4HB Monomers Research Articles

Development and physiochemical assessment of graphene-bioactive glass-P(3HB-co-4HB) composite scaffold as prospect biomaterial for wound healing

The clinical management of wounds presents a considerable challenge because dressing selection must prioritise the provision of appropriate barrier and the healing properties, consider patient’s compliance factors such as comfort, functionality and practicality. This study primarily aimed to develop a composite scaffold patch for potential application in wound healing. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is a biopolymer that originated from bacteria. It is well-recognised owing to its distinctive mechanical and physical characteristics suitable for biomedical applications. Graphene (G) and bioactive glass (BG) are biocompatible towards humans, and enhanced properties are achievable by adding biopolymer. In this study, composite scaffolds were developed by combining P(3HB-co-4HB) at a distinct proportion of 4HB monomer reinforced with G (3.0 wt.%) and BG (2.5 wt.%) by using solvent casting, resulting in two types of composite scaffolds: P(3HB-co-25%4HB)/G/BG and P(3HB-co-37%4HB)/G/BG. A successful composite scaffold as a unified structure was achieved based on chemical assessments of organic and inorganic elements within the composites. The pure polymer displayed a smooth surface, and the BG and G addition into the composite scaffolds increased surface roughness, forming irregular pores and protuberances. The wettability and hydrophilicity of the composites significantly improved up to 40% in terms of water uptake. An increment in crystallisation temperature diminished the flexibility of the composite’s scaffolds. Evaluation of Presto Blue biocompatibility demonstrated nontoxic behaviour with a dosage of less than 25.00 mg ml−1 of composite scaffold-conditioned media. The L929 fibroblast cells displayed excellent adhesion to both types of composite scaffolds, as evidenced by the increased percentage of cell viability observed throughout 14 d of exposure. These findings demonstrate the importance of optimising each component within the composite scaffolds and their interrelation, paving the way for excellent material properties and enhancing the potential for wound healing applications.

The Development and Characterisation of a P(3HB-co-4HB)-Bioactive Glass-Graphene Hydrogel as a Potential Formulation for Biomedical and Therapeutical Translation.

The clinical management of wounds is known to be a significant challenge: not only does the dressing need to ensure and provide the appropriate barrier and healing characteristics, but consideration of patient compliance concerning comfort, functionality, and practicality also needs to be included. The poly(3-hydroxybutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) copolymer, isolated from Cupriavidus malaysiensis USM1020 (C. malaysiensis USM1020), was produced in the presence of excess carbon sources (1,4-butanediol and 1,6-hexanediol) using either a shake flask cultivation process or a bioreactor fermentation system. P(3HB-co-4HB) is widely known to be biodegradable and highly biocompatible and contains a tuneable 4HB monomer molar fraction, which is known to affect the final physicochemical properties of the intracellular copolymer. In this paper, we describe not only the fabrication of the polymeric gel but also its optimised profiling using a range of physical and mechanical techniques, i.e., SEM, FTIR, DMA, DSC, and WCA. The further enhancement of the gel through additional functionalisation with sol-gel-derived bioactive glass and liquid-exfoliated graphene was also investigated. The biocompatibility and biological characterisation of the substrates was assessed using murine osteoblasts (MC3T3), human primary dermal fibroblasts (HDFs), human fibroblast (BJ) cells, and standard cell culture assays (i.e., metabolic activity, LDH release, and live/dead staining). In short, P(3HB-co-4HB) was successfully isolated from the bacteria, with the defined physico-chemical profiles dependent on the culture substrate and culturing platform used. The additional enhancement of the copolymer with bioactive glass and/or graphene was also demonstrated by varying the combination loading of the materials, i.e., graphene resulted in an increase in tensile strength (~11 MPa) and the wettability increased following the incorporation of bioactive glass and 0.01 wt% graphene (WCA ~46.3°). No detrimental effects in terms of biocompatibility were noticed during the 7 days of culture in the primary and established cell lines. This study demonstrates the importance of optimising each of the individual components within the biocomposite and their relationship concerning the fine-tuning of the material's properties, thus targeting and impacting the endpoint application.

