Experimental investigation of the governing parameters in the electrospinning of poly(3-hydroxybutyrate) scaffolds : structural characteristics of the pores

We sought to determine the impact of electrospinning parameters on a trustworthy criterion that could evidently improve the maximum applicability of fibrous scaffolds for tissue regeneration. We used an image analysis technique to elucidate the web permeability index (WPI) by modeling the formation of electrospun scaffolds. Poly(3-hydroxybutyrate) (P3HB) scaffolds were fabricated according to predetermined conditions of levels in a Taguchi orthogonal design. The material parameters were the polymer concentration, conductivity, and volatility of the solution. The processing parameters were the applied voltage and nozzle-to-collector distance. With a law to monitor the WPI values when the polymer concentration or the applied voltage was increased, the pore interconnectivity was decreased. The quality of the jet instability altered the pore numbers, areas, and other structural characteristics, all of which determined the scaffold porosity and aperture interconnectivity. An initial drastic increase was observed in the WPI values because of the chain entanglement phenomenon above a 6 wt % P3HB content. Although the solution mixture significantly (p < 0.05) changed the scaffold architectural characteristics as a function of the solution viscosity and surface tension, it had a minor impact on the WPI values. The solution mixture gained the third place of significance, and the distance was approved as the least important factor.

The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.

Modified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using hydrogen bonding monomers

Properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were significantly modified by a hydrogen bonding (H-bond) monomer-bisphenol A (BPA). BPA lowered the T-m of PHBV and widened the heat-processing window of PHBV. At the same time, a dynamic H-bond network in the blends was observed indicating that BPA acted as a physical cross-link agent. BPA enhanced the T, of PHBV and reduced the crystallization rate of PHBV. It resulted in larger crystallites in PHBV/BPA blends showed by WAXD. However, the crystallinity of PHBV was hardly reduced. SAXS results suggested that BPA molecules distributed in the inter-lamellar region of PHBV. Finally, a desired tension property was obtained, which had an elongation at break of 370% and a yield stress of 16 MPa. By comparing the tension properties of PHBV/BPA and PHBV/tert-butyl phenol blends, it was concluded that the H-bond network is essential to the improvement of ductility.

Crosslinking of poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate)] using dicumyl peroxide as initiator

In order to modify poly [(3-hydroxybutyrate)-co-(3-hydroxyvalerate)] (PHBV), the crosslinking of this copolymer was carried out at 160degreesC using dicumyl peroxide (DCP) as the initiator. The torque of the PHBV melt showed an abrupt upturn when DCP was added. Appropriate values for the gel fraction and crosslink density were obtained when the DCP content was up to 1 wt% of the PHBV. According to the NMR spectroscopic data, the location of the free radical reaction was determined to be at the tertiary carbons in the PHBV chains. The melting point, crystallization temperature and crystallinity of PHBV decreased significantly with increasing DCP content. The effect of crosslinking on the melt viscosity of PHBV was confirmed as being positive. Moreover, the mechanical properties of PHBV were improved by curing with DCP. When 1 wt% DCP was used, the ultimate elongation of PHBV increased from 4 to 11 %. A preliminary biodegradation study confirmed the total biodegradability of crosslinked PHBV.

Thermal analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) irradiated under vacuum

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was irradiated by Co-60 gamma-rays (doses of 50, 100 and 200kGy) under vacuum. The thermal analysis of control and irradiated PHBV, under vacuum was carried out by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The tensile properties of control and irradiated PHBV were examined by using an Instron tensile testing machine. In the thermal degradation of control and irradiated PHBV, a one-step weight loss was observed. The derivative thermogravimetric curves of control and irradiated PHBV confirmed only one weight-loss step change. The onset degradation temperature (T-o) and the temperature of maximum weight-loss rate (T-p) of control and irradiated PHBV were in line with the heating rate (degreesC min(-1)). T-o and T-p of PHBV decreased with increasing radiation dose at the same heating rate. The DSC results showed that Co-60 gamma-radiation significantly affected the thermal properties of PHBV. With increasing radiation dose, the melting temperature (T-m) of PHBV shifted to a lower value, due to the decrease in crystal size. The tensile strength and fracture strain of the irradiated PHBV decreased, hence indicating an increased brittleness.

