The polyhydroxyalkanoate (PHA) accumulation dynamics in aerobic granules that undergo the growth-disintegration cycle were investigated. Four sequencing batch reactors (SBR) were inoculated with aerobic granules at different stages of development (different sizes). Different sizes of aerobic granules showed varying PHA contents. Thus, further study was conducted to investigate the diffusion of substrate and oxygen on PHA accumulation using various organic loading rates (OLR) and aeration rates (AR). An increase in OLR from 0.91 to 3.64kg COD/m(3)day increased the PHA content from 0.66 to 0.87g PHA/g CDW. Meanwhile, an AR increase from 1 to 4L/min only accelerated the maximum PHA accumulation without affecting the PHA content. However, the PHA composition only changes with AR, while the hydroxyvalerate (HV) content increased at a higher AR.
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.
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 effects of variable aeration in the famine period on polyhydroxyalkanoate (PHA) accumulation in aerobic granules were investigated. Results showed that regardless of the aeration rates used during famine period, all aerobic granules achieved a similar chemical oxygen demand removal and PHA content. The decrease in famine-period aeration rates accelerated the maximum PHA accumulation together with increase in granular size and settling ability. The PHA-accumulating microorganisms were found to have shifted closer to the surface of the granules when the aeration rate was reduced. Moreover, PHA compositional changes occurred, where the hydroxyvalerate content had increased with the reduction in aeration rate. Ultimately, the results indicate that the requirement of aeration for PHA accumulation in aerobic granules is highly insignificant in the famine phase. PHA production in aerobic granules under zero aeration in the famine period may result in an energy input reduction of up to 74%.
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.
A simple procedure for recovering biodegradable polymer from bacterial cells has been developed using economical and environmentally friendly solvent or chemicals. Recombinant bacterium, Cupriavidus necator harboring pBBR1MCS-C2 plasmid polyhydroxyalkanoate (PHA) synthase gene was used for the production of copolymer P(3HB-co-3HHx) from crude palm kernel oil (CPKO). NaOH was chosen in this study as it could give high purity and recovery yield. Increase of NaOH concentration had resulted in an increase of the PHA purity, but the recovery yield had decreased. The greater improvement of PHA purity and recovery were achieved by incubating the freeze-dried cells (10-30 g/L) in NaOH (0.1 M) for 1-3 h at 30°C and polishing using 20% (v/v) of ethanol. The treatment caused negligible degradation of the molecular weight of PHA recovered from the bacterial cells. The present review also highlights other extraction methods to provide greater insights into economical and sustainable recovery of PHA from bacterial cells.
Polyhydroxyalkanoate (PHA) recovery from aerobic granules was investigated using four cell digestion agents, namely, sodium hypochlorite, sodium hydroxide, acetone and sodium chloride. Simultaneously, the removal of extracellular polymeric substances (EPS) and its effect on PHA yield were investigated. The highest PHA recovery yield was obtained using sodium hypochlorite, accounting for 89% cell dry weight (CDW). The highest PHA was recovered after the sodium hypochlorite completely removed the EPS from the aerobic granules. The average molecular weight (Mw) of the PHA recovered using sodium hypochlorite was 5.31 × 10(5)g/mol with only 1.8% molecular weight degradation. The energy and duration analysis for PHA recovery revealed that the sodium hypochlorite method required the least amount of energy and time at 0.0561 MJ/g PHA and 26 h, respectively. The PHA that was recovered was a P3(HB-co-HV) co-polymer.
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.
Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.
A microbial fuel cell is a sustainable and environmental-friendly device that combines electricity generation and wastewater treatment through metabolic activities of microorganisms. However, low power output from inadequate electron transfer to the anode electrode hampers its practical implementation. Nanocomposites of oxidized carbon nanotubes and medium-chain-length polyhydroxyalkanoates (mcl-PHA) grafted with methyl acrylate monomers enhance the electrochemical function of electrodes in microbial fuel cell. Extensive polymerization of methyl acrylate monomers within mcl-PHA matrix, and homogenous dispersion of carbon nanotubes within the graft matrix are responsible for the enhancement. Modified electrodes exhibit high conductivities, better redox peak and reduction of cell internal resistance up to 76%. A stable voltage output at almost 700 mV running for 225 H generates maximum power and current density of 351 mW/m2 and 765 mA/m2 , respectively. Superior biofilm growth on modified surface is responsible for improved electron transfer to the anode hence stable and elevated power output generation.
In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.
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.
An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well.
The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.
Poly(3-hydroxybutyrate) [P(3HB)], a polymer belonging to the polyhydroxyalkanoate (PHA) family, is accumulated by numerous bacteria as carbon and energy storage material. The mobilization of accumulated P(3HB) is associated with increased stress and starvation tolerance. However, the potential function of accumulated copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] remained unknown. In this study, Delftia acidovorans DS 17 was used to evaluate the contributions of P(3HB) and P(3HB-co-3HV) granules during simulated exogenous carbon deprivation on cell survival by transferring cells with PHAs to carbon-free mineral salt medium supplemented with 1% (w/v) nitrogen source. By mobilizing the intracellular P(3HB) and P(3HB-co-3HV) at 11 and 40 mol% 3HV compositions, the cells survived starvation. Surprisingly, D. acidovorans containing P(3HB-co-94 mol% 3HV) also survived although the mobilization was not as effective. Similarly, recombinant Escherichia coli pGEM-T::phbCAB(Cn) (harboring the PHA biosynthesis genes of Cupriavidus necator) containing P(3HB) granules had a higher viable cell counts compared to those without P(3HB) granules but without any P(3HB) mobilization when exposed to oxidative stress by photoactivated titanium dioxide. This study provided strong evidence that enhancement of stress tolerance in PHA producers can be achieved without mobilization of the previously accumulated granules. Instead, PHA biosynthesis may improve bacterial survival via multiple mechanisms.
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.
The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. (1)H NMR and (13)C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties.
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.
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.
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.