Search Results (29)

Polyhydroxyalkanoates (PHAs) are polyesters synthesized as a carbon and energy reserve material by a wide number of bacteria. These polymers are characterized by their thermoplastic properties similar to those of plastics derived from the petrochemical industry, such as polyethylene and polypropylene. PHAs are widely used in the medical field and have the potential to be used in other applications due to their biocompatibility and biodegradability. Among PHAs, P(3HB-co-3HV) copolymers are thermo-elastomeric polyesters that are typically soft and flexible with low to no crystallinity, which can expand the range of applications of these bioplastics. Several bacterial species, such as Cupriavidus necator, Azotobacter vinelandii, Halomonas sp. and Bacillus megaterium, have been successfully used for P(3HB-co-3HV) production, both in batch and fed-batch cultures using different low-cost substrates, such as vegetable and fruit waste. Nevertheless, in recent years, several fermentation strategies using other microbial models, such as methanotrophic bacterial strains as well as halophilic bacteria, have been developed in order to improve PHA production in cultivation conditions that are easily implemented on a large scale. This review aims to summarize the recent trends in the production and recovery of PHA copolymers by fermentation, including different cultivation modalities, low-cost raw materials, as well as downstream strategies that have recently been developed with the purpose of producing copolymers, such as P(3HB-co-3HV), with suitable mechanical properties for applications in the biomedical field. Full article

The Bacillus megaterium LVN01 species native to Colombia has demonstrated the ability to metabolize different coproducts or industrial waste (such as fique juice, cane molasses, and residual glycerol) and accumulate polyhydroxybutyrate (PHB), giving it potential in the bioplastics industry. In this research, the potential of liquid digestate as a carbon source for the production of PHA polymers in fermentation processes with this bacterial strain was evaluated. Favorably, it was found that B. megaterium utilizes the nutrients from this residual substrate to multiply appropriately and efficiently synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Bench-scale aerobic batch fermentation, under the operational conditions of this research [volume: 3 L; temperature: 30.8 °C; agitation: 400 rpm; pH: 7.0 ± 0.2; dissolved oxygen: 100% saturation; antifoam: 10% (v/v)], generated maximum values of dry cell weight (DCW) (0.56 g cell L⁻¹) at 60 h, while the maximum PHBV yield (360 mg PHBV L⁻¹) occurred at 16 h, which is very favorable for sustainable degradable bioplastics production. Additionally, GC–MS and NMR analyses confirmed that the PHBV copolymer synthesized by B. megaterium is made up of the monomers 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Furthermore, the thermal properties determined by TGA (Tonset = 283.1 °C; Tendset = 296.98 °C; Td = 290.114 °C) and DSC (Tm = °C 155.7 °C; ΔHf = 19.80 J g⁻¹; Xcr = 18.17%) indicate that it is a thermally stable biopolymer with low percentages of crystallinity, providing flexibility that facilitates molding, adaptation, and application in various industrial sectors. Full article

Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO₂ as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB⁻4, cannot produce PHA. Strain 1F2, derived from strain PHB⁻4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO_2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO₂. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 10⁶ on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA. Full article

The synthesis of PHA was first investigated using WFOs obtained from smoked-sprat heads, substandard fresh sprats, and fresh mackerel heads and backbones. All the WFOs ensured the growth of the wild-type strain Cupriavidus necator B-10646 and the synthesis of PHA, regardless of the degree of lipid saturation (from 0.52 to 0.65) and the set and ratio of fatty acids (FA), which was represented by acids with chain lengths from C14 to C24. The bacterial biomass concentration and PHA synthesis were comparable (4.1–4.6 g/L and about 70%) when using WFO obtained from smoked-sprat heads and fresh mackerel, and it was twice as high as the bacterial biomass concentration from the fresh sprat waste. This depended on the type of WFO, the bacteria synthesized P(3HB) homopolymer or P(3HB-co-3HV-co-3HHx) copolymer, which had a lower degree of crystallinity (C_x 71%) and a lower molecular weight (M_n 134 kDa) compared to the P(3HB) (M_n 175–209 kDa and C_x 74–78%) at comparable temperatures (T_melt and T_degr of 158–168 °C and 261–284 °C, respectively). The new types of WFO, studied for the first time, are suitable as a carbon substrates for PHA synthesis. The WFOs obtained in the production of canned Baltic sprat and Baltic mackerel can be considered a promising and renewable substrate for PHA biosynthesis. Full article