Synthesis and characterization of maleated poly(3-hydroxybutyrate)

Graft copolymerization of maleic anhydride (MA) onto poly(3-hydroxybutyrate) (PHB) was carried out by use of benzoyl peroxide as initiator. The effects of various polymerization conditions on graft degree were investigated, including solvents, monomer and initiator concentrations, reaction temperature, and time. The monomer and initiator concentrations played an important role in graft copolymerization, and graft degree could be controlled in the range from 0.2 to 0.85% by changing the reaction conditions. The crystallization behavior and the thermal stability of PHB and maleated PHB were studied by DSC, WAXD, optical microscopy, and TGA. The results showed that, after grafting MA, the crystallization behavior of PHB was obviously changed. The cold crystallization temperature from the glass state increased, the crystallization temperature from the melted state decreased, and the growth rate of spherulite decreased. With the increase in graft degree, the banding texture of spherulites became more distinct and orderly. Moreover, the thermal stability of maleated PHB was obviously improved, compared with that of pure PHB.

Isothermal crystallization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Isothermal crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was investigated by means of differential scanning calorimetry and polarized optical microscopy (POM). The Avrami analysis can be used successfully to describe the isothermal crystallization kinetics of PHBV, which indicates that the Avrami exponent n = 3 is good for all the temperatures investigated. The spherulitic growth rate, G, was determined by POM. The result shows that the G has a maximum value at about 353 K. Using the equilibrium melting temperature (448 K) determined by the Flory equation for melting point depression together with U-* = 1500 cal mol(-1), T-infinity = 30 K and T-g = 278 K, the nucleation parameter K-g was determined, which was found to be 3.14+/-0.07 x 10(5) (K-2), lower than that for pure PHB. The surface-free energy sigma = 2.55 x 10(-2) J m(-2) and sigma(e) = 2.70+/-0.06 x 10-2 J m(-2) were estimated and the work of chain-folding (q = 12.5+/-0.2 kJ mol(-1)) was derived from sigma(e), and found to be lower than that for PHB. This implies that the chains of PHBV are more flexible than that of PHB.

Effect of nucleating agents on the crystallization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The effect of nucleating agents on the crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was studied. A differential scanning calorimeter was used to monitor the energy of the crystallization process from the melt and melting behavior. During the crystallization process from the melt, nucleating agent led to an increase in crystallization temperature (T-c) of PHBV compared with that for plain PHBV (without nucleating agent). The melting temperature of PHBV changed little with addition of nucleating agent. However, the areas of two melting peaks changed considerably with added nucleating agent. During isothermal crystallization, dependence of the relative degree of crystallization on time was described by the Avrami equation. The addition of nucleating agent caused an increase in the overall crystallization rate of PHBV, but did not influence the mechanism of nucleation and growth of the PHB crystals. The equilibrium melting temperature of PHBV was determined as 187degreesC. Analysis of kinetic data according to nucleation theories showed that the increase in crystallization rate of PHBV in the composite is due to the decrease in surface energy of the extremity surface.

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) (PHB) and maleated PHB were investigated by differential scanning calorimetry using various cooling rates. The results show that the crystallization behavior of maleated PHB from the melt greatly depends on cooling rates and its degree of grafting. With the increase in cooling rate, the crystallization process for PHB and maleated PHB begins at lower temperature. For maleated PHB, the introduction of maleic anhydride group hinders its crystallization, causing crystallization and nucleation rates to decrease, and crystallite size distribution becomes wider. The Avrami analysis, modified by Jeziorny, was used to describe the nonisothermal crystallization of PHB and maleated PHB. Double melting peaks for maleated PHB were observed, which was caused by recrystallization during the heating process.

