Search Results (83)

To combat the growing issue of petroleum plastic waste, alternative bio-based polymers are being developed. Many of these biopolymers are made from bio-derived materials, or are biodegradable, but the most promising polymers fall in both categories. Polyhydroxyalkanoates (PHAs) are one such class of polymers, and poly-3-hydroxybutyrate (P3HB), the most popular PHA, has shown great potential. This study utilized two types of cotton-derived glucose, alongside commercial glucose, as a feedstock for the biosynthesis of P3HB by Cupriavidus necator (also known as Ralstonia eutropha). The fermentation took place in a 2-L bioreactor, showing potential for scale-up. A single-solvent extraction method was created and utilized to reduce process complexity and chemical consumption of the polymer extraction. Both cotton-derived glucoses were shown to produce more P3HB than commercial glucose. The resulting P3HB samples were compared to each other and to the literature based on polymer yield and thermal characteristics. While all samples averaged a smaller yield than seen in the literature (indicating the need for optimization of the bacterial growth and metabolism with a growth curve in our future work), the cotton-derived glucose was shown to yield more P3HB than commercial glucose. Further, cotton-derived P3HB had very similar thermal properties to the commercial glucose-derived P3HB (and to values from the literature) with onset of thermal degradation ranging from 185 °C to 263 °C, cold crystallization temperatures ranging from 24 °C to 28 °C, and melting temperatures ranging from 147 °C to 151 °C. Lastly, all samples were shown to have a similar percentage crystallinity, ranging from 38% to 45%, which is slightly lower than that reported in the literature. P3HB made from cotton-derived glucose was shown to have potential as a scalable, sustainable alternative process. Full article

The environmental burden of conventional plastics has sparked interest in sustainable alternatives such as polyhydroxyalkanoates (PHAs). However, despite ample research in bioprocess development and the use of inexpensive waste streams, production costs remain a barrier to widespread commercialization. Complementary to this, genetic engineering offers another avenue for improved productivity. Cupriavidus necator stands out as a model host for PHA production due to its substrate flexibility, high intracellular polymer accumulation, and tractability to genetic modification. This review delves into metabolic engineering strategies that have been developed to enhance the production of poly(3-hydroxybutyrate) (PHB) and related copolymers in C. necator. Strategies include the optimization of central carbon flux, redox and cofactor balancing, adaptation to oxygen-limiting conditions, and fine-tuning of granule-associated protein expression and the regulatory network. This is followed by outlining engineered pathways improving the synthesis of PHB copolymers, PHBV, PHBHHx, and other emerging variants, emphasizing genetic modifications enabling biosynthesis based on unrelated single-carbon sources. Among these, enzyme engineering strategies and the establishment of novel artificial pathways are widely discussed. In particular, this review offers a comprehensive overview of promising engineering strategies, serving as a resource for future strain development and positioning C. necator as a valuable microbial chassis for biopolymer production at an industrial scale. Full article

Engineering of Cupriavidus necator could enable the production of various polyhydroxyalkanoates (PHAs); particularly, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HH)), a biopolymer with enhanced mechanical and thermal properties compared to poly(3-hydroxybutyrate) (PHB), can be efficiently produced from vegetable oils. However, challenges remain in the recovery process, particularly in removing residual oil and minimizing degradation of the polymer structure during extraction steps. This study investigated the effects of ethanol-based defatting on the recovery and polymeric properties of P(3HB-co-3HH). The proposed method involves the addition of ethanol to the cell broth to effectively remove residual oil. Ethanol improved the separation of microbial cells from the broth, thereby streamlining the downstream recovery process. Using ethanol in the washing step increased the recovery yield and purity to 95.7% and 83.4%, respectively (compared to 87.4% and 76.2% for distilled water washing), representing improvements of 8.3% and 7.2%. Ethanol washing also resulted in a 19% higher molecular weight compared to water washing, indicating reduced polymer degradation. In terms of physical properties, the elongation at break showed a significant difference: 241.9 ± 27.0% with ethanol washing compared to water (177.7 ± 10.3%), indicating ethanol washing retains flexibility. Overall, an ethanol washing step for defatting could simplify the recovery steps, increase yield and purity, and retain mechanical properties, especially for P(3HB-co-3HH) from oils. Full article

