Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

Microbial Degradation Behavior in Seawater of Polyester Blends Containing Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx).

The microbial degradation behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and its compound with several polyesters such as poly(butylene adipate-co-telephtharate) (PBAT), poly(butylene succinate) (PBS), and polylactic acid (PLA) in seawater was tested by a biological oxygen demand (BOD) method. PHBHHx showed excellent biodegradation in seawater in this study. In addition, the biodegradation rate of several blends was much influenced by the weight ratio of PHBHHx in their blends and decreased in accordance with the decrement of PHBHHX ratio. The surface morphology of the sheet was important factor for controlling the biodegradation rate of PHBHHx-containing blends in seawater.

Production of (3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer from coffee waste oil using engineered Ralstonia eutropha.

Polyhydroxyalkonate (PHA) is a type of polymer that has the potential to replace petro-based plastics. To make PHA production more economically feasible, there is a need to find a new carbon source and engineer microbes to produce a commercially valuable polymer. Coffee waste is an inexpensive raw material that contains fatty acids. It can act as a sustainable carbon source and seems quite promising with PHA production in Ralstonia eutropha, which is a well-known microbe for PHA accumulation, and has the potential to utilize fatty acids. In this study, to make poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)), which has superior properties in terms of biodegradability, biocompatibility, and mechanical strength, engineered strain Ralstonia eutropha Re2133 overexpressing (R)-specific enoyl coenzyme-A hydratase (phaJ) and PHA synthetase (phaC2) with deletion of acetoacetyl Co-A reductases (phaB1, phaB2, and phaB3) was used to produce PHA from coffee waste oil. At a coffee oil concentration of 1.5%, and C/N ratio of 20, the R. eutropha Re2133 fermentation process results in 69% w/w of DCW PHA accumulation and consists of HB (78 mol%) and HHx (22 mol%). This shows the feasibility of using coffee waste oil for P(HB-co-HHx) production, as it is a low-cost fatty acid enriched waste material.

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Insights into biofilm development on polyhydroxyalkanoate biofilm carrier for anoxic azo dye decolourization of acid orange 7.

Polyhydroxyalkanoates (PHAs) are promising alternatives to non-degradable polymers in various applications. This study explored the use of biologically recovered PHA as a biofilm carrier in a moving bed biofilm reactor for acid orange 7 treatment. The PHA was comprised of 86 ± 1 mol% of 3-hydroxybutyrate and 14 ± 1 mol% of 3-hydroxyhexanoate and was melt-fused at 140 °C into pellets. The net positive surface charge of the PHA biocarrier facilitated attachment of negatively charged activated sludge, promoting biofilm formation. A 236-µm mature biofilm developed after 26 days. The high polysaccharides-to-protein ratio (>1) in the biofilm's extracellular polymeric substances indicated a stable biofilm structure. Four main microbial strains in the biofilm were identified as Leclercia adecarboxylata, Leuconostoc citreum, Bacillus cereus, and Rhodotorula mucilaginosa, all of which exhibited decolourization abilities. In conclusion, PHA holds promise as an effective biocarrier for biofilm development, offering a sustainable alternative in wastewater treatment applications.

Fabrication of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/ZnO Nanocomposite Films for Active Packaging Applications: Impact of ZnO Type on Structure-Property Dynamics.

Bio-based and biodegradable polyhydroxyalkanoates (PHAs) have great potential as sustainable packaging materials. The incorporation of zinc oxide nanoparticles (ZnO NPs) could further improve their functional properties by providing enhanced barrier and antimicrobial properties, although current literature lacks details on how the characteristics of ZnO influence the structure-property relationships in PHA/ZnO nanocomposites. Therefore, commercial ZnO NPs with different morphologies (rod-like, spherical) and silane surface modification are incorporated into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) via extrusion and compression molding. All ZnO NPs are homogeneously distributed in the PHBHHx matrix at 1, 3 and 5 wt.%, but finer dispersion is achieved with modified ZnO. No chemical interactions between ZnO and PHBHHx are observed due to a lack of hydroxyl groups on ZnO. The fabricated nanocomposite films retain the flexible properties of PHBHHx with minimal impact of ZnO NPs on crystallization kinetics and the degree of crystallinity (53 to 56%). The opacity gradually increases with ZnO loading, while remaining translucent up to 5 wt.% ZnO and providing an effective UV barrier. Improved oxygen barrier and antibacterial effects against S. aureus are dependent on the intrinsic characteristics of ZnO rather than its morphology. We conclude that PHBHHx retains its favorable processing properties while producing nanocomposite films that are suitable as flexible active packaging materials.

