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 T _max 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.     

Design and Evaluation of Self-Nanoemulsifying Pellets of Repaglinide

The aim of study was to develop self-nanoemulsifying pellets (SNEP) for oral delivery of poorly water soluble drug, repaglinide (RPG). Solubility of RPG in oily phases and surfactants was determined to identify components of self-nanoemulsifying drug delivery system (SNEDDS). The surfactants and cosurfactants were screened for their ability to emulsify oily phase. Ternary phase diagrams were constructed to identify nanoemulsification area for the selected systems. SNEDDS formulations with globule size less than 100 nm were evaluated for in vivo anti-hyperglycemic activity in neonatal streptozotocin rat model. A significant reduction in glucose levels was produced by optimized SNEDDS formulation in comparison to the control group. The optimized SNEDDS formulations were pelletized via extrusion/spheronization technique using microcrystalline cellulose and lactose. SNEP were characterized by X-ray powder diffraction and scanning electron microscopy. X-ray diffraction study indicated loss of crystallinity of RPG in SNEP. The SNEP exhibited good flow properties, mechanical strength and formed nanoemulsion with globule size less than 200 nm. SNEP showed in vitro release of more than 80% RPG in 10 min which was significantly higher than RPG containing reference pellets. In conclusion, our studies illustrated that RPG, a poorly water soluble drug can be successfully formulated into SNEP which can serve as a promising system for the delivery of poorly water soluble drugs.     

Production of nanocellulose in miniature-bioreactor: Optimization and characterization

Bacterial cellulose (BC) is a very fascinating microbial biopolymer which is mainly produced by Gluconacetobacter xylinum. Optimization of BC production by G. xylinum was performed based on scale-down studies in miniature-bioreactor and response surface methodology in which the optimum pH value (6.5) and shaking rate (50?rpm) were obtained. The static culture condition for BC production has newly been defined. Nanostructure of BC includes nanofibers up to (60?nm) and nanoporosity up to (265?nm) was observed by scanning electron microscopy. By Fourier transform infrared spectroscopy study, the most expected BC interaction is nucleophilic interaction. MTT assay showed high biocompatibility. Appropriate mechanical strength (0.37?MPa) and Young’s modulus (3.36?MPa) evinced BC scaffold utilization for skin tissue. The results indicate that BC sheets can be utilized in biomedical application and nanotechnology approaches.     

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Comparisons of physical properties of bacterial celluloses produced in different culture conditions using saccharified food wastes

The saccharogenic liquid (SFW) obtained by the enzymatic saccharification of food wastes was used as a medium for production of bacterial cellulose (BC). The enzymatic saccharification of food wastes was carried out by the cultivation supernatant ofTrichoderma harziaum FJ1 culture.Acetobacter xylinum KJ1 was employed for the BC production culture. The physical properties, such as polymerization, crystallinity, Young's modulus, and tensile strength, of BCs produced by three culture methods: the static cultures using HS (Hestrin-Schramm) as a reference medium (A) or the SFW medium (B), the shaking culture (C) or the air circulation culture (D) using the SFW medium, were investigated. The degrees of polymerization of BCs produced under the different culture conditions (A∼D) showed 11000, 9500, 8500, and 9200, respectively. Young's modulus was 4.15, 5.0, 4.0, and 4.6 GPa, respectively. Tensile strength was 124, 200, 80, and 184 MPa, respectively. All of the BC had a form of cellulose I representing pure cellulose. In the case of the shaking culture, the degree of crystallinity was 51.2%, the lowest degree. Under the other culturing conditions, the trend should remain in the range of 89.7–84%. Overall, the physical properties of BC produced from SFW were similar to those of BC from HS medium, a commercial complex medium, and BC production by the air circulation culture mode brought more favorable results in terms of the physical properties and its ease of scale-up. Therefore, it is expected that a new BC production method, like air circulation culture using SFW, would contribute greatly to BC-related manufacturing.     

Impact of Carboxymethylcellulose on Morphology and Antibiotic Production by Streptomyces hygroscopicus

A chemically defined media consisting of carboxymethylcellulose (CMC) was developed to maximize the production of antibiotics, hexaene H-85 and azalomycine, by Streptomyces hygroscopicus CH-7. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. By adding the polymer to the fermentation medium, the growth was changed from a single large glob to small reproducible pellets, and wall growth was diminished to a minimum. Maximum concentrations of hexaene H-85 (146.7 mg/dm(3)) and azalomycine (188.6 mg/dm(3)) were reached at 3.0% and 1.0% (w/v) CMC, respectively.     