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) is a representative PHA copolymer that can improve the mechanical limitations of polyhydroxybutyrate (P(3HB)). Although genetic engineering can facilitate 3HHx incorporation, it often compromises cell growth and reduces polymer molecular weight owing to metabolic disruptions caused by the deletion of acetoacetyl coenzyme A (acetyl-CoA) reductase (PhaB). To address this issue, native strains capable of producing high levels of 3HHx were identified via oil-based Cupriavidus screening. Eight PHA-producing strains were isolated from various samples and Cupriavidus sp. Oh_1 exhibited the highest polyhydroxybutyrate (PHB) production at 15.23 g/L from 17.2 g/L of biomass using soybean oil. Moreover, Oh_1/phaC_RaJ_Pa, containing enoyl-CoA hydratase (phaJ) and PHA synthetase (phaC), was identified as the most effective novel strain producing the highest 3HHx mole fraction, 48.93 g/L of PHA from 52.3 g/L of biomass and achieving a maximum 3HHx accumulation of 27.2 mol%. The resulting P(3HB-co-3HHx) showed a higher Mw (12.3 × 10⁵) compared with P(3HB-co-3HHx) produced by the phaB-deleted strain (14.6 × 10⁴). Higher production of 3HHx was attributed to the higher expression of phaC_Ra and phaJ_Pa in Oh_1, with log2 fold changes of 2.94 and 8.2, respectively, as well as the upregulation of certain β-oxidation encoding operons. Collectively, these findings highlight a strain capable of synthesizing a substantial 3HHx fraction without requiring gene deletions or extensive genetic modifications. Full article

Poly(3-hydroxybutyrate) is a biodegradable plastic produced by various microbes. Considering the emerging environmental problems caused by plastics, P(3HB) has gained attention as a substitute for conventional plastics. In this study, we isolated a novel P(3HB)-producing microbe, Limimaricola sp. YI8, which utilized sucrose as a cost-effective carbon source for P(3HB) production. Under optimized conditions, Limimaricola sp. YI8 produced 6.2 g/L P(3HB) using sucrose as the sole carbon source. P(3HB) extracted from YI8 exhibited a pinkish color derived from a dye produced naturally by YI8. Films fabricated from extracted P(3HB) polymer were subjected to analyses, including gel permeation chromatography, universal test machine, and differential scanning calorimetry, to determine their physical properties. The obtained values were almost identical to those of P(3HB) films extracted from Escherichia coli and Cupriavidus necator H16. Overall, this study presents the potential of Limimaricola spp. YI8 as a P(3HB)-producing strain and the P(3HB) films extracted from this strain. Full article

A copolymer of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a practical biodegradable plastic, and at present, the copolymer is produced at commercial scale via heterotrophic cultivation of an engineered strain of a facultative hydrogen-oxidizing bacterium, Cupriavidus necator, using vegetable oil as the carbon source. In our previous report, we investigated PHBHHx production from CO₂ via pH-stat jar cultivation of the newly created recombinants of C. necator under autotropic conditions, feeding the inorganic substrate gas mixture (H₂/O₂/CO₂ = 80:10:10 v/v%) into a recycled-gas closed-circuit (RGCC) culture system. The dry cell weight (DCW) and PHBHHx concentration with the best strain MF01/pBPP-ccr_MeJ_Ac-emd increased to 59.62 ± 3.18 g·L⁻¹ and 49.31 ± 3.14 g·L⁻¹, respectively, after 216 h. In this study, we investigated the high-concentration production of PHBHHx with a shorter cultivation time by using a jar fermenter equipped with a basket-shaped agitator to enhance oxygen transfer in the culture medium and by continuously supplying the gases with higher O₂ concentrations to maintain the gas composition within the reservoir at a constant ratio. The concentrations of ammonium and phosphate in the culture medium were maintained at low levels. As a result, the DCW and PHBHHx concentrations increased to 109.5 ± 0.30 g·L⁻¹ and 85.2 ± 0.62 g·L⁻¹ after 148 h, respectively. The 3HHx composition was 10.1 ± 0.693 mol%, which is suitable for practical applications. Full article

The increasing global demand for natural substances such as the sesquiterpene α-humulene makes optimizing microbial production essential. A production process using the versatile host Cupriavidus necator has been recently improved by adjusting the minimal media and process parameters. Understanding microbial and process robustness is key to ensuring consistent performance under different conditions. This study is the first to investigate and quantify the robustness of microbial α-humulene production and biomass formation using C. necator pKR-hum. Established process improvements and the impact of common or individual precultures were analyzed and quantified for their effect on the robustness of product and biomass formation. We report a robust α-humulene production process with even more consistent biomass formation using C. necator pKR-hum. Even with a simulated process disturbance, 79% of the maximum α-humulene level was still produced. Overall, our results show that the α-humulene production process using C. necator pKR-hum is highly robust, demonstrating its resilience to process disturbances and suitability for further industrial applications. Full article