Tuning the Structure-Property Relationships in Binary and Ternary Blends of PLA/PBAT/PHBH.

While the brittle polylactide (PLA) has a high durability among bioplastics, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) with certain ductility exhibits facile compostability. The addition of polybutylene adipate terephthalate (PBAT) may also be used to improve the ductility and toughness of brittle bioplastics. Binary and ternary blends of PLA/PBAT/PHBH based on either PLA or PHBH as the matrix have been manufactured using a twin-screw extruder. The melt rheological, mechanical, and morphological properties of the processed samples were examined. Binary blends of PLA/PHBH show superior strength, with the PLA75/PHBH25 blend exhibiting a tensile strength of 35.2 ± 3.0 MPa, which may be attributed to miscible-like morphology. In contrast, blends of PLA with PBAT demonstrate low strength, with the PLA50/PBAT50 blend exhibits a tensile strength of 9.5 ± 2.0 MPa due to the presence of large droplets in the matrix. PBAT-containing blends exhibit lower impact strengths compared to PHBH-containing blends. For instance, a PLA75/PBAT25 blend displays an impact strength of 1.76 ± 0.1 kJ/m2, whereas the PHBH75/PBAT25 blend displays an impact strength of 2.61 ± 0.3 kJ/m2, which may be attributed to uniformly dispersed PBAT droplets.

Gas fermentation combined with water electrolysis for production of polyhydroxyalkanoate copolymer from carbon dioxide by engineered Ralstonia eutropha.

A recycled-gas closed-circuit culture system was developed for safe autotrophic cultivation of a hydrogen-oxidizing, polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha, using a non-combustible gas mixture with low-concentration of H2 supplied by water electrolysis. Automated feedback regulation of gas flow enabled input of H2, CO2, and O2 well balanced with the cellular demands, leading to constant gas composition throughout the cultivation. The engineered strain of R. eutropha produced 1.71 g/L of poly(3-hydroxybutyrate-co-12.5 mol% 3-hydroxyhexanoate) on a gas mixture of H2/CO2/O2/N2 = 4:12:7:77 vol% with a 69.2 wt% cellular content. Overexpression of can encoding cytosolic carbonic anhydrase increased the 3HHx fraction up to 19.6 mol%. The yields of biomass and PHA on input H2 were determined to be 72.9 % and 63.1 %, corresponding to 51.0 % and 44.2 % yield on electricity, respectively. The equivalent solar-to-biomass/PHA efficiencies were estimated to be 2.1-3.8 %, highlighting the high energy conversion capability of R. eutropha.

Optimization of Cellulose Nanofiber Loading and Processing Conditions during Melt Extrusion of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Bionanocomposites.

This present study optimized the cellulose nanofiber (CNF) loading and melt processing conditions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(HB-co-11% HHx) bionanocomposite fabrication in twin screw extruder by using the response surface methodology (RSM). A face-centered central composite design (CCD) was applied to statistically specify the important parameters, namely CNF loading (1-9 wt.%), rotational speed (20-60 rpm), and temperature (135-175 °C), on the mechanical properties of the P(HB-co-11% HHx) bionanocomposites. The developed model reveals that CNF loading and temperature were the dominating parameters that enhanced the mechanical properties of the P(HB-co-11% HHx)/CNF bionanocomposites. The optimal CNF loading, rotational speed, and temperature for P(HB-co-11% HHx) bionanocomposite fabrication were 1.5 wt.%, 20 rpm, and 160 °C, respectively. The predicted tensile strength, flexural strength, and flexural modulus for these optimum conditions were 22.96 MPa, 33.91 MPa, and 1.02 GPa, respectively, with maximum desirability of 0.929. P(HB-co-11% HHx)/CNF bionanocomposites exhibited improved tensile strength, flexural strength, and modulus by 17, 6, and 20%, respectively, as compared to the neat P(HB-co-11% HHx). While the crystallinity of P(HB-co-11% HHx)/CNF bionanocomposites increased by 17% under the optimal fabrication conditions, the thermal stability of the P(HB-co-11% HHx)/CNF bionanocomposites was not significantly different from neat P(HB-co-11% HHx).

