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.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Ring Opening Polymerization of Six- and Eight-Membered Racemic Cyclic Esters for Biodegradable Materials.

The low percentage of recyclability of the polymeric materials obtained by olefin transition metal (TM) polymerization catalysis has increased the interest in their substitution with more eco-friendly materials with reliable physical and mechanical properties. Among the variety of known biodegradable polymers, linear aliphatic polyesters produced by ring-opening polymerization (ROP) of cyclic esters occupy a prominent position. The polymer properties are highly dependent on the macromolecule microstructure, and the control of stereoselectivity is necessary for providing materials with precise and finely tuned properties. In this review, we aim to outline the main synthetic routes, the physical properties and also the applications of three commercially available biodegradable materials: Polylactic acid (PLA), Poly(Lactic-co-Glycolic Acid) (PLGA), and Poly(3-hydroxybutyrate) (P3HB), all of three easily accessible via ROP. In this framework, understanding the origin of enantioselectivity and the factors that determine it is then crucial for the development of materials with suitable thermal and mechanical properties.

Environmental sustainability assessment of biodegradable bio-based poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from agro-residues: Production and end-of-life scenarios.

In the context of a circular bio-based economy, more public attention has been paid to the environmental sustainability of biodegradable bio-based plastics, particularly plastics produced using emerging biotechnologies, e.g. poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV. However, this has not been thoroughly investigated in the literature. Therefore, this study aimed to address three aspects regarding the environmental impact of PHBV-based plastic: (i) the potential environmental benefits of scaling up pellet production from pilot to industrial scale and the environmental hotspots at each scale, (ii) the most favourable end-of-life (EOL) scenario for PHBV, and (iii) the environmental performance of PHBV compared to benchmark materials considering both the pellet production and EOL stages. Life cycle assessment (LCA) was implemented using Cumulative Exergy Extraction from the Natural Environment (CEENE) and Environmental Footprint (EF) methods. The results show that, firstly, when upscaling the PHBV pellet production from pilot to industrial scale, a significant environmental benefit can be achieved by reducing electricity and nutrient usage, together with the implementation of better practices such as recycling effluent for diluting feedstock. Moreover, from the circularity perspective, mechanical recycling might be the most favourable EOL scenario for short-life PHBV-based products, using the carbon neutrality approach, as the material remains recycled and hence environmental credits are achieved by substituting recyclates for virgin raw materials. Lastly, PHBV can be environmentally beneficial equal to or even to some extent greater than common bio- and fossil-based plastics produced with well-established technologies. Besides methodological choices, feedstock source and technology specifications (e.g. pure or mixed microbial cultures) were also identified as significant factors contributing to the variations in LCA of (bio)plastics; therefore, transparency in reporting these factors, along with consistency in implementing the methodologies, is crucial for conducting a meaningful comparative LCA.

Comparison effect of collagen/P3HB composite scaffold and human amniotic membrane loaded with mesenchymal stem cells on colon anastomosis healing in male rats.

Covering surgical wounds with biomaterials, biologic scaffolds, and mesenchymal stem cells (MSCs) improves the healing process and reduces postoperative complications. This study was designed to evaluate and compare the effect of MSC-free/MSC-seeded new collagen/poly(3-hydroxybutyrate) (COL/P3HB) composite scaffold and human amniotic membrane (HAM) on the colon anastomosis healing process. COL/P3HB scaffold was prepared using freeze-drying method. MSCs were isolated and characterized from rat adipose tissue. After biocompatibility evaluation by MTT assay, MSCs were seeded on the scaffold and HAM by micro-mass seeding technique. In total, 35 male rats were randomly divided into five groups. After the surgical procedure, cecum incisions were covered by the MSC-free/MSC-seeded scaffold or HAM. Incisions in the control group were only sutured. One month later, the healing process was determined by stereological analysis. The Kruskal-Wallis followed by Dunn's tests were utilized for statistical outcome analysis (SPSS software version 21). COL/10% P3HB scaffold showed the best mechanical and structural properties (7.86 MPa strength, porosity more than 75%). MTT assay indicated that scaffold and especially HAM have suitable biocompatibility. Collagenization and neovascularization were significantly higher, and necrosis was considerably lower in all treated groups in comparison with the controls. MSC-seeded scaffold and HAM significantly decrease inflammation and increase gland volume compared with other groups. The MSC-seeded HAM was significantly successful in decreasing edema compared with other groups. Newly synthesized COL/P3HB scaffold improves the colon anastomosis healing; however, the major positive effect belonged to HAM. MSCs remarkably increase their healing process. Further investigations may contribute to confirming these results in other wound healing.

Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

IMPORTANCE: Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Production of poly (3-hydroxybutyrate) and extracellular polymeric substances from glycerol by the acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic, and metallotolerant bacteria able to use dissolved oxygen or Fe(III) as an electron sink. The ability of this extremophile to accumulate poly(3-hydroxybutyrate) (PHB) and secrete extracellular polymeric substances (EPS) has also been reported. Hence, the aim of this work is to characterize the production of PHB and EPS by the wild strain DSM2389 using glycerol in shaken flasks and bioreactor. Results showed that maximum PHB accumulation (37-42% w/w) was obtained using glycerol concentrations of 9 and 15 g L-1, where maximum dry cell weight titers reached 3.6 and 3.9 g L-1, respectively. The culture in the bioreactor showed that PHB accumulation takes place under oxygen limitation, while the redox potential of the culture medium could be used for online monitoring of the PHB production. Recovered EPS was analyzed by Fourier-transform infrared spectroscopy and subjected to gas chromatography-mass spectrometry after cleavage and derivatization steps. These analyses showed the presence of sugars which were identified as mannose, rhamnose and glucose, in a proportion near to 3.2:2.3:1, respectively. Since glycerol had not been used in previous works, these findings suggest the potential of A. cryptum to produce biopolymers from this compound at a large scale with a low risk of microbial contamination due to the low pH of the fermentation process.

Rapid combinatorial rewiring of metabolic networks for enhanced poly(3-hydroxybutyrate) production in Corynebacterium glutamicum.

BACKGROUND: The disposal of plastic waste is a major environmental challenge. With recent advances in microbial genetic and metabolic engineering technologies, microbial polyhydroxyalkanoates (PHAs) are being used as next-generation biomaterials to replace petroleum-based synthetic plastics in a sustainable future. However, the relatively high production cost of bioprocesses hinders the production and application of microbial PHAs on an industrial scale. RESULTS: Here, we describe a rapid strategy to rewire metabolic networks in an industrial microorganism, Corynebacterium glutamicum, for the enhanced production of poly(3-hydroxybutyrate) (PHB). A three-gene PHB biosynthetic pathway in Rasltonia eutropha was refactored for high-level gene expression. A fluorescence-based quantification assay for cellular PHB content using BODIPY was devised for the rapid fluorescence-activated cell sorting (FACS)-based screening of a large combinatorial metabolic network library constructed in C. glutamicum. Rewiring metabolic networks across the central carbon metabolism enabled highly efficient production of PHB up to 29% of dry cell weight with the highest cellular PHB productivity ever reported in C. glutamicum using a sole carbon source. CONCLUSIONS: We successfully constructed a heterologous PHB biosynthetic pathway and rapidly optimized metabolic networks across central metabolism in C. glutamicum for enhanced production of PHB using glucose or fructose as a sole carbon source in minimal media. We expect that this FACS-based metabolic rewiring framework will accelerate strain engineering processes for the production of diverse biochemicals and biopolymers.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Aerobic-anaerobic transition boosts poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Rhodospirillum rubrum: the key role of carbon dioxide.

