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The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not from inexpensive carbon sources such as sugars. In previous studies, we demonstrated that engineered strains of a hydrogen-oxidizing bacterium, Cupriavidus necator, synthesized PHBHHx with a high cellular content not only from sugars but also from CO2 as the sole carbon source in the flask culture. In this study, the highly efficient production of PHBHHx from CO2 was investigated via pH-stat jar cultivation of recombinant C. necator strains while feeding the substrate gas mixture (H2/O2/CO2 = 80:10:10 v/v%) to a complete mineral medium in a recycled-gas, closed-circuit culture system. As a result, the dry cell mass and PHBHHx concentration with the strain MF01/pBPP-ccrMeJAc-emd reached up to 59.62 ± 3.18 g·L−1 and 49.31 ± 3.14 g·L−1, respectively, after 216 h of jar cultivation with limited addition of ammonia and phosphate solutions. The 3HHx composition was close to 10 mol%, which is suitable for practical applications. It is expected that the autotrophic cultivation of the recombinant C. necator can be feasible for the mass production of PHBHHx from CO2.

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L−1 in septum flasks. However, an increase in final product titer and space–time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space–time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0–4.2.

Green waste, e.g., grass clippings, is currently insufficiently recycled and has untapped potential as a valuable resource. Our aim was to use juice from grass clippings as a growth medium for microorganisms. Herein, we demonstrate the production of the sesquiterpene α-humulene with the versatile organism Cupriavidus necator pKR-hum on a growth medium from grass clippings. The medium was compared with established media in terms of microbial growth and terpene production. C. necator pKR-hum shows a maximum growth rate of 0.43 h−1 in the grass medium and 0.50 h−1 in a lysogeny broth (LB) medium. With the grass medium, 2 mg/L of α-humulene were produced compared to 10 mg/L with the LB medium. By concentrating the grass medium and using a controlled bioreactor in combination with an optimized in situ product removal, comparable product concentrations could likely be achieved. To the best of our knowledge, this is the first time that juice from grass clippings has been used as a growth medium without any further additives for microbial product synthesis. This use of green waste as a material represents a new bioeconomic utilization option of waste materials and could contribute to improving the economics of grass biorefineries. [ABSTRACT FROM AUTHOR]

Traditional plastics represent a tremendous threat to the environment because of increases in polluting manufacturing as well as their very extended degradation time. Polyhydroxyalkanoates (PHAs) are polymers with similar performance to plastic but are compostable and synthesizable from renewable sources and therefore could be a replacement for fossil-based plastics. However, their production costs are still too high, thus demanding the investigation of new and cheap substrates. In this sense, agricultural wastes are attractive because they are inexpensive and largely available. Specifically, fruit and vegetables are rich in sugars that could be fermented into PHAs. In this work two strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, well-known PHA-producing microbes, were screened for their ability to grow and accumulate PHAs. Ten different fruit and vegetable processing waste streams, never before reported in combination with these strains, were tested. Residues from red apple and melon were found to be the most suitable feedstocks for PHA production. Under specific selected conditions, C. necator DSM 545 accumulated up to 7.4 and 4.3 g/L of 3-hydroxybutyrate (3HB) from red apple and melon, respectively. Copolymer production was also obtained from melon. These results confirm the attractiveness of food processing waste as a promising candidate for PHA production. Ultimately, these novel substrates draw attention for future studies on process optimization and upscaling with C. necator. [ABSTRACT FROM AUTHOR]

The chemical industry is transitioning to more sustainable and biobased processes. One key element of this transition is coupling energy fluxes and feedstock utilization for optimizing processes, routes and efficiencies. Here, we show for the first time the coupling of the Kolbe electrolysis at the anode with a subsequent microbial conversion of the cathodically produced co‐product hydrogen. Kolbe electrolysis of valeric acid yields the liquid drop‐in fuel additive n‐octane. Subsequently, the solvent isopropanol is produced by resting Cupriavidus necator cells using gaseous electrolysis products (esp. CO2 and H2). The resting microbial cells show carbon efficiencies of up to 41 % and Coulombic/Faradaic efficiencies of 60 % and 80 % for anodic and cathodic reactions, respectively. The implementation of a paired electrolyser resulted in superior process performances with overall efficiencies of up to 64.4 %. [ABSTRACT FROM AUTHOR]