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Fibers Using Centrifugal Fiber Spinning: Structure, Properties and Application Potential.

Biobased and biodegradable polyhydroxyalkanoates (PHAs) are currently gaining momentum. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) polymer has a useful processing window for extrusion and injection molding of packaging, agricultural and fishery applications with required flexibility. Processing PHBHHx into fibers using electrospinning or centrifugal fiber spinning (CFS) can further broaden the application area, although CFS remains rather unexplored. In this study, PHBHHx fibers are centrifugally spun from 4-12 wt.% polymer/chloroform solutions. Beads and beads-on-a-string (BOAS) fibrous structures with an average diameter (ϕav) between 0.5 and 1.6 µm form at 4-8 wt.% polymer concentrations, while more continuous fibers (ϕav = 3.6-4.6 µm) with few beads form at 10-12 wt.% polymer concentrations. This change is correlated with increased solution viscosity and enhanced mechanical properties of the fiber mats (strength, stiffness and elongation values range between 1.2-9.4 MPa, 11-93 MPa, and 102-188%, respectively), though the crystallinity degree of the fibers remains constant (33.0-34.3%). In addition, PHBHHx fibers are shown to anneal at 160 °C in a hot press into 10-20 µm compact top-layers on PHBHHx film substrates. We conclude that CFS is a promising novel processing technique for the production of PHBHHx fibers with tunable morphology and properties. Subsequent thermal post-processing as a barrier or active substrate top-layer offers new application potential.

Ternary Blends from Biological Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), Poly(propylene carbonate) and Poly(vinyl acetate) with Balanced Properties.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has gained significant attention because of its biodegradability and sustainability. However, its expanded application in some fields is limited by the brittleness and low melt viscoelasticity. In this work, poly(vinyl acetate) (PVAc) was introduced into PHBH/poly(propylene carbonate) (PPC) blends via melt compounding with the aim of obtaining a good balance of properties. Dynamic mechanical analysis results suggested that PPC and PHBH were immiscible. PVAc was miscible with both a PHBH matrix and PPC phase, while it showed better miscibility with PHBH than with PPC. Therefore, PVAc was selectively localized in a PHBH matrix, reducing interfacial tension and refining dispersed phase morphology. The crystallization rate of PHBH slowed down, and the degree of crystallinity decreased with the introduction of PPC and PVAc. Moreover, the PVAc phase significantly improved the melt viscoelasticity of ternary blends. The most interesting result was that the remarkable enhancement of toughness for PHBH/PPC blends was obtained by adding PVAc without sacrificing the strength markedly. Compared with the PHBH/PPC blend, the elongation at the break and yield strength of the PHBH/PPC/10PVAc blend increased by 1145% and 7.9%, respectively. The combination of high melt viscoelasticity, toughness and strength is important for the promotion of the practical application of biological PHBH.

Effects of Marine Sand on the Microbial Degradation of Biodegradable Plastics in Seawater and Biofilm Communities that Formed on Plastic Surfaces.

Four types of biodegradable plastics were evaluated for their biodegradability in seawater collected at Ajigaura coast, Japan, in the presence or absence of marine sand. One of the plastics, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), showed a degree of biodegradation in a seawater sample, and the addition of marine sand markedly accelerated its biodegradation. The addition of marine sand did not affect the bacterial composition of the biofilm that formed on PHBH, and the family Rhodobacteraceae, which was predicted to contribute to the degradation of PHBH, was dominant in biofilm communities regardless of the addition of marine sand. Marine sand may serve as a bacterial source, resulting in the accelerated degradation of PHBH.

Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) From Glucose by Escherichia coli Through Butyryl-CoA Formation Driven by Ccr-Emd Combination.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is a practical kind of bacterial polyhydroxyalkanoates (PHAs). A previous study has established an artificial pathway for the biosynthesis of P(3HB-co-3HHx) from structurally unrelated sugars in Ralstonia eutropha, in which crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) are a key combination for generation of butyryl-CoA and the following chain elongation. This study focused on the installation of the artificial pathway into Escherichia coli. The recombinant strain of E. coli JM109 harboring 11 heterologous genes including Ccr and Emd produced P(3HB-co-3HHx) composed of 14 mol% 3HHx with 41 wt% of dry cellular weight from glucose. Further investigations revealed that the C6 monomer (R)-3HHx-CoA was not supplied by (R)-specific reduction of 3-oxohexanoyl-CoA but by (R)-specific hydration of 2-hexenoyl-CoA formed through reverse ß-oxidation after the elongation from C4 to C6. While contribution of the reverse ß-oxidation to the conversion of the C4 intermediates was very limited, crotonyl-CoA, a precursor of butyryl-CoA, was generated by dehydration of (R)-3HB-CoA. Several modifications previously reported for enhancement of bioproduction in E. coli were examined for the copolyester synthesis. Elimination of the global regulator Cra or PdhR as well as the block of acetate formation resulted in poor PHA synthesis. The strain lacking RNase G accumulated more PHA but with almost no 3HHx unit. Introduction of the phosphite oxidation system for regeneration of NADPH led to copolyester synthesis with the higher cellular content and higher 3HHx composition by two-stage cultivation with phosphite than those in the absence of phosphite.

A review on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] and genetic modifications that affect its production.

Polyhydroxyalkanoates (PHAs) have garnered global attention to replace petroleum-based plastics in certain applications due to their biodegradability and sustainability. Among the different types of PHAs, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] copolymer has similar properties to commodity plastics, making them a suitable candidate to replace certain types of single-use plastics, medical devices, and packaging materials. The degradation rate of P(3HB-co-3HHx) is faster than the commercial petroleum-based plastics which take a very long time to be degraded, causing harmful pollution to both land and marine ecosystem. The biodegradability of the P(3HB-co-3HHx) is also dependent on its 3HHx molar composition which in turn influences the crystallinity of the material. Various metabolic pathways like the common PHA biosynthesis pathway, which involves phaA, phaB, and phaC, ß-oxidation, and fatty acids de novo synthesis are used by bacteria to produce PHA from different carbon sources like fatty acids and sugars, respectively. There are various factors affecting the 3HHx molar composition of P(3HB-co-3HHx), like PhaCs, the engineering of PhaCs, and the metabolic engineering of strains. It is crucial to control the 3HHx molar composition in the P(3HB-co-3HHx) as it will affect its properties and applications in different fields.

Development and Characterization of Fully Renewable and Biodegradable Polyhydroxyalkanoate Blends with Improved Thermoformability.

Poly(3-hydroxybutyrate-co-3-valerate) (PHBV), being one of the most studied and commercially available polyhydroxyalkanoates (PHAs), presents an intrinsic brittleness and narrow processing window that currently hinders its use in several plastic applications. The aim of this study was to develop a biodegradable PHA-based blend by combining PHBV with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), another copolyester of the PHA family that shows a more ductile behavior. Blends of PHBV with 20% wt., 30% wt., and 40% wt. of PHBH were obtained by melt mixing, processed by cast extrusion in the form of films, and characterized in terms of their morphology, crystallization behavior, thermal stability, mechanical properties, and thermoformability. Full miscibility of both biopolymers was observed in the amorphous phase due to the presence of a single delta peak, ranging from 4.5 °C to 13.7 °C. Moreover, the incorporation of PHBH hindered the crystallization process of PHBV by decreasing the spherulite growth rate from 1.0 µm/min to 0.3 µm/min. However, for the entire composition range studied, the high brittleness of the resulting materials remained since the presence of PHBH did not prevent the PHBV crystalline phase from governing the mechanical behavior of the blend. Interestingly, the addition of PHBH greatly improved the thermoformability by widening the processing window of PHBV by 7 s, as a result of the increase in the melt strength of the blends even for the lowest PHBH content.

Effect of Graphene Oxide on the Properties of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate).

