Project description:Due to increased environmental pressures, significant research has focused on finding suitable biodegradable plastics to replace ubiquitous petrochemical-derived polymers. Polyhydroxyalkanoates (PHAs) are a class of polymers that can be synthesized by microorganisms and are biodegradable, making them suitable candidates. The present study looks at the degradation properties of two PHA polymers: polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-polyhydroxyvalerate (PHBV; 8 wt.% valerate), in two different soil conditions: soil fully saturated with water (100% relative humidity, RH) and soil with 40% RH. The degradation was evaluated by observing the changes in appearance, chemical signatures, mechanical properties, and molecular weight of samples. Both PHB and PHBV were degraded completely after two weeks in 100% RH soil conditions and showed significant reductions in mechanical properties after just three days. The samples in 40% RH soil, however, showed minimal changes in mechanical properties, melting temperatures/crystallinity, and molecular weight over six weeks. By observing the degradation behavior for different soil conditions, these results can pave the way for identifying situations where the current use of plastics can be replaced with biodegradable alternatives.
Project description:In modern life petroleum-based plastic has become indispensable due to its frequent use as an easily available and a low cost packaging and moulding material. However, its rapidly growing use is causing aquatic and terrestrial pollution. Under these circumstances, research and development for biodegradable plastic (bioplastics) is inevitable. Polyhydroxybutyrate (PHB), a type of microbial polyester that accumulates as a carbon/energy storage material in various microorganisms can be a good alternative. In this study, 23 cyanobacterial strains (15 heterocystous and 8 non-heterocystous) were screened for PHB production. The highest PHB (6.44% w/w of dry cells) was detected in Nostoc muscorum NCCU- 442 and the lowest in Spirulina platensis NCCU-S5 (0.51% w/w of dry cells), whereas no PHB was found in Cylindrospermum sp., Oscillatoria sp. and Plectonema sp. Presence of PHB granules in Nostoc muscorum NCCU- 442 was confirmed microscopically with Sudan black B and Nile red A staining. Pretreatment of biomass with methanol: acetone: water: dimethylformamide [40: 40: 18: 2 (MAD-I)] with 2 h magnetic bar stirring followed by 30 h continuous chloroform soxhlet extraction acted as optimal extraction conditions. Optimized physicochemical conditions viz. 7.5 pH, 30°C temperature, 10:14 h light:dark periods with 0.4% glucose (as additional carbon source), 1.0 gl-1 sodium chloride and phosphorus deficiency yielded 26.37% PHB on 7th day instead of 21st day. Using FTIR, 1H NMR and GC-MS, extracted polymer was identified as PHB. Thermal properties (melting temperature, decomposition temperatures etc.) of the extracted polymer were determined by TGA and DSC. Further, the polymer showed good tensile strength and young's modulus with a low extension to break ratio comparable to petrochemical plastic. Biodegradability potential tested as weight loss percentage showed efficient degradation (24.58%) of PHB within 60 days by mixed microbial culture in comparison to petrochemical plastic.
Project description:Polyhydroxybutyrate (PHB)/polycaprolactone (PCL)/stearate Mg-Al layered double hydroxide (LDH) nanocomposites were prepared via solution casting intercalation method. Coprecipitation method was used to prepare the anionic clay Mg-Al LDH from nitrate salt solution. Modification of nitrate anions by stearate anions between the LDH layers via ion exchange reaction. FTIR spectra showed the presence of carboxylic acid (COOH) group which indicates that stearate anions were successfully intercalated into the Mg-Al LDH. The formation of nanocomposites only involves physical interaction as there are no new functional groups or new bonding formed. X-ray diffraction (XRD) and transmission electron microscopy (TEM) indicated that the mixtures of nanocomposites are intercalated and exfoliated types. XRD results showed increasing of basal spacing from 8.66 to 32.97 Å in modified stearate Mg-Al LDH, and TEM results revealed that the stearate Mg-Al LDH layers are homogeneously distributed in the PHB/PCL polymer blends matrix. Enhancement in 300% elongation at break and 66% tensile strength in the presence of 1.0 wt % of the stearate Mg-Al LDH as compare with PHB/PCL blends. Scanning electron microscopy (SEM) proved that clay improves compatibility between polymer matrix and the best ratio 80PHB/20PCL/1stearate Mg-Al LDH surface is well dispersed and stretched before it breaks.
