Jeffrey M. Catchmark

Pullulan: biosynthesis, production, and applications

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan’s chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed.

Biosynthesis, production and applications of bacterial cellulose

Bacterial cellulose (BC) as a never-dried biopolymer synthesized in abundance by Gluconacetobacter xylinus is in a pure form which requires no intensive processing to remove unwanted impurities and contaminants such as lignin, pectin and hemicellulose. In contrast to plant cellulose, BC, with several remarkable physical properties, can be grown to any desired shape and structure to meet the needs of different applications. BC has been commercialized as diet foods, filtration membranes, paper additives, and wound dressings. This review article presents an overview of BC structure, biosynthesis, applications, state-of-the-art advances in enhancing BC production, and its material properties through the investigations of genetic regulations, fermentation parameters, and bioreactor design. In addition, future prospects on its applications through chemical modification as a new biologically active derivative will be discussed.

Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis.

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 Youngs 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.

Advances in biofilm reactors for production of value-added products

Biofilms are defined as microbial cell layers, which are irreversibly or reversibly attached on solid surfaces. These attached cells are embedded in a self-produced exopolysaccharide matrix, and exhibit different growth and bioactivity compared with suspended cells. With their high biomass density, stability, and potential for long-term fermentation, biofilm reactors are employed for the fermentation and bioconversion, which need large amount of biomass. During the past decade, biofilm reactors have been successfully applied for production of many value-added products. This review article summarizes the applications of biofilm reactors with different novel designs. Advantages and concerns using biofilm reactors, potential uses for industrial-scale production, and further investigation needs are discussed.

Formation and characterization of spherelike bacterial cellulose particles produced by Acetobacter xylinum JCM 9730 strain.

Spherelike cellulose formation as a function of agitated culture rotational speeds and flask sizes for two different cellulose producing Acetobacter xylinum strains, JCM 9730 (ATCC 700178) and NCIMB (ATCC 23769), has been studied in this work. Results showed that the JCM 9730 strain could form spherelike cellulose particles in the agitated culture with a rotational speed above 100 rpm. The NCIMB strain, however, formed no spherelike cellulose particles under any culture condition examined. For the JCM 9730 strain, approximately 10 mm diameter spheres were produced at a rotational speed of 150 rpm in 100 mL of culture solution in a 150 mL Erlenmeyer flask, while 0.5-1 mm diameter particles were produced in 100 mL of agitated culture with a rotational speed of 200 rpm in a 250 mL Erlenmeyer flask. Data from the measurement of biomass concentration and bacterial cellulose concentration revealed that the JCM 9730 strain exhibited higher cellulose yield (up to 6.8 times) as compared to the NCIMB strain. Scanning electron microscopy analysis of lyophilized spherelike cellulose particles indicated that culture rotational speed had an impact on the internal structure of the spherelike particles. Smaller spherelike particles produced at 150 rpm were hollow and the cellulose shell exhibited a layered structure. Larger particles produced at 125 rpm were solid where the cellulose in the central region did not exhibit a layered structure, but the outer layer was similar in structure to the particles produced at 150 rpm.

Quantification of cellulose nanowhiskers sulfate esterification levels.

Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, combustion gas analysis and N(2) adsorption were used to quantify the degree of desulfation of cellulose nanowhiskers (CNWs). CNWs were produced by hydrolyzing cotton cellulose with sulfuric acid or hydrochloric acid. Hydrochloric acid treatment did not result in any cellulose chemical modification. Hydrolysis using H(2)SO(4) introduced sulfate groups onto the cellulose surface. Our results indicate that commercial cotton cellulose as received contained sulfur. The sulfur content of H(2)SO(4)-prepared CNWs was higher than that exhibited by the original cellulose due to the esterification process. Two desulfation methods, acid-catalyzed and solvolytic desulfation, have been explored to remove the sulfate groups. Neither desulfation method examined removed the sulfate groups from H(2)SO(4)-prepared CNWs completely. An estimation of surface sulfate esterification levels was made based on a model of the cellulose structure and available surface area of CNWs. According to these models, more than one third of hydroxyl groups on the surface were substituted by sulfate.

Effects of CMC addition on bacterial cellulose production in a biofilm reactor and its paper sheets analysis.