Polyhydroxybutyrate (PHB) has emerged as a novel material for replacing various plastics used in the medical field. However, its application as a drug-delivery carrier for colitis-targeted delivery has not been explored. In this study, we used biosynthesized PHB combined with Eudragit FS (EFS) and cyclosporine A (CSA) to develop pH-responsive controlled CSA-releasing nanoparticles (CSA-PENPs) for colitis-targeted drug delivery and demonstrated its enhanced therapeutic efficacy in a dextran sulfate sodium (DSS)-induced murine colitis model. PHB was successfully biosynthesized in the bacterium Cupriavidus necator, as demonstrated by 1H-NMR and FT-IR analyses. CSA-PENPs were fabricated via the oil-in-water emulsion solvent evaporation method. Owing to the potent pH-responsive and sustained drug release properties provided by PHB and EFS, CSA-PENPs could deliver a sufficient amount of CSA to inflamed tissues in the distal colon; in contrast, CSA-loaded EFS nanoparticles displayed premature burst release before reaching the target site. Due to enhanced CSA delivery to colitis tissues, CSA-PENPs exhibited potent anti-inflammatory effects in the DSS-induced murine colitis model. Overall, CSA-PENPs could be a promising drug-delivery system for treating ulcerative colitis. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized by certain bacteria and archaea with functions comparable to conventional plastics. Previously, our research group reported a newly PHA-producing bacterial strain, Rhodococcus pyridinivorans BSRT1-1, from the soil in Thailand. However, this strain's PHA synthase (phaCRp) gene has not yet been characterized. Thus, this study aims to synthesize PHA using a newly engineered bacterial strain, Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp, which harbors the phaCRp from strain BSRT1-1, and characterize the properties of PHA for skin tissue engineering application. To the best of our knowledge, this is the first study on the characterization of the PhaC from R. pyridinivorans species. The results demonstrated that the expression of the phaCRp in C. necator PHB−4 had developed in PHA production up to 3.1 ± 0.3 g/L when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source. Interestingly, the engineered strain produced a 3-hydroxybutyrate (3HB) with 2 mol% of 3-hydroxyhexanoate (3HHx) monomer without adding precursor substrates. In addition, the 70 L stirrer bioreactor improved P(3HB-co-2 mol% 3HHx) yield 1.4-fold over the flask scale without altering monomer composition. Furthermore, the characterization of copolymer properties showed that this copolymer is promising for skin tissue engineering applications. [ABSTRACT FROM AUTHOR]

Poly[(R)-3-hydroxybutyrate- co -(R)-3-hydroxyhexanoate] [P(3HB- co -3HHx)] has a high potential to serve as a commercial bioplastic due to its biodegradability, thermoplastic and mechanical properties. The properties of this copolymer are greatly affected by the composition of 3HHx monomer. One of the most efficient ways to modulate the composition of 3HHx monomer in P(3HB- co -3HHx) is by manipulating the (R)-3HHx-CoA monomer supply. In this study, a new (R)-specific enoyl-CoA hydratase originating from a non-PHA producer, Streptomyces sp. strain CFMR 7 (PhaJ S s), was characterized and found to be effective in supplying 3HHx monomer during in vivo production of P(3HB -co- 3HHx) copolymer. The P(3HB- co -3HHx) copolymer produced from the Cupriavidus necator transformant that harbors p haJ Ss , PHB−4/pBBR1- C BP-M-CPF4 J Ss , showed enhanced 3HHx incorporation of up to 11 mol% without affecting the P(3HB- co -3HHx) production when palm oil was used as the carbon source. In addition, both k cat and k cat / K m of PhaJ S s were higher toward the C6 than the shorter C4 substrates, underscoring the preference for 3-hydroxyhexanoyl-CoA. These results suggest that PhaJ S s has a significant ability to supply 3HHx monomers for PHA biosynthesis via β-oxidation and can be applied for metabolic engineering of robust PHA-producing strains. [ABSTRACT FROM AUTHOR]

Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting nonmodel bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in nonmodel bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO 2 and hydrogen. However, its low electroporation efficiency hampers the high-throughput genetic engineering required to develop C. necator into an industrially relevant host organism. Thus, conjugation has often been the method of choice for introducing exogenous DNA, especially when introducing large plasmids or suicide plasmids. We first propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of nonmodel bacteria.

The growing demand for sustainable food packaging and the increasing concerns regarding environmental pollution have driven interest in biodegradable materials. This paper presents an in-depth review of the production of Polyhydroxyalkanoates (PHA), a biodegradable polymer, from food waste. PHA-based bioplastics, particularly when derived from low-cost carbon sources such as volatile fatty acids (VFAs) and waste oils, offer a promising solution for reducing plastic waste and enhancing food packaging sustainability. Through optimization of microbial fermentation processes, PHA production can achieve significant efficiency improvements, with yields reaching up to 87 % PHA content under ideal conditions. This review highlights the technical advancements in using PHA for food packaging, emphasizing its biodegradability, biocompatibility, and potential to serve as a biodegradable alternative to petroleum-based plastics. However, challenges such as high production costs, mechanical limitations, and the need for scalability remain barriers to industrial adoption. The future of PHA in food packaging hinges on overcoming these challenges through further research and innovation in production techniques, material properties, and cost reduction strategies, along with necessary legislative support to promote widespread use., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

The expanding field of synthetic biology requires diversification of microbial chassis to expedite the transition from a fossil fuel-dependent economy to a sustainable bioeconomy. Relying exclusively on established model organisms such as Escherichia coli and Saccharomyces cerevisiae may not suffice to drive the profound advancements needed in biotechnology. In this context, Cupriavidus necator, an extraordinarily versatile microorganism, has emerged as a potential catalyst for transformative breakthroughs in industrial biomanufacturing. This comprehensive book chapter offers an in-depth review of the remarkable technological progress achieved by C. necator in the past decade, with a specific focus on the fields of molecular biology tools, metabolic engineering, and innovative fermentation strategies. Through this exploration, we aim to shed light on the pivotal role of C. necator in shaping the future of sustainable bioprocessing and bioproduct development., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C0) and the specific growth rate (µ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C0 = 2.9–23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C0 = 19.1 g/L) saponified SCG oil. Peak µ-values of 0.139 h−1 and 0.145 h−1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates (µ = 0.35 h−1) than on the crude SCG oil (µ = 0.23 h−1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA. Key points: • The bioavailability of plant oil substrates can be improved via saponification. • Cell growth and inhibition were accurately described by the Han-Levenpsiel model. • Mixing crude and saponified oils enable variation of free fatty acid content. [ABSTRACT FROM AUTHOR]