Preparation and physicochemical properties of a novel hydroxyapatite/chitosan–silk fibroin composite

A novel hydroxyapatite/chitosan–silk fibroin (HA/CTS–SF) composite was prepared for bone repair and replacement by a coprecipitation method. It was revealed that the inorganic phase in the composite was carbonate-substituted HA with low crystallinity. The HA crystallites were found to be needle-like in shape with a typical size of 20–50 nm in length and around 10 nm in width. The composite exhibited a higher compressive strength than the precipitated HA without any organic source involved, which was closely related to the perfect incorporation of chitosan and SF macromolecules into the composite. The chemical interactions occurring between the mineral phase and the organic matrix were thought to improve the interfacial bonding and thus resulted in the enhanced mechanical property of the composite.     

Effects of carboxymethylcellulose and carboxypolymethylene on morphology of Aspergillus fumigatus NRRL 2346 and fumagillin production

Aspergillus fumigatus NRRL 2346 is the producer of fumagillin, an antitumor antibiotic that inhibits angiogenesis. This strain is very difficult to grow reproducibly in shake flasks owing to an extreme form of pellet growth and extensive wall growth. The effects of carboxymethylcellulose (CMC) and carboxypolymethylene (Carbopol) on growth and fumagillin production by A. fumigatus were investigated. By adding the polymers to the fermentation medium, the growth form of the mold was changed from a single large glob to small reproducible pellets, and wall growth was diminished to a minimum. Carbopol, at a lower concentration, was more effective than CMC in improving both morphology and production. Small pellets were produced which favored fumagillin biosynthesis. 1.5% (wt/vol) CMC and 0.3% (wt/vol) Carbopol were found to be the optimum concentrations; higher levels increased viscosity to an unacceptable level.     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

一株产细菌纤维素菌株的筛选鉴定及其产物分析

从红茶菌液中筛选获得一株产细菌纤维素的菌株BC-41,经生理生化分析和分子生物学鉴定,现证实该菌株为中间葡糖酸醋杆菌(Gluconacetobacter intermedius)。对该菌株所产生的细菌纤维素进行了物理特性的表征和分析,获得以下数据:BC-41所产的纤维素纯度达到91.32%,湿纤维素膜含水率达99.16%,每克干纤维素膜能吸水28.59 g;扫描电子显微镜观察,显示该纤维素具有网状结构,且纤维束宽度分布在40-100 nm之间;X射线衍射分析,证实该纤维素的晶型为纤维素I型,结晶指数为48.8%;通过黏度测定法,得出该纤维素的平均聚合度达2 100。     

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.     

Novel stimuli-responsive micelle self-assembled from Y-shaped P(UA-Y-NIPAAm) copolymer for drug delivery

A new amphiphilic Y-shaped copolymer, comprised of hydrophobic poly(undecylenic acid) (PUA) and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm), was designed and synthesized. A cytotoxicity study revealed that P(UA-Y-NIPAAm) copolymers did not exhibit apparent inhibition impact on the proliferation of cells when the concentration of the copolymer was below 1000 mg/L. Characterization demonstrated that the P(UA-Y-NIPAAm) copolymer is thermosensitive with a lower critical solution temperature (LCST) of 31 degrees C. In water, the P(UA-Y-NIPAAm) copolymer would self-assemble into micelles with a critical micelle concentration (CMC) of 20 mg/L. Self-assembled P(UA-Y-NIPAAm) micelles exhibited a nanospherical morphology of 40 to approximately 80 nm in size. The controlled drug release behavior of the P(UA-Y-NIPAAm) micelles was further investigated, and self-assembled micelles exhibited improved properties in controlled drug release.     

Effects of Carboxymethylcellulose and Carboxypolymethylene on Morphology of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> NRRL 2346 and Fumagillin Production

Aspergillus fumigatus NRRL 2346 is the producer of fumagillin, an antitumor antibiotic that inhibits angiogenesis. This strain is very difficult to grow reproducibly in shake flasks owing to an extreme form of pellet growth and extensive wall growth. The effects of carboxymethylcellulose (CMC) and carboxypolymethylene (Carbopol) on growth and fumagillin production by A. fumigatus were investigated. By adding the polymers to the fermentation medium, the growth form of the mold was changed from a single large glob to small reproducible pellets, and wall growth was diminished to a minimum. Carbopol, at a lower concentration, was more effective than CMC in improving both morphology and production. Small pellets were produced which favored fumagillin biosynthesis. 1.5% (wt/vol) CMC and 0.3% (wt/vol) Carbopol were found to be the optimum concentrations; higher levels increased viscosity to an unacceptable level. Received: 1 November 2001 / Accepted: 27 March 2002     

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.     

