A moderately halophilic bacterium isolated from fermenting shrimp paste, Salinivibrio sp. M318 was found capable of using fish sauce and mixtures of waste fish oil and glycerol as nitrogen and carbon sources, respectively, for poly(3-hydroxybutyrate) (PHB) production. A cell dry weight (CDW) of up to 10 g/L and PHB content of 51.7 wt% were obtained after 48 h of cultivation in flask experiment. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was synthesized when 1,4-butanediol, γ-butyrolactone, or sodium 4-hydroxybutyrate was added as precursors to the culture medium. The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was achieved by supplying precursors such as sodium valerate, sodium propionate, and sodium heptanoate. Salinivibrio sp. M318 was able to accumulate the above mentioned PHAs during the growth phase. High CDW of 69.1 g/L and PHB content of 51.5 wt% were obtained by strain Salinivibrio sp. M318 after 78 h of cultivation in fed-batch culture. The results demonstrate Salinivibrio sp. M318 to be a promising wild-type bacterium for the production of PHA from aquaculture residues.
Polyhydroxyalkanoates (PHAs) have attracted the attention of academia and industry because of their plastic-like properties and biodegradability. However, practical applications as a commodity material have not materialized because of their high production cost and unsatisfactory mechanical properties. PHAs are also believed to have high-value applications as an absorbable biomaterial for tissue engineering and drug-delivery devices because of their biocompatibility. However, research in these areas is still in its very early stages. The main problem faced by proponents of PHAs is the lack of a niche area where PHAs will be the most desired material in terms of its function during use rather than because of its eco-friendly virtues after use. Here, we report on the oil-absorbing property of PHA films and its potential applications. By comparing with some of the existing commercial products, the potential application of PHAs as cosmetic oil-blotting films is revealed for the first time. Besides having the ability to rapidly absorb and retain oil, PHA films also have a natural oil-indicator property, showing obvious changes in opacity following oil absorption. Surface analysis revealed that the surface structures such as porosity and smoothness exert great influence on the rapid oil-absorption properties of the PHA films. These newly discovered properties could be exploited to create a niche area for the practical applications of PHAs.
This research explicitly investigates the utilization of Chlorella Vulgaris sp. microalgae as a renewable source for lipid production, focusing on its application in bioplastic manufacturing. This study employed the supercritical fluid extraction technique employing supercritical CO2 (sCO2) as a green technology to selectively extract and produce PHA's precursor utilizing CO2 solvent as a cleaner solvent compared to conventional extraction method. The study assessed the effects of three extraction parameters, namely temperature (40-60 °C), pressure (15-35 MPa), and solvent flow rate (4-8 ml/min). The pressure, flowrate, and temperature were found to be the most significant parameters affecting the sCO2 extraction. Through Taguchi optimization, the optimal parameters were determined as 60 °C, 35 MPa, and 4 ml/min with the highest lipid yield of 46.74 wt%; above-average findings were reported. Furthermore, the pretreatment process involved significant effects such as crumpled and exhaustive structure, facilitating the efficient extraction of total lipids from the microalgae matrix. This study investigated the microstructure of microalgae biomatrix before and after extraction using scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). Fourier-transform infrared spectroscopy (FTIR) was utilized to assess the potential of the extracted material as a precursor for biodegradable plastic production, with a focus on reduced heavy metal content through inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis. The lipid extracted from Chlorella Vulgaris sp. microalgae was analysed using gas chromatography-mass spectrometry (GC-MS), identifying key constituents, including oleic acid (C18H34O2), n-Hexadecanoic acid (C16H32O2), and octadecanoic acid (C18H36O2), essential for polyhydroxyalkanoate (PHA) formation.
Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world's second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.
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.
Dielectric spectroscopy is employed to study the relaxation phenomena in natural polyhydroxyalkanoates (PHAs) upon temperature and frequency variations. Effects of PHAs molecular structure on the relaxation, arising from the differences in monomeric composition, are investigated under identical conditions in a frequency range of 10-2-106 Hz, and at different temperatures. All PHA samples showed different dielectric response at different temperature. Primary α-relaxation signals are observed at temperature corresponding to the glass transition temperature. On the other hand, secondary β- and γ-relaxations are detected at low temperatures, and attributed to local motions of polar groups and small segments of the polymer chain. The dielectric properties of representative PHA samples are compared and discussed.
At present, polyhydroxyalkanoates (PHAs) have been considered as a promising alternative to conventional plastics due to their diverse variability in structure and rapid biodegradation. To ensure cost competitiveness in the market, thermoseparating aqueous two-phase extraction (ATPE) with the advantages of being mild and environmental-friendly was suggested as the primary isolation and purification tool for PHAs. Utilizing two-level full factorial design, this work studied the influence and interaction between four independent variables on the partitioning behavior of PHAs. Based on the experimental results, feed forward neural network (FFNN) was used to develop an empirical model of PHAs based on the ATPE thermoseparating input-output parameter. In this case, bootstrap resampling technique was used to generate more data. At the conditions of 15 wt % phosphate salt, 18 wt % ethylene oxide⁻propylene oxide (EOPO), and pH 10 without the addition of NaCl, the purification and recovery of PHAs achieved a highest yield of 93.9%. Overall, the statistical analysis demonstrated that the phosphate concentration and thermoseparating polymer concentration were the most significant parameters due to their individual influence and synergistic interaction between them on all the response variables. The final results of the FFNN model showed the ability of the model to seamlessly generalize the relationship between the input⁻output of the process.