Bacterial cellulose (BC) can be grown into any desired shape such as pellicles, pellets, and spherelike balls, depending on the cultivation method, additives, and cell population. In this study, Acetobacter xylinum (ATCC 700178) was grown in the production medium with different concentrations of carboxylmethylcellulose (CMC) and were evaluated for BC production by using a PCS biofilm reactor. The results demonstrated that BC production was enhanced to its maximum (∼13 g/L) when 1.5% of CMC was applied, which was 1.7-fold higher than the result obtained from control culture. The major type of the produced BC was also switched from BC pellicle to small pellets. The ratio of BC pellets in suspension increased from 0 to 93%. Fourier transform infrared (FTIR) spectroscopy demonstrated that CMC was incorporated into BC during fermentation and resulted in the decreased crystallinity and crystal size. The X-ray diffraction (XRD) patterns indicated that CMC-BC exhibited both lower crystallinity (80%) and crystal size (4.2 nm) when compared with control samples (86% and 5.3 nm). The harvested BC was subjected to paper formation and its mechanical strength was determined. Dynamic mechanical analysis (DMA) results demonstrated that BC paper sheets exhibited higher tensile strength and Youngs modulus when compared with regular paper.

In vitro biodegradability and mechanical properties of bioabsorbable bacterial cellulose incorporating cellulases.

Bacterially produced cellulose is being actively studied as a novel scaffold material for wound care and tissue engineering applications. Bioabsorbability of the scaffold material is desired to enable improved restoration of targeted tissue. Recently, a bioabsorbable bacterial cellulose (BBC) incorporating cellulase enzymes has been demonstrated. It was revealed that some cellulases may lose up to 90% of their activity if present in a suboptimal pH environment. Therefore, a key challenge in the practical implementation of this approach rests in compensating for the variation in the wound or tissue pH, which may significantly reduce the activity of some enzymes. In this work, buffer ingredients were incorporated into the bacterial cellulose in order to create a more optimal pH microenvironment for the preferred acid cellulases, which are significantly less active at the biological pH 7.4. The results demonstrated that incorporation of buffer ingredients helped to retain the activity of the cellulases. The glucose released from degraded materials was also increased from 30% without incorporation of buffer ingredients to 97% in the presence of incorporated buffer ingredients at the suboptimal pH environment of 7.4. The use of simulated body fluid and simulated tissue padding, both mimicking the real wound environment, also demonstrated some improvements in terms of material degradation. Measurements of mechanical properties of materials revealed that BBC materials have tensile strength and extensibility similar to human skin, especially when hydrated with saline water prior to use.

Integration of cellulases into bacterial cellulose: Toward bioabsorbable cellulose composites.

Cellulose biodegradation resulting from enzymolysis generally occurs in nature rather than in the human body because of the absence of cellulose degrading enzymes. In order to achieve in-vivo degradation in human body for in-vivo tissue regeneration applications, we developed a bioaborbable bacterial cellulose (BBC) material, which integrates one or more cellulose degrading enzymes (cellulases), and demonstrated its degradability in vitro using buffers with pH values relevant to wound environments. We introduced a double lyophilizing process to retain the microstructure of the bacterial cellulose as well as the activity of embedded enzymes allowing for long-term storage of the material, which only requires hydration before use. Enzymes and their combinations have been examined to optimize the in-vitro degradation of the BBC material. In-vitro studies revealed that acidic cellulases from Trichoderma viride showed reasonable activity for pH values ranging from 4.5 to 6.0. A commercial cellulase (cellulase-5000) did not show good activity at pH 7.4, but its degrading ability increased when used in conjunction with a β-glucosidase from Bacillus subtilis or a β-glucosidase from Trichoderma sp. Given the harmless glucose product of the enzymatic degradation of cellulose, the BBC material may be ideal for many wound care and tissue engineering applications for the bioabsorbable purpose.

The impact of cellulose structure on binding interactions with hemicellulose and pectin

Four cellulose substrates including highly crystalline cellulose nanowhiskers (CNWs) from Gluconacetobacter xylinus (cellulose Iα) or cotton (cellulose Iβ) and amorphous cellulose derived from CNWs (phosphoric acid swollen cellulose nanowhiskers, PASCNWs) were used to explore the interaction between cellulose and well-defined xyloglucan, xylan, arabinogalactan and pectin. The binding behavior was characterized by adsorption isotherm and Langmuir models. The maximum adsorption and the binding constant of xyloglucan, xylan and pectin to any CNWs were always higher than to PASCNWs derived from the same source. The binding affinity of xyloglucan, xylan and pectin to G. xylinus cellulose was generally higher than to cotton cellulose, showing that binding interactions depended on the biological origin of cellulose and associated differences in its structure. The surface area, porosity, crystal plane and degree of order of cellulose substrate may all impact the interactions.