BACKGROUND: Microbially produced bioplastics are specially promising materials since they can be naturally synthesized and degraded, making its end-of-life management more amenable to the environment. A prominent example of these new materials are polyhydroxyalkanoates. These polyesters serve manly as carbon and energy storage and increase the resistance to stress. Their synthesis can also work as an electron sink for the regeneration of oxidized cofactors. In terms of biotechnological applications, the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV, has interesting biotechnological properties due to its lower stiffness and fragility compared to the homopolymer poly(3-hydroxybutyrate) (P3HB). In this work, we explored the potentiality of Rhodospirillum rubrum as a producer of this co-polymer, exploiting its metabolic versatility when grown in different aeration conditions and photoheterotrophically. RESULTS: When shaken flasks experiments were carried out with limited aeration using fructose as carbon source, PHBV production was triggered reaching 29 ± 2% CDW of polymer accumulation with a 75 ± 1%mol of 3-hydroxyvalerate (3HV) (condition C2). Propionate and acetate were secreted in this condition. The synthesis of PHBV was exclusively carried out by the PHA synthase PhaC2. Interestingly, transcription of cbbM coding RuBisCO, the key enzyme of the Calvin-Benson-Bassham cycle, was similar in aerobic and microaerobic/anaerobic cultures. The maximal PHBV yield (81% CDW with 86%mol 3HV) was achieved when cells were transferred from aerobic to anaerobic conditions and controlling the CO2 concentration by adding bicarbonate to the culture. In these conditions, the cells behaved like resting cells, since polymer accumulation prevailed over residual biomass formation. In the absence of bicarbonate, cells could not adapt to an anaerobic environment in the studied lapse. CONCLUSIONS: We found that two-phase growth (aerobic-anaerobic) significantly improved the previous report of PHBV production in purple nonsulfur bacteria, maximizing the polymer accumulation at the expense of other components of the biomass. The presence of CO2 is key in this process demonstrating the involvement of the Calvin-Benson-Bassham in the adaptation to changes in oxygen availability. These results stand R. rubrum as a promising producer of high-3HV-content PHBV co-polymer from fructose, a PHBV unrelated carbon source.

Co-Processing Agricultural Residues and Wet Organic Waste Can Produce Lower-Cost Carbon-Negative Fuels and Bioplastics.

Scalable, low-cost biofuel and biochemical production can accelerate progress on the path to a more circular carbon economy and reduced dependence on crude oil. Rather than producing a single fuel product, lignocellulosic biorefineries have the potential to serve as hubs for the production of fuels, production of petrochemical replacements, and treatment of high-moisture organic waste. A detailed techno-economic analysis and life-cycle greenhouse gas assessment are developed to explore the cost and emission impacts of integrated corn stover-to-ethanol biorefineries that incorporate both codigestion of organic wastes and different strategies for utilizing biogas, including onsite energy generation, upgrading to bio-compressed natural gas (bioCNG), conversion to poly(3-hydroxybutyrate) (PHB) bioplastic, and conversion to single-cell protein (SCP). We find that codigesting manure or a combination of manure and food waste alongside process wastewater can reduce the biorefinery's total costs per metric ton of CO2 equivalent mitigated by half or more. Upgrading biogas to bioCNG is the most cost-effective climate mitigation strategy, while upgrading biogas to PHB or SCP is competitive with combusting biogas onsite.

High performance filtration membranes from electrospun poly (3-hydroxybutyrate)-based fiber membranes for fine particulate protection.

PM2.5 (particulate matter with a size of <2.5 µm) pollution has become a critical issue owing to its adverse health effects, including bronchitis, pneumonopathy, and cardiovascular diseases. Globally, around 8.9 million premature casualties related to exposure to PM2.5 were reported. Face masks are the only option that may restrict exposure to PM2.5. In this study, a PM2.5 dust filter was developed via the electrospinning technique using the poly (3-hydroxybutyrate) (PHB) biopolymer. Smooth and continuous fibers without beads were formed. The PHB membrane was further characterized, and the effects of the polymer solution concentration, applied voltage, and needle-to-collector distance were analyzed via the design of experiments technique, with three factors and three levels. The concentration of the polymer solution had the most significant effect on the fiber size and the porosity. The fiber diameter increased with increasing concentration, but decreases the porosity. The sample with a fiber diameter of ∼600 nm exhibited a higher PM2.5 filtration efficiency than the samples with a diameter of 900 nm, according to an ASTM F2299-based test. The PHB fiber mats fabricated at a concentration of 10%w/v, applied voltage of 15 kV, and needle tip-to-collector distance of 20 cm exhibited a high filtration efficiency of 95% and a pressure drop of <5 mmH2O/cm2. The tensile strength of the developed membranes ranged from 2.4 to 5.01 MPa, higher than those of the mask filters available in the market. Therefore, the prepared electrospun PHB fiber mats have great potential for the manufacture of PM2.5 filtration membranes.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxvalerate) from volatile fatty acids by Cupriavidus necator.

