Renewable resource-based green composites from recycled cellulose fiber and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) bioplastic

Novel "green" composites were successfully fabricated from recycled cellulose fibers (RCF) and a bacterial polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by melt mixing technique. Various weight contents (15%, 30%, and 40%) of the fibers were incorporated in the PHBV matrix. The effect of the fiber weight contents on the thermal, mechanical, and dynamic-mechanical thermal properties of PHBV was investigated and a comparative property analysis was performed with RCF-reinforced polypropylene (PP) composites. The tensile and storage moduli of the PHBV-based composites were improved by 220% and 190%, respectively, by reinforcement with 40 wt % RCF. Halpin-Tsai and Tsai-Pagano's equations were applied for the theoretical modeling of the tensile modulus of PHBV-based composites. The heat deflection temperature (HDT) of the PHBV-based composites was increased from 105 to 131 degrees C, while the coefficient of linear thermal expansion (CLTE) value was reduced by 70% upon reinforcement with 40 wt % RCF. The PHBV-based composites had also shown better tensile and storage moduli and lower CLTE values than PP-based composites. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were used to study the melting behavior, thermal stability, and morphology of the composite systems, respectively.     

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) improved osteogenic differentiation of the human induced pluripotent stem cells while considered as an artificial extracellular matrix

Cocell polymers can be the best implants for replacing bone defects in patients. The pluripotent stem cells produced from the patient and the nanofibrous polymeric scaffold that can be completely degraded in the body and its produced monomers could be also usable are the best options for this implant. In this study, electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated and characterized and then osteogenic differentiation of the human-induced pluripotent stem cells (iPSCs) was investigated while cultured on PHBV scaffold. MTT results showed that cultured iPSCs on PHBV proliferation were increased compared to those cultured on tissue culture polystyrene (TCPS) as the control. Alkaline phosphatase (ALP) activity and calcium content were also significantly increased in iPSCs cultured on PHBV compared to the cultured on TCPS under osteogenic medium. Gene expression evaluation demonstrated that Runx2, collagen type I, ALP, osteonectin, and osteocalcin were upregulated in iPSCs cultured on PHBV scaffold in comparison with those cultured on TCPS for 2 weeks. Western blot analysis have shown that osteocalcin and osteopontin expression as two major osteogenic markers were increased in iPSCs cultured on PHBV scaffold. According to the results, nanofiber-based PHBV has a promising potential to increase osteogenic differentiation of the stem cells and iPSCs-PHBV as a cell-co-polymer construct demonstrated that has a great efficiency for use as a bone tissue engineered bioimplant.     

Introducing amine functionalities on a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) surface: comparing the use of ammonia plasma treatment and ethylenediamine aminolysis

Amine functionalities were introduced onto the surface of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films by applying radio frequency ammonia plasma treatment and wet ethylenediamine treatment. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS) for chemical composition and Raman microspectroscopy for the spatial distribution of the chemical moieties. The relative amount of amine functionalities introduced onto the PHBV surface was determined by exposing the treated films to the vapor of trifluoromethylbenzaldehyde (TFBA) prior to XPS analysis. The highest amount of amino groups on the PHBV surface could be introduced by use of ammonia plasma at short treatment times of 5 and 10 s, but no effect of plasma power within the range of 2.5-20 W was observed. Ethylenediamine treatment yielded fewer surface amino groups, and in addition an increase in crystallinity as well as degradation of PHBV was evident from Fourier transform infrared spectroscopy. Raman maps showed that the coverage of amino groups on the PHBV surfaces was patchy with large areas having no amine functionalities.     

