Search Results (88)

The escalating global demand for large-scale, cost-effective, and sustainable high-quality protein has positioned single-cell protein (SCP) production from one-carbon (C1) gases as a highly promising solution. In this study, eight chemolithoautotrophic hydrogen-oxidizing bacteria (HOB) were isolated from mangrove sediments. Based on the 16S rRNA gene sequence analysis, they belonged to genera Sulfurimonas, Sulfurovum, Thiomicrolovo, and Marinobacterium. Among these, Thiomicrolovo sp. ZZH C-3 was identified as the most promising candidate for SCP production based on the highest biomass and protein content, and was selected for further characterization. Strain ZZH C-3 is a Gram-negative, short rod-shaped bacterium with multiple flagella. It can grow chemolithoautotrophically by using molecular hydrogen as an energy source and molecular oxygen as an electron acceptor. Genomic analysis further confirmed that ZZH C-3 harbors a complete reverse tricarboxylic acid (rTCA) cycle gene set for carbon fixation, and diverse hydrogenases (Group I, II, IV) for hydrogen oxidation. Subsequently, its cultivation conditions and medium composition for SCP production were systematically optimized using single-factor experiments and response surface methodology (RSM). Results showed that the optimal growth conditions were 28 °C, pH 7.0, and with 1 g/L (NH₄)₂SO₄ as the nitrogen source, 5–10% oxygen concentration, 9.70 mg/L FeSO₄·7H₂O, 0.17 g/L CaCl₂·2H₂O, and 1.90 mg/L MnSO₄·H₂O. Under the optimized conditions, strain ZZH C-3 achieved a maximum specific growth rate of 0.46 h⁻¹. After 28 h of cultivation, the optical density at 600 nm (OD₆₀₀) reached 0.94, corresponding to a biomass concentration of 0.60 g/L, and the protein content ranked at 73.56%. The biomass yield on hydrogen (Y_H2) was approximately 3.01 g/g H₂, with an average H₂-to-CO₂ consumption molar ratio of about 3.78. Compared to the model HOB Cupriavidus necator, strain ZZH C-3 exhibited a lower H₂/CO₂ consumption ratio, superior substrate conversion efficiency, and high protein content. Overall, this study not only validated the potential of mangrove HOB for SCP production but also offers new insights for future metabolic engineering strategies designed to enhance CO₂-to-biomass conversion efficiency. Full article

Cupriavidus necator is a well-studied microorganism with potential application in bioregenerative life support systems for single-cell protein and bioplastic production. Most studies have been carried out in autotrophy or heterotrophy, requiring O₂ as the final electron acceptor. In the context of inhabited missions, access to O₂ will primarily be limited to the crew. In this study, we investigated the capacity of C. necator to carry out nitrate respiration as a strategy to limit oxygen supply to the cultures by providing nitrate from another compartment of the Bioregenerative Life Support System (BLSS). Batch bioreactor experiments were carried out to determine the best conditions for nitrate utilization in terms of pH and aeration. Continuous cultures were then performed under two carbon sources (glucose vs. acetic acid) and two substrate limitations (nitrate vs. carbon). The optimal conditions were found to be pH 7.5 under anaerobiosis. They were applied in chemostats, where three steady-states were obtained at a low dilution rate. In all cases, the biomass consisted of a mixture of protein (from 29 ± 1% Cell Dry Weight (CDW) to 39 ± 2% CDW) and polyhydroxybutyrate (from 45 ± 2% CDW to 57 ± 3% CDW), which was found to be a key component for nitrate respiration metabolism. Microaerobic conditions were also tested in batch culture, reporting for the first time aerobic nitrate respiration in C. necator. Under these conditions, growth parameters improved during the nitrate phase; however, the specific growth rate during the nitrite phase was lower than that observed under strictly anaerobic conditions. Full article