The phytopathogenic fungus Sclerotinia sclerotiorum has a wide host range and causes significant economic losses in crops worldwide. This pathogen uses oxalic acid as a virulence factor; for this reason, the degradation of this organic acid by oxalotrophic bacteria has been proposed as a biological control approach. However, previous studies on the potential role of oxalotrophy in biocontrol did not investigate the differential effect of oxalic acid consumption and the subsequent pH alkalinisation on fungal growth. In this study, confrontation experiments on different media using a wild-type (WT) strain of S. sclerotiorum and an oxalate-deficient mutant (strain Δoah) with the soil oxalotrophic bacteria Cupriavidus necator and Cupriavidus oxalaticus showed the combined effect of media composition on oxalic acid production, pH, and fungal growth control. Oxalotrophic bacteria were able to control S. sclerotiorum only in the medium in which oxalic acid was produced. However, the deficient Δoah mutant was also controlled, indicating that the consumption of oxalic acid is not the sole mechanism of biocontrol. WT S. sclerotiorum acidified the medium when inoculated alone, while for both fungi, the pH of the medium changed from neutral to alkaline in the presence of bacteria. Therefore, medium alkalinisation independent of oxalotrophy contributes to fungal growth control. Full article

The use of polyhydroxybutyrate (PHB) can help diminish fossil chemical dependency because it can partially replace petrochemical plastics due to its biodegradability and similar mechanical properties. However, its production costs are high compared with fossil-based plastics. Alternative carbon sources can be used in the fermentation media because they are renewable and low-cost. Vegetable oils are especially attractive due to their high carbon content, contributing to high production rates per gram of substrate. This work aimed to produce PHB from Cupriavidus necator LPB1421 using either corn or soybean oil as the sole carbon source. Urea was the best nitrogen source, enabling a DCW production of 4.35 g/L (corn oil) and 10.4 g/L (soybean oil). After media optimization, the DCW of corn oil reached 22.13 g/L, with 57.46% PHB accumulation (12.71 g PHB/L), whereas soybean oil led to a DCW of 19.83 g/L, with 54.91% PHB accumulation (10.89 g PHB/L). This media composition was employed in a kinetics assay, revealing similar fermentation parameters among both oils and a yield of 0.2118 g PHB/g for corn oil and 0.1815 g PHB/g for soybean oil. These results open the possibility of integrating PHB production with biofuel manufacturing in a bioethanol/biodiesel biorefinery concept. Full article

Cupriavidus necator can produce single-cell proteins (SCPs) using electrons produced by hydrogen as energy, oxygen as electron acceptors, and CO₂ as carbon sources. Gas fermentation is a process of microbial fermentation that uses gas substrates (such as hydrogen, carbon dioxide, etc.) which faces several challenges, mainly including the low solubility of gas substrates, the danger of hydrogen and oxygen mixing, and the optimization of fermentation conditions. To overcome these challenges, this article explores a variety of strategies—including the design of a self-developed bioreactor—to reduce the risk of static electricity. Without the addition of filler material, the results showed that the maximum cell dry weight (CDW) of 30% secondary seed inoculation was 20.41% higher than that of 10% secondary seed inoculum, and 5.99% higher than that of 20% secondary seed inoculum. Combined with the filler material and with the use of high-efficiency continuous fermentation technology, the average yield of continuous fermentation was 23.31 g/day, while the average yield of batch fermentation was 14.33 g/day. The daily yield of continuous fermentation is 1.63 times that of batch fermentation. These efforts are aimed at improving the efficiency and safety of gas fermentation. Full article

