
The authors thank Dr. Anthony L. Pometto III from Iowa State University for kindly supplying the PCS tubes used in this research. Funding for this project was supported in part by a seed grant from the College of Agricultural Sciences at the Pennsylvania State University and the Pennsylvania Agricultural Experiment station. The authors are very grateful to Nichole Wonderling from the Materials Research Institute of the Pennsylvania State University for her assistance with x-ray diffraction measurements. Thanks also to Dr. Xuelian Zhang and Yufen Zeng at the Department of Forest Resources for their help for DMA and TGA analysis.
- 11.Watanabe K, Hori Y, Tabuchi M, Morinaga Y, Yoshinaga F, Horii F, Sugiyama J, Okano T: Structural features of bacterial cellulose vary depending on the cultural conditions. Proc Cellulose Society of Japan, Kyoto, Japan 1994, 45-50.Google Scholar
- 15.Characklis WG, Wilderer PA: Structure and Function of Biofilms. Wiley, New York; 1989:6-9.Google Scholar
- 16.Pometto AL III, Demirci A, Johnson KE: Immobilization of microorganisms on a support made of synthetic polymer and plant material. U.S. Patent No. 5595893 1997.Google Scholar
- 19.Demirci A, Pometto AL III, Ho KLG: Continuous ethanol production in biofilm reactors containing plastic composite rings and disks. In Proceedings of theSecond Biomass Conference of the Americas: Energy, Environment, Agricultural and Industry, Portland, OR, 21–24 Aug 1995. U.S. Department of Energy, National Renewable Energy Laboratory: Golden, CO; 1995.Google Scholar
- 22.Ho KLG, Pometto AL III, Hinz PN: Ingredient selection for plastic composite supports for L-(+)-lactic acid biofilm fermentation by Lactobacillus casei subsp. Rhamnosus . Appl Environ Microbiol 1997, 63: 2516-2523.Google Scholar
- 23.Ho KLG, Pometto AL III, Hinz PN, Demirci A: Nutrient leaching and end product accumulation in plastic composite supports for L-(+)-lactic acid biofilm fermentation. Appl Environ Microbiol 1997, 63: 2524-2532.Google Scholar
- 24.Ho KLG, Pometto AL III, Hinz PN: Optimization of L-(+)-lactic acid production by ring and disc plastic composite supports through repeated-batch biofilm fermentation. Appl Environ Microbiol 1997, 63: 2533-2542.Google Scholar
- 30.Zhang LN, Xi Q, Mo ZS, Jin XG: Current researching methods on polymer physics. Wuhan College Press 2003, 194-195.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.