This review article will discuss the ways in which various polymeric materials, such as polyethylene (PE), polypropylene (PP), polystyrene (PS), and poly(ethylene terephthalate) (PET) can potentially be used to produce bioplastics, such as polyhydroxyalkanoates (PHAs) through microbial cultivation. We will present up-to-date information regarding notable microbial strains that are actively used in the biodegradation of polyolefins. We will also review some of the metabolic pathways involved in the process of plastic depolymerization and discuss challenges relevant to the valorization of plastic waste. The aim of this review is also to showcase the importance of methods, including oxidative degradation and microbial-based processes, that are currently being used in the fields of microbiology and biotechnology to limit the environmental burden of waste plastics. It is our hope that this article will contribute to the concept of bio-upcycling plastic waste to value-added products via microbial routes for a more sustainable future. [ABSTRACT FROM AUTHOR]

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria. • Elementary flux modes of C. necator for PHB synthesis from fructose and formate. • Metabolite sampling identified common reactions among EFMs with low driving force. • PHB from formate shows low driving forces for aconitase, citrate lyase, NADPH synthesis. • Phosphoketolase and ATP citrate lyase expression increased driving forces and PHB production. [ABSTRACT FROM AUTHOR]

Presented herein are the results of a novel recycling method for waste Tetra Pak® packaging materials. The polyethylene (PE-T) component of this packaging material, obtained via a separation process using a "solvents method", was used as a carbon source for the biosynthesis of polyhydroxyalkanoates (PHAs) by the bacterial strain Cupriavidus necator H16. Bacteria were grown for 48–72 h, at 30 °C, in TSB (nitrogen-rich) or BSM (nitrogen-limited) media supplemented with PE-T. Growth was monitored by viable counting. It was demonstrated that C. necator utilised PE-T in both growth media, but was only able to accumulate 40% w/w PHA in TSB supplemented with PE-T. Only 1.5% w/w PHA was accumulated in the TSB control, and no PHA was detected in the BSM control. Extracted biopolymers were characterised by nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR) spectroscopy, electrospray tandem mass spectrometry (ESI-MS/MS), gel permeation chromatography (GPC), and accelerator mass spectrometry (AMS). The characterisation of PHA by ESI-MS/MS revealed that PHA produced by C. necator in TSB supplemented with PE-T contained 3-hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxyhexanoate co-monomeric units. AMS analysis also confirmed the presence of 96.73% modern carbon and 3.27% old carbon in PHA derived from Tetra Pak®. Thus, this study demonstrates the feasibility of our proposed recycling method for waste Tetra Pak® packaging materials, alongside its potential for producing value-added PHA, and the ability of 14C analysis in validating this bioconversion process. [ABSTRACT FROM AUTHOR]

Waste paper holds great potential as a substrate for the microbial production of bioplastic (Poly-3-hydroxybutyrate (PHB)). This study aimed to produce PHB by utilizing office paper as a substrate using Cupriavidus necator through batch and fed-batch simultaneous saccharification and fermentation (SSF) approach. For the batch experiment, different loadings of shredded office paper (3, 5 and 10%) with two different pretreatments H2O2 (OPH) and H2O2 and Triton X-100 (OPTH) were carried out. For the fed-batch experiment, paper loading started with 3% and two more additions were made at 36 and 84 h. Both experiments were conducted at 30 °C, 200 rpm and pH 7 using 55.5 FPU/g of cellulase and 37.5 CBU/g of β-glucosidase with a fixed amount of nitrogen source. High PHB yield was observed with OPH in all loadings, though the OPHT showed a better hydrolysis. Maximum PHB yield (4.27 g/L) was achieved with 10% OP on the sixth day of fermentation in batch SSF. Whereas, maximum PHB yield (4.19 g/L) was obtained within a shorter time (66 h)with OPH in the fed-batch experiment. The extracted PHB showed well-matched characteristic features to the standard PHB. Finally, this study proves the feasibility of employing the SSF process for PHB production using waste paper as an alternative approach to overcome the shortcoming of the separate hydrolysis and fermentation (SHF) process. [ABSTRACT FROM AUTHOR]

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction with a kcat of 11 ± 0.4 s-1 We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reactions are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme and on the basis of the present work, we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalyzing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.

The present study focuses on sustainable production of biodegradable polymers by Cupriavidus necator DSMZ 545 using glycerol as substrate. The batch growth and biopolymer production kinetics were established in a 7-L bioreactor, which resulted in a total biomass of 8.88 g L–1 and poly(3-hydroxybutyrate) (PHB) accumulation of 6.76 g L–1. The batch kinetic and independently acquired substrate inhibition data were then used to develop a mathematical model for PHB production process. This was eventually used to design different nutrient feeding strategies under constant feed rate, decreasing feed rate, and pseudo steady state of substrate (glycerol) to optimize the PHB production during fed-batch cultivation. Among all the fed-batch cultivation strategies, the highest PHB accumulation and productivity of 13.12 g L–1 and 0.27 g L–1 h–1, respectively, was achieved in fed-batch bioreactor cultivation where a pseudo steady state with respect to glycerol was maintained.

