The sustainability in polyhydroxyalkanoates (PHA) production is drawing increasing attention as the effort to increase the economic feasibility for commercialization pursues. Oleic acid is widely preferred by bacteria but its employment for PHA production makes sustainability rather dubious. This study showed promising poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] content of 68 wt % by lipase genes-harbouring Cupriavidus malaysiensis USMAA2-4 transformant from palm olein and 1-pentanol. High oleic acid content and low oil saturation caused palm olein to outperform crude palm oil, crude palm kernel oil and soybean oil due to its preference for oleic acid shown by previous screening. The transformant showed 8-fold and 40-fold higher lipase activity compared to C. necator H16 and its wild-type respectively. The transformant was unaffected by Co2+ but the growth of C. necator H16 was inversely proportional to Co2+ concentration and the employment of 1-pentanol also ceased its growth and PHA accumulation. Although the inhibitory effect of Fe2+, Cu2+ and Zn2+ at high molarity on LipA decreased PHA content of C. malaysiensis USMAA2-4 transformant by 23-24 wt %, the lipase activity was restorable with high molarity of Ca2+, thus resulted in higher PHA content. The transformant enabled the employment of low-cost 1-pentanol as the precursor for cost-effective PHA production and its preference for palm olein contributed to higher sustainability.
Polyhydroxyalkanoate (PHA) is a type of polyesters produced in the form of accumulated intracellular granules by many microorganisms. It is viewed as an environmentally friendly bioproduct due to its biodegradability and biocompatibility. The production of the PHA using oil substrates such as waste oil and plant oil, has gained considerable attention due to the high product yield and lower substrate cost. Nevertheless, the PHA fermentation using oil substrate is complicated due to the heterogenous fatty acid composition, varied bio-accessibility and possible inhibitory effect on the bacterial culture. This review presents the current state-of-the-art of PHA production from oil-based substrates. This paper firstly discusses the technical details, such as the choice of bacteria strain and fermentation conditions, characteristic of the oil substrate as well as the PHA composition and application. Finally, the paper discusses the challenges and prospects for up-scaling towards a cleaner and effective bioprocess. From the literature review, depending on the cell culture and the type of PHA produced, the oil platform can have a PHA yield of 0.2-0.8 g PHA/g oil substrate, with PHA content mostly from 40 to 90% of the cell dry weight. There is an on-going search for more effective oil-utilising PHA producers and lower cost substrate for effective PHA production. The final application of the PHA polymer influences the treatment needed during downstream processing and its economic performance. PHA with different compositions exhibits varied decomposition behaviour under different conditions, requiring further insight towards its management towards a sustainable circular economy.
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.
The exponential increase in the use and careless discard of synthetic plastics has created an alarming concern over the environmental health due to the detrimental effects of petroleum based synthetic polymeric compounds. Piling up of these plastic commodities on various ecological niches and entry of their fragmented parts into soil and water has clearly affected the quality of these ecosystems in the past few decades. Among the many constructive strategies developed to tackle this global issue, use of biopolymers like polyhydroxyalkanoates as sustainable alternatives for synthetic plastics has gained momentum. Despite their excellent material properties and significant biodegradability, polyhydroxyalkanoates still fails to compete with their synthetic counterparts majorly due to the high cost associated with their production and purification thereby limiting their commercialization. Usage of renewable feedstocks as substrates for polyhydroxyalkanoates production has been the thrust area of research to attain the sustainability tag. This review work attempts to provide insights about the recent developments in the production of polyhydroxyalkanoates using renewable feedstock along with various pretreatment methods used for substrate preparation for polyhydroxyalkanoates production. Further, the application of blends based on polyhydroxyalkanoates, and the challenges associated with the waste valorization based polyhydroxyalkanoates production strategy is elaborated in this review work.
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.
Cupriavidus sp. USMAA1020, USMAA2-4, and USMAHM13 are capable of producing polyhydroxyalkanoate (PHA). This biopolymer is an alternative solution to synthetic plastics, whereby polyhydroxyalkanoate synthase is the key enzyme involved in PHA biosynthesis. Here, we report the complete genomes of three Cupriavidus sp. strains: USMAA1020, USMAA2-4, and USMAHM13.
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.
Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.
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.
Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8 g L-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1 g L-1 , and 48 H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2 M Tris-HCl buffer (pH 9, strength = 0.2 M, 30 °C) at 0.21 g L-1 H-1 and 98.6 ± 1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.
Polyhydroxyalkanoate (PHA) synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace petroleum-based plastics. While two structures have been determined, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. A model of the class I PhaC from Aquitalea sp. USM4, characterised with Km of 394 μM and kcat of 476 s-1 on 3-(R)-hydroxybutyryl-CoA, revealed a three-branched channel at the dimeric interface. Two of them are opened to the solvent and are expected to serve as the putative routes for substrate entrance and product exit, while the third is elongated in the class II PhaC1 model from Pseudomonas aeruginosa, indicating a role in accommodating the hydroxyalkanoate (HA) moiety of a HA-CoA substrate. Docking of the two tetrahedral intermediates, formed during the transfer of the growing PHA chain from the catalytic Cys to a new molecule of substrate and back to Cys, suggests a common elongation mechanism requiring the HA moiety of the ligand to rotate ~180°. Substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates.
