Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture.
Polyhydroxyalkanoates (PHAs), a family of biodegradable and renewable biopolymers show a huge potential as an alternative to conventional plastics. Extractive bioconversion (in situ product recovery) is a technique that integrates upstream fermentation and downstream purification. In this study, extractive bioconversion of PHAs from Cupriavidus necator H16 was performed via a thermo-separating aqueous two-phase system to reduce the cost and environmental impacts of PHAs production. Key operating parameters, such as polymer concentration, temperature, and pH, were optimized. The strategy achieved a yield and PF of 97.6% and 1.36-fold, respectively at 5% EOPO 3900 concentration, 30 °C fermentation temperature and pH 6. The PHAs production process was also successfully scaled up in a 2 L bioreactor. To the best of our knowledge, this is the first report on extractive fermentation of PHAs from Cupriavidus necator utilizing a thermo-separation system to achieve a better productivity and purity of the target product.
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.
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.
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 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 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.
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.
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.
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 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.
Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 77 kDa.
The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C(8:0)) to oleic acid (C(18:1)) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1)H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d)) of 264.6 to 318.8 (± 0.2) (o)C, melting temperature (T(m)) of 43. (± 0.2) (o)C, glass transition temperature (T(g)) of -1.0 (± 0.2) (o)C and apparent melting enthalpy of fusion (ΔH(f)) of 100.9 (± 0.1) J g(-1).
The effects of organic solvents and their binary mixture in the glucose functionalization of bacterial poly-3-hydroxyalkanoates catalyzed by Lecitase™ Ultra were studied. Equal volume binary mixture of DMSO and chloroform with moderate polarity was more effective for the enzyme catalyzed synthesis of the carbohydrate polymer at ≈38.2 (±0.8)% reactant conversion as compared to the mono-phasic and other binary solvents studied. The apparent reaction rate constant as a function of medium water activity (aw) was observed to increase with increasing solvent polarity, with optimum aw of 0.2, 0.4 and 0.7 (±0.1) observed in hydrophilic DMSO, binary mixture DMSO:isooctane and hydrophobic isooctane, respectively. Molecular sieve loading between 13 to 15gL(-1) (±0.2) and reaction temperature between 40 to 50°C were found optimal. Functionalized PHA polymer showed potential characteristics and biodegradability.
Wound management and healing in several physiological or pathological conditions, particularly when comorbidities are involved, usually proves to be difficult. This presents complications leading to socio-economic and public health burdens. The accelerative wound healing potential of biocompatible poly(3-hydroxyalkanoates)-co-(6-hydroxyhexanoate) (PHA-PCL) composite hydrogel is reported herein. The biosynthesized PHA-PCL macromer was cross-linked with PEGMA to give a hydrogel. Twenty-four rats weighing 200-250 g each were randomly assigned to four groups of six rats. Rats in group I (negative control) were dressed with sterilized gum acacia paste in 10% normal saline while PEGMA-alone hydrogel (PH) was used to dress group II (secondary control) rats. Group III rats were dressed with PHAs-PCL cross-linked PEGMA hydrogel (PPH). For the positive control (group IV), the rats were dressed with Intrasite(®) gel. Biochemical, histomorphometric and immunohistomorphometric analyses revealed a significant difference in area closure and re-epithelialization on days 7 and 14 in PPH or Intrasite(®) gel groups compared to gum acacia or PEGMA-alone groups. Furthermore, wounds dressed with PPH or Intrasite(®) gel showed evident collagen deposition, enhanced fibrosis and extensively organized angiogenesis on day 14 compared to the negative control group. While improvement in wound healing of the PH dressed group could be observed, there was no significant difference between the negative control group and the PH dressed group in any of the tests. The findings suggested that topical application of PPH accelerated the rats' wound healing process by improving angiogenesis attributed to the increased microvessel density (MVD) and expressions of VEGF-A in tissue samples. Thus, PPH has been demonstrated to be effective in the treatment of cutaneous wounds in rats, and could be a potential novel agent in the management and acceleration of wound healing in humans and animals.
A significant source of microplastics is from the usage of microbeads in the market since petrochemical plastic bead is a material used in cosmetic scrubs. A possible way to counteract the problem is by the substitution of synthetic plastic to natural biodegradable polymer. Polyhydroxyalkanoate (PHA) is a general class of thermoplastic microbial polymer and it is the best alternative to some petrochemical plastics due to its biodegradability. Some PHA has earned its way into cosmetic application due to its biocompatibility. This data article reports data on the development of biodegradable microbeads by using the double emulsion solvent evaporation technique. Our data describe the extraction of biopolymer from marine bacteria that was cultivated in shaken flask culture, removal of endotoxins using oxidizing agent, the production of microbeads using a peristaltic pump with a specific flowrate and silicon tubing, and the cytotoxicity of the microbeads.
Polyhydroxyalkanoate (PHA) is a family of biopolymers produced by some bacteria and is accumulated intracellularly as carbon and energy storage material. Fifteen PHA-producing bacterial strains were identified from bacteria isolated from Antarctic soils collected around Casey Station (66°17'S, 110°32'E) and Signy Island (60°45'S, 45°36'W). Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 0.5% (w/v) sodium octanoate or glucose. 16S rRNA gene sequence analysis revealed that the isolated PHA-producing strains were mainly Pseudomonas spp. and a few were Janthinobacterium spp. All the isolated Pseudomonas strains were able to produce medium-chain-length (mcl) PHA using fatty acids as carbon source, while some could also produce mcl-PHA by using glucose. The Janthinobacterium strains could only utilize glucose to produce polyhydroxybutyrate (PHB). A Pseudomonas isolate, UMAB-40, accumulated PHA up to 48% cell dry mass when utilizing fatty acids as carbon source. This high accumulation occurred at between 5°C and 20°C, then decreased with increasing temperatures. Highly unsaturated mcl-PHA was produced by UMAB-40 from glucose. Such characteristics may be associated with the ability of UMAB-40 to survive in the cold.
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.
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).
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.