A promising strategy to alleviate the plastic pollution from traditional petroleum-based plastics is the application of biodegradable plastics, in which polyhydroxyalkanoates (PHAs) have received increasing interest owing to their considerable biodegradability. In the PHAs family, poly(3-hydroxybutyrate-co-3-hydroxvalerate) (PHBV) has better mechanical properties, which possesses broader application prospects. With this purpose, the present study adopted Cupriavidus necator to synthesize PHBV utilizing volatile fatty acids (VFAs) as sole carbon sources. Results showed that the concentration and composition of VFAs significantly influenced the production of PHAs. Especially, even carbon VFAs (acetate and butyrate) synthesized only poly(3-hydroxybutyrate) (PHB), while the addition of odd carbon VFAs (propionate and valerate) resulted in PHBV production. The 3-hydroxyvalerate (3HV) contents in PHBV were directly determined by the specific VFAs compositions, in which valerate was the preferred substrate for 3HV accumulation. After optimization by response surface methodology, the highest PHBV accumulation achieved 79.47% in dry cells, and the conversion efficiency of VFAs to PHBV reached 40%, with the PHBV production of 1.20 ± 0.05 g/L. This study revealed the metabolic rule of VFAs converting into PHAs by C. necator and figured out the optimal VFAs condition for PHBV accumulation, which provides a valuable reference for developing downstream strategies of PHBV production in industrial applications in future.

Poly(3-hydroxybutyrate) 3D-Scaffold-Conduit for Guided Tissue Sprouting.

Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique. In flat scaffolds (scaffold-1), one side was more porous (pore size 100-300 µm), while the other side was smoother (pore size 10-50 µm). Such scaffolds are suitable for the in vitro cultivation of rat mesenchymal stem cells and 3T3 fibroblasts, and, upon subcutaneous implantation to older rats, they cause moderate inflammation and the formation of a fibrous capsule. Scaffold-2s are homogeneous volumetric hard sponges (pore size 30-300 µm) with more structured pores. They were suitable for the in vitro culturing of 3T3 fibroblasts. Scaffold-2s were used to manufacture a conduit from the PHB/PHBV tube with scaffold-2 as a filler. The subcutaneous implantation of such conduits to older rats resulted in gradual soft connective tissue sprouting through the filler material of the scaffold-2 without any visible inflammatory processes. Thus, scaffold-2 can be used as a guide for connective tissue sprouting. The obtained data are advanced studies for reconstructive surgery and tissue engineering application for the elderly patients.

Biological and Oxidative Degradation of Ultrathin-Fibrous Nonwovens Based on Poly(lactic Acid)/Poly(3-Hydroxybutyrate) Blends.

Developing biodegradable materials based on polymer blends with a programmable self-destruction period in the environmental conditions of living systems is a promising direction in polymer chemistry. In this work, novel non-woven fibrous materials obtained by electrospinning based on the blends of poly(lactic acid) (PLA) and poly(3-hydroxybutyrate) (PHB) were developed. The kinetics of biodegradation was studied in the aquatic environment of the inoculum of soil microorganisms. Oxidative degradation was studied under the ozone gaseous medium. The changes in chemical composition and structure of the materials were studied by optical microscopy, DSC, TGA, and FTIR-spectroscopy. The disappearance of the structural bands of PHB in the IR-spectra of the blends and a significant decrease in the enthalpy of melting after 90 days of exposure in the inoculum indicated the biodegradation of PHB while PLA remained stable. It was shown that the rate of ozonation was higher for PLA and the blends with a high content of PLA. The lower density of the amorphous regions of the blends determined an increased rate of their oxidation by ozone compared to homopolymers. The optimal composition in terms of degradation kinetics is a fibrous material based on the blend of 30PLA/70PHB that can be used as an effective ecosorbent, for biopackaging, and as a highly porous covering material for agricultural purposes.