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
The nutrition-versatility of Burkholderia sp. strain USM (JCM 15050) has initiated the studies on the use of this bacterium for polyhydroxyalkanoate (PHA) production. To date, the Burkholderia sp. has been reported to synthesize 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxy-4-methylvalerate monomers. In this study, the PHA biosynthetic genes of this strain were successfully cloned and characterized. The PHA biosynthetic cluster of this strain consisted of a PHA synthase (phaC), β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The translated products of these genes revealed identities to corresponding proteins of Burkholderia vietnamiensis (99-100 %) and Cupriavidus necator H16 (63-89%). Heterologous expression of phaCBs conferred PHA synthesis to the PHA-negative Cupriavidus necator PHB¯4, confirming that phaCBs encoded functionally active protein. PHA synthase activity measurements revealed that the crude extracts of C. necator PHB¯4 transformant showed higher synthase activity (243 U/g) compared to that of wild-types Burkholderia sp. (151 U/g) and C. necator H16 (180 U/g). Interestingly, the transformant C. necator PHB¯4 harbouring Burkholderia sp. PHA synthase gene accumulated poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 4-hydroxybutyrate monomer as high as up to 87 mol% from sodium 4-hydroxybutyrate. The wild type Burkholderia sp. did not have the ability to produce this copolymer.
The feasibility of using waste glycerol from the biodiesel industry for biosynthesis of polyhydroxyalkanoate (PHA) by Burkholderia cepacia BPT1213 was evaluated. Culture conditions were optimized by growing B. cepacia BPT1213 in mineral salt medium supplemented with 2% waste glycerol in a 2.5 L bioreactor. Response surface methodology was used to determine the influence of aeration rate (0.6-1.8 vvm), agitation speed (100-300 rpm), and cultivation period (48-72 hr) on PHA production. The optimum conditions for the growth and PHA accumulation were 1.5 vvm, 300 rpm, and 72 hr, with predicted values of 5.08 g/L cell dry weight (CDW), 66.07% PHA content, and 3.35 g/L total PHA concentration. Using these conditions, the experimental system produced 5.63 g/L of CDW with 64.00% wt/wt PHA content, which is threefold higher PHA concentration (3.60 g/L) compared to the non-optimized conditions. The melting temperature (Tm ) of purified PHA was 173.45 ± 1.05°C. In conclusion, the statistical approach was significantly increased the PHA production using waste glycerol as the sole carbon source.
A new halogen-free and environmental-friendly method using water and ethanol is developed as an alternative for the recovery of polyhydroxyalkanoates (PHA) from recombinant Cupriavidus necator in comparison to the established chloroform extraction method. After optimisation, our results showed that the halogen-free method produced a PHA with 81% purity and 96% recovery yield, in comparison to the chloroform extraction system which resulted in a highly pure PHA with 95% yield. Although the purity of the PHA using the new method is lower, the molecular weight of the extracted PHA is not compromised. This new method can be further developed as an alternative and more environmental-friendly method for industrial application.
PHAs (polyhydroxyalkanoates) have emerged as biodegradable plastics more strongly in the 20th century. A wide range of bacterial species along with fungi, plants, oilseed crops and carbon sources have been used extensively to synthesize PHA on large scales. Alteration of PHA monomers in their structures and composition has led to the development of biodegradable and biocompatible polymers with highly specific mechanical properties. This leads to the incorporation of PHA in numerous biomedical applications within the previous decade. PHAs have been fabricated in various forms to perform tissue engineering to repair liver, bone, cartilage, heart tissues, cardiovascular tissues, bone marrow, and to act as drug delivery system and nerve conduits. A large number of animal trials have been carried out to assess the biomedical properties of PHA monomers, which also confirms the high compatibility of PHA family for this field. This review summarizes the synthesis of PHA from different sources, and biosynthetic pathways and biomedical applications of biosynthesized polyhydroxyalkanoates.
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyester which are biosynthesized from the intracellular cells of microalgae through the cultivation of organic food waste medium. Before cultivation process, food waste must undergo several pre-treatment techniques such as chemical, biological, physical or mechanical in order to solubilize complex food waste matter into simpler micro- and macronutrients in which allow bio-valorisation of microalgae and food waste compound during the cultivation process. This work reviews four microalgae genera namely Chlamydomonas, Chlorella, Spirulina, and Botryococcus, are selected as suitable species due to rapid growth rate, minimal nutrient requirement, greater adaptability and flexibility prior to lower the overall production cost and maximized the production of PHAs. This study also focuses on the different mode of cultivation for the accumulation of PHAs followed by cell wall destabilization, extraction, and purification. Nonetheless, this review provides future insights into enhancing the productivity of bioplastic derived from microalgae towards low-cost, large-scale, and higher productivity of PHAs.
