Electrospinning is a promising approach to fabricate desirable electropsun nanofibrous scaffold that could be applied in the medical fields. In this study, bacterial copolymer poly(3-hydroxybutyrate-co-68 mol% 4-hydroxybutyrate) [P(3HB-co-68mol% 4HB)] copolymer produced was fabricated into electrospun nanofibers using various combination of electrospinning parameters including the polymer solution, applied voltage and injection speed. The morphology of the fabricated scaffolds were observed using scanning electron microscope (SEM). The SEM images were analysed for the fibre diameter distribution of the scaffolds using Image Analyser. The results revealed that the 8 wt% of polymer solution, 25 kV/cm of the applied voltage and 1.5 mL/h of the injection speed was the most suitable combination. This electrospinning parameters combination fabricated nanofibrous P(3HB-co-4HB) scaffold with smooth, beadles and uniform nanofibers with small fibre diameter distribution.
Bacteria capable of degrading polyhydroxyalkanoates (PHA) by secreting extracellular depolymerase enzymes were isolated from water and soil samples collected from various environments in Malaysia. A total of 8 potential degraders exhibited clear zones on poly(3-hydroxybutyrate) [P(3HB)] based agar, indicating the presence of extracellular PHA depolymerase. Among the isolates, DP5 exhibited the largest clearing zone with a degradation index of 6.0. The highest degradation activity of P(3HB) was also observed with depolymerase enzyme of DP5 in mineral salt medium containing P(3HB). Based on biochemical characterization and 16S rRNA cloning and sequencing, isolate DP5 was found to belong to the genus Acidovorax and subsequently named as Acidovorax sp. DP5. The highest extracellular depolymerase enzyme activity was achieved when 0.25% (w/v) of P(3HB) and 1 g/L of urea were used as carbon and nitrogen source, respectively, in the culture media. The most suitable assay condition of the depolymerase enzyme in response to pH and temperature was tested. The depolymerase produced by strain Acidovorax sp. DP5 showed high percentage of degradation with P(3HB) films in an alkaline condition with pH 9 and at a temperature of 40°C.
This paper investigates the degradation of polyhydroxyalkanoates and its biofiber composites in both soil and lake environment. Time-dependent changes in the weight loss of films were monitored. The rate of degradation of poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-23 mol% 4HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-9 mol% 3HV-co-19 mol% 4HB)] were investigated. The rate of degradation in the lake is higher compared to that in the soil. The highest rate of degradation in lake environment (15.6% w/w week(-1)) was observed with P(3HB-co-3HV-co-4HB) terpolymer. Additionally, the rate of degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-38 mol% 3HV)] was compared to PHBV biofiber composites containing compatibilizers and empty fruit bunch (EFB). Here, composites with 30% EFB displayed the highest rate of degradation both in the lake (25.6% w/w week(-1)) and soil (15.6% w/w week(-1)) environment.
The main focus of this study is the incorporation of collagen peptides to fabricate P(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] nano-fiber construct to further enhance surface wettability and support cell growth while harbouring desired properties for biodegradable wound dressing. Simultaneous electrospinning of nanofiber P(3HB-co-4HB)/collagen peptides construct was carried out using dual syringe system. The wettability of the constructs increased with the increase in 4HB molar fraction from 20mol% 4HB [53.2°], P(3HB-co-35mol%4HB)[48.9°], P(3HB-co-50mol%4HB)[44.5°] and P(3HB-co-82mol%4HB) [37.7°]. In vitro study carried out using mouse fibroblast cells (L929) grown on nanofiber P(3HB-co-4HB)/collagen peptides construct showed an increase in cell proliferation. In vivo study using animal model (Sprague Dawley rats) showed that nanofibrous P(3HB-co-4HB)/collagen peptides construct had a significant effect on wound contractions with the highest percentage of wound closure of 79%. Hence, P(3HB-co-4HB)/collagen peptides construct suitable for wound dressing have been developed using nano-fabrication technique.