Here a multiple heavy metal and antibiotic resistant bacterium Cupriavidus necator C39 (C. necator C39) was isolated from a Gold-Copper mine in Zijin, Fujian, China. C. necator C39 was able to tolerate intermediate concentrations of heavy metal(loid)s in Tris Minimal (TMM) Medium (Cu(II) 2 mM, Zn(II) 2 mM, Ni(II) 0.2 mM, Au(III) 70 μM and As(III) 2.5 mM). In addition, high resistance to multiple antibiotics was experimentally observed. Moreover, strain C39 was able to grow on TMM medium containing aromatic compounds such as benzoate, phenol, indole, p-hydroxybenzoic acid or phloroglucinol anhydrous as the sole carbon sources. The complete genome of this strain revealed 2 circular chromosomes and 1 plasmid, and showed the closest type strain is C. necator N-1T based on Genome BLAST Distance Phylogeny. The arsenic-resistance (ars) cluster GST-arsR-arsICBR-yciI and a scattered gene encoding the putative arsenite efflux pump ArsB were identified on the genome of strain C39, which thereby may provide the bacterium a robust capability for arsenic resistance. Genes encoding multidrug resistance efflux pump may confer high antibiotic resistance to strain C39. Key genes encoding functions in degradation pathways of benzene compounds, including benzoate, phenol, benzamide, catechol, 3- or 4-fluorobenzoate, 3- or 4-hydroxybenzoate and 3,4-dihydroxybenzoate, indicated its potential for degrading those benzene compounds.

[Display omitted] • Nutrient stresses were applied to Cupriavidus necator growing on carbon dioxide. • Nitrogen starvation induced a PHB accumulation of 54% of total cell dry weight. • The carbon conversion yield into PHB reached a maximum of 85% under nitrogen stress. • Single-stage starvation was more effective than double-stages for PHB production. • Gas transfer enhancement in the fed-batch mode could improve the polymer yield. Cupriavidus necator is a facultative chemolithoautotrophic bacterium able to convert carbon dioxide into poly-3-hydroxybutyrate. This is highly promising as the conversion process allows the production of sustainable and biodegradable plastics. Poly-3-hydroxybutyrate accumulation is known to be induced by nutrient starvation, but information regarding the optimal stress conditions controlling the process is still heterogeneous and fragmentary. This study presents a comprehensive comparison of the effects of nutrient stress conditions, namely nitrogen, hydrogen, phosphorus, oxygen, and magnesium deprivation, on poly-3-hydroxybutyrate accumulation in C. necator DSM545. Nitrogen starvation exhibited the highest poly-3-hydroxybutyrate accumulation, achieving 54% of total cell dry weight after four days of nutrient stress, and a carbon conversion efficiency of 85%. The gas consumption patterns indicated flexible physiological mechanisms underlying polymer accumulation and depolymerization. These findings provide insights into strategies for efficient carbon conversion into bioplastics, and highlight the key role of C. necator for future industrial-scale applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Toxicological analysis of common redox mediators with Cupriavidus necator. • Anodic respiration of C. necator with 4 different redox mediators in a BES. • Current densities of up to 260 µA cm−2 are possible with ferricyanide. The non-pathogenic β-proteobacterium Cupriavidus necator has the ability to switch between chemoorganotrophic, chemolithoautotrophic and electrotrophic growth modes, making this microorganism a widely used host for cellular bioprocesses. Oxygen usually acts as the terminal electron acceptor in all growth modes. However, several challenges are associated with aeration, such as foam formation, oxygen supply costs, and the formation of an explosive gas mixture in chemolithoautotrophic cultivation with H 2 , CO 2 and O 2. Bioelectrochemical systems in which O 2 is replaced by an electrode as a terminal electron acceptor offer a promising solution to these problems. The aim of this study was to establish a mediated electron transfer between the anode and the metabolism of living cells, i.e. anodic respiration, using fructose as electron and carbon source. Since C. necator is not able to transfer electrons directly to an electrode, redox mediators are required for this process. Based on previous observations on the extracellular electron transfer enabled by a polymeric mediator, we tested 11 common biological and non-biological redox mediators for their functionality and inhibitory effect for anodic electron transfer in a C. necator -based bioelectrochemical system. The use of ferricyanide at a concentration of 15 mM resulted in the highest current density of 260.75 µA cm−2 and a coulombic efficiency of 64.1 %. [ABSTRACT FROM AUTHOR]