The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
Polyhydroxyalkanoates (PHAs) has been paid great attention because of its useful thermoplastic properties and complete degradation in various natural environments. But, at industrial level, the successful commercialization of PHAs is limited by the high production cost due to the expensive carbon source and recovery processes. Pseudomonas mendocina PSU cultured for 72 h in mineral salts medium (MSM) containing 2% (v/v) biodiesel liquid waste (BLW) produced 79.7 wt% poly(3-hydroxybutyrate) (PHB) at 72 h. In addition, this strain produced 43.6 wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 8.6 HV mol% at 60 h when added with 0.3% sodium propionate. The synthesized intracellular PHA granules were recovered and purified by the recently reported biological method using mealworms. The weight average molecular weight (Mw ) and number average molecular weight (Mn ) of the biologically extracted PHA were higher than that from the chloroform extraction with comparable melting temperature (Tm ) and high purity. This study has successfully established a low-cost process to synthesize PHAs from BLW and subsequently confirmed the ability of mealworms to extract PHAs from various kinds of bacterial cells.
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.
A natural biodegradable polymer, polyhydroxyalkanoate (PHA), was adjuvanted with a vaccine seed to observe the biomaterial's ability in enhancing an immune response in rats. The adjuvant potential of PHA was tested using the whole-killed Pasteurella multocida B:2 (PMB2) vaccine in Sprague Dawley (SD) rats to detect changes in serum immunoglobulin G (IgG) and immunoglobulin M (IgM) responses. A common PHA, poly(3-hydroxybutyrate) [P(3HB)], from Bacillus megaterium UMTKB-1 was constructed into microparticles using the solvent evaporation method. Twelve SD rats were divided into four treatment groups: 1) non-treatment as negative control, 2) P(3HB) adjuvant, 3) PMB2 vaccine, and 4) adjuvanted-P(3HB)/PMB2 vaccine groups, which were intramuscularly vaccinated twice. Immunoglobulins IgG and IgM levels were used as markers of the immune response induced by the adjuvanted-P(3HB)/PMB2 vaccine and analysed over an eight-week study period. The group vaccinated specifically with adjuvanted-P(3HB)/PMB2 vaccine had higher concentrations of immunoglobulins compared to other treatment groups, hence demonstrating the potential of the adjuvant to enhance immune response. Findings showed a need to delay the delivery of the second booster dose to determine the appropriate regime for the adjuvanted-P(3HB)/PMB2 vaccine.
Polyhydroxyalkanoates (PHAs) has been investigated for more than eighty years. Ever since then, the scientists are kept on synthesizing and developing new polymers and application to suit human interests nowadays. The resourcefulness of PHAs has made them a good candidates for the study of their potential in a variety of areas from biomedical/medical fields to food, packaging, textile and household material. In medical field (specifically in tissue engineering application), producing a biocompatible 3-D scaffold with adaptable physical properties are essential. However, to the best of our knowledge, scaffolds from PHB and PHBV with thickness greater than 1 mm have not been produced yet. In this work, PHB and PHBV porous 3-D scaffolds with an improved thickness greater than 4 mm was fabricated using conventional method of solvent-casting particulate-leaching (SCPL). A preliminary assessment on the improved thickness 3-D scaffolds was carried out to examine its pore interconnectivity by using non-invasive color staining method. The vertical cross sections image of the stained scaffolds was analyzed by image analyzer software. This technique was considered simple, fast and cost effective method prior to the usage of super accurate analytical instruments (micro-computed tomography). The results showed that over 80% of the pores have been interconnected with the adjacent pores. Moreover, there was a good correlation between the predicted pore interconnectivity and porosity. These results indicated how well a simple technique can do by giving an overview of the internal morphology of a porous 3-D structure material.
Oil palm empty fruit bunch (OPEFB), contains abundant cellulose and hemicelluloses and can be used as a renewable resource for fuel and chemical production. This study, as the first attempt, aims to convert OPEFB derived sugars to polyhydroxybutyrate (PHB). OPEFB collected from a Malaysia palm oil refinery plant was chemically pretreated and enzymatically hydrolyzed by an in-house prepared cellulase cocktail. The PHB producer, Bacillus megaterium R11, was isolated in Singapore and could accumulate PHB up to 51.3% of its cell dry weight (CDW) from both glucose and xylose. Tryptone was identified as its best nitrogen source. PHB content and production reached 58.5% and 9.32 g/L, respectively, for an overall OPEFB sugar concentration of 45 g/L. These respectively reached 51.6% and 12.48 g/L for OPEFB hydrolysate containing 60 g/L sugar with a productivity of 0.260 g/L/h.