Biosynthesis of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from Different 4-Hydroxybutyrate Precursors by New Wild-Type Strain Cupriavidus necator IBP/SFU-1

The study addresses the growth of the new wild-type strain Cupriavidus necator IBP/SFU-1 and the synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB) on media containing fructose and three different precursors of 4HB (ε-caprolactone 1,4-butanediol and 1,6-hexanediol). It was found that ε-caprolactone is the best precursor for the synthesis of P(3HB-co-4HB) copolymers. By varying the concentration and number of doses of ε-caprolactone added into the bacterial culture, it was possible to find conditions that ensured the synthesis of P(3HB-co-4HB) copolymers with different contents of 4HB (from 3–5 to 22.4 mol.%). The physicochemical properties of the copolymers were investigated depending on the proportions of 4HB monomers. The effect of 4HB monomers was manifested in a certain decrease in the weight-average molecular weight (Mw) (272–353 kDa), number-average molecular weight (Mn) (47–67 kDa) of the samples, and an increase in polydispersity (5.09–6.71) compared with P(3HB). The crystallinity degree decreased with an increasing fraction of the 4HB units (from 72 to 59%, as the 4HB content increased from 0 to 22.4 mol.%). In addition, the increase in 4HB content affected the temperature parameters (melting point, glass transition temperature, crystallization temperature, and thermal degradation temperature).

Effective production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by engineered Halomonas bluephagenesis grown on glucose and 1,4-Butanediol

Halomonas bluephagenesis has been engineered to produce flexible copolymers P34HB or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose and petrol-chemical precursor, γ-butyrolactone. Herein, gene cluster aldD-dhaT was constructed in recombinant H. bluephagenesis for catalyzing 1,4-butanediol (BDO) into 4-hydroxybutyrate, which could grow to 86 g L−1 dry cell mass (DCM) containing 77 wt% P(3HB-co-14 mol% 4HB) in 7-L bioreactor fed with glucose and bio-based BDO. Furthermore, 4HB monomer ratio could be increased to 16 mol% by engineered H. bluephagenesis TDH4-WZY254 with defected outer-membrane. Upon deletion of 4HB degradation pathway, followed by aldD-dhaT integration, the resulted H. bluephagenesis TDB141ΔAC was grown to 95 g L−1 DCM containing 79 wt% P(3HB-co-14 mol% 4HB) with a BDO conversion efficiency of 86% under fed-batch fermentation. Notably, 4HB molar ratio can be significantly improved to 21 mol% with negligible effects on cell growth and P34HB synthesis by adding 50% more BDO. This study successfully demonstrated a fully bio-based P34HB effectively produced by H. bluephagenesis.

Surface Modification of Sponge-like Porous Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/Gelatine Blend Scaffolds for Potential Biomedical Applications.

In this study, we described the preparation of sponge-like porous scaffolds that are feasible for medical applications. A porous structure provides a good microenvironment for cell attachment and proliferation. In this study, a biocompatible PHA, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was blended with gelatine to improve the copolymer’s hydrophilicity, while structural porosity was introduced into the scaffold via a combination of solvent casting and freeze-drying techniques. Scanning electron microscopy results revealed that the blended scaffolds exhibited higher porosity when the 4HB compositions of P(3HB-co-4HB) ranged from 27 mol% to 50 mol%, but porosity decreased with a high 4HB monomer composition of 82 mol%. The pore size, water absorption capacity, and cell proliferation assay results showed significant improvement after the final weight of blend scaffolds was reduced by half from the initial 0.79 g to 0.4 g. The pore size of 0.79g-(P27mol%G10) increased three-fold while the water absorption capacity of 0.4g-(P50mol%G10) increased to 325%. Meanwhile, the cell proliferation and attachment of 0.4g-(P50mol%G10) and 0.4g-(P82mol%G7.5) increased as compared to the initial seeding number. Based on the overall data obtained, we can conclude that the introduction of a small amount of gelatine into P(3HB-co-4HB) improved the physical and biological properties of blend scaffolds, and the 0.4g-(P50mol%G10) shows great potential for medical applications considering its unique structure and properties.

Determination of substrate addition order strategy for bespoke Polyhydroxyalkanoates production: A molecular modeling based approach

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible materials with a wide range of biotechnological applications. An integrated study of the thermodynamic and kinetic behavior of the polymerization initiation process was developed for fine-tuning bespoke PHA production. Simplified molecular scale models were established to describe the initiation of PHA formation which cannot be captured with current physical tracking techniques in cells. A thermodynamic study and ab initio calculations demonstrate that the 4HB monomer exhibits higher reactivity in terms of PHA formation compared to 3HB, 3HV, 3HHx and 5HV. In order to produce terpolymer P(3HB-co-3HV-co-4HB) with a high fraction of 4HB, different scale fed-batch fermentation experiments using glucose, γ-butyrolactone, and propionic acid as carbon sources/precursors in Cupriavidus necator proved feeding in order of monomer reactivity led to double biomass and more than 50% of 4HB accumulation in terpolymer. The molecular model and substrate addition order strategy demonstrated in this work provide an efficient and practical fermentation method for controlling biopolymer production.