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

Low-density polyethylene (LDPE) is among the most abundant synthetic plastics in the world, contributing significantly to the plastic waste accumulation problem. A variety of microorganisms, such as Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas chlororaphis PA2361, can form biofilms on the surface of LDPE polymers and cause damage to the exterior structure. However, the damage is not extensive and complete degradation has not been achieved. The changes in polymer structure were analyzed using Time-domain Nuclear Magnetic Resonance (TD-NMR), High-Temperature Size-Exclusion Chromatography (HT-SEC), Differential Scanning Calorimetry (DSC), and Gas Chromatography with a Flame Ionization Detector (GC-FID). Limited degradation of the LDPE powder was seen in the first 30 days of incubation with the bacteria. Degradation can be seen in the LDPE weight loss percentage, LDPE degradation products in the supernatant, and the decrease in the percentage of amorphous regions (from >47% to 40%). The changes in weight-average molar mass (Mw), number-average molar mass (Mn), and the dispersity ratio (Đ) indicate that the low-molar mass fractions of the LDPE were preferentially degraded. The results here confirmed that LDPE degradation is heavily dependent on the presence of amorphous content and that only the amorphous content was degraded via bacterial enzymatic action. Full article

Although efforts have been undertaken to produce polyhydroxyalkanoates (PHA) with various monomers, the low yield of PHAs because of complex metabolic pathways and inhibitory substrates remains a major hurdle in their analyses and applications. Therefore, we investigated the feasibility of mass production of PHAs containing 5-hydroxyvalerate (5HV) using δ-valerolactone (DVL) without any pretreatment along with the addition of plant oil to achieve enough biomass. We identified that PhaC_BP-M-CPF4, a PHA synthase, was capable of incorporating 5HV monomers and that C. necator PHB⁻⁴ harboring phaC_BP-M-CPF4 synthesized poly(3HB-co-3HHx-co-5HV) in the presence of bean oil and DVL. In fed-batch fermentation, the supply of bean oil resulted in the synthesis of 49 g/L of poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol%5HV) from 66 g/L of biomass. Thermophysical studies showed that 3HHx was effective in increasing the elongation, whereas 5HV was effective in decreasing the melting point. The contact angles of poly(3HB-co-3HHx-co-5HV) and poly(3HB-co-3HHx) were 109 and 98°, respectively. In addition, the analysis of microbial degradation confirmed that poly(3HB-co-3HHx-co-5HV) degraded more slowly (82% over 7 days) compared to poly(3HB-co-3HHx) (100% over 5 days). Overall, the oil-based fermentation strategy helped produce more PHA, and the mass production of novel PHAs could provide more opportunities to study polymer properties. Full article

The production of polyhydroxyalkanoates using submerged cultures of Cupriavidus necator DSM 428 was evaluated using low-cost substrates from agroindustry: (i) dextrose from cassava starch and (ii) a mixture of sugarcane vinasse from the bioethanol industry and dextrose from cassava starch. The effects of vinasse composition (2.5, 5.0, 7.5, 25, 50, and 75% v/v) and the use of raw and activated carbon-pre-treated vinasse were assessed. The results indicate that cultivations using only cassava starch dextrose reached 4.33 g/L of biomass as the dry cell weight and a poly(3-hydroxybutyrate) (PHB) production of 47.1%. Raw vinasse proportions of 25, 50, and 75% in the culture medium resulted in total inhibition. Vinasse treated at the same ratios led to biomass production in the range 1.7–4.44 g/L. The higher PHB production scenario was obtained in a medium containing dextrose and treated vinasse (7.5%), yielding 5.9 g/L of biomass and 51% of PHB accumulation. The produced PHB was characterized by XRD and FTIR for an analysis of crystalline structure and chemical functional groups, respectively. EDS was employed for a semi-quantitative analysis of the chemical composition, and SEM was used to analyze the morphology of the microgranules. The results of DSC and TGA analyses demonstrated the thermal stability of the obtained PHB. Full article

This study focuses on mitigating the socio-economic and environmental damage of the invasive macroalga Rugulopteryx okamurae and counteracting the pollution from petroleum-based plastics by using the alga as a feedstock for polyhydroxybutyrate (PHB) production. The enzymatic hydrolysis of R. okamurae, non-pretreated and hydrothermally acid-pretreated (0.2 N HCl, 15 min), was carried out, reaching reducing sugar (RS) concentrations of 10.7 g/L and 21.7 g/L, respectively. The hydrolysates obtained were used as a culture medium for PHB production with Cupriavidus necator, a Gram-negative soil bacterium, without supplementation with any external carbon and nitrogen sources. The highest yield (0.774 g PHB/g RS) and biopolymer accumulation percentage (89.8% cell dry weight, CDW) were achieved with hydrolysates from pretreated macroalga, reaching values comparable to the highest reported in the literature. Hence, it can be concluded that hydrolysates obtained from algal biomass hydrothermally pretreated with acid have a concentration of sugars and a C/N ratio that favour PHB production. Full article