Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.
In this study, the ability of Cupriavidus sp. USMAA2-4 to synthesize polyhydroxyalkanoates (PHA) containing 4-hydroxyvalerate monomer (4HV) was studied through one-stage cultivation using γ-valerolactone as the carbon precursor. The presence of 4HV monomer unit in the polymer was detected through gas chromatography analysis, proving the capability of this wild strain bacterium to produce poly(3-hydrxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] terpolymer. Existence of a 4HV monomer unit in the PHA produced was further confirmed through (13)C and (1)H NMR analysis. P(3HB-co-88 % 3HV-co-1 % 4HV) terpolymer with the highest PHA content of 63 wt% was obtained through combination of 0.14 wt% C of γ-valerolactone with 0.42 wt% C of oleic acid. Various compositions of P(3HB-co-3HV-co-4HV) terpolymer with 3HV and 4HV compositions ranging from 11 to 94 mol% and from 1 to 4 mol%, respectively, were acquired by manipulating γ-valerolactone and oleic acid concentrations. The molecular weight and the thermal and mechanical properties of four different compositions of terpolymers-P(3HB-co-91 % 3HV-co-1 % 4HV), P(3HB-co-55 % 3HV-co-2 % 4HV), P(3HB-co-27 % 3HV-co-2 % 4HV), and P(3HB-co-9 % 3HV-co-1 % 4HV)-were characterized. Among these terpolymers, P(3HB-co-27 % 3HV-co-2 % 4HV) terpolymer with a molecular weight of 5.7 (10(5) Da) exhibited the highest elongation to break (264 %). The monomer unit compositional distributions of these terpolymers were investigated through acetone-water fractionation analysis. The results suggested that these produced terpolymers had broad 3HV compositional distribution and narrow 4HV compositional distribution.
This work aims to shed light in the fabrication of poly(3-hydroxybutyrate-co-44%-4-hydroxybutyrate)[P(3HB-co-44%4HB)]/chitosan-based silver nanocomposite material using different contents of silver nanoparticle (SNP); 1-9 wt%. Two approaches were applied in the fabrication; namely solvent casting and chemical crosslinking via glutaraldehyde (GA). A detailed characterization was conducted in order to yield information regarding the nanocomposite material. X-ray diffraction analysis exhibited the nature of the three components that exist in the nanocomposite films: P(3HB-co-4HB), chitosan, and SNP. In term of mechanical properties, tensile strength, and elongation at break were significantly improved up to 125% and 22%, respectively with the impregnation of the SNP. The melting temperature of the nanocomposite materials was increased whereas their thermal stability was slightly changed. Scanning electron microscopy images revealed that incorporation of 9 wt% of SNP caused agglomeration but the surface roughness of the material was significantly improved with the loading. Staphylococcus aureus and Escherichia coli were completely inhibited by the nanocomposite films with 7 and 9 wt% of SNP, respectively. On the other hand, degradation of the nanocomposite materials outweighed the degradation of the pure copolymer. These bioactive and biodegradable materials stand a good chance to serve the vast need of biomedical applications namely management and care of wound as wound dressing.
Microbial pigments are gaining intensive attention due to increasing awareness of the toxicity of synthetic colours. In this study, a novel polymer-producing bacterium designated as Cupriavidus sp. USMAHM13 was also found to produce yellow pigment when cultivated in nutrient broth. Various parameters such as temperature, pH and ratio of culture volume to flask volume were found to influence the yellow pigment production. UV-Visible, Fourier transform infrared and (13)C-nuclear magnetic resonance analyses revealed that the crude yellow pigment might probably represent new bioactive compound in the carotenoid family. The crude yellow pigment also exhibited a wide spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria with their inhibition zones and minimal inhibitory concentrations ranged from 25 to 38 mm and from 0.63 to 2.5 mg/ml, respectively. To the best of our knowledge, this is the first report on the identification and characterization of yellow pigment produced by bacterium belonging to the genus Cupriavidus.
Copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been the center of attention in the bio-industrial fields, as it possesses superior mechanical properties compared to poly(3-hydroxybutyrate) [P(3HB)]. The usage of oleic acid and 1-pentanol was exploited as the carbon source for the production of P(3HB-co-3HV) copolymer by using a locally isolated strain Cupriavidus sp. USMAA2-4. In this study, the productivity of polyhydroxyalkanoate (PHA) was improved by varying the frequency of feeding in fed-batch culture. The highest productivity (0.48 g/L/h) that represents 200 % increment was obtained by feeding the carbon source and nitrogen source three times and also by considering the oxygen uptake rate (OUR) and oxygen transfer rate (OTR). A significantly higher P(3HB-co-3HV) concentration of 25.7 g/L and PHA content of 66 wt% were obtained. The 3-hydroxyvalerate (3HV) monomer composition obtained was 24 mol% with the growth of 13.3 g/L. The different frequency of feeding carried out has produced a blend copolymer and has broadened the monomer distribution. In addition, increase in number of granules was also observed as the frequency of feeding increases. In general, the most glaring increment in productivity offer advantage for industrial P(3HB-co-3HV) production, and it is crucial in developing cost-effective processes for commercialization.