The main shortcomings of polyhydroxybutyrate (PHB), which is a biodegradable and biocompatible polymer used for biomedical and food packaging applications, are its low thermal stability, poor impact resistance and lack of antibacterial activity. This issue can be improved by blending with other biodegradable polymers such as polyhydroxyhexanoate to form poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which is a copolymer with better impact strength and lower melting point. However, PHBHHx shows reduced stiffness than PHB and poorer barrier properties against moisture and gases, which is a drawback for use in the food industry. In this regard, novel biodegradable PHBHHx/graphene oxide (GO) nanocomposites have been prepared via a simple, cheap and environmentally friendly solvent casting method to enhance the mechanical properties and antimicrobial activity. The morphology, mechanical, thermal, barrier and antibacterial properties of the nanocomposites were assessed via several characterization methods to show the enhancement in the biopolymer properties. The stiffness and strength of the biopolymer were enhanced up to 40% and 28%, respectively, related to the strong matrix-nanofiller interfacial adhesion attained via hydrogen bonding interactions. Moreover, the nanocomposites showed superior thermal stability (as far as 40 °C), lower water uptake (up to 70%) and better gas and vapour barrier properties (about 45 and 35% reduction) than neat PHBHHx. They also displayed strong biocide action against Gram positive and Gram negative bacteria. These bio-based nanocomposites with antimicrobial activity offer new perspectives for the replacement of traditional petroleum-based synthetic polymers currently used for food packaging.

Oxygen Gas and UV Barrier Properties of Nano-ZnO-Coated PET and PHBHHx Materials Fabricated by Ultrasonic Spray-Coating Technique.

Ultrasonic spray-coating (USSC)-a wet chemical deposition method to deposit ultrathin (down to 20 nm) coatings-is being applied as a promising alternative deposition method for functional coatings due to an economical, simple, and precise coating process with easy control over its operating parameters. In this research, zinc oxide nanoparticles (ZnO NPs) were ultrasonically spray-coated on commercial-grade polyethylene terephthalate (PET) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) films. The most suitable parameters for the ink composition, the ultrasonic spray-coating process, and the number of coating passes (up to 50×) were selected on the basis of a series of experiments. The oxygen gas barrier properties in terms of the oxygen transmission rate (OTR) of neat PET, and 3×, 5×, 10×, and 50× ZnO NP-coated PET and PHBHHx substrates were investigated. The OTR values for neat PET, and 3×, 5×, and 10× ZnO NP-coated PET substrates were found to be the same; however, a 5% reduction in OTR for 50× ZnO NP-coated PET substrate was observed compared to the neat PET substrate. No reduction in OTR was found for any above number of coating passes on PHBHHx substrates against the neat PHBHHx substrate. However, the ultraviolet (UV) tests of 3×, 5×, and 10× ZnO NP-coated PET and PHBHH× substrates revealed a significant decrease in percentage transmission for 10× coated PET and PHBHHx substrates as compared to their 3× and 5× ZnO NP-coated substrates, respectively. It was revealed from the study that the 50× ZnO NP coating of the PET substrate created a slight difference in OTR as compared to the reference substrate. However, the ultrasonic spray-coating method created a significant UV barrier effect for 3×, 5×, and 10× ZnO NP-coated PET and PHBHHx substrates, which demonstrates that the optimized coating method cannot be used to create a high oxygen barrier but can certainly be applied for UV barrier applications in food packaging. It is concluded that ultrasonic spray deposition of ZnO NPs on PET and PHBHHx materials has shown promising results for UV barrier properties, demonstrating the advantages of using this method compared to other coating methods with regard to cost-effectiveness, precise coating, and better process control.

Extrusion and Injection Molding of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx): Influence of Processing Conditions on Mechanical Properties and Microstructure.

Biobased and biodegradable polyhydroxyalkanoates (PHAs) have great potential as sustainable packaging materials. However, improvements in their processing and mechanical properties are necessary. In this work, the influence of melt processing conditions on the mechanical properties and microstructure of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is examined using a full factorial design of experiments (DoE) approach. We have found that strict control over processing temperature, mold temperature, screw speed, and cooling time leads to highly increased elongation at break values, mainly under influence of higher mold temperatures at 80 °C. Increased elongation of the moldings is attributed to relaxation and decreased orientation of the polymer chains together with a homogeneous microstructure at slower cooling rates. Based on the statistically substantiated models to determine the optimal processing conditions and their effects on microstructure variation and mechanical properties of PHBHHx samples, we conclude that optimizing the processing of this biopolymer can improve the applicability of the material and extend its scope in the realm of flexible packaging applications.