Molecular systematics of basal subfamilies of ants using 28S rRNA (Hymenoptera: Formicidae)

For many years, the ant subfamily Ponerinae was hypothesized to contain the basal (early branching) lineages of ants. Recently the Ponerinae were reclassified into six poneromorph subfamilies based on morphological analysis. We evaluate this new poneromorph classification using 1240 base pairs of DNA sequence data obtained from 28S rRNA gene sequences of 68 terminal taxa. The molecular tree supported the monophyly of the ant family Formicidae, with 100% parsimony bootstrap (PB) support and posterior probabilities (PP) of 1.00, with the ant subfamily Leptanillinae as a sister group to all other ants (PB=62, PP=93). However, our analyses strongly support the polyphyly of the Poneromorph subfamilies (sensu Bolton). The Ectatomminae and Heteroponerinae are more closely related to the Formicoid subfamilies than to the rest of the poneromophs (PB=96, PP=100). The Amblyoponinae (PB=52, PP=96), Paraponerinae (PB=100, PP=100), Ponerinae (PB<50, PP=71), and Proceratiinae (PB=98, PP=100) appear as distinct lineages at the base of the tree and are identified as a poneroid grade. Monophyletic origins for the poneroid subfamilies Amblyoponinae, Paraponerinae, Ponerinae and Proceratiinae are supported in our analysis. However, the genus Platythyrea forms a distinct sister group to the Ponerini within the Ponerinae. The Heteroponerinae, based on our sample of Heteroponera, are associated with the subfamily Ectatomminae (PB=98, PP=100). Furthermore, our data indicate the genus Probolomyrmex belongs to the Proceratiinae as suggested by recent morphological analysis (PB=98, PP=100).     

Sequence distribution in microbial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polyesters determined by NMR and MS

The microstructure of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters (PHBV) as well as a mixture of two PHBV copolyesters of different comonomer composition and sequence distribution was studied by 13C NMR based on dyad and triad analysis and multistage electrospray ionization mass spectrometry (ESI-MSn). Both techniques gave results that were in good agreement for all investigated samples. The effect of microstructure on PHBV thermal properties was investigated from the melting behavior of samples. A PHBV copolyester with randomly distributed hydroxyvalerate units (12.0 mol % HV) showed a single melting peak, whereas samples with nonrandom composition distribution showed multiple melting peaks in their thermograms. Such complex melting behavior suggested that the 12.9 and 27.1 mol % PHBV copolyesters were actually blends of several copolymers with widely different comonomer-unit composition.     

Crystallization behavior and hydrophobic properties of biodegradable ethyl cellulose-g-poly(3-hydroxybutyrate-co-3-hydroxyvalerate): The influence of the side-chain length and grafting density

The copolymerization of grafting poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) onto ethyl cellulose (EC) was carried out through the homogeneous acylation reaction between EC as a backbone and telechelic OH-terminated PHBV oligomer as side chains in 1,2-dichloroethane by using 1,6-hexamethylene diisocyanate (HDI) as a coupling agent and dibutyltin dilaurate as catalyst. The resulting copolymers were studied by using NMR, FT-IR, WAXD, DSC, and contact angle measurements. It is found that with the increasing of the HDI/PHBV fraction, a transition exhibition occurred on crystallization behavior and hydrophobic properties, which could be modulated through controlling the lengths and grafting densities of PHBV side chains. Compared with those of neat PHBV, the degree of crystallinity for EC-g-PHBV_1.8 decreased from 58.1% to 39.1%, the maximum decomposition temperature increased from 259.6 to 266.3 °C, and the contact angle increased from 60.1° to 95.7°.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Fabrication,microstructure characterization,and degradation performance of electrospun mats based on poly(3-hydroxybutyrate-co-3 hydroxyvalerate)/polyethylene glycol blend for potential tissue engineering

There have been strong demands for nanofibrous scaffolds fabricated by electrospinning for various fields due to their various advantages. Electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) fibre mats were prepared. The effects of processing variables as well as the inclusion of poly(ethylene glycol) (PEG) on the morphologies of generated fibres were investigated using Fourier-transform infrared spectroscopy and scanning electron microscopy. The average fibrous diameter was monitored in the range 400–3000 nm relying on the total content of PEG. The fluorescence cell imaging of electrospun mats was also explored. The results of cell viability demonstrated that skin fibroblast BJ-1 cells showed different adhesions and growth rates for the three kinds of PHBV fibres. Electrospun PHBV mats with low amount of PEG offer a high-quality medium for cell growth. Therefore, those mats exhibited high potential for soft tissue engineering, in particular wound healing.     

Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.     

Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitosan scaffolds for skin regeneration

The purpose of this study was to evaluate hybrid poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/chitosan nanofibrous mats as scaffolds for skin engineering. In vitro studies were carried out to test the potential of the scaffolds for fibroblasts adhesion, viability, and proliferation (L929 cell line). The in vivo performance was also studied in a full-thickness wound healing model. PHBV/chitosan 4:1 (w/w) exhibited a higher in vitro biocompatibility and a better ability for cell adhesion and growth, compared to PHBV/chitosan 2:3 (w/w). The in vivo assay also revealed the better performance of this scaffold, improving the wound healing process in rats.     

PHBV‐TiO2 mats prepared by electrospinning technique: Physico‐chemical properties and cytocompatibility

One of the most important challenges in tissue engineering research is the development of biomimetic materials. In this present study, we have investigated the effect of the titanium dioxide (TiO₂) nanoparticles on the properties of electrospun mats of poly (hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV), to be used as scaffold. The morphology of electrospun fibers was observed by scanning electron microscopy (SEM). Both pure PHBV and nanocomposites fibers were smooth and uniform. However, there was an increase in fiber diameter with the increase of TiO₂ concentration. Thermal properties of PHBV and nanocomposite mats were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). DSC analysis showed that the crystallization temperature for PHBV shifts to higher temperature in the presence of the nanoparticles, indicating that TiO₂ nanoparticles change the process of crystallization of PHBV due to heterogeneous nucleation effect. TGA showed that in the presence of the nanoparticles, the curves are shifted to lower temperatures indicating a decreasing in thermal stability of nanocomposites compared to pure PHBV. To produce scaffolds for tissue engineering, it is important to evaluate the biocompatibility of the material. Cytotoxicity assay showed that TiO₂ nanoparticles were not cytotoxic for cells at the concentration used to synthesize the mats. The proliferation of cells on the mats was evaluated by the MTT assay. Results showed that the nanocomposite samples increased cell proliferation compared to the pure PHBV. These results indicate that continuous electrospun fibrous scaffolds may be a good substrate for tissue regeneration.     

Degradation of polypropylene–poly-L-lactide blend by bacteria isolated from compost

Abstract

Polypropylene (PP) degrading bacteria (P1 to P16) were isolated from compost using enrichment technique. Five isolates (P3, P6, P8, P10, and P13) were selected based on their degradation abilities. These isolates were identified as Bacillus spp. through biochemical characteristics and 16S rDNA sequence analysis. The isolates were tested for their ability to degrade blends of PP and poly-L-lactide (PLLA) (PP80 and PP80C6) in minimal media as well as in soil. In minimal media, the growth of bacteria increased with time, showing utilization of blend as carbon source. The protein content was estimated at the end of 15?days and maximum amount was secreted by isolate P8 indicating maximum potential to degrade polymers compared to other isolates. Scanning electron microscopy (SEM) results revealed the formation of biofilm on the polymer surface. Fourier-transform infrared spectroscopy (FTIR) analysis showed the formation of new bond at 2123?cm^?1 and breakage of old C=O ester bond at 1757?cm^?1 in case of polymer PP80C6. Thermogravimetric analysis (TGA) showed decrease in thermal stability of polymers after degradation. The carbon dioxide evolved from sample was measured and biodegradation degree was also calculated. The degree of biodegradation shown by the isolate P8 was 12% and the P6 was 10%. The results demonstrated that Bacillus species isolated from composted samples in this study provided promising evidence for the biodegradation of polypropylene and poly-L-lactide (PP-PLLA) blends in the environment.     