The increasing demand for rapid identification of bacteria capable of degrading environmentally relevant organic compounds highlights the need for scalable and selective analytical tools. Cupriavidus necator catabolizes several hydroxybenzoic acids, including 2-hydroxybenzoate (salicylate, 2-HBA), 4-hydroxybenzoate (4-HBA), and 3-hydroxybenzoate (3-HBA), funneling them into central aromatic catabolism via monooxygenation to 2,5-dihydroxybenzoate (gentisate, 2,5-dHBA) and 3,4-dihydroxybenzoate (protocatechuate, 3,4-dHBA) followed by the oxidative cleavage reaction, enabling complete conversion to tricarboxylic acid (TCA) cycle intermediates. To quantify how readily C. necator is able to activate catabolic genes in response to hydroxybenzoic acid, an extracellular ligand, we applied an approach centered on a transcription-factor (TF)-based biosensor that combines ligand-bound regulator activity with a fluorescent reporter. This approach allowed to evaluate the ligand sensitivity by determining gene activation threshold AC_min and half-maximal effective concentration EC₅₀. Amongst studied hydroxybenzoic acids, 2-HBA and 4-HBA sensors from C. necator showed very low thresholds 4.8 and 2.4 μM and EC₅₀ values of 19.91 and 13.06 μM, indicating high sensitivity to these compounds and implicating a scavenging characteristic of associated catabolism. This study shows that the TF-based-biosensor approach applied for mapping functional sensing ranges of hydroxybenzoates combined with the research and informatics of catabolism can advance our understanding of how gene expression regulation systems have evolved to respond differentially to the availability and concentration of carbon sources. Furthermore, it can inform metabolic engineering strategies in the prevention of premature pathway activation or in predicting competitive substrate hierarchies in complex mixed environments. Full article

Hydrogenases are key enzymes in microbial energy metabolism, catalyzing the reversible conversion between molecular hydrogen and protons. Among them, [NiFe]-hydrogenases are particularly attractive for biocatalytic applications due to the oxygen tolerance of several members of this class and their ability to couple hydrogen oxidation with redox cofactor regeneration. In this study, a recombinant soluble [NiFe]-hydrogenase from Cupriavidus necator H16 was successfully expressed in Escherichia coli BL21 (DE3), purified, and characterised with a focus on its applicability for NAD⁺ regeneration. Unlike previous studies that primarily used native C. necator extracts or complex maturation systems, this work provides the first quantitative demonstration that an aerobically purified recombinant soluble [NiFe]-hydrogenase expressed in E. coli can function effectively as an NAD⁺ regeneration catalyst and operate within multi-enzymatic cascade reactions under application-relevant conditions. The crude recombinant enzyme displayed a volumetric activity of 0.273 ± 0.024 U/mL and a specific activity of 0.018 ± 0.002 U/mg_cells in the hydrogen oxidation assay, while purification yielded a specific activity of 0.114 ± 0.001 U/mg with an overall recovery of 79.2%. The enzyme exhibited an optimal temperature of 35 °C and a pH optimum of 7.00. Thermal stability analysis revealed rapid deactivation at 40 °C (k_d = 0.4186 ± 0.0788 h⁻¹, t_1/2 ≈ 1.7 h) and substantially slower deactivation at 4 °C (k_d = 0.1141 ± 0.0139 h⁻¹, t_1/2 ≈ 6.1 h). Batch NADH oxidation experiments confirmed efficient cofactor turnover and high specificity towards NADH over NADPH. Finally, integration of the hydrogenase into a one-pot two-enzyme glucose oxidation system demonstrated its capacity for in situ NAD⁺ regeneration, although the reaction stopped after approximately 5 min due to acidification from gluconic acid formation, highlighting pH control as a key requirement for future process optimization. Full article

Polyhydroxybutyrate (PHB), as a biodegradable and green polymer, holds significant potential for replacing traditional petroleum-based plastics. However, its production efficiency and cost remain bottlenecks limiting large-scale application. In recent years, hybrid systems constructed from photosensitive nanomaterials and microorganisms have provided a novel pathway for enhancing PHB synthesis efficiency. These systems augment the supply of intracellular reducing power through efficient photo-generated electron injection, thereby driving microbial carbon fixation and PHB anabolic metabolism. This review systematically summarizes the mechanisms and performance of various types of photosensitive materials (including g-C₃N₄, CdS, polymer dots, etc.) in regulating PHB synthesis in microorganisms, such as Cupriavidus necator H16. It focuses on the influence of material composition, structure, energy band characteristics, and their interfacial interactions with microorganisms on electron transfer efficiency and biocompatibility. Furthermore, the article outlines the current challenges faced by these hybrid systems in key energy and mass transfer processes, including light energy conversion, transmembrane electron transport, and NADPH regeneration. It also prospects the design principles of novel bio-inspired multi-level heterojunction materials and their application potential in constructing efficient “material microbe” collaborative synthesis systems. This review aims to provide a material-level theoretical foundation and design strategies for developing high-performance and sustainable light-driven biomanufacturing technologies for PHB. Full article

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