P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).
A Gram-staining-negative, aerobic, rod-shaped, yellow-orange-pigmented, gliding bacterium, designated as strain ST2L12(T), was isolated from estuarine mangrove sediment from Matang Mangrove Forest, Perak, Malaysia. Strain ST2L12(T) grew at 15-39 °C, pH 6-8 and in 1-6 % (w/v) NaCl. This strain was able to degrade xylan and casein. 16S rRNA gene sequence analysis showed 95.3-92.8 % similarity to members of the genera Mangrovimonas, Meridianimaribacter, Sediminibacter, Gaetbulibacter and Hoppeia. Phylogenetic analysis indicated that it belonged to the family Flavobacteriaceae. Respiratory quinone present was menaquinone-6 (MK-6), and the DNA G+C content was 38.3 mol%. The predominant fatty acids were iso-C15:0, iso-C15:1, C15:0 and iso-C17:0 3-OH. Moreover, previous genome comparison study showed that the genome of ST2L12(T) is 1.4 times larger compared to its closest relative, Mangrovimonas yunxiaonensis LYYY01(T). Phenotypic, fatty acid, 16S rRNA gene sequence and previous genome data indicate that strain ST2L12(T) represents a novel species of the genus Mangrovimonas in the family Flavobacteriaceae, for which the name Mangrovimonas xylaniphaga sp. nov. is proposed. The type strain of Mangrovimonas xylaniphaga is ST2L12(T) (=LMG 28914(T)=JCM 30880(T)).
This study reports the production of P(3HB-co-4HB) [Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)] in possession of high molecular weight and elastomeric properties by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate cultivation system. 1,4-butanediol and 1,6-hexanediol are found to be efficient substrate mixture that has resulted in high copolymer yield, occupying a maximum of 70wt% of the total biomass and producing higher 4HB monomer composition ranging from 31mol% to 41mol%. In substrate mixtures involving 1,6-hexanediol, cleavage of the 6-hydroxyhexanoyl-CoA produces Acetyl-CoA and 4-hydroxybutyryl-CoA. Acetyl-CoA is instrumental in initiating the cell growth in the single-stage fermentation system, preventing 4-hydroxybutyryl-CoA from being utilized via β-oxidation and retained the 4HB monomer at higher ratios. Macroscopic kinetic models of the bioprocesses have revealed that the P(3HB-co-4HB) formation appears to be in the nature of mixed-growth associated with higher formation rate during exponential growth phase; evidenced by higher growth associated constants, α, from 0.0690g/g to 0.4615g/g compared to non-growth associated constants, β, from 0.0092g/g/h to 0.0459g/g/h. The P(3HB-co-31mol% 4HB) produced from the substrate mixture exhibited high weight-average molecular weight, Mwof 927kDa approaching a million Dalton, and possessed elongation at break of 1637% upon cultivation at 0.56wt% C. This is the first report on such properties for the P(3HB-co-4HB) copolymer. The copolymer is highly resistant to polymer deformation after being stretched.
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.
Polyhydroxyalkanoate (PHA) is a biogenic polymer that has the potential to substitute synthetic plastic in numerous applications. This is due to its unique attribute of being a biodegradable and biocompatible thermoplastic, achievable through microbial fermentation from a broad utilizable range of renewable resources. Among all the PHAs discovered, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] stands out as a next generation healthcare biomaterial for having high biopharmaceutical and medical value since it is highly compatible to mammalian tissue. This review provides a critical assessment and complete overview of the development and trend of P(3HB-co-4HB) research over the last few decades, highlighting aspects from the microbial strain discovery to metabolic engineering and bioprocess cultivation strategies. The article also outlines the relevance of P(3HB-co-4HB) as a material for high value-added products in numerous healthcare-related applications.
A novel, rod-shaped, Gram-stain-negative, halophilic and non-motile bacterium, designated CCB-MM1T, was isolated from a sample of estuarine sediment collected from Matang Mangrove Forest, Malaysia. The cells possessed a rod-coccus cell cycle in association with growth phase and formed aggregates. Strain CCB-MM1T was both catalase and oxidase positive, and able to degrade starch. Optimum growth occurred at 30 °C and pH 7.0 in the presence of 2-3 % (w/v) NaCl. The 16S rRNA gene sequence of strain CCB-MM1T showed 98.12, 97.46 and 97.33 % sequence similarity with Microbulbifer rhizosphaerae Cs16bT, Microbulbifer maritimus TF-17T and Microbulbifergwangyangensis GY2T respectively. Strain CCB-MM1T and M. rhizosphaerae Cs16bT formed a cluster in the phylogenetic tree. The major cellular fatty acids were iso-C17 : 1 ω9c and iso-C15 : 0, and the total polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphoaminolipid, two unidentified lipids, an unidentified glycolipid and an unidentified aminolipid. The major respiratory quinone was ubiquinone Q-8 and the genomic DNA G+C content of the strain was 58.9 mol%. On the basis of the phylogenetic, phenotypic and genotypic data presented here, strain CCB-MM1T represents a novel species of the genus Microbulbifer, for which the name Microbulbiferaggregans sp. nov. is proposed. The type strain is CCB-MM1T (=LMG 29920T=JCM 31875T).
