Cellulose nanocomposites production through co-culture fermentation
Open Access
- Author:
- Liu, Ke
- Graduate Program:
- Agricultural and Biological Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 14, 2018
- Committee Members:
- Jeffrey M Catchmark, Dissertation Advisor/Co-Advisor
Jeffrey M Catchmark, Committee Chair/Co-Chair
Ali Demirci, Committee Member
Cibin Raj, Committee Member
Edward G Dudley, Outside Member - Keywords:
- Bacterial cellulose
Exopolysaccharides
Escherichia coli
Hyaluronic acid
Lactococcus lactis
Co-culturing
Bioreactor
Biofilm
Ribbon assembly
Crystallinity - Abstract:
- Bacterial cellulose (BC) is a water-insoluble polysaccharide which consists of β-linked 1, 4 glucose residues. It exhibits several advantageous properties such as high purity, high crystallinity, a high degree of polymerization, high water content, and excellent biocompatibility comparing to other natural or regenerated cellulose. The extensively studied strains in the lab are Gluconacetobacter xylinus and Gluconacetobacter hansenii due to their relatively high yield of BC. Under static culture, cellulose in the pellicle form is synthesized by these strains at the air-liquid interface. The physiological structures behind the BC production are the linearly arranged terminal complexes (TCs) that distributed along the longitudinal surface of the strains. Glucan chains are secreted into the culture medium through the TCs while coupling with the crystallization process. Meanwhile, higher order cellulose fibrils are self-assembled through the process of co-crystallization, physical aggregation, and bundling. The presence of other materials in the culture medium such as exopolysaccharides (EPS), proteins, or inorganic chemicals could affect the crystallization or self-assembly process and result in the modifications of BC microstructure including the crystallinity, crystalline polymorphism, crystallite size, and ribbon width or endow new properties to BC. Therefore, this study was focused on achieving the modifications on BC through co-culturing G. hansenii with Escherichia coli or Lactococcus lactis, both of which were able to secrete EPS. The BC-based nanocomposites were directly produced through co-culturing without the need of supplying additives into the culture media or further modification processes. The effects of EPS on the assembly processes of BC were also explored. The EPS extracted from E. coli ATCC 35860 under agitation conditions were chosen to study. After harvesting and purifying the EPS, its composition was analyzed by gas chromatography-mass (GC-MS) spectrometry. When adding the purified EPS into the culture media, another kind of EPS, composed of fructose, was produced by G. xylinus ATCC 23769 and a minor portion of the added EPS was incorporated into cellulose fibrillar network. The characteristics of BC nanocomposites synthesized in the presence of purified EPS was systematically studied through tensile testing, x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and field emission scanning electron microscopy (FESEM). The results revealed that the EPS affected the cellulose-cellulose interactions during the physical aggregation of crystal microfibrils, but did not impact the co-crystallization process during BC synthesis. The addition of 4 mg/L or 8 mg/L purified EPS into the culture media, was found to significantly improve the tensile strength of BC nanocomposites while maintaining BC crystallinity and crystal size. To simplify the production processes of BC pellicles with enhanced mechanical properties, a novel fermentation procedure, which is to co-culture G. hansenii with E. coli under static conditions, was developed. During co-culture, the mannose-rich EPS synthesized by E. coli were incorporated into the BC network and affected the aggregation of co-crystallized microfibrils without significantly changing the crystal sizes of BC. When co-culturing G. hansenii ATCC 23769 with E. coli ATCC 700728, which produced a low concentration of EPS at 3.3 ± 0.7 mg/L, but the BC pellicles exhibited a Young’s modulus of 4,874 ± 1144 MPa and a stress at break of 80.7 ± 21.1 MPa, which are 81.9% and 79.3% higher than those of pure BC, respectively. The growth dynamics of the two co-cultured strains suggested that the production of EPS and BC were enhanced through co-culturing fermentation. While co-culturing under static can manufacture BC-based nanocomposites, the incorporated amounts of EPS is difficult to adjust due to the static conditions. A novel two-vessel circulating system was developed in order to provide relatively static conditions for BC production while controlling the growth conditions for the co-cultured strain. L. lactis APJ3, which was genetically modified for the synthesis of hyaluronic acid (HA), was selected. The concentration of HA secreted by L. lactis APJ3 was controlled by adjusting the constant feed rate of glucose. The dynamic growth of the two strains revealed that L. lactis APJ3 was mainly growing within the first 48 hours while G. hansenii ATCC 23769 became active after 48 hours. The FTIR-ATR spectroscopy proved the incorporation of HA into the cellulose network. XRD analysis indicated that the presence of HA would not affect the crystallinity of BC/HA but increase the crystalline sizes. The FESEM images showed that more ribbons within the range of 20 – 40 nm diameter and larger ribbons between the range of 180 – 360 nm diameter were observed in BC/HA composites. The strain at break and the water holding capacity of BC/HA increased with the concentration of HA. The designed two-vessel circulating system provided a new method to directly produce BC-based nanocomposites. Since the production of HA by L. lactis APJ3 was genetically engineered based on the P170 expression system, and the P170 promoter would be up-regulated as lactic acid accumulated, controlling the pH values of culture media was a straightforward strategy to achieve the regulation on the yield of HA. The BC/HA nanocomposites were synthesized by co-culturing G. hansenii ATCC 23769 with L. lactis APJ3 under static conditions with different initial pH values of culture media. The HA concentration produced by L. lactis and the dry weight of BC/HA during co-culture were regulated by the initial pH values of culture media. The incorporation of HA into the cellulose network increased the crystal sizes when the initial pH values were at 7.0, 6.2, and 5.5. The strain at break was also increased while Young’s modulus was decreased when comparing BC/HA to pure BC produced under the initial pH values of culture media at 7.0 and 6.2. When the initial pH value was 4.0, the HA concentration in the culture media exhibited the lowest level observed, which was 20.4 ± 2.3 mg/L. The BC/HA composite synthesized under this condition exhibited an improved Young’s modulus of 5029 ± 1743 MPa from 2705 ± 656 MPa associated with the pure BC. The FESEM images showed that the presence of HA dramatically changed the distribution of ribbon width in BC/HA compared to that of pure BC.