Current Advances towards 4-Hydroxybutyrate Containing Polyhydroxyalkanoates Production for Biomedical Applications.

Polyhydroxyalkanoates (PHA) are polyesters having high promise in biomedical applications. Among different types of PHA, poly-4-hydroxybutyrate (P4HB) is the only polymer that has received FDA approval for medical applications. However, most PHA producing microorganisms lack the ability to synthesize P4HB or PHA comprising 4-hydroxybutyrate (4HB) monomer due to their absence of a 4HB monomer supplying pathway. Thus, most microorganisms require supplementation of 4HB precursors to synthesize 4HB polymers. However, usage of 4HB precursors incurs additional production cost. Therefore, researchers have adopted strategies to reduce the cost, such as utilizing low-cost substrate as well as constructing 4HB monomer supplying pathways in microorganisms. We herein summarize the biomedical applications of P4HB, the natural producers of 4HB polymer, and the various strategies that have been applied in producing 4HB polymers in non-4HB producing microorganisms. It is expected that the readers would gain a vivid idea on the different strategic developments in the field of 4HB polymer production.

In Vivo Characterization and Application of the PHA Synthase from Azotobacter vinelandii for the Biosynthesis of Polyhydroxyalkanoate Containing 4-Hydroxybutyrate.

Polyhydroxyalkanoate (PHA) is a biodegradable thermoplastic naturally synthesized by many microorganisms, and the PHA synthase (PhaC) is known to be the key enzyme involved in determining the material properties and monomer composition of the produced PHA. The ability to exploit widely distributed, commonly found soil microorganisms such as Azotobacter vinelandii to synthesize PHA containing the lipase-degradable 4-hydroxybutyrate (4HB) monomer will allow for convenient production of biocompatible and flexible PHA. Comparisons between the A. vinelandii wild type and mutant strains, with and without a surface layer (S-layer), respectively, in terms of gene or amino acid sequences, synthase activity, granule morphology, and PHA productivity, revealed that the S-layer is the sole factor affecting PHA biosynthesis by A. vinelandii. Based on PHA biosynthesis using different carbon sources, the PhaC of A. vinelandii showed specificity for short-chain-length PHA monomers, making it a member of the Class I PHA synthases. In addition, it was proven that the PhaC of A. vinelandii has the inherent ability to polymerize 4-hydroxybutyrate (4HB) and the mediated accumulation of PHA with 4HB fractions ranging from 10 mol% to as high as 22 mol%. The synthesis of biocompatible PHA containing tailorable amounts of 4HB with an expanded range of elasticity and lipase-degradability will enable a wider range of applications in the biomedical field.

Properties of degradable polyhydroxyalkanoates with different monomer compositions

PurposeTo synthesize and investigate polyhydroxyalkanoates (PHAs) with different monomer composition and percentages and polymer films prepared from them. ResultsVarious PHAs: homopolymer poly-3-hydroxybutyrate P(3HB) and 2-, 3-, and 4-component copolymers comprising various combinations of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), and 3-hydroxyhexanoate (3HHx) monomers were synthesized under specialized conditions. Relationships were found between the monomer composition of PHAs and their molecular-weight and thermal properties and degree of crystallinity. All copolymers had decreased weight average molecular weights, Mw (to 390–600 kDa), and increased values of polydispersity (3.2–4.6) compared to the P(3HB). PHA copolymers showed different thermal behavior: an insignificant decrease in Tmelt and the presence of the second peak in the melting region and changes in parameters of crystallization and glass transition. At the same time, they retained thermostability, and the difference between Tmelt and Tdegr was at least 100–120 °C. Incorporation of 4HB, 3HV, and 3HHx monomer units into the 3-hydroxybutyrate chain caused changes in the amorphous to crystalline ratio and decreased the degree of crystallinity (Cx) to 20–40%. According to the degree to which the monomers reduced crystallinity, they were ranked as follows: 4HB – 3HHx – 3HV. A unique set of films was produced; their surface properties and physical/mechanical properties were studied as dependent on PHA composition; monomers other than 3-hydroxybutyrate were found to enhance hydrophilicity, surface development, and elasticity of polymer films. ConclusionAn innovative set of PHA copolymers was synthesized and solution-cast films were prepared from them; the copolymers and films were investigated as dependent on polymer chemical composition. Results obtained in the present study contribute to the solution of a critical issue of producing degradable polymer materials.