The intracellular nature of microbial polyhydroxyalkanoates (PHAs) requires two main steps in their production: bioconversion and extraction. The most widely used method for recovering poly(3-hydroxybutyrate) (PHB) is solvent extraction. Traditional approaches involve the use of halogenated solvents, whose environmental and health hazards are well known. New alternatives advocate the use of sustainable agents, which can be an effective and economical choice. In this work, the bioconversion process of starch-based water into PHB by Cupriavidus necator DSM 545 was first scaled-up using previously optimized conditions. A statistical model was then developed to optimize the best conditions for polymer extraction using NaOH by response surface methodology with a central composite design. The optimized and validated conditions were: 0.05 M NaOH at 29.6 °C for 1 h, resulting in 87.31 ± 0.33 % polymer yield, 104.13 ± 2.22 % polymer purity and 104.76 ± 2.41 % polymer recovery. These conditions were validated for operation at three different biomass-to-NaOH concentrations: 5.7 g/L, 20 g/L and 79.5 g/L, and scaled-up without sacrificing polymer yield, with high levels of PHB purity and recovery. In addition, this work proposed an approach for the scale-up of a halogen-free process for PHB extraction using NaOH, considering parameters that can be easily integrated into the common ranges used in the industry. [Display omitted] • PHB production by Cupriavidus necator DSM 545 using potato wastewater is scaled. • NaOH solution could be a sustainable and cost-effective method for PHA extraction. • PHB extraction is performed using the lowest concentration of the halogen-free agent. • Optimal conditions for PHB extraction are 0.05 M NaOH at 29.6 °C for 1 h. • NaOH extraction yields a polymer with a purity of over 99%. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are biopolymer synthesized as storage granules by microbes in the cell's cytoplasm under excess carbon and limited nitrogen conditions. This study aims to examine the production and optimization of PHA from Ceiba pentandra oil, a non-edible feedstock, by Cupriavidus necator. Response surface methodology was used to optimize the concentrations of carbon, nitrogen, and inoculum for the production of PHA. To improve the physical, mechanical, and thermal properties of PHA, it was blended with polylactic acid (PLA) which possesses high bio-compatibility and bio-absorbability. Furthermore, batch mode cultivation of PHA in a 3-L bioreactor was performed under the optimized conditions (Ceiba pentandra oil—17.13 g/L, urea—1.34 g/L, and inoculum—53.42 mL/L), and 8.51 g/L (0.47 g PHA/g of Ceiba pentandra oil) of PHA product was obtained. The PHA was characterized by FTIR and the presence of functional groups such as C=O, OH, and CH were observed. 1H and 13C NMR spectroscopy revealed the existence of hydroxy valerate (HV) and hydroxybutyrate (HB), and maximal decomposition temperature was identified to be 217.7 °C from TGA analysis. Therefore, Ceiba pendantra oil appears to be an attractive alternate carbon source for PHA production. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are abundant microbial polyesters accumulated in the form of intracellular granules by numerous prokaryotes primarily as storage of carbon and energy. Apart from their storage function, the presence of PHA also enhances the robustness of the microbial cells against various stressors. In this work, we investigated the role of PHA in Cupriavidus necator , a model organism concerning PHA metabolism, for adaptation to osmotic pressure and copper ions. In long-term laboratory evolution experiments, the bacterial culture was cultivated in presence of elevated doses of sodium chloride or copper ions (incubations lasted 78 passages for Cu2+ and 68 passages for NaCl) and the evolved strains were compared with the wild-type strain in terms of growth and PHA production capacity, cell morphology (investigated by various electron microscopy techniques), activities of selected enzymes involved in PHA metabolism and other crucial metabolic pathways, the chemical composition of bacterial biomass (determined by infrared and Raman spectroscopy) and also considering robustness against various stressors. The results confirmed the important role of PHA metabolism for adaptation to both tested stressors. [ABSTRACT FROM AUTHOR]

Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O2-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O2 content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L–1 of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [<0.1 U (L d) –1]. Our strategy has important implications for the use of E. coli K-12 and B strains in the recombinant production of complex metalloenzymes, and provides a blueprint for the production of catalytically active [NiFe]-hydrogenases in biotechnologically relevant quantities. [ABSTRACT FROM AUTHOR]

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active β-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important β-ketothiolases for C6 metabolism in β-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. Key points: • Role of six β-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter. [ABSTRACT FROM AUTHOR]

Recombinant Cupriavidus necator H16/pMPJAS03, expressing a P. putida KT2440 enoyl-CoA hydratase (phaJ), was able to synthesize short-chain-length/ medium-chain-length (scl-mcl) PHA copolymers with a high content of mcl subunits using its native poly(3-hydroxyalkanoate) synthase. The cells were cultivated on fructose with canola oil or canola oil/decanoic acid (DA) mixtures in fed-batch fermentations. The recombinant C. necator H16 (without any synthase modification) produced a polymer composed of 3-hydroxybutyrate (3HB) with mcl-subunits, including 3-hydroxyhexanoate (3HHx), and about 300-fold more 3-hydroxyoctanoate (3HO) than the yields reported in previous studies, as well as a significant amount of 3-hydroxydecanoate (3HD). Increasing the DA content in the feed from 0% to 15% v/v increased the molar content of the 3HD subunits from 1.2 to 2.1 mol%. The presence of larger monomers, such as 3HO and 3HD, decreased the crystallinity and melting temperature and modified the mechanical properties of the polymers. Thus, replacing either of the two gene products (phaJ or phaC1) required to produce PHA from CoA-3-hydroxy fatty acids with broader spectrum enzymes, is suitable for the production of commercially useful scl-mcl-PHA. • PHA synthase of wild type C. necator H16 can accept medium-chain-length substrates if provided with a suitable enoyl-CoA hydratase (phaJ). • Subunits as high as C10 can be incorporated into the polymer. • Resulting materials have thermal and mechanical properties comparable to scl-mcl PHAs produced by strains with "broad spectrum" synthases. [ABSTRACT FROM AUTHOR]

