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The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzymes molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

The International Space Station currently requires four annual replenishments for food supply, a practice that won't be feasible for deep space missions due to the greater distances. Based on the design of closed ecological life support systems, two waste streams were identified: urea from the crew urine, volatile fatty acids (VFAs) from a first stage of anaerobic digestion of waste. The objective of this study was to assess the ability of bacterium Cupriavidus necator to produce single cell protein on urea and VFAs. Thus, the effect of carbon sources (glucose vs VFAs) and the dilution rate on the biomass composition was determined in continuous cultures. Complete transformation of the carbon source into protein-rich biomass was achieved up to 78 % cell dry weight (CDW). For both carbon sources, the protein content increased from 55.0 %CDW to 78 %CDW with a decrease in the dilