Two formulations of lambda-cyhalothrin (EC-Emulsion concentrate and MC-Microencapsulated) were impregnated into bednets made of polyethylene and polyester. The nets were treated at a dosage of 15 mg/m2. For bioassay of insecticidal efficacy, female Anopheles maculatus and Aedes aegypti were exposed to the nets for two minutes and mortality was scored 24 hours later. The nets were also tested after repeated washings with water and with soap and water. Microencapsulated (2.5CS) formulation was more effective than emulsion concentrate (2.5EC) formulation on both net materials--polyethylene and polyester. Repeated washing with water and soap reduces the efficacy of all bednet treatment combinations. Microencapsulated formulation on polyethylene gave best results; it could sustain up to five washes with water and two with soap and water.
A locally isolated soil microorganism identified as Erwinia sp. USMI-20 has been found to produce poly(3-hydroxybutyrate), P(3HB), from either palm oil or glucose and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from a combination of palm oil and a second carbon source of either one of the following compounds: propionic acid, n-propanol, valeric acid and n-pentanol. It was found that Erwinia sp. USMI-20 could produce P(3HB) up to 69 wt.% polymer content with a dry cell weight of 4.4 g/l from an initial amount of 14.5 g/l of glucose followed by a feeding rate of glucose at 0.48 g/h glucose. On the other hand, the bacteria can achieve 46 wt.% of P(3HB) and a dry cell weight of 3.6 g/l from a batch fermentation in a 10-l fermentor from an initial concentration of 4.6 g/l of palm oil. Further characterisation of the polymer production was also carried out by using different types of palm oil. Among the different palm oils that were used, crude palm oil was the best lipid source for P(3HB) production as compared to palm olein and palm kernel oil. In the production of the copolymer, P(3HB-co-3HV), the highest mole fraction of 3-HV units could be as high as 47 mol% from a single feeding of valeric acid upon initial growth on palm oil.
catena-Poly[dicyclohexylammonium [tributyltin-mu-(4-oxo-4H-pyran-2,6-dicarboxylato-O(2):O( 6))]], (C(12)H(24)N)[Sn(C(7)H(2)O(6))(C(4)H(9))(3)], consists of 4-oxo-4H-pyran-2,6-dicarboxylato groups that axially link adjacent tributyltin units into a linear polyanionic chain. The ammonium counter-ions surround the chain, and each cation forms a pair of hydrogen bonds to the double-bond carbonyl O atoms of the same dianionic group. The chain propagates in a zigzag manner along the c axis of the monoclinic cell. In catena-poly[methyl(phenyl)ammonium [tributyltin-mu-(pyridine-2,6-dicarboxylato-O(2):O(6))]], (C(7)H(10)N)[Sn(C(7)H(3)NO(4))(C(4)H(9))(3)], the pyridine-2, 6-dicarboxylato groups also link the tributyltin groups into a chain, but the hydrogen-bonded chain propagates linearly on the ac face of the monoclinic cell.
The medium-chain-length polyhydroxyalkanoate (PHA(MCL)) produced by Pseudomonas putida PGA1 using saponified palm kernel oil as the carbon source could degrade readily in water taken from Kayu Ara River in Selangor, Malaysia. A weight loss of 71.3% of the PHA film occurred in 86 d. The pH of the river water medium fell from 7.5 (at d 0) to 4.7 (at d 86), and there was a net release of CO2. In sterilized river water, the PHA film also lost weight and the pH of the water fell, but to lesser extents. The C8 monomer of the PHA was completely removed after 6 d of immersion in the river water, while the proportions of the other monomers (C10, C12, and C14) were reversed from that of the undegraded PHA. By contrast, the monomer composition of the PHA immersed in sterilized river water did not change significantly from that of the undegraded PHA. Scanning electron microscopy showed physical signs of degradation on the PHA film immersed in the river water, but the film immersed in sterilized river water was relatively unblemished. The results thus indicate that the PHA(MCL) was degraded in tropical river water by biologic as well as nonbiologic means. A significant finding is that shorter-chain monomers were selectively removed throughout the entire PHA molecule, and this suggests enzymatic action.
Bacterial polyhydroxyalkanoates (PHAs) are perceived to be a suitable alternative to petrochemical plastics because they have similar material properties, are environmentally degradable, and are produced from renewable resources. In this study, the in situ degradation of medium-chain-length PHA (PHAMCL) films in tropical forest and mangrove soils was assessed. The PHAMCL was produced by Pseudomonas putida PGA1 using saponified palm kernel oil (SPKO) as the carbon source. After 112 d of burial, there was 16.7% reduction in gross weight of the films buried in acidic forest soil (FS), 3.0% in the ones buried in alkaline forest soil by the side of a stream (FSst) and 4.5% in those buried in mangrove soil (MS). There was a slight decrease in molecular weight for the films buried in FS but not for the films buried in FSst and in MS. However, no changes were observed for the melting temperature, glass transition temperature, monomer compositions, structure, and functional group analyses of the films from any of the burial sites during the test period. This means that the integral properties of the films were maintained during that period and degradation was by surface erosion. Scanning electron microscopy of the films from the three sites revealed holes on the film surfaces which could be attributed to attack by microorganisms and bigger organisms such as detritivores. For comparison purposes, films of polyhydroxybutyrate (PHB), a short-chain-length PHA, and polyethylene (PE) were buried together with the PHAMCL films in all three sites. The PHB films disintegrated completely in MS and lost 73.5% of their initial weight in FSst, but only 4.6% in FS suggesting that water movement played a major role in breaking up the brittle PHB films. The PE films did not register any weight loss in any of the test sites.
In early attempts to isolate palm oil-utilising bacteria from palm oil mill effluent (POME), diluted liquid samples of POME were spread on agar containing POME as primary nutrient. 45 purified colonies were screened for intracellular lipids by staining with Sudan Black B. Of these, 10 isolates were positively stained. The latter were grown in a nitrogen-limiting medium with palm olein (a triglyceride) or saponified palm olein (salts of fatty acids) as carbon source. None of the isolates grew in the palm olein medium but all grew well in the saponified palm olein medium. Of the latter however, only one isolate was positively stained with Nile Blue A, indicating the presence of PHA. This method did not successfully generate bacterial isolates which could metabolise palm olein to produce PHA. An enrichment technique was therefore developed whereby a selective medium was designed. The latter comprised minerals and palm olein (1% w/v) as sole carbon source to which POME (2.5% v/v) was added as the source of bacteria. The culture was incubated with shaking at 30 degrees C for 4 weeks. Out of seven isolates obtained from the selective medium, two isolates, FLP1 and FLP2, could utilise palm olein for growth and production of the homopolyester, poly(3-hydroxybutyrate). FLP1 is gram-negative and is identified (BIOLOG) to have 80% similarity to Burkholderia cepacia. When grown with propionate or valerate, FLP1 produced a copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate).
Three strains of Spirulina platensis isolated from different locations showed capability of synthesizing poly(3-hydroxybutyrate) [P(3HB)] under nitrogen-starved conditions with a maximum accumulation of up to 10 wt.% of the cell dry weight (CDW) under mixotrophic culture conditions. Intracellular degradation (mobilization) of P(3HB) granules by S. platensis was initiated by the restoration of nitrogen source. This mobilization process was affected by both illumination and culture pH. The mobilization of P(3HB) was better under illumination (80% degradation) than in dark conditions (40% degradation) over a period of 4 days. Alkaline conditions (pH 10-11) were optimal for both biosynthesis and mobilization of P(3HB) at which 90% of the accumulated P(3HB) was mobilized. Transmission electron microscopy (TEM) revealed that the mobilization of P(3HB) involved changes in granule quantity and morphology. The P(3HB) granules became irregular in shape and the boundary region was less defined. In contrast to bacteria, in S. platensis the intracellular mobilization of P(3HB) seems to be faster than the biosynthesis process. This is because in cyanobacteria chlorosis delays the P(3HB) accumulation process.
Fourier transform infrared (FT-IR) spectroscopic studies have been undertaken to investigate the interactions among components in a system of hexanoyl chitosan-lithium trifluoromethanesulfonate (LiCF(3)SO(3))-diethyl carbonate (DEC)/ethylene carbonate (EC). LiCF(3)SO(3) interacts with the hexanoyl chitosan to form a hexanoyl chitosan-salt complex that results in the shifting of the N(COR)(2), CONHR and OCOR bands to lower wavenumbers. Interactions between EC and DEC with LiCF(3)SO(3) has been noted and discussed. Evidence of interaction between EC and DEC has been obtained experimentally. Studies on polymer-plasticizer spectra suggested that there is no interaction between the polymer host and plasticizers. Competition between plasticizer and polymer on associating with Li(+) ions was observed from the spectral data for gel polymer electrolytes. The obtained spectroscopic data has been correlated with the conductivity performance of hexanoyl chitosan-based polymer electrolytes.
The ability of Delftia acidovorans to incorporate a broad range of 3-hydroxyvalerate (3HV) monomers into polyhydroxyalkanoate (PHA) copolymers was evaluated in this study. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] containing 0-90 mol% of 3HV was obtained when a mixture of sodium 3-hydroxybutyrate and sodium valerate was used as the carbon sources. Transmission electron microscopy analysis revealed an interesting aspect of the P(3HB-co-3HV) granules containing high molar ratios of 3HV whereby, the copolymer granules were generally larger than those of poly(3-hydroxybutyrate) [P(3HB)] granules, despite having almost the same cellular PHA contents. The large number of P(3HB-co-3HV) granules occupying almost the entire cell volume did not correspond to a higher amount of polymer by weight. This indicated that the granules of P(3HB-co-3HV) contain polymer chains that are loosely packed and therefore have lower density than P(3HB) granules. It was also interesting to note that a decrease in the length of the side chain from 3HV to 4-hydroxybutyrate (4HB) corresponded to an increase in the density of the respective PHA granules. The presence of longer side chain monomers (3HV) in the PHA structure seem to exhibit steric effects that prevent the polymer chains in the granules from being closely packed. The results reported here have important implications on the maximum ability of bacterial cells to accumulate PHA containing monomers with longer side chain length.
Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates offer the most diverse range of thermal and mechanical properties. In this study, the biocompatibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB); containing 50 mol % of 4-hydroxybutyrate] copolymer produced by Delftia acidovorans was evaluated. The cytotoxicity, mode of cell death, and genotoxicity of P(3HB-co-4HB) extract against V79 and L929 fibroblast cells were assessed using MTT assay, acridine orange/propidium iodide staining, and alkaline comet assay, respectively. Our results demonstrate that P(3HB-co-4HB) treated on both cell lines were comparable with clinically-used Polyglactin 910, where more than 60% of viable cells were observed following 72-h treatment at 200 mg/mL. Further morphological investigation on the mode of cell death showed an increase in apoptotic cells in a time-dependent manner in both cell lines. On the other hand, P(3HB-co-4HB) at 200 mg/mL showed no genotoxic effects as determined by alkaline comet assay following 72-h treatment. In conclusion, our study indicated that P(3HB-co-4HB) compounds showed good biocompatibility in fibroblast cells suggesting that it has potential to be used for future medical applications.
Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid) (PLGA) hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.
Cupriavidus sp. USMAA1020 was isolated from Malaysian environment and able to synthesize poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)] when grown on gamma-butyrolactone as the sole carbon source. The polyester was purified from freeze-dried cells and analyzed by nuclear magnetic resonance (NMR) spectroscopy. 1H and 13C NMR results confirmed the presence of 3HB and 4HB monomers. In a one-step cultivation process, P(3HB-co-4HB) accumulation by Cupriavidus sp. USMAA1020 was affected by carbon to nitrogen ratio (C/N). A two-step cultivation process accumulated P(3HB-co-4HB) copolyester with a higher 4HB fraction (53 mol%) in nitrogen-free mineral medium containing gamma-butyrolactone. The biosynthesis of P(3HB-co-4HB) was also achieved by using 4-hydroxybutyric acid and alkanediol as 1,4-butanediol. The composition of copolyesters varied from 32 to 51 mol% 4HB, depending on the carbon sources supplied. The copolyester produced by Cupriavidus sp. USMAA1020 has a random sequence distribution of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) units when analyzed by nuclear magnetic resonance (NMR) spectroscopy. When gamma-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 25 to 60 mol% as the concentration of gamma-butyrolactone in the culture medium increased from 2.5 g/L to 20.0 g/L.
Copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Cupriavidus sp. (USMAA2-4) (DSM 19379) from carbon sources of 1,4-butanediol and gamma-butyrolactone. The composition of copolyesters produced varied from 0 to 45 mol% 4HB, depending on the combination of carbon sources supplied. The P(3HB-co-4HB) films containing Mitragyna speciosa crude extract were prepared with the ratio varying from 10 to 40% (w/w). The in vitro crude extract release of the films was studied in 0.1M phosphate buffer (pH 7.4) at 37 degrees C. Although the release rate was slow, it was maintained at a constant rate. This suggests that the crude extract release was due to the polymer degradation because the amount of crude extract released was consistent. The amount of degradation was based on the films' dry weight loss, decrease in molecular weight and surface morphology changes. The degradation rate increased with the 4HB content. This showed that the polymer degradation is dependant on the molecular weight, crystallinity, thermal properties and water permeability. The different drug loading ratio which led to surface morphology changes also gave an effect on polymer degradation.
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.
Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(epsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.
One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.
Polyhydroxyalkanoates (PHA) are naturally occurring biopolyesters that have great potential in the medical field. However, the leachables resulting from sterilization process of the biomaterials may exert toxic effect including genetic damage. Here, we demonstrate that although gamma-irradiation of poly(3-hydroxybutyrate-co-50 mol % 4-hydroxybutyrate) [P(3HB-co-4HB)] did not cause any change in the morphology by scanning electron microscopy, there was a significant degradation of this copolymer where the molecular weight was reduced by 37% after sterilization indicating the generation of leachables. Therefore, further investigation on the ability of the extract of this poststerilized copolymer to induce mutagenic effect was performed using Ames test (S. typhimurium strains TA1535 and TA1537) and umu test (S. typhimurium strain TA1535/pSK1002). Additionally, the capability of the extract to induce clastogenic effect was determined using Chinese hamster lung V79 fibroblast cells. Our results showed that with and without the presence of S9 metabolic activation, no mutagenic effects were observed in both Ames and umu tests when treated with P(3HB-co-4HB) extract. Similarly, treatment of P(3HB-co-4HB) extract in V79 fibroblast cells showed no significant production of micronuclei when compared with the positive control (Mitomycin C). Together, these results indicate that leachables of poststerilized P(3HB-co-4HB) cause no mutagenic and clastogenic effects.
Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).
Chemical recycling of bio-based polymers polyhydroxyalkanoates (PHAs) by thermal degradation was investigated from the viewpoint of biorefinery. The thermal degradation resulted in successful transformation of PHAs into vinyl monomers using alkali earth compound (AEC) catalysts. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) were smoothly and selectively depolymerized into crotonic (CA) and 2-pentenoic (2-PA) acids at lower degradation temperatures in the presence of CaO and Mg(OH)(2) as catalysts. Obtained CA from 3-hydroxybutyrate sequences in PHBV was copolymerized with acrylic acid to produce useful water-soluble copolymers, poly(crotonic acid-co-acrylic acid) that have high glass-transition temperatures. The copolymerization of CA derived from PHA pyrolysis is an example of cascade utilization of PHAs, which meets the idea of sustainable development.