Polyhydroxyalkanoate (PHA) is a microbial polymer that has been at the forefront of many attempts at tissue engineering. However, the surface of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is hydrophobic with few recognition sites for cell attachment. Various concentrations of fish-scale collagen peptides (FSCPs) were incorporated into P(3HB-co-4HB) copolymer by aminolysis. Later, FSCPs were introduced onto the aminolyzed P(3HB-co-4HB) scaffolds. Introduction of the FSCP groups was verified using Fourier transform infrared spectroscopy and the ninhydrin method. The effect of the incorporation of FSCPs on hydrophilicity was investigated using the water contact angle. As the concentration of FSCPs increased, the water contact angle decreased. In vitro study demonstrated that P(3HB-co-4HB)/FSCP scaffolds provided better cell attachment and growth of L929 mouse fibroblast cells and better cell proliferation. In vivo study showed that P(3HB-co-4HB)/1.5 wt% FSCPs had a significant effect on wound contractions, with the highest percentage of wound closure (61%) in 7 d.
Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M (n)) of copolymers ranged from 260 x 10(3) to 590 x 10(3)Da, and the polydispersities (M (w)/M (n)) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T (m)), glass transition temperature (T (g)), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.
Nanobiotechnology has undoubtedly influenced major breakthroughs in medical sciences. Application of nanosized materials has made it possible for researchers to investigate a broad spectrum of treatments for diseases with minimally invasive procedures. Silver nanoparticles (AgNPs) have been a subject of investigation for numerous applications in agriculture, water treatment, biosensors, textiles, and the food industry as well as in the medical field, mainly due to their antimicrobial properties and nanoparticle nature. In general, AgNPs are known for their superior physical, chemical, and biological properties. The properties of AgNPs differ based on their methods of synthesis and to date, the biological method has been preferred because it is rapid, nontoxic, and can produce well-defined size and morphology under optimized conditions. Nevertheless, the common issue concerning biological or biobased production is its sustainability. Researchers have employed various strategies in addressing this shortcoming, such as recently testing agricultural biowastes such as fruit peels for the synthesis of AgNPs. The use of biowastes is definitely cost-effective and eco-friendly; moreover, it has been reported that the reduction process is simple and rapid with reasonably high yield. This review aims to address the developments in using fruit- and vegetable-based biowastes for biologically producing AgNPs to be applied as antimicrobial coatings in biomedical applications.
Biomaterial scaffolds play crucial role to promote cell proliferation and foster the regeneration of new tissues. The progress in material science has paved the way for the generation of ingenious biomaterials. However, these biomaterials require further optimization to be effectively used in existing clinical treatments. It is crucial to develop biomaterials which mimics structure that can be actively involved in delivering signals to cells for the formation of the regenerated tissue. In this research we nanoengineered a functional scaffold to support the proliferation of myoblast cells. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is chosen as scaffold material owing to its desirable mechanical and physical properties combined with good biocompatibility, thus eliciting appropriate host tissue responses. In this study P(3HB-co-4HB) copolymer was biosynthesized using Cupriavidus malaysiensis USMAA1020 transformant harboring additional PHA synthase gene, and the viability of a novel P(3HB-co-4HB) electrospun nanofiber scaffold, surface functionalized with RGD peptides, was explored. In order to immobilize RGD peptides molecules onto the P(3HB-co-4HB) nanofibers surface, an aminolysis reaction was performed. The nanoengineered scaffolds were characterized using SEM, organic elemental analysis (CHN analysis), FTIR, surface wettability and their in vitro degradation behavior was evaluated. The cell culture study using H9c2 myoblast cells was conducted to assess the in vitro cellular response of the engineered scaffold. Our results demonstrated that nano-P(3HB-co-4HB)-RGD scaffold possessed an average fiber diameter distribution between 200 and 300 nm, closely biomimicking, from a morphological point of view, the structural ECM components, thus acting as potential ECM analogs. This study indicates that the surface conjugation of biomimetic RGD peptide to the nano-P(3HB-co-4HB) fibers increased the surface wettability (15 ± 2°) and enhanced H9c2 myoblast cells attachment and proliferation. In summary, the study reveals that nano-P(3HB-co-4HB)-RGD scaffold can be considered a promising candidate to be further explored as cardiac construct for building cardiac construct.