The kinetics of the thermal decomposition of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)

The thermal decomposition mechanism of maleated poly(3-hydroxybutyrate) (PHB) was investigated by FTIR and H-1 NMR. The results of experiments showed that the random chain scission of maleated PHB obeyed the six-membered ring ester decomposition process. The thermal decomposition behavior of PHB and maleated PHB with different graft degree were studied by thermogravimetry (TGA) using various heating-up rates. The thermal stability of maleated PHB was evidently better than that of PHB. With increase in graft degree, the thermal decomposition temperature of maleated PHB gradually increased and then declined. Activation energy E. as a kinetic parameter of thermal decomposition was estimated by the Flynn-Wall-Ozawa and Kissinger methods, respectively. It could be seen that approximately equal values of activation energy were obtained by both methods.

Study on the biodegradation process of poly (3-hydroxybutyrate)

Poly (3-hydroxybutyrate) (PHB) films were biodegraded by DS9701. The degradation process was monitored by using SEM. It was shown that the PHB degradation occurred firstly in the amorphous part of PHB and then in the crystalline part, especially from the center of PHB spherulites. PHB deplymerase produced by DS9701 mainly attacked the second ester bond of PHB and the degraded product was dimmer, determined by using mass spectrometer.

Controlled delivery of gentamicin using poly(3-hydroxybutyrate) microspheres

Poly(3-hydroxybutyrate), P(3HB), produced from Bacillus cereus SPV using a simple glucose feeding strategy was used to fabricate P(3HB) microspheres using a solid-in-oil-water (s/o/w) technique. For this study, several parameters such as polymer concentration, surfactant and stirring rates were varied in order to determine their effect on microsphere characteristics. The average size of the microspheres was in the range of 2 μm to 1.54 μm with specific surface areas varying between 9.60 m(2)/g and 6.05 m(2)/g. Low stirring speed of 300 rpm produced slightly larger microspheres when compared to the smaller microspheres produced when the stirring velocity was increased to 800 rpm. The surface morphology of the microspheres after solvent evaporation appeared smooth when observed under SEM. Gentamicin was encapsulated within these P(3HB) microspheres and the release kinetics from the microspheres exhibiting the highest encapsulation efficiency, which was 48%, was investigated. The in vitro release of gentamicin was bimodal, an initial burst release was observed followed by a diffusion mediated sustained release. Biodegradable P(3HB) microspheres developed in this research has shown high potential to be used in various biomedical applications.

Inverse Metabolic Engineering of Synechocystis PCC 6803 for Improved Growth Rate and Poly-3-hydroxybutyrate Production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) with a broad range of 3HV content at high yields by Burkholderia sacchari IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y-3HV/prop) increasing from 1.10 to 1.34 g g(-1). Copolymer productivity of 1 g l(-1) h(-1) was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y-3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).

Biosynthesis and characterization of biodegradable Poly(3-hydroxybutyrate) from renewable sources

Poly(3-hydroxybutyrate) was produced in fed-batch cultures of Ralstonia eutropha DSM 428 and Alcaligenes latus ATCC 29712 on a mineral medium with different carbon sources such as sucrose, sodium lactate, lactic acid, soybean oil and fatty acid. The bacteria converted the different carbon sources supplied into P3HB. The best results were obtained when lactate or soybean oil were supplied as the sole carbon source. The range of number average molar mass (Mn) for the polymers, analyzed by Gel Permeation Chromatography was 1.65 to 0.79 x 10(5) g mol(-1). FTIR spectroscopy revealed a characteristic absorbance associated with polyester structures. The crystallinity degree, determinate from X-ray diffractograms, was about 69% in all synthesized polymers. The thermal properties associated to semicrystalline polymers indicated a glass transition at 0.1 degrees C and a melting point at about 175 degrees C and enthalpy of 63-89 J g(-1). The (1)H-NMR and (13)C-NMR spectra of the polymers were in agreement with the calculated chemical shifts associated with P3HB structures.