Adhesion of Escherichia coli and Lactobacillus fermentum to Films and Electrospun Fibrous Scaffolds from Composites of Poly(3-hydroxybutyrate) with Magnetic Nanoparticles in a Low-Frequency Magnetic Field.

The ability of materials to adhere bacteria on their surface is one of the most important aspects of their development and application in bioengineering. In this work, the effect of the properties of films and electrospun scaffolds made of composite materials based on biosynthetic poly(3-hydroxybutyrate) (PHB) with the addition of magnetite nanoparticles (MNP) and their complex with graphene oxide (MNP/GO) on the adhesion of E. coli and L. fermentum under the influence of a low-frequency magnetic field and without it was investigated. The physicochemical properties (crystallinity; surface hydrophilicity) of the materials were investigated by X-ray structural analysis, differential scanning calorimetry and "drop deposition" methods, and their surface topography was studied by scanning electron and atomic force microscopy. Crystal violet staining made it possible to reveal differences in the surface charge value and to study the adhesion of bacteria to it. It was shown that the differences in physicochemical properties of materials and the manifestation of magnetoactive properties of materials have a multidirectional effect on the adhesion of model microorganisms. Compared to pure PHB, the adhesion of E. coli to PHB-MNP/GO, and for L. fermentum to both composite materials, was higher. In the magnetic field, the adhesion of E. coli increased markedly compared to PHB-MNP/GO, whereas the effect on the adhesion of L. fermentum was reversed and was only evident in samples with PHB-MNP. Thus, the resultant factors enhancing and impairing the substrate binding of Gram-negative E. coli and Gram-positive L. fermentum turned out to be multidirectional, as they probably have different sensitivity to them. The results obtained will allow for the development of materials with externally controlled adhesion of bacteria to them for biotechnology and medicine.

Overview of Antimicrobial Biodegradable Polyester-Based Formulations.

As the clinical complications induced by microbial infections are known to have life-threatening side effects, conventional anti-infective therapy is necessary, but not sufficient to overcome these issues. Some of their limitations are connected to drug-related inefficiency or resistance and pathogen-related adaptive modifications. Therefore, there is an urgent need for advanced antimicrobials and antimicrobial devices. A challenging, yet successful route has been the development of new biostatic or biocide agents and biomaterials by considering the indisputable advantages of biopolymers. Polymers are attractive materials due to their physical and chemical properties, such as compositional and structural versatility, tunable reactivity, solubility and degradability, and mechanical and chemical tunability, together with their intrinsic biocompatibility and bioactivity, thus enabling the fabrication of effective pharmacologically active antimicrobial formulations. Besides representing protective or potentiating carriers for conventional drugs, biopolymers possess an impressive ability for conjugation or functionalization. These aspects are key for avoiding malicious side effects or providing targeted and triggered drug delivery (specific and selective cellular targeting), and generally to define their pharmacological efficacy. Moreover, biopolymers can be processed in different forms (particles, fibers, films, membranes, or scaffolds), which prove excellent candidates for modern anti-infective applications. This review contains an overview of antimicrobial polyester-based formulations, centered around the effect of the dimensionality over the properties of the material and the effect of the production route or post-processing actions.

Biomedical Applications of the Biopolymer Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV): Drug Encapsulation and Scaffold Fabrication.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biodegradable and biocompatible biopolymer that has gained popularity in the field of biomedicine. This review provides an overview of recent advances and potential applications of PHBV, with special emphasis on drug encapsulation and scaffold construction. PHBV has shown to be a versatile platform for drug delivery, offering controlled release, enhanced therapeutic efficacy, and reduced side effects. The encapsulation of various drugs, such as anticancer agents, antibiotics, and anti-inflammatory drugs, in PHBV nanoparticles or microspheres has been extensively investigated, demonstrating enhanced drug stability, prolonged release kinetics, and increased bioavailability. Additionally, PHBV has been used as a scaffold material for tissue engineering applications, such as bone, cartilage, and skin regeneration. The incorporation of PHBV into scaffolds has been shown to improve mechanical properties, biocompatibility, and cellular interactions, making them suitable for tissue engineering constructs. This review highlights the potential of PHBV in drug encapsulation and scaffold fabrication, showing its promising role in advancing biomedical applications.