Synthesis and Characterization of Multicomponent PHAs

Cupriavidus necator B10646 bacterial cells were cultivated in the mode of synthesis of the reserve polyhydroxyalkanoates (PHAs) in the growth medium that contained, in addition to glucose as the main substrate, precursor substrates of the monomers of various monocarboxylic acids – salts of valeric and hexanoic acids, propionate, and γ-butyrolactone. PHA terpolymers and quaterpolymers with different compositions and proportions of monomers were synthesized, and their physicochemical properties were studied. The terpolymers were composed of monomers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), or 3-hydroxyhexanoate (3HHx) and had the following compositions: P(3HB/3HB/4HHx) and P(3HB/3HV/4HHx). The quaterpolymers had the following composition: P(3HB/3HV/4HB/3HHx). All copolymer samples, regardless of the composition and proportions of monomers, had lower molecular weights and higher polydispersity values compared to the highly crystalline 3-hydroxybutyrate homopolymer, but retained thermal stability properties, with a difference between the melting point and thermal degradation of at least 100-110 °С. The inclusion of 3HV, 4HB, and 3HHx monomers in the C-chain of 3HB caused changes in the crystalline to amorphous phase ratio and a significant decrease in the degree of crystallinity (Cx), which depended on the type of monomers and their contents in the copolymer. The maximum decrease in Cx (9-17 %) was detected in the P(3HB/3HV/4HB) terpolymer and the P(3HB/3HV/4HB/3HHx) quaterpolymer (30-36 %). The study confirms that there is the possibility of synthesizing polymers with various compositions, including new ones, which differ significantly in their basic properties

Production of P(3HB-co-4HB) copolymer with high 4HB molar fraction by Burkholderia contaminans Kad1 PHA synthase

Burkholderia sp. has been reported to synthesize polyhydroxyalkanoates (PHAs) with different monomers such as 3-hydroxybutyrate and 3-hydroxyvalerate. In this study, the PHA synthase gene of B. contaminans Kad1 (phaCBcon) was successfully cloned and characterized. Heterologous expression of phaCBcon conferred PHA synthesis to the mutant Cupriavidus necator PHB¯4, confirming that phaCBcon encoded a functional active protein. The transformant C. necator PHB¯4 (Cn-Kad1) harboring phaCBcon was able to synthesize PHA when grown on sodium-4-hydroxybutyrate (sodium-4-HB) as the sole carbon source in a two-stage cultivation. NMR results confirmed the presence of poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB). Cn-Kad1 has the ability to produce a broad range of copolymer with a molecular weight (Mw) in the range of 8.0 × 105 to 10 × 105 Dalton. However, the number average molecular weight (Mn) decreased from 5.0 × 105 to 4.7 × 105 Da, when the sodium-4-HB concentration increased from 0.1 to 0.4 M. Higher concentration of sodium-4-HB, resulted in higher 4HB compositions in the polymer and consequently gave lower values for both the melting temperature (Tm) and the glass transition temperature (Tg). Interestingly, Cn-Kad1 accumulated P(3HB-co-4HB) with 4HB monomer up to 51 mol% from sodium-4-HB, while, the wild type B. contaminans Kad1 has limited ability to accumulate PHA containing 4HB monomer.

Biotechnological Production of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate-co-3-Hydroxyvalerate) Terpolymer by Cupriavidus sp. DSM 19379.

The terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 4-hydroxybutyrate (4HB) was produced employing Cupriavidus sp. DSM 19379. Growth in the presence of γ-butyrolactone, ε-caprolactone, 1,4-butanediol, and 1,6-hexanediol resulted in the synthesis of a polymer consisting of 3HB and 4HB monomers. Single and two-stage terpolymer production strategies were utilized to incorporate the 3HV subunit into the polymer structure. At the single-stage cultivation mode, γ-butyrolactone or 1,4-butanediol served as the primary substrate and propionic and valeric acid as the precursor of 3HV. In the two-stage production, glycerol was used in the growth phase, and precursors for the formation of the terpolymer in combination with the nitrogen limitation in the medium were used in the second phase. The aim of this work was to maximize the Polyhydroxyalkanoates (PHA) yields with a high proportion of 3HV and 4HB using different culture strategies. The obtained polymers contained 0–29 mol% of 3HV and 16–32 mol% of 4HB. Selected polymers were subjected to a material properties analysis such as differential scanning calorimetry (DSC), thermogravimetry, and size exclusion chromatography coupled with multi angle light scattering (SEC-MALS) for determination of the molecular weight. The number of polymers in the biomass, as well as the monomer composition of the polymer were determined by gas chromatography.

