Effect of Plasma Treatment on Bamboo Fiber-Reinforced Epoxy Composites.

Bamboo cellulose fiber (BF)-reinforced epoxy (EP) composites were fabricated with BF subjected to plasma treatment using argon (Ar), oxygen (O2), and nitrogen (N2) gases. Optimal mechanical properties of the EP/BF composites were achieved with BFs subjected to 30 min of plasma treatment using Ar. This is because Ar gas improved the plasma electron density, surface polarity, and BF roughness. Flexural strength and flexural modulus increased with O2 plasma treatment. Scanning electron microscopy images showed that the etching of the fiber surface with Ar gas improved interfacial adhesion. The water contact angle and surface tension of the EP/BF composite improved after 10 min of Ar treatment, owing to the compatibility between the BFs and the EP matrix. The Fourier transform infrared spectroscopy results confirmed a reduction in lignin after treatment and the formation of new peaks at 1736 cm-1, which indicated a reaction between epoxy groups of the EP and carbon in the BF backbone. This reaction improved the compatibility, mechanical properties, and water resistance of the composites.

Fabrication of Fish Scale-Based Gelatin Methacryloyl for 3D Bioprinting Application.

Gelatin methacryloyl (GelMA) is an ideal bioink that is commonly used in bioprinting. GelMA is primarily acquired from mammalian sources; however, the required amount makes the market price extremely high. Since garbage overflow is currently a global issue, we hypothesized that fish scales left over from the seafood industry could be used to synthesize GelMA. Clinically, the utilization of fish products is more advantageous than those derived from mammals as they lower the possibility of disease transmission from mammals to humans and are permissible for practitioners of all major religions. In this study, we used gelatin extracted from fish scales and conventional GelMA synthesis methods to synthesize GelMA, then tested it at different concentrations in order to evaluated and compared the mechanical properties and cell responses. The fish scale GelMA had a printing accuracy of 97%, a swelling ratio of 482%, and a compressive strength of about 85 kPa at a 10% w/v GelMA concentration. Keratinocyte cells (HaCaT cells) were bioprinted with the GelMA bioink to assess cell viability and proliferation. After 72 h of culture, the number of cells increased by almost three-fold compared to 24 h, as indicated by many fluorescent cell nuclei. Based on this finding, it is possible to use fish scale GelMA bioink as a scaffold to support and enhance cell viability and proliferation. Therefore, we conclude that fish scale-based GelMA has the potential to be used as an alternative biomaterial for a wide range of biomedical applications.

Fabrication and biological properties of artificial tendon composite from medium chain length polyhydroxyalkanoate.

Medium chain length polyhydroxyalkanoate (MCL-PHA), a biodegradable and biocompatible material, has a mechanical characteristic of hyper-elasticity, comparable to elastomeric material with similar properties to human tendon flexibility. These MCL-PHA properties gave rise to applying this material as an artificial tendon or ligament implant. In this study, the material was solution-casted in cylinder and rectangular shapes in the molds with the designated small holes. A portion of the torn human tendon was threaded into the holes as a suture to generate a composite tendon graft. The tensile testing of the three types of MCL-PHA/tendon composite shows that the cylinder material shape with the zigzag threaded three holes has the highest value of maximum tensile strength at 56 MPa, closing to the ultimate tendon tensile stress (50-100 MPa). Fibroblast cells collected from patients were employed as primary tendon cells for growing to attach to the surface of the MCL-PHA material to prove the concept of the composite tendon graft. The cells could attach and proliferate with substantial viability and generate collagen, leading to chondrogenic induction of tendon cells. An in vivo biocompatibility was also conducted in a rat subcutaneous model in comparison with medical-grade silicone. The MCL-PHA material was found to be biocompatible with the surrounding tissues. For surgical application, after the MCL-PHA material is decomposed, tendon cells should develop into an attached tendon and co-generated as a tendon graft.

Enhancing protein trapping efficiency of graphene oxide-polybutylene succinate nanofiber membrane via molecular imprinting.

Filtration of biological liquids has been widely employed in biological, medical, and environmental investigations due to its convenience; many could be performed without energy and on-site, particularly protein separation. However, most available membranes are universal protein absorption or sub-fractionation due to molecule sizes or properties. SPMA, or syringe-push membrane absorption, is a quick and easy way to prepare biofluids for protein evaluation. The idea of initiating SPMA was to filter proteins from human urine for subsequent proteomic analysis. In our previous study, we developed nanofiber membranes made from polybutylene succinate (PBS) composed of graphene oxide (GO) for SPMA. In this study, we combined molecular imprinting with our developed PBS fiber membranes mixed with graphene oxide to improve protein capture selectivity in a lock-and-key fashion and thereby increase the efficacy of protein capture. As a model, we selected albumin from human serum (ABH), a clinically significant urine biomarker, for proteomic application. The nanofibrous membrane was generated utilizing the electrospinning technique with PBS/GO composite. The PBS/GO solution mixed with ABH was injected from a syringe and transformed into nanofibers by an electric voltage, which led the fibers to a rotating collector spinning for fiber collection. The imprinting process was carried out by removing the albumin protein template from the membrane through immersion of the membrane in a 60% acetonitrile solution for 4 h to generate a molecular imprint on the membrane. Protein trapping ability, high surface area, the potential for producing affinity with proteins, and molecular-level memory were all evaluated using the fabricated membrane morphology, protein binding capacity, and quantitative protein measurement. This study revealed that GO is a controlling factor, increasing electrical conductivity and reducing fiber sizes and membrane pore areas in PBS-GO-composites. On the other hand, the molecular imprinting did not influence membrane shape, nanofiber size, or density. Human albumin imprinted membrane could increase the PBS-GO membrane's ABH binding capacity from 50 to 83%. It can be indicated that applying the imprinting technique in combination with the graphene oxide composite technique resulted in enhanced ABH binding capabilities than using either technique individually in membrane fabrication. The suitable protein elution solution is at 60% acetonitrile with an immersion time of 4 h. Our approach has resulted in the possibility of improving filter membranes for protein enrichment and storage in a variety of biological fluids.

Enhancement in mechanical and antimicrobial properties of epoxidized natural rubber via reactive blending with chlorhexidine gluconate.

An epoxidized natural rubber (ENR) blend with chlorhexidine gluconate (CHG) was prepared using a two-roll mill at 130 °C. CHG was added at concentrations of 0.2, 0.5, 1, 2, 5, and 10% (w/w) as an antimicrobial additive. The ENR blend with 10% (w/w) CHG showed the best tensile strength, elastic recovery, and Shore A hardness. The ENR/CHG blend exhibited a smooth fracture surface. The appearance of a new peak in the Fourier transform infrared spectrum confirmed that the amino groups of CHG reacted with the epoxy groups of ENR. The ENR with 10% CHG exhibited an inhibition zone against Staphylococcus aureus. The proposed blending improved the mechanical properties, elasticity, morphology, and antimicrobial properties of the ENR.

Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol.

BACKGROUND: This study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions. RESULTS: A newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH4Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate. CONCLUSION: Cupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

Preparation, Characterization, and Biological Properties of Hydroxyapatite from Bigeye Snapper (Priancanthus tayenus) Bone.

The optimum condition of acid hydrolysis for hydroxyapatite extraction from bigeye snapper (Priancanthus tayenus) bone and the effects of extraction time (10-60 min) and HCl concentration (2.0-5.0% w/v) on yield and hydroxyapatite properties were determined. The optimum extracted condition was found using 5% HCl for 60 min, which was 13.4% yield; 19.8 g/100 g Ca content; 9.6 g/100 g P content; 2.1 Ca/P ratio; L*, a*, b*; and ΔE as 84.5, 2.8, 16.5, and 15.6, respectively. The using of 5% NaOH solution was optimum for hydroxyapatite precipitation from the extracted solution. The characteristic and biological properties of the obtained hydroxyapatite were studied. Fourier transform infrared spectroscopy and X-ray diffraction results showed a good comparison between the extracted and commercial hydroxyapatite. The microstructure of the extracted hydroxyapatite from a scanning electron microscope showed an irregular and flat-plate shape, large surface area, and roughness. The extracted hydroxyapatite was non- and low-cytotoxicity at a concentration of 50 and 100-400 µg/mL, respectively. Bovine serum albumin (BSA) adsorption and desorption of hydroxyapatite was studied. An increasing BSA concentration, hydroxyapatite amount, and adsorption time significantly increased protein adsorption on hydroxyapatite. Protein desorption from BSA-loaded hydroxyapatite showed an increase of release initially in the first 4 days and became a steady release rate until 14 days.

Modified Poly(Lactic Acid) Epoxy Resin Using Chitosan for Reactive Blending with Epoxidized Natural Rubber: Analysis of Annealing Time.

Poly(lactic acid) was melt-blended with epoxy resin without hardener and chitosan (CTS) to prepare modified PLA (PLAEC). Epoxy resin 5% and CTS 1-20% (wt/wt) were incorporated into PLA during melt mixing. PLAEC was melt-blended with an epoxidized natural rubber (ENR) 80/20 wt. The PLAEC CTS 1% blended with ENR (PLAEC1/ENR) showed a high tensile strength (30 MPa) and elongation at break (7%). The annealing process at 80 °C for 0-15 min maintained a tensile strength of approximately 30 MPa. SEM images of the PLAE/ENR blend showed phase inversion from co-continuous to ENR particle dispersion in the PLA matrix with the addition of CTS, whereas the annealing time reduced the hole sizes of the extracted ENR phase due to the shrinkage of PLA by crystallization. Thermal properties were observed by DSC and a Vicat softening test. The annealing process increased the crystallinity and Vicat softening temperature of the PLAEC1/ENR blend. Reactions of -COOH/epoxy groups and epoxy/-NH2 groups occurred during PLAE and PLAEC preparation, respectively. FTIR confirmed the reaction between the -NH2 groups of CTS in PLAEC and the epoxy groups of ENR. This reaction increased the mechanical properties, while the annealing process improved the morphology and thermal properties of the blend.