Project description:Natural polysaccharide pectin has for the first time been grafted with polyhydroxybutyrate (PHB) via ring-opening polymerization of ?-butyrolactone. This copolymer, pectin-polyhydroxybutyrate (pec-PHB), was blended with PHB in various proportions and electrospun to produce nanofibers that exhibited uniform and bead-free nanostructures, suggesting the miscibility of PHB and pec-PHB. These nanofiber blends exhibited reduced fiber diameters from 499 to 336-426 nm and water contact angles from 123.8 to 88.2° on incorporation of pec-PHB. They also displayed 39-335% enhancement of elongation at break relative to pristine PHB nanofibers. pec-PHB nanofibers were found to be noncytotoxic and biocompatible. Human retinal pigmented epithelium (ARPE-19) cells were seeded onto pristine PHB and pec-PHB nanofibers as scaffold and showed good proliferation. Higher proportions of pec-PHB (pec-PHB10 and pec-PHB20) yielded higher densities of cells with similar characteristics to normal RPE cells. We propose, therefore, that nanofibers of pec-PHB have significant potential as retinal tissue engineering scaffold materials.
Project description:Application of polyhydroxybutyrate (PHB) to plastic industry has expanded over the last decades due to its attracting features over petro-based plastic, and therefore, its waste accumulation in nature is inevitable. In the present study, a total of four bacterial strains, viz., MK3, PN12, PW1, and Lna3, were formulated into a consortium and subsequently used as biological tool for degradation of biopolymers. The consortium was tested through ? max shifts under in vitro conditions for utilization of PHB as sole carbon source. Talc-based bioformulations of consortium were used for the degradation of PHB film composites under in situ conditions. After 9 months of incubation, the recovered samples were monitored through Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM), respectively. Analytical data, viz., changes in ? max shifts (212-219 nm), FT-IR spectra, and SEM micrographs, revealed the biodegradation potential of developed consortium against PHB film composites, i.e., higher degradation of copolymer films was found over blend films. The used consortium had enhanced the rate of natural degradation and can be further used as a natural tool to maintain and restore global environmental safety.
Project description:The waste generated by single-use plastics is often non-recyclable and non-biodegradable, inevitably ending up in our landfills, ecosystems, and food chain. Through the introduction of biodegradable polymers as substitutes for common plastics, we can decrease our impact on the planet. In this study, we evaluate the changes in mechanical and thermal properties of polyhydroxybutyrate-based composites with various additives: Microspheres, carbon fibers or polyethylene glycol (2000, 10,000, and 20,000 MW). The mixtures were injection molded using an in-house mold attached to a commercial extruder. The resulting samples were characterized using microscopy and a series of spectroscopic, thermal, and mechanical techniques. We have shown that the addition of carbon fibers and microspheres had minimal impact on thermal stability, whereas polyethylene glycol showed slight improvements at higher molecular weights. All of the composite samples showed a decrease in hardness and compressibility. The findings described in this study will improve our understanding of polyhydroxybutyrate-based composites prepared by injection molding, enabling advancements in integrating biodegradable plastics into everyday products.
Project description:The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, "frozen-hydrated" state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts.
Project description:Polyhydroxybutyrate (PHB) is a natural polyester synthesized by several microorganisms. Moreover, it has excellent biodegradability and is an eco-friendly material because it converts water and carbon dioxide as final decomposition products. However, the applications of PHB are limited because of its stiffness and brittleness. Because cellulose nanocrystals (CNCs) have excellent intrinsic mechanical properties such as high specific strength and modulus, they may compensate for the insufficient physical properties of PHB by producing their nanocomposites. In this study, natural polyesters were extracted from Cupriavidus necator fermentation with CNCs, which were well-dispersed in nitrogen-limited liquid culture media. Fourier-transform infrared spectroscopy results revealed that the additional O-H peak originating from cellulose at 3500-3200 cm-1 was observed for PHB along with the C=O and -COO bands at 1720 cm-1. This suggests that PHB-CNC nanocomposites could be readily obtained using C. necator fermented in well-dispersed CNC-supplemented culture media.
Project description:Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps.
Project description:Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.