The properties, features of thermal behavior and crystallization of copolymers containing various types of valerate monomers were studied depending on the set and ratio of monomers. We synthesized and studied the properties of three-component copolymers containing unusual monomers 4-hydroxyvalerate (4HV) and 3-hydroxy-4-methylvalerate (3H4MV), in addition to the usual 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomers. The results showed that P(3HB-co-3HV-co-4HV) and P(3HB-co-3HV-co-3H4MV) terpolymers tended to increase thermal stability, especially for methylated samples, including an increase in the gap between melting point (T_melt) and thermal degradation temperature (T_degr), an increase in the melting point and glass transition temperature, as well as a lower degree of crystallinity (40–46%) compared with P(3HB-co-3HV) (58–66%). The copolymer crystallization kinetics depended on the set and ratio of monomers. For terpolymers during exothermic crystallization, higher rates of spherulite formation (G_max) were registered, reaching, depending on the ratio of monomers, 1.6–2.0 µm/min, which was several times higher than the G_max index (0.52 µm/min) for the P(3HB-co-3HV) copolymer. The revealed differences in the thermal properties and crystallization kinetics of terpolymers indicate that they are promising polymers for processing into high quality products from melts. Full article

The results of constructing 3D scaffolds from degradable poly(3-hydrosbutyrpate-co-3-hydroxyvalerate) using FDM technology and studying the structure, mechanical properties, biocompatibility in vitro, and osteoplastic properties in vivo are presented. In the process of obtaining granules, filaments, and scaffolds from the initial polymer material, a slight change in the crystallization and glass transition temperature and a noticeable decrease in molecular weight (by 40%) were registered. During the compression test, depending on the direction of load application (parallel or perpendicular to the layers of the scaffold), the 3D scaffolds had a Young’s modulus of 207.52 ± 19.12 and 241.34 ± 7.62 MPa and compressive stress tensile strength of 19.45 ± 2.10 and 22.43 ± 1.89 MPa, respectively. SEM, fluorescent staining with DAPI, and calorimetric MTT tests showed the high biological compatibility of scaffolds and active colonization by NIH 3T3 fibroblasts, which retained their metabolic activity for a long time (up to 10 days). The osteoplastic properties of the 3D scaffolds were studied in the segmental osteotomy test on a model defect in the diaphyseal zone of the femur in domestic Landrace pigs. X-ray and histological analysis confirmed the formation of fully mature bone tissue and complete restoration of the defect in 150 days of observation. The results allow us to conclude that the constructed resorbable 3D scaffolds are promising for bone grafting. Full article

With the growing interest in bioplastics, there is an urgent need to develop rapid analysis methods linked to production technology development. This study focused on the production of a commercially non-available homopolymer, poly(3-hydroxyvalerate) (P(3HV)), and a commercially available copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), through fermentation using two different bacterial strains. The bacteria Chromobacterium violaceum and Bacillus sp. CYR1 were used to produce P(3HV) and P(3HB-co-3HV), respectively. The bacterium Bacillus sp. CYR1 produced 415 mg/L of P(3HB-co-3HV) when incubated with acetic acid and valeric acid as the carbon sources, whereas the bacterium C. violaceum produced 0.198 g of P(3HV)/g dry biomass when incubated with sodium valerate as the carbon source. Additionally, we developed a fast, simple, and inexpensive method to quantify P(3HV) and P(3HB-co-3HV) using high-performance liquid chromatography (HPLC). As the alkaline decomposition of P(3HB-co-3HV) releases 2-butenoic acid (2BE) and 2-pentenoic acid (2PE), we were able to determine the concentration using HPLC. Moreover, calibration curves were prepared using standard 2BE and 2PE, along with sample 2BE and 2PE produced by the alkaline decomposition of poly(3-hydroxybutyrate) and P(3HV), respectively. Finally, the HPLC results obtained by our new method were compared using gas chromatography (GC) analysis. Full article