Global increase in demand for food supply has resulted in surplus generation of wastes. What was once considered wastes, has now become a resource. Studies were carried out on the conversion of biowastes into wealth using methods such as extraction, incineration and microbial intervention. Agro-industry biowastes are promising sources of carbon for microbial fermentation to be transformed into value-added products. In the era of circular economy, the goal is to establish an economic system which aims to eliminate waste and ensure continual use of resources in a close-loop cycle. Biowaste collection is technically and economically practicable, hence it serves as a renewable carbon feedstock. Biowastes are commonly biotransformed into value-added materials such as bioethanol, bioplastics, biofuels, biohydrogen, biobutanol and biogas. This review reveals the recent developments on microbial transformation of biowastes into biotechnologically important products. This approach addresses measures taken globally to valorize waste to achieve low carbon economy. The sustainable use of these renewable resources is a positive approach towards waste management and promoting circular economy.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is a bacterial derived biopolymer widely known for its unique physical and mechanical properties to be used in biomedical application. In this study, antimicrobial agent silver sulfadiazine (SSD) coat/collagen peptide coat-P(3HB-co-4HB) (SCCC) and SSD blend/collagen peptide coat-P(3HB-co-4HB) scaffolds (SBCC) were fabricated using a green salt leaching technique combined with freeze-drying. This was then followed by the incorporation of collagen peptides at various concentrations (2.5-12.5 wt.%) to P(3HB-co-4HB) using collagen-coating. As a result, two types of P(3HB-co-4HB) scaffolds were fabricated, including SCCC and SBCC scaffolds. The increasing concentrations of collagen peptides from 2.5 wt.% to 12.5 wt.% exhibited a decline in their porosity. The wettability and hydrophilicity increased as the concentration of collagen peptides in the scaffolds increased. In terms of the cytotoxic results, MTS assay demonstrated the L929 fibroblast scaffolds adhered well to the fabricated scaffolds. The 10 wt.% collagen peptides coated SCCC and SBCC scaffolds displayed highest cell proliferation rate. The antimicrobial analysis of the fabricated scaffolds exhibited 100% inhibition towards various pathogenic microorganisms. However, the SCCC scaffold exhibited 100% inhibition between 12 and 24 h, but the SBCC scaffolds with SSD impregnated in the scaffold had controlled release of the antimicrobial agent. Thus, this study will elucidate the surface interface-cell interactions of the SSD-P(3HB-co-4HB)-collagen peptide scaffolds and controlled release of SSD, antimicrobial agent.
Separation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco-balance as P(3HB-co-4HB) is widely used for biomedical applications. Therefore, an efficient and eco-based extraction of P(3HB-co-4HB) using a combination of NaOH and Lysol in digesting the non-polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB-co-4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.
Polyhydroxyalkanoates (PHA) are biodegradable polymers that are considered able to replace synthetic plastic because their biochemical characteristics are in some cases the same as other biodegradable polymers. However, due to the disadvantages of costly and non-renewable carbon sources, the production of PHA has been lower in the industrial sector against conventional plastics. At the same time, first-generation sugar-based cultivated feedstocks as substrates for PHA production threatens food security and considerably require other resources such as land and energy. Therefore, attempts have been made in pursuit of suitable sustainable and affordable sources of carbon to reduce production costs. Thus, in this review, we highlight utilising waste lignocellulosic feedstocks (LF) as a renewable and inexpensive carbon source to produce PHA. These waste feedstocks, second-generation plant lignocellulosic biomass, such as maize stoves, dedicated energy crops, rice straws, wood chips, are commonly available renewable biomass sources with a steady supply of about 150 billion tonnes per year of global yield. The generation of PHA from lignocellulose is still in its infancy, hence more screening of lignocellulosic materials and improvements in downstream processing and substrate pre-treatment are needed in the future to further advance the biopolymer sector.