Ultrastructural alterations associated with the growth of resistant Pseudomonas aeruginosa in the presence of benzalkonium chloride

Cells of Pseudomonas aeruginosa resistant to benzalkonium chloride (BC) underwent unique ultrastructural reorganizations when they were grown in the presence of 1 mg of BC/ml. The resistant cells usually contained a single, centrally positioned pseudovacuole. The pseudovacuole was surrounded by a diffuse substance that spread irregularly throughout the cytoplasm. The presence of the pseudovacuole seemed to cause a physical compartmentalization of the cytoplasm into random pockets of ribosomes and nuclear material. Contained within the pseudovacuole was a horseshoe-shaped, electron-dense body which was bounded by a trilaminar membrane 5.2 nm in width. These bodies averaged 77 nm when measured through the long axis. The surfaces of resistant cells were covered by an additional layer not found in sensitive cells. Thin sections of sensitive cells which had been treated with 1 mg of BC/ml showed little or no lysis. The cytoplasm appeared to be deeply stained and coagulated. Ribosomes were no longer distinctly visible. Although the cell wall remained intact, the cell membrane was dissolved and fragmented. BC-grown resistant cells could not be successfully stained by standard techniques; however, details were demonstrated with the aid of a combination of 1.5% glutaraldehyde, 1% osmium tetroxide, and 1% phosphotungstic acid prepared in 0.1 m sodium dimethylarsonate buffer (pH 6.8).     

Structure study of cellulose fibers wet-spun from environmentally friendly NaOH/urea aqueous solutions

In this study, structure changes of regenerated cellulose fibers wet-spun from a cotton linter pulp (degree of polymerization approximately 620) solution in an NaOH/urea solvent under different conditions were investigated by simultaneous synchrotron wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS). WAXD results indicated that the increase in flow rate during spinning produced a better crystal orientation and a higher degree of crystallinity, whereas a 2-fold increase in draw ratio only affected the crystal orientation. When coagulated in a H2SO4/Na2SO4 aqueous solution at 15 degrees C, the regenerated fibers exhibited the highest crystallinity and a crystal orientation comparable to that of commercial rayon fibers by the viscose method. SAXS patterns exhibited a pair of meridional maxima in all regenerated cellulose fibers, indicating the existence of a lamellar structure. A fibrillar superstructure was observed only at higher flow rates (>20 m/min). The conformation of cellulose molecules in NaOH/urea aqueous solution was also investigated by static and dynamic light scattering. It was found that cellulose chains formed aggregates with a radius of gyration, Rg, of about 232 nm and an apparent hydrodynamic radius, Rh, of about 172 nm. The NaOH/urea solvent system is low-cost and environmentally friendly, which may offer an alternative route to replace more hazardous existing methods for the production of regenerated cellulose fibers.     

Bacterial cellulose production from the litchi extract by Gluconacetobacter xylinus

Although litchi has both nutrient and edible value, the extremely short preservation time limited its further market promotion. To explore processed litchi products with longer preservation time, litchi extract was selected as an alternative feedstock for production of bacterial cellulose (BC). After 2 weeks of static fermentation, 2.53 g/L of the BC membrane was obtained. The trace elements including magnesium (Mg) and sodium (Na) in the litchi extract were partly absorbed in the BC membrane, but no potassium (K) element was detected in it, curiously. Scanning electron microscope (SEM) photographs exhibited an ultrafine network nanostructure for the BC produced in the litchi extract. Analysis of the fourier-transform infrared spectroscopy (FTIR) confirmed the pellicles to be a cellulosic material. Interestingly, X-ray diffraction (XRD) results showed the BC membrane obtained from litchi extract had higher crystallinity of 94.0% than that from HS medium. Overall, the work showed the potential of producing high value-added polymer from litchi resources.     

Study of the Conditions for the Biosynthesis of Bacterial Cellulose by the Producer <Emphasis Type="Italic">Medusomyces gisevii</Emphasis> Sa-12

The effect of culture conditions on the bacterial cellulose (BC) yield synthesized by symbiotic culture Medusomyces gisevii Sa-12 grown in synthetic nutrient medium was studied for the first time. The conditions providing the maximum yield of bacterial cellulose were evaluated: the initial glucose concentration in the culture medium was 20–25 g/L, the temperature was 24–27°C, the ratio of the nutrient medium to the air volume was 1: 10, and the content of the black tea extracts comprised 1.6–4.8 g/L. A sample of chemically pure BC with the following characteristics was obtained under the selected culture conditions: moisture— 99.0%, degree of polymerization—4800, average width of microfibrillar ribbons—30.6 nm, degree of crystallinity— 86%, and the content of triclinic modification Iα—98%.     

细菌纤维素在生物医学材料中应用的研究进展

细菌纤维素是一种天然的生物高聚物,具有生物活性、生物可降解性、生物适应性,具有独特的物理、化学和机械性能,例如高的结晶度、高的持水性、超细纳米纤维网络、高抗张强度和弹性模量等,因而成为近来国际上新型生物医学材料的研究热点。本文概括了细菌纤维素的性质、研究历史以及在生物医学材料上的应用,重点阐述了细菌纤维素在组织工程支架、人工血管、人工皮肤和治疗皮肤损伤方面的应用以及当前研究现状。