In this study, we fabricated three dimensional (3D) porous scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content. P(HB-50HV) was biosynthesized from bacteria Cupriavidus necator H16 and the in vitro proliferation of dental cells for tissue engineering application was evaluated. Comparisons were made with scaffolds prepared by poly(hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and polycaprolactone (PCL). The water contact angle results indicated a hydrophobic character for all polymeric films. All fabricated scaffolds exhibited a high porosity of 90% with a sponge-like appearance. The P(HB-50HV) scaffolds were distinctively different in compressive modulus and was the material with the lowest stiffness among all scaffolds tested between the dry and wet conditions. The human gingival fibroblasts (HGFs) and periodontal ligament stem cells (PDLSCs) cultured onto the P(HB-50HV) scaffold adhered to the scaffold and exhibited the highest proliferation with a healthy morphology, demonstrating excellent cell compatibility with P(HB-50HV) scaffolds. These results indicate that the P(HB-50HV) scaffold could be applied as a biomaterial for periodontal tissue engineering and stem cell applications. Full article

The results of surface modification of solvent casting films made from polyhydroxyalkanoates (PHAs) of various compositions are presented: homopolymer poly-3-hydroxybutyrate P(3HB) and copolymers comprising various combinations of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate(4HB), and 3-hydroxyhexanoate (3HHx) monomers treated with a CO₂ laser in continuous and quasi-pulsed radiation modes. The effects of PHAs film surface modification, depending on the composition and ratio of monomers according to the results of the study of SEM and AFM, contact angles of wetting with water, adhesion and growth of fibroblasts have been revealed for the laser radiation regime used. Under continuous irradiation with vector lines, melted regions in the form of grooves are formed on the surface of the films, in which most of the samples have increased values of the contact angle and a decrease in roughness. The quasi-pulse mode by the raster method causes the formation of holes without pronounced melted zones, the total area of which is lower by 20% compared to the area of melted grooves. The number of viable fibroblasts NIH 3T3 on the films after the quasi-pulse mode is 1.5–2.0 times higher compared to the continuous mode, and depends to a greater extent on the laser treatment mode than on the PHAs’ composition. The use of various modes of laser modification on the surface of PHAs with different compositions makes it possible to influence the morphology and properties of polymer films in a targeted manner. The results that have been obtained contribute to solving the critical issue of functional biodegradable polymeric materials. Full article

The aim of this study was to obtain biocomposites consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), bacterial cellulose (BC) and α-tocopherol by a melt processing technique for potential use in biomedical applications. The melt processing and roughness of biocomposites were evaluated and compared to sample without BC. The degradation rate of PHBV/BC biocomposites was measured in phosphate buffer saline (PBS) by determining the mass variation and evidencing of thermal and structural changes by differential scanning calorimetry (DSC) and attenuated total reflectance-Fourier transformed infrared spectrometry (ATR-FTIR). The cell viability, cell morphology, cell cycle distribution and total collagen content were investigated on murine NCTC fibroblasts. Overall, the adding of BC to polyester matrix led to an adequate melt processing of biocomposites and increased surface roughness and cytocompatibility, allowing the cells to secrete the extracellular matrix (collagen) and stimulate cell proliferation. Results showed that the PHBV/BC biocomposites were favorable for long-term degradation and could be used for the design of medical devices with controlled degradability. Full article

From a series of biodegradable natural polymers of polyhydroxyalkanoates (PHAs)—poly-3-hydroxybutyrate (P(3HB) and copolymers containing, in addition to 3HB monomers, monomers of 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), and 4-hydroxybutyrate (4HB), with different ratios of monomers poured—solvent casting films and nanomembranes with oriented and non-oriented ultrathin fibers were obtained by electrostatic molding. With the use of SEM, AFM, and measurement of contact angles and energy characteristics, the surface properties and mechanical and biological properties of the polymer products were studied depending on the method of production and the composition of PHAs. It has been shown in cultures of mouse fibroblasts of the NIH 3T3 line and diploid human embryonic cells of the M22 line that elastic films and nanomembranes composed of P(3HB-co-4HB) copolymers have high biocompatibility and provide adhesion, proliferation and preservation of the high physiological activity of cells for up to 7 days. Polymer films, namely oriented and non-oriented nanomembranes coated with type 1 collagen, are positively evaluated as experimental wound dressings in experiments on laboratory animals with model and surgical skin lesions. The results of planimetric measurements of the dynamics of wound healing and analysis of histological sections showed the regeneration of model skin defects in groups of animals using experimental wound dressings from P(3HB-co-4HB) of all types, but most actively when using non-oriented nanomembranes obtained by electrospinning. The study highlights the importance of nonwoven nanomembranes obtained by electrospinning from degradable low-crystalline copolymers P(3HB-co-4HB) in the effectiveness of the skin wound healing process. Full article