Biosynthesis and characterization of co and ter-polyesters of polyhydroxyalkanoates containing high monomeric fractions of 4-hydroxybutyrate and 5-hydroxyvalerate via a novel PHA synthase

Polyhydroxyalkanoates (PHAs) are a group of microbial polyesters that are widely studied due to their versatile physicochemical and mechanical properties. The biosynthesis of PHAs is governed by the enzyme PHA synthase. We have previously reported the discovery of a novel wide substrate specificity PHA synthase isolated from a mangrove environment. In this study, we report the biosynthesis, via the novel PHA synthase, of various PHA co- and terpolymers containing 4-hydroxybutyrate (4HB) and 5-hydroxyvalerate (5HV) monomers, which are lipase degradable. The biosynthesized polymers have varying 4HB and 5HV contents between 5 and 70 mol % depending on the concentration of precursor compounds used. All the polymers exhibit low melting temperature (Tm) and crystallinity and elastomeric behaviour due to high 4HB and 5HV monomer contents. Supplementation of crude palm kernel oil (CPKO) as carbon source with appropriate precursors enabled the biosynthesis of interesting terpolymers and copolymers containing 3-hydroxyhexanoate (3HHx) along with 5HV and/or 4HB monomers. Lipase degradation assays indicated that co- and ter-polymers containing 4HB and 5HV monomers were successfully hydrolysed by various lipases, but addition of 3HHx monomers in the polymer chain reduced its degradability. These properties of the polymers suggest potential uses as biomaterials.

High PHA density fed-batch cultivation strategies for 4HB-rich P(3HB-co-4HB) copolymer production by transformant Cupriavidus malaysiensis USMAA1020

P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74–92 wt% and 4HB monomer composition of 92–99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose

Poly(3-hydroxybutyrate-co−4-hydroxybutyrate) [P(3HB-co−4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co−4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co−4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co−17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co−4HB) from only glucose using Halomonas.

Properties of PHA bi-, ter-, and quarter-polymers containing 4-hydroxybutyrate monomer units

The present study investigates physicochemical, mechanical, and biological properties of polyhydroxyalkanoate (PHA) copolymers containing 4-hydroxybutyrate (4HB) synthesized in Cupriavidus eutrophus B10646 culture. In poly(3-hydroxybutyrate/4-hydroxybutyrate) [P(3HB/4HB)] bipolymers, 4HB varied between 10.4 and 75.0 mol%; in poly(3-hydroxybutyrate/3-hydroxyvalerate/4-hydroxybutyrate) terpolymers, 4HB constituted 28.7–55.6 mol%; and in poly(3-hydroxybutyrate/3-hydroxyvalerate/4-hydroxybutyrate/3-hydroxyhexanoate) quaterpolymers, 4HB varied between 9.3 and 13.3 mol%. The degree of crystallinity of P(3HB/4HB) copolymers decreased consistently with an increase in 4HB content, reaching 38%. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate into copolymers enhanced that effect. The effect of 4HB monomer units on temperature properties of copolymers was exhibited as lowering of the melting temperature and crystallization temperature, which improved the processing-related properties of the copolymers. All copolymers containing 4HB showed enhanced elongation at break compared to poly(3-hydroxybutyrate). Polymer films prepared from PHAs with different chemical composition had similar microstructure and porosity and had no toxic effect on mouse fibroblast NIH 3 T3 cells, proving their high biocompatibility.

Synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with high 4HB composition and PHA content using 1,4-butanediol and 1,6-hexanediol for medical application