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.
A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50-60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3-5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80-90 wt% were obtained when 10-30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60-180 min at 30 °C and polished using 20 % (v/v) of ethanol.
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.
The agarolytic bacterium Persicobacter sp. CCB-QB2 was isolated from seaweed (genus Ulva) collected from a coastal area of Malaysia. Here, we report a high-quality draft genome sequence for QB2. The Rapid Annotation using Subsystem Technology (RAST) annotation server identified four β-agarases (PdAgaA, PdAgaB, PdAgaC, and PdAgaD) as well as galK, galE, and phosphoglucomutase, which are related to the Leloir pathway. Interestingly, QB2 exhibited a diauxic growth in the presence of two kinds of nutrients, such as tryptone and agar. In cells grown with agar, the profiles of agarase activity and growth rate were very similar. galK, galE, and phosphoglucomutase genes were highly expressed in the second growth phase of diauxic growth, indicating that QB2 cells use galactose hydrolyzed from agar by its agarases and exhibit nutrient prioritization. This is the first report describing diauxic growth for agarolytic bacteria. QB2 is a potential novel model organism for studying diauxic growth in environmental bacteria.
Two-stage fermentation was normally employed to achieve a high poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] productivity with higher 4HB molar fraction. Here, we demonstrated single-stage fermentation method which is more industrial feasible by implementing mixed-substrate cultivation strategy. Studies on bioreactor scale show a remarkably high PHA accumulation of 73 wt%, contributing to a high PHA concentration and product yield of 8.6 g/L and 2.7 g/g, respectively. This fermentation strategy has resulted in copolymers with wider range of 4HB monomer composition, which ranges from 12 to 55 mol%. These copolymers show a broad range of weight average molecular weight (M w ) from 119.5 to 407.0 kDa. The copolymer characteristics were found to be predominantly affected by the nature of the substrates and the mixture strategies, regardless of the 4HB monomer compositions. This was supported by the determination of copolymer randomness using (13)C-NMR analysis. The study warrants significantly in the copolymer scale-up and modeling at industrial level.
Marine sponges are acknowledged as a bacterial hotspot and resource of novel natural products or genetic material with industrial or commercial potential. However, sponge-associated bacteria are difficult to be cultivated and the production of their desirable metabolites is inadequate in terms of rate and quantity, yet bioinformatics and metagenomics tools are steadily progressing. Bacterial diversity profiles of high-microbial-abundance wild tropical marine sponges Aaptos aaptos and Xestospongia muta were obtained by sample collection at Pulau Bidong and Pulau Redang islands, 16S rRNA amplicon sequencing on Illumina HiSeq2500 platform (250 bp paired-end) and metagenomics analysis using Ribosomal Database Project (RDP) classifier. Raw sequencing data in fastq format and relative abundance histograms of the dominant 10 species are available in the public repository Discover Mendeley Data (http://dx.doi.org/10.17632/zrcks5s8xp). Filtered sequencing data of operational taxonomic unit (OTU) with chimera removed is available in NCBI accession numbers from MT464469 to MT465036.
Paederus fuscipes, a vector of Paederus dermatitis in most tropical and subtropical countries of the world have a high prevalence in human dwellings due to their positively phototaxic behaviour which has caused a tremendous impact on human health. In this paper, P. fuscipes dispersal flights were studied for two seasons of the rice cultivation phases in residential premises built close to rice field areas (≈32-60 m and 164 m) in mainland Penang, Malaysia. We examined the effects of different light illuminance, building floor level and their association with rice stages as a focal cause of P. fuscipes dispersion from the rice fields towards human dwellings. The present study showed a significant interaction between different light illuminances and rice cultivation phases in attracting P. fuscipes to disperse and invade human dwellings. The highest number of P. fuscipes was captured near the bright light. P. fuscipes flights increased in line with each floor level, and the highest captures took place at higher building floor levels (levels 2 and 3) compared to lower building floor levels (ground floor and level 1) of a three storey apartment in both rice seasons. This finding not only conveys a better understanding on P. fuscipes dispersal pattern, but also draws public attention on the occurrence of dermatitis linearis caused by the Paederus beetles.
Pararhodobacter-like strain CCB-MM2 is a halophilic alphaproteobacterium isolated from estuarine sediment collected from Matang Mangrove Forest in Malaysia. Here, we present the draft genome sequence of CCB-MM2 and provide insights into its physiological roles and metabolic potential.