Synthesis of P(3HB-co-3HV-co-4HV) copolymers by the wild-type strain Cupriavidus necator B-10646 on fructose or sodium butyrate as the main C-substrate with the addition of γ-valerolactone as a precursor of 3HV and 4HV monomers was studied. Bacterial cells were cultivated in the modes that enabled production of a series of copolymers with molar fractions of 3HV (from 7.3 to 23.4 mol.%) and 4HV (from 1.9 to 4.7 mol.%) with bacterial biomass concentration (8.2 ± 0.2 g/L) and PHA content (80 ± 2%). Using HPLC, DTA, DSC, X-Ray, SEM, and AFM, the physicochemical properties of copolymers and films prepared from them have been investigated as dependent on proportions of monomers. Copolymers are characterized by a reduced degree of crystallinity (C_x 38–49%) molecular weight characteristics M_n (45–87 kDa), and M_w (201–248 kDa) compared with P(3HB). The properties of the films surface of various composition including the porosity and surface roughness were studied. Most of the samples showed a decrease in the average pore area and an increase in their number with a total increase in 3HV and 4HV monomers. The results allow scaling up the productive synthesis of P(3HB-co-3HV-co-4HV) copolymers using Cupriavidus necator B-10646. Full article

Polybutylene succinate (PBS) is a bioplastic substitute for synthetic plastics that are made from petroleum-based products such as polyethylene and polypropylene. However, the biodegradation rate of PBS is still low and similar to that of polylactic acid (PLA). Moreover, our knowledge about degrader species is limited to a few fungi and mixed consortia. Here, to identify a bacterial degrader to accelerate PBS degradation, we screened and isolated Terribacillus sp. JY49, which showed significant degradability. In order to optimize solid and liquid culture conditions, the effect of factors such as temperature, additional carbon sources, and salt concentrations on degradation was confirmed. We observed a degradation yield of 22.3% after 7 days when adding 1% of glucose. Additionally, NaCl was added to liquid media, and degradation yield was decreased but PBS films were broken into pieces. Comparing the degree of PBS degradation during 10 days, the degradation yield was 31.4% after 10 days at 30 °C. Alteration of physical properties of films was analyzed by using scanning electron microscopy (SEM), gel permeation chromatography (GPC), and Fourier transform infrared (FT-IR). In addition, Terribacillus sp. JY49 showed clear zones on poly(butylene adipate-co-terephthalate) (PBAT), polycaprolactone (PCL), and copolymers such as P(3HB-co-3HV) and P(3HV-co-4HB), exhibiting a broad spectrum of degradation activities on bioplastics. However, there was no significant difference in absorbance when esterase activity was examined for different types of bioplastics. Overall, Terribacillus sp. JY49 is a potential bacterial strain that can degrade PBS and other bioplastics, and this is the first report of Terribacillus sp. as a bioplastic degrader. Full article

Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (M_w) and higher polydispersity of the extracted samples. Although such changes in the biopolymer’s molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-M_w oligomers are sought. Full article

To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a high conversion of sucrose (88.9%) to hexoses. We showed the necessity to adjust the chemical composition of molasses hydrolysate to balance with the physiological needs of C. necator B-10646 and reduce excess sugars and nitrogen and eliminate phosphorus deficiency. The modes of cultivation of bacteria on diluted hydrolyzed molasses with the controlled feeding of phosphorus and glucose were implemented. Depending on the ratio of sugars introduced into the bacterial culture due to the molasses hydrolysate and glucose additions, the bacterial biomass concentration was obtained from 20–25 to 80–85 g/L with a polymer content up to 80%. The hydrolysates of molasses containing trace amounts of propionate and valerate were used to synthesize a P(3HB-co-3HV) copolymer with minor inclusions of 3-hydroxyvlaerate monomers. The introduction of precursors into the medium ensured the synthesis of copolymers with reduced values of the degree of crystallinity, containing, in addition to 3HB, monomers 3HB, 4HB, or 3HHx in an amount of 12–16 mol.%. Full article