Aerobic dynamic feeding (ADF) strategy was applied in sequencing batch reactor (SBR) to accumulate polyhydroxyalkanoate (PHA) in aerobic granules. The aerobic granules were able to remove 90% of the COD from palm oil mill effluent (POME). The volatile fatty acids (VFAs) in the POME are the sole source of the PHA accumulation. In this work, 100% removal of propionic and butyric acids in the POME were observed. The highest amount of PHA produced in aerobic granules was 0.6833mgPHA/mgbiomass. The PHA formed was identified as a P (hydroxybutyrate-co-hydroxyvalerate) P (HB-co-HV).
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.
High PHA production and storage using palm oil mill effluent (POME) was investigated using a laboratory batch Bio-PORec® system under aerobic-feeding conditions. Results showed that maximum PHA was obtained at a specific rate (q(p)) of 0.343 C-mol/C-molh when air was supplied at 20 ml/min. The PHA yield was found to be 0.80 C-mol/C-mol acetic acid (HAc) at microaerophilic condition and the mass balance calculation showed that PHA production increased up to 15.68±2.15 C-mmol/cycle. The experiments showed that short feeding rate, limited requirements for electron acceptors (e.g. O(2), NO(3)) and nutrients (N and P) showed lower tendency of glycogen accumulation and contributed more to PHA productivity.
We attempted to synthesize a polyhydroxyalkanoate (PHA) containing newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer by using wild type Burkholderia sp. USM (JCM15050) and its transformed strain harboring the PHA synthase gene of Aeromonas caviae (phaCAc). The introduction of 3H4MV as a second monomer will improve the material properties of 3HB-based polymers. To promote the accumulation of PHA containing 3H4MV monomer, isocaproic acid was provided as co-carbon source. Approximately 1mol% of 3H4MV was detected in wild type Burkholderia sp. cultures when they were fed glucose or fructose together with isocaproic acid. Thus, the wild type strain can synthesize the 3H4MV monomer. High 3H4MV fractions, of about 40mol%, were obtained when the transformed strain was cultivated on glucose or fructose together with isocaproic acid. In addition, the ability of the transformed strain to mobilize accumulated PHA containing 3H4MV monomer was demonstrated in this study. This is the first report on mobilization of the 3H4MV monomer.
The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL(-1)) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0-23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (Mw) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M(w) compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (Tm), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate.
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.
Polymeric nanoparticles gain a widespread interest in food and pharmaceutical industries as delivery systems that encapsulate, protect, and release lipophilic compounds such as omega-3 fatty acids, fat-soluble vitamins, carotenoids, carvedilol, cyclosporine, and ketoprofen. In this study, medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA)-incorporated nanoparticle was developed via facile organic solvent-free nanoemulsion templating technique. The water content (W/surfactant-to-oil (S/O)), S/O, and Cremophor EL-to-Span 80 (Cremo/Sp80) ratios were first optimized using response surface methodology (RSM) to obtain nanoemulsion template prior to incorporation of mcl-PHA. Their effects on nanoemulsion formation were investigated. The mcl-PHA-incorporated nanoparticle system showed a good preservation capability of β-carotene and extended storage stability.
Polyhydroxyalkanoate (PHA) is a type of biopolymer produced by most bacteria and archaea, resembling thermoplastic with biodegradability and biocompatibility features. Here, we report the complete genome of a PHA producer, Aquitalea sp. USM4, isolated from Perak, Malaysia. This bacterium possessed a 4.2 Mb circular chromosome and a 54,370 bp plasmid. A total of 4067 predicted protein-coding sequences, 87 tRNA genes, and 25 rRNA operons were identified using PGAP. Based on ANI and dDDH analysis, the Aquitalea sp. USM4 is highly similar to Aquitalea pelogenes. We also identified genes, including acetyl-CoA (phaA), acetoacetyl-CoA (phaB), PHA synthase (phaC), enoyl-CoA hydratase (phaJ), and phasin (phaP), which play an important role in PHA production in Aquitalea sp. USM4. The heterologous expression of phaC1 from Aquitalea sp. USM4 in Cupriavidus necator PHB-4 was able to incorporate six different types of PHA monomers, which are 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), 3-hydroxyhexanoate (3HHx) and isocaproic acid (3H4MV) with suitable precursor substrates. This is the first complete genome sequence of the genus Aquitalea among the 22 genome sequences from 4 Aquitalea species listed in the GOLD database, which provides an insight into its genome evolution and molecular machinery responsible for PHA biosynthesis.
Halophilic micro-organisms have received much interest because of their potential biotechnological applications, among which is the capability of some strains to synthesize polyhydroxyalkanoates (PHA). Halomonas sp. SK5, which was isolated from hypersaline microbial mats, accumulated intracellular granules of poly(3-hydroxybutyrate) [P(3HB)] in modified accumulation medium supplemented with 10% (w/v) salinity and 3% (w/v) glucose.