Because of their lower environmental impact, biodegradable polymers such as polyhydroxyalkanoates (PHAs) produced within cultured bacteria represent promising alternatives to petroleum-based plastics. PHA production in flasks yielded optimal results with a carbon-to-nitrogen ratio of 22. The 5-L scale experimental results revealed that when glucose was used as the carbon source, Cupriavidus necator could produce 4.74 g/L PHA with 77.6% of PHA content in the microorganism 72 h after the initiation of the experiment. When the hydrolysis liquid from inedible rice was used as the carbon source, the highest concentration of PHA and ratio of PHA content in the microorganism were 4.82 g/L and 68.6%, respectively, after 72 h. Using the hydrolysis liquid from inedible rice as the carbon source reduced the culture cost and shortened the culture time, without affecting the structure of the PHA during production. Using the hydrolysis liquid from rice as the carbon source in PHA production (by C. necator) yielded optimal results, and the results may serve as a reference for applications involving other PHA-producing bacteria. Employing alternative carbon sources to culture bacteria might become a means of increasing the productivity and ensuring the quality of PHA products in the future. [ABSTRACT FROM AUTHOR]

The photocatalyst graphitic carbon nitride (g-C 3 N 4) is known to photostimulate the production of the bioplastic polyhydroxybutyrate (PHB) by Cupriavidus necator. In previous studies, the combination of C. necator and g-C 3 N 4 increased PHB yield from either an organic or inorganic carbon substrate under a light intensity of 4200 lx. Here, different parameters including light intensity, pH, temperature, nitrogen and carbon concentrations, aeration, and inoculum size were explored to maximize PHB production by hybrid photosynthesis from fructose and visible light. A g-C 3 N 4 / C. necator culture grown with a lower light intensity of 2100 lx, an inoculum size of 128.30 × 106 CFU ml−1, and constant aeration produced 7.16 g l−1 d−1 PHB with a product yield from fructose of 60.94%. Furthermore, the ratio of incident photons harvested by g-C 3 N 4 converted into NADPH+H+ by C. necator for PHB production was improved to 19.74% after the process optimization. In comparison, the PHB production rate of a non-optimized g-C 3 N 4 / C. necator system exposed to 4200 lx was only 2.94 g l−1 d−1 with a product yield from fructose of 33.29%. These results demonstrate that hybrid photosynthesis productivity can be significantly augmented by decreasing light intensity and adjusting other parameters, which is promising for future bioproduction applications. [Display omitted] • Visible light and g-C 3 N 4 augment heterotrophic PHB production by Cupriavidus necator. • g-C 3 N 4 / C. necator synthesizes more PHB at a lower light intensity of 2100 lx. • A constant aeration and a larger inoculum also improve PHB production under light. • After optimization, g-C 3 N 4 / C. necator reaches a PHB production titer of 12.19 g l−1. • The PHB production ield from fructose was 60.94% with a QE NADPH of 19.74%. [ABSTRACT FROM AUTHOR]

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.%. [ABSTRACT FROM AUTHOR]

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. Key points: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization. [ABSTRACT FROM AUTHOR]

Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O2-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O2 content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L–1 of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

Cupriavidus necator DSM 545 was cultivated on a range of crude and emulsified plant oils (olive, rapeseed, sesame seed, spent coffee grounds (SCGs) and sunflower) of varying fatty acid (FA) composition to assess their influence on the organism’s growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] biopolymer production. Initially five different surfactants (gum arabic, dimethyl sulfoxide (DSMO), Tween 80, Triton X-100, and sodium dodecyl sulfate) were screened for their influence on cell growth and ability to form stable oil-in-water emulsions. The highest total biomass (11.3 ± 0.2 g L−1) and P(3HB-co-3HV) (10.0 ± 0.6 g L−1) concentrations were achieved with SCGs oil emulsified with DMSO. At 88.9 ± 5.2%, the corresponding polymer fraction of the total biomass is among the highest values reported in the literature. During the initial growth phase (0–24 h), the organism consumed all fatty acids in broadly the same proportion, but preference was then displayed in the subsequent P(3HB-co-3HV) production phase (24–120 h) for saturated FAs (particularly palmitic acid) over unsaturated FAs. SCG oil was an ideal substrate in comparison to the other plant oils due to its high acid value (17.9 ± 0.1 mgKOH goil−1) and palmitic acid content (31.4 ± 0.5%, w/w). Emulsifying the plant oil cultures initially increased substrate bioavailability, but the influence diminished as the fermentations progressed. Overall, the use of SCGs as a feedstock for PHA production will help reduce the manufacturing costs of the biopolymer and generate a valorisation strategy for an otherwise highly unutilised resource. [ABSTRACT FROM AUTHOR]