Evaluation of Biodegradabilities of Biosynthetic Polyhydroxyalkanoates in Thailand Seawater and Toxicity Assessment of Environmental Safety Levels.

Every year, thousands of tons of non-biodegradable plastic products are dumped into marine environments in Thailand's territorial seawater, impacting various marine animals. Recently, there has been a surge in interest in biodegradable plastics as a solution for aquatic environments. However, in Thailand's coastal waters, no suitable biodegradable plastic has been used as ocean-biodegradable packaging. Among them, polyhydroxyalkanoates (PHAs) have excellent biodegradability even in seawater, which is the desired property for packaging applications in tourist places such as plastic bags and bottles. In this report, we assess the environment's safety and study the biodegradation in Thailand seawater of polyhydroxybutyrate (PHB) and PHA copolymer (PHBVV) that were successfully synthesized by bacteria with similar molecular weight. The two types of extracted PHA samples were preliminary biodegradability tested in the marine environment compared with cellulose and polyethylene. Within 28 days, PHB and PHBVV could be biodegraded in both natural and synthetic seawater with 61.2 and 96.5%, respectively. Furthermore, we assessed residual toxicity after biodegradation for environmental safety using seawater samples containing residual digested compounds and the standard guide for acute toxicity tests. It was discovered that marine water mites (Artemia franciscana) have 100 percent viability, indicating that they are non-toxic to the marine environment.

Electrospun Fibers of Polybutylene Succinate/Graphene Oxide Composite for Syringe-Push Protein Absorption Membrane.

The adsorption of proteins on membranes has been used for simple, low-cost, and minimal sample handling of large volume, low protein abundance liquid samples. Syringe-push membrane absorption (SPMA) is an innovative way to process bio-fluid samples by combining a medical syringe and protein-absorbable membrane, which makes SPMA a simple, rapid protein and proteomic analysis method. However, the membrane used for SPMA is only limited to commercially available protein-absorbable membrane options. To raise the method's efficiency, higher protein binding capacity with a lower back pressure membrane is needed. In this research, we fabricated electrospun polybutylene succinate (PBS) membrane and compared it to electrospun polyvinylidene fluoride (PVDF). Rolling electrospinning (RE) and non-rolling electrospinning (NRE) were employed to synthesize polymer fibers, resulting in the different characteristics of mechanical and morphological properties. Adding graphene oxide (GO) composite does not affect their mechanical properties; however, electrospun PBS membrane can be applied as a filter membrane and has a higher pore area than electrospun PVDF membrane. Albumin solution filtration was performed using all the electrospun filter membranes by the SPMA technique to measure the protein capture efficiency and staining of the protein on the membranes, and these membranes were compared to the commercial filter membranes-PVDF, nitrocellulose, and Whatman no. 1. A combination of rolling electrospinning with graphene oxide composite and PBS resulted in two times more captured protein when compared to commercial membrane filtration and more than sixfold protein binding than non-composite polymer. The protein staining results further confirmed the enhancement of the protein binding property, showing more intense stained color in compositing polymer with GO.

Evaluation of 3D-Printing Scaffold Fabrication on Biosynthetic Medium-Chain-Length Polyhydroxyalkanoate Terpolyester as Biomaterial-Ink.

Currently, the selection of materials for tissue engineering scaffolds is still limited because some tissues require flexible and compatible materials with human cells. Medium-chain-length polyhydroxyalkanoate (MCL-PHA) synthesized in microorganisms is an interesting polymer for use in this area and has elastomeric properties compatible with the human body. MCL-PHAs are elastomers with biodegradability and cellular compatibility, making them an attractive material for fabricating soft tissue that requires high elasticity. In this research, MCL-PHA was produced by fed-batch fermentation that Pseudomonas Putida ATCC 47054 was cultured to accumulate MCL-PHA by using glycerol and sodium octanoate as carbon sources. The amounts of dry cell density, MCL-PHA product per dry cells, and MCL-PHA productivity were at 15 g/L, 27%, and 0.067 g/L/h, respectively, and the components of MCL-PHA consisting of 3-hydroxydecanoate (3HD) 64.5%, 3-hydroxyoctanoate (3HO) 32.2%, and 3-hydroxyhexanoate (3HHx) 3.3%. The biosynthesized MCL-PHA terpolyester has a relatively low melting temperature, low crystallinity, and high ductility at 52 °C, 15.7%, and 218%, respectively, and considering as elastomeric polyester. The high-resolution scaffold of MCL-PHA terpolyester biomaterial-ink (approximately 0.36 mm porous size) could be printed in a selected condition with a 3D printer, similar to the optimum pore size for cell attachment and proliferation. The rheological characteristic of this MCL-PHA biomaterial-ink exhibits shear-thinning behavior, leading to good shape fidelity. The study results yielded a condition capable of fabricating an elastomer scaffold of the MCL-PHA terpolyester, giving rise to the ideal soft tissue engineering application.

Proteomic Examination for Gluconeogenesis Pathway-Shift during Polyhydroxyalkanoate Formation in Cupriavidus necator Grown on Glycerol.

Because of availability and inexpensive, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by Cupriavidus necator was found to produce lower PHA with low bacterial growth rates. According to our study, C. necator was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the Cupriavidus necator H16 experiment give rise to the break down the bonds of α,α-1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (C6H14O6), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs.

Microbial synthesis of polyhydroxybutyrate from glycerol: gluconeogenesis, molecular weight and material properties of biopolyester.

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3-hydroxybutyrate) (PHB) production from glycerol. Compared to glucose-based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h(-1) . We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non-fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol-based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol-based PHB is lower than those of glucose-based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar.

Comonomer compositional distribution, physical properties, and enzymatic degradability of bacterial poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) copolyesters.

Poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (P(3HB-co-3H4MV)) was synthesized by using Ralstonia eutropha strain PHB(-)4 (PHA-negative mutant) harboring with the N149S/D171G double mutation of PHA synthase gene from Aeromonas caviae (PhaC(Ac) NSDG mutant) with 4-methylvaleric acid and fructose as the carbon sources. Because the microbially synthesized P(3HB-co-3H4MV) samples were found to have broad comonomer compositional distribution, P(3HB-co-3H4MV) samples with 3H4MV content of 7, 11, and 35 mol % were fractionated into several fractions with different comonomer composition ranging from 2 to 47 mol % by using a chloroform/n-hexane mixture. It was confirmed that a series of well-fractionated P(3HB-co-3H4MV) samples had a statistically random distribution. Physical properties and structure of the fractionated P(3HB-co-3H4MV) random copolymers were investigated. Both the melting temperature and glass-transition temperature of P(3HB-co-3H4MV) copolymers decreased with an increase in the 3H4MV composition. The degree of X-ray crystallinity of fractionated P(3HB-co-3H4MV) films decreased from 60 to 13% as the 3H4MV fraction increased from 0 to 39 mol %. Enzymatic degradation test for fractionated P(3HB-co-3H4MV) films was carried out at 37 degrees C in the presence of PHB depolymerase from Ralstonia pickettii T1. The rates of enzymatic erosion markedly increased with an increase in the 3H4MV composition to reach the highest one at 7 mol % of 3H4MV, followed by a decrease in erosion rate.

Identification, biosynthesis, and characterization of polyhydroxyalkanoate copolymer consisting of 3-hydroxybutyrate and 3-hydroxy-4-methylvalerate.

In this study, we found that Ralstonia eutropha strain PHB(-)4 expressing the polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps)) from Pseudomonas sp. 61-3 synthesized a PHA copolymer containing a 3-hydroxybutyrate (3HB) and small amounts of 3-hydroxy-4-methylvalerate (3H4MV) and 3-hydroxyvalerate (3HV) from fructose as a sole carbon source. 3H4MV is a monomer unit that has hitherto not been reported as a naturally occurring component of PHAs. To increase the 3H4MV fraction in PHA copolymers, the culture medium was supplemented with four structural analogs that served as 3H4MV precursors. Of these, 4-methylvalerate (4MV) was the most efficient in increasing the 3H4MV content of PHA. The R. eutropha strain PHB(-)4 expressing Aeromonas cavaie PHA synthase (PhaC(Ac)) and its mutant (PhaC(Ac) NSDG mutant) were also able to synthesize 3H4MV-containing PHA from 4MV, increasing 3H4MV molar fraction in PHA copolymer up to 38 mol %. The structure and physical properties of P(3HB-co-3H4MV) copolymers were characterized by gel permeation chromatography, NMR spectroscopy, differential scanning calorimetry, and tensile testing. It can be shown that P(3HB-co-3H4MV) samples synthesized by PhaC(Ac) and its mutant NSDG were of higher molecular weights than those by PhaC1(Ps) and were a flexible and ductile material with moderate toughness. Additionally, the ductility can be kept for at least 180 days without significant deterioration caused by secondary crystallization when 3H4MV molar fraction was higher than 14 mol %. The newly identified 3H4MV unit is a promising monomer for improving material property and stability of 3HB-based copolymers.