ENHANCED PRODUCTION OF MICROBIAL EXTRACELLULAR POLYSACCHARIDES AND MATERIALS PROPERTY ANALYSIS
Open Access
- Author:
- Cheng, Kuan-Chen
- Graduate Program:
- Agricultural and Biological Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 22, 2010
- Committee Members:
- Ali Demirci, Dissertation Advisor/Co-Advisor
Ali Demirci, Committee Chair/Co-Chair
Jeffrey M Catchmark, Committee Chair/Co-Chair
Virendra Puri, Committee Member
Nicole Robitaille Brown, Committee Member - Keywords:
- pullulan
bacterial cellulose
fermentation
biofilm reactor
bioreactor design
polysaccharide - Abstract:
- Bacterial cellulose, a water-insoluble exopolysaccharide (EPS) produced by Acetobacter xylinum, 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. Pullulan, a water soluble EPS synthesized by a yeast-like fungus Aureobasidium pullulans, is often described as an ƒÑ-1, 6 linked maltotriose polymer. With this unique linkage pattern, pullulan demonstrates distinctive physical properties, such as adhesive ability, the capacity to form fibers, and the ability to be formed into thin and biodegradable films which are transparent and impermeable to oxygen. As a result, pullulan has been used for a wide range of applications in food, pharmaceutical, chemical, and environmental remediation applications. The purpose of this research is to improve the rate of cellulose and pullulan production and their material properties by exploring new culture methods including a new biofilm reactor design and the addition of different additives. The physical and chemical properties of these novel polysaccharide composites were evaluated using several characterization approaches. First, a novel biofilm bioreactor configuration was implemented, including a solid substrate (plastic composite support, PCS) to promote effective nutrient delivery for biofilm development. PCS degrades slowly with time offering a distinct advantage: disruption of biofilm formation preventing thick film formation, which results in an oxygen and substrate diffusion barrier. Second, several additives were used to make EPS composite and the mechanical properties of this new EPS composite were evaluated. The broader impacts of this work will benefit industries which need cellulose fiber and/or pullulan such as the forest products industry and other industries utilizing the medical value of this material. Also, the molecular weight distribution of pullulan plays an important role relative to its bioactive potential. For example, pullulan, which is suitable for intravenous injection should have a narrow molecule weight distribution (MWD) with Mw/Mn = 1.2 and MW about 60 kDa. This work focused on the development of new processes which have potential to be environmentally benign and sustainable. Enhancement of bacterial cellulose (BC) production by Acetobacter xylinum was explored through the addition of different additives into the fermentation medium in agitated culture including agar, carboxymethylcellulose (CMC), microcrystalline cellulose, and sodium alginate. 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 less than 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 tensile strength in both stress at break and Young¡¦s modulus when compared to BC pellicle since the former were in pellet form. As for BC production in biofilm reactors, the type SFYR+ PCS 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. For pullulan production, the optimal cultivation medium was first evaluated for Aureobasidium pullulans. The optimal growth condition for pullulan production by A. pullulans was found as 75 g/l of sucrose as carbon source, 3 g/l of yeast extract, and cultivation temperature at 30oC. Under these conditions with an initial pH at 5, the 20.7 g/l of final pullulan concentration and 0.22 g/l/h maximum production rate were observed. The optimal kinetic parameter was as follows: initial pH at 2.0, switched to pH 5.0 after 72 h and kept constant; agitation speed at 200 rpm; aeration at 1.5 vvm. Using the optimal medium, a biofilm reactor with plastic composite support (PCS) was then evaluated for pullulan production using A. pullulans. In test tube fermentations, PCS with soybean hulls, defatted soybean flour, yeast extract, dried bovine red blood cells, and mineral salts was selected for biofilm reactor fermentation (due to its high nitrogen content, moderate nitrogen leaching rate, and high biomass attachment). Three pH profiles were later applied to evaluate their effects on pullulan production in a PCS biofilm reactor. The results demonstrated that when a constant pH at 5.0 was applied, the time course of pullulan production was advanced and the concentration of pullulan reached 32.9 g/L after 7-day cultivation, which is 1.8-fold higher than its respective suspension culture. Response surface methodology using Box-Behnken design was then employed to study the effects of sucrose and nitrogen concentrations on pullulan production. After 7-day fermentation with optimum condition, the pullulan production reached 60.7 g/L, which was 1.8 times higher than the result from initial medium, and was the highest yield reported to date. The quality analysis demonstrated that the purity of produced pullulan was 95.2% and its viscosity was 2.5 centipoise (cP). Fourier Transform Infrared Spectroscopy (FTIR) also suggested that the produced exopolysaccharide was pullulan. Pullulan fermentations by A. pullulans with various initial ammonium ion concentrations were evaluated in a 2-L bioreactor. The results demonstrated that A. pullulans produced highest pullulan (23.1 g/L) when the initial ammonium sulfate was at 5 g/L. The purity of produced pullulan was 94.6%. Seven g/L of ammonium sulfate produced more biomass due to the higher level of nitrogen source, but the pullulan-degrading enzyme activity was detected after the depletion of sucrose, which reduced pullulan concentration. From the results of fed-batch fermentation, addition of 10 g/L of sucrose suppressed A. pullulans from producing pullulan-degrading enzyme. Using the results from suspended-cell, batch fermentation, the modified Gompertz model can serve as a universal equation to fit biomass production, pullulan production, and sucrose consumption. Furthermore, validation of this modified Gompertz model indicated that biomass production (slope = 1.00, R2 = 0.991), pullulan production (slope = 1.10, R2 = 0.991), and sucrose consumption (slope = 0.96, R2 = 0.991) were all predicted accurately. Additionally, the modified Gompertz equation demonstrated its generality to fit all pullulan production, biomass production, and sucrose consumption curves at three ammonium sulfate levels. After incorporating the degrading factor, a re-modified Gompertz equation was obtained that can adequately describe the decrease of pullulan; which resulted from the presence of pullulan-degrading enzyme, at the late fermentation stage. In summary, excess ammonium ions with the depletion of carbon source resulted in the presence of pullulan-degrading enzyme that subsequently reduced pullulan concentration. A re-modified Gompertz equation can serve as a generalized equation to describe pullulan fermentation. In conclusion, these studies clearly demonstrated that the biofilm reactor can be employed to enhance microbial extracellular polysaccharide production. BC production and its material property are highly affected by additives during fermentation. BC produced from PCS biofilm reactor exhibited tensile strength comparable to that produced in pellicle form. For pullulan production, optimal cultivation parameters and solid support for biofilm formation were determined. The produced pullulan maintained its high purity around 95%. The mathematical models proposed in this study can pave the way for further studies, for example, an online recovery of BC and/or pullulan from existing biofilm reactor.