Many homologous genes encoding b-oxidation enzymes have been found in the genome of Cupriavidus necator H16 (synonym Ralstonia eutropha H16). By proteome analysis, the degradation of adipic acid was investigated and showed differences from the degradation of hexanoic acid. During b-oxidation of adipic acid, activation with coenzyme A (CoA) is catalyzed by the two-subunit acyl-CoA ligase encoded by B0198 and B0199. The operon is completed by B0200 encoding a thiolase catalyzing the cleavage of acetyl-CoA at the end of the β-oxidation cycle. C. necator DB0198-B0200 strain showed improved growth on adipic acid. Potential substitutes are B1239 for B0198-B0199 and A0170 as well as A1445 for B0200. A deletion mutant without all three thiolases showed diminished growth. The deletion of detected acyl-CoA dehydrogenase encoded by B2555 has an altered phenotype grown with sebacic acid but not adipic acid. With hexanoic acid, acyl-CoA dehydrogenase encoded by B0087 was detected on two-dimensional (2D) gels. Both enzymes are active with adipoyl-CoA and hexanoyl-CoA as substrates, but specific activity indicates a higher activity of B2555 with adipoyl-CoA. 2D gels, growth experiments, and enzyme assays suggest the specific expression of B2555 for the degradation of dicarboxylic acids. In C. necator H16, the degradation of carboxylic acids potentially changes with an increasing chain length. Two operons involved in growth with long-chain fatty acids seem to be replaced during growth on medium-chain carboxylic acids. Only two deletion mutants showed diminished growth. Replacement of deleted genes with one of the numerous homologous is likely. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points: • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. [ABSTRACT FROM AUTHOR]

Over the past decade, formic acid and acetic acid have gained increasing attention as alternative feedstocks for poly-3-hydroxybutyrate (PHB) production as these potentially CO 2 -derived molecules are naturally assimilated by Cupriavidus necator. Both organic acids were individually evaluated in fed-batch fermentations at bioreactor scale. Acetic acid was revealed as the most promising carbon source yielding 42.3 g L−1 PHB, whereas no significant amount of PHB was produced from formic acid. Hence, acetic acid was further used as the substrate during process intensification. Key performance characteristics, including process stability, PHB titer, and productivity were optimized by introducing NH 4 -acetate as the nitrogen source, extending the growth phase, and implementing a repeated fed-batch procedure, respectively. These advanced fermentation strategies resulted in the establishment of a stable fermentation process reaching 58.5 g L−1 PHB, while doubling the productivity to 0.93 g L−1 h−1 PHB. [Display omitted] • Acetic acid was revealed as most promising CO 2 -derived feedstock over formic acid. • Ammonium acetate as nitrogen source enables stable pH-static feeding of acetic acid. • Extension of the growth phase by linear feeding increases final PHB concentration. • Repeated fed-batch strategy enhances PHB productivity. • 58.5 g L−1 PHB was produced with a productivity of 0.93 g L−1 h−1 PHB. [ABSTRACT FROM AUTHOR]

Abstract Background To establish bioplastics as a real alternative to conventional plastics, high production costs must be constrained by using different kinds of wastewater streams as organic substrates and novel microbial strains as material-accumulating bacteria with high performance. Volatile fatty acids (VFAs) from the effluent of dairy wastewater biodigestion represent a new and inexpensive feedstock, which was used in this study for biopolymer production through microbial processes. Results Cupriavidus necator DSM 13513 was particularly able to accumulate PHAs when operating in fed-batch mode by limiting the oxygen level together with intermittent feeding of a carbon source; maximum poly-β-hydroxybutyrate (PHB) accumulation was achieved in 48 h without compromising microbial growth. The complex VFAs mixture from the digestate did not influence PHA homopolymer accumulation. In fact, structural characterization by NMR analysis revealed PHB synthesis by C. necator DSM 13513 grown with different VFAs mixtures. Moreover, the bioplastic disk obtained from C. necator DSM 13513 cells grown on VFAs from digested dairy wastewater effluent presented good thermic properties and low affinity to water. Conclusions Overall, the results make digested dairy wastewater effluent suitable for PHB production for specific biobased industrial applications.

Polyhydroxyalkanoates (PHAs) are polymers of hydroxy derived fatty acids synthesized by various organisms. These polymers can be effectively used as a biocompatible and biodegradable alternative to the chemically synthesized plastic. PHA cost, however, still limits the increase in PHA production. One of the ways to reduce PHA cost is to use inexpensive carbon sources such as fatty acids. The aim of this work was to study the effect of various concentrations of oleic acid (5-50 g/L) on the growth of the Cupriavidus necator B-10646 bacterium, polymer synthesis and properties. Cells were grown for 48 h in Schlegel mineral medium at 30°C and 200 rpm on an incubator shaker. The content and the composition of the polymer were determined by chromatography of fatty acid methyl esters using a chromatographymass spectrometer. The molecular weight distribution of the polymer was determined using gel permeation chromatography. Thermal analysis was performed using a differential scanning calorimeter. The maximum yield of biomass (6.4-6.7 g/L) and the highest polymer content (64- 71% of the weight of dry biomass) were obtained from 10-20 g/L of oleic acid after 48 hours of cultivation. In addition to 3-hydroxybutyrate, which is the dominant monomer (more than 98 mol.%), 3-hydroxyvalerate (0.7-1.7 mol.%) and 3-hydroxyhexanoate (0.1-0.4 mol.%) were identified in the polymer synthesized by the bacterium. As the concentration of oleic acid was increased, both the weight average and the number average molecular weights decreased (from 803 to 381 kDa and from 292 to 94 kDa, respectively) but polydispersity of the polymers increased (from 2.8 to 4.1). The results obtained in the present study provide the basis for the next stage of scaling up the process of PHA synthesis from oleic acid