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] with high 4HB monomer harbors enhanced biocompatibility and mechanical properties, which are useful as implantable and absorbable biomaterial in medical and pharmaceutical fields. Transformant Cupriavidus sp. USMAA1020 with an additional PHA synthase gene, phaC was found to produce P(3HB-co-4HB) with 86 mol% of 4HB monomer composition and high PHA content of 69 wt% in shake flask cultivation using mixed substrate of 1,6-hexanediol and 1,4-butanediol. Single-stage cultivation in 3 L fermentation has confirmed the ability of this strain to produce high 4HB monomer composition of 95 mol% in large scale fermentation with 75 wt% PHA content and high PHA concentration of 18.7 g/L. Interestingly, this strain was capable of surviving higher carbon concentration (1.05 wt% C) than the wild-type strain (0.69 wt% C). The present study results in P(3HB-co-4HB) with 1.3 and 2.3-fold PHA content and concentration respectively, with the ability to accumulate 2.1-fold 4HB monomer composition higher compared to the wild-type strain. A higher specific growth rate of 0.123 h−1 accompanied by high product yield, Yp/x of 3.8 times more than the wild-type strain were also obtained. Image from TEM showed cells with PHA content of 75 wt%, whichwere occupied with significant PHA granule. This copolymer possesses an average molecular weight (M w) and a polydispersity index of 156 kDa and 3.5 respectively with a tensile strength, elongation at break and Young’s modulus of 22.9 MPa, 463.2% and 187.3 MPa respectively. This polymer has a glass transition temperature (T g) and melting temperature (T m) of −48.9 °C and 61.9 °C respectively.

A fed-batch strategy to produce high poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) terpolymer yield with enhanced mechanical properties in bioreactor.

This study reports an efficient fed-batch strategy to improve poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer production by Cupriavidus sp. USMAA2-4 with enhanced mechanical properties in bioreactor. The cultivations have been performed by combining oleic acid with γ-butyrolactone at different concentration ratios with 1-pentanol at a fixed concentration. The batch and fed-batch fermentations have resulted in P(3HB-co-3HV-co-4HB) with compositions of 9-35mol% 3HV and 4-24mol% 4HB monomers. The DO-stat fed-batch fermentation strategies have significantly improved the production with a maximum 4.4-fold increment of cell dry weight (CDW). Besides, appropriate feeding of the substrates has resulted inan increment of terpolymer productivity from 0.086-0.347 g/L/h, with a significantly shortened cultivation time. The bacterial growth and terpolymer formation have been found to be affected by the concentration of carbon sources supplied. Characterization of P(3HB-co-3HV-co-4HB) has demonstrated that incorporation of 3HV and 4HB monomer has significantly improved the physical and thermodynamic properties of the polymers, by reducing the polymer's crystallinity. The tensile strength, Young's modulus of the terpolymer has been discovered to increase with the increase of M w. The fed-batch fermentation strategies employed in this study have resulted in terpolymers with a range of flexible materials having improved tensile strength and Young's modulus as compared to the terpolymer produced from batch fermentation. Possession of lower melting temperature indicates an enhanced thermal stability which broadens the polymer processing window.

En route to economical eco-friendly solvent system in enhancing sustainable recovery of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer.

Separation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco-balance as P(3HB-co-4HB) is widely used for biomedical applications. Therefore, an efficient and eco-based extraction of P(3HB-co-4HB) using a combination of NaOH and Lysol in digesting the non-polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB-co-4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.

Fed-Batch Synthesis of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate) from Sucrose and 4-Hydroxybutyrate Precursors by Burkholderia sacchari Strain DSM 17165.

Based on direct sucrose conversion, the bacterium Burkholderia sacchari is an excellent producer of the microbial homopolyester poly(3-hydroxybutyrate) (PHB). Restrictions of the strain’s wild type in metabolizing structurally related 3-hydroxyvalerate (3HV) precursors towards 3HV-containing polyhydroxyalkanoate (PHA) copolyester calls for alternatives. We demonstrate the highly productive biosynthesis of PHA copolyesters consisting of 3-hydroxybuytrate (3HB) and 4-hydroxybutyrate (4HB) monomers. Controlled bioreactor cultivations were carried out using saccharose from the Brazilian sugarcane industry as the main carbon source, with and without co-feeding with the 4HB-related precursor γ-butyrolactone (GBL). Without GBL co-feeding, the homopolyester PHB was produced at a volumetric productivity of 1.29 g/(L·h), a mass fraction of 0.52 g PHB per g biomass, and a final PHB concentration of 36.5 g/L; the maximum specific growth rate µmax amounted to 0.15 1/h. Adding GBL, we obtained 3HB and 4HB monomers in the polyester at a volumetric productivity of 1.87 g/(L·h), a mass fraction of 0.72 g PHA per g biomass, a final PHA concentration of 53.7 g/L, and a µmax of 0.18 1/h. Thermoanalysis revealed improved material properties of the second polyester in terms of reduced melting temperature Tm (161 °C vs. 178 °C) and decreased degree of crystallinity Xc (24% vs. 71%), indicating its enhanced suitability for polymer processing.