Effect of organosoluble salts on the nanofibrous structure of electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Electrospinning of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in chloroform was investigated to develop non-woven biodegradable nanofibrous structures for tissue engineering. Ultrafine PHBV fibers were obtained by electrospinning of 20 wt.% PHBV solution in chloroform and the resulting fiber diameters were in the range of 1.0-4.0 microm. When small amounts of benzyl trialkylammonium chlorides were added to the PHBV solution, the average diameter was decreased to 1.0 microm and the fibers were amounted in a straight shape. Conductivity of the PHBV solution was a major parameter affecting the morphology and diameter of the electrospun PHBV fibers. PHBV non-woven structures electrospun with salt exhibited a higher degradation rate than those prepared without salt probably due to the increase of surface area of PHBV fibers.     

Production in Escherichia coli of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources

The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.     

Purification and characterization of an extracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Acidovorax sp. HB01

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading strain Acidovorax sp. HB01 was isolated from an activated sludge sample. A novel PHBV depolymerase with a molecular weight of 43.4 kDa was purified to homogeneity from the culture supernatant of the HB01 strain. The optimum pH and temperature of the PHBV depolymerase were 7.0 and 50 °C, respectively. The PHBV depolymerase can also degrade polyhydroxybutyrate, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(caprolactone); however, the PHBV degradation activity of the depolymerase is higher than its activity against the other polymers. Effect of metal ions and various inhibitors on the PHBV depolymerase activity was examined. The addition of Na(+), K(+), and Ca(2+) markedly increased the hydrolysis rate, whereas the enzyme activity was inhibited by Zn(2+), Mg(2+), Mn(2+), and particularly by Cu(2+) and Fe(2+). Ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of depolymerase was identified as the 3-hydroxybutyric acid monomer and 3-hydroxyvaleric acid monomers via mass spectrometry.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.     

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO₂ production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.     

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from activated sludge

Poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) was produced using a co-culture of activated sludge. When butyric acid was used as sole carbon source, PHB was produced. When valeric acid was added to the medium, PHBV was produced. The 3-hydroxyvalerate (3HV) mole fraction in the PHBV reached a maximum of 54% when valeric acid was used as sole carbon source. When the 3HV units in the co-polymer increased from 0.0 to 54.0 mol%, the melting temperature ( T m ) decreased from 178 to 99°C. The composition, and hence the mechanical properties, of the co-polymer produced by activated sludge can be controlled by adjusting the medium composition.     

Fungal contribution to in situ biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film in soil

The contribution of fungi to the microbial degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films in soil was studied. Various traces, cavities, and grooves observed on the dented surface of PHBV films demonstrated that the degradation was a concerted effect of a microbial consortium colonizing the film surface, including fungi, bacteria, and actinomycetes. The succession of microbial consortia in the soil around the PHBV films during the degradation showed a distinctive increase in the fungal population, resulting in its dominance. Comparison of the degradation ability of microbial strains isolated from soil where PHBV films were degraded, revealed that fungi showed the highest contribution to PHBV degradation, growing very rapidly along the film surface with their high degradation ability and then expanding their hyphae in a three-dimensional manner.     

Intralobular localization of different cytochrome P-450 form dependent monooxygenase activities in the liver of normal and inducer-treated rats.

1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. 2. 7-Ethoxyresorufin O-deethylase (7EROD), 7-pentoxyresorufin O-dealkylase (7PROD) and N-nitrosodimethylamine N-demethylase (NAND) activities of PP hepatocytes were not significantly different from those of PV hepatocytes. 3. Ethylmorphine N-demethylase (EMND) activity was significantly higher in PV hepatocytes than in PP hepatocytes of normal rats. 4. EMND activity was induced by PCN and PB treatments. The response of EMND activity to PCN treatment was higher in PP hepatocytes than that in PV hepatocytes, and as a result the PV dominance disappeared following PCN treatment. 5. Extents of the response of this activity to PB treatment were similar in PP and PV hepatocytes, and PV dominance remained unchanged even after induction.