Polyhydroxyalkanoates have attracted much attention as biodegradable alternative to petroleum-based synthetic plastics. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer is one of the best characterized PHA copolymers because of its high commercial potential. However, commercial use of PHAs has been limited by their high price. One approach to reducing the cost of PHA production is to use inexpensive carbon sources (fatty acids, plant oils, etc.). The aim of this work was to study synthesis of P(3HB-co-3HV) by the Cupriavidus necator B-10646 bacterium grown on oleic acid and different biochemical precursors of 3HV. Bacterial cells were grown for 72 h at 30°C and 200 rpm on an incubator shaker. Salts of propionic or valeric acids were used as precursors of 3HV. The content and the composition of the polymer were determined by gas chromatography of fatty acid methyl esters. Lipids and polymer were extracted from biomass using the method of Folch. The addition of potassium propionate and valerate did not inhibit bacterial growth and polymer synthesis, the cell concentration and polymer content reaching 9.3-9.5 g/L and 80-83%, respectively. The addition of potassium valerate or propionate led to the synthesis of (P(3HB-co-3HV)) copolymer containing 21.2 and 14.3 mol% of 3HV, respectively. The number average molecular weight (Mn) of the polymer synthesized by the bacterium on oleic acid alone was 220 kDa; the polydispersity of the polymer was 3.5. The polymer synthesized in the presence of potassium valerate and propionate was characterized by a lower Mn (156-178 kDa) and a higher polydispersity of the polymer (4.4-4.9). The main fatty acids (FA) of intracellular lipids were oleic (33.26% of the total FA) and palmitic acid (27.48% of the total FA). The addition of potassium propionate or valerate did not cause any significant changes in the composition of the FA of intracellular lipids of the strain studied. This study demonstrates the ability of C. necator B-10646 to synthesize P(3HB-co-3HV) from mixtures of oleic acid and 3HV precursors. The data obtained can be used to develop and implement an economically feasible process of the P(3HB-co-3HV) production

Abstract Background Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones. Results In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

The utilization of waste cooking oil (WCO) or waste fish oil (WFO) as inexpensive carbon substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 was investigated. Fed-batch cultivation mode in bioreactor was applied in this study. High cell dry weight (CDW) of 135.1 g/L, PHB content of 76.9 wt%, PHB productivity of 1.73 g/L/h, and PHB yield of 0.8 g/g were obtained from WCO. In the case of WFO, the CDW, PHB content, PHB productivity, and PHB yield were 114.8 g/L, 72.5 wt%, 1.73 g/L/h, and 0.92 g/g, respectively. The PHB productivity and yield obtained in the current study from WCO or WFO are among the highest reported so far for PHA production using oils as sole carbon substrate, suggesting that both WCO and WFO can be used as inexpensive carbon substrates for the production of PHA on an industrial scale.

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situ to investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator (MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

[Display omitted] • Ineffective electroporation of Cupriavidus necator H16 with pBBR1MCS-2 is explained. • Minimal, modular vectors (pCAT) with broad host range replication machinery were constructed. • pCAT vectors transform C. nector via electroporation with high efficiency. • Genes cloned in pCAT vectors are stably propagated. • pCAT one-pot multi-fragment assemblies can be electroporated directly into C. necator. Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/ μ g DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator , circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features. [ABSTRACT FROM AUTHOR]

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogenoxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01DB1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPPccrMeJAc- emd harboring short-chain-length-specific PhaJ. [ABSTRACT FROM AUTHOR]

Chemolithoautotrophic bacterium Ralstonia eutropha is a versatile host for production of various useful compounds including polyhydroxyalkanoates (PHAs) under both heterotrophic and autotrophic conditions. In this bacterium, Calvin–Benson–Bassham (CBB) cycle is functional even under heterotrophic conditions on sugars and reutilizes CO 2 emitted through sugar metabolisms into PHA, leading to increase in yield of the storage polyester. This study focused on isopropanol production from glucose by engineered strains of R. eutropha. The isopropanol-producing strains were constructed by introduction of codon-optimized genes of acetoacetate decarboxylase (adc) and primary-secondary alcohol dehydrogenase (adh) from clostridia into glucose-utilizing and PHA-negative (Δ phaC1) strain of R. eutropha. Several genetic modifications showed that high expression of the isopropanol synthesis genes by using a strong synthetic promoter and deletion of NAD+-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase genes (paaH1 and had) in addition to NADPH-dependent acetoacetyl-CoA reductase genes (phaB1 and phaB3) were effective for improving isopropanol production with low by-production of acetone. Isopropanol titer of 4.13 g/L was achieved by two-stage cultivation of the strain IP-007/pBj5c2-adh-adc, corresponding to overall yield of 0.6 mol mol-glucose−1. The fixation of sugar-derived CO 2 during isopropanol synthesis was evaluated by 13C-labelling of the isopropanol produced from [1–13C]-glucose. The 13C-abundance in isopropanol synthesized by the engineered strain was significantly increased up to 4.8%, demonstrating actual reassimilation of CO 2 emitted from glucose moiety by decarboxylation and potential contribution towards increase in the carbon yield of isopropanol on glucose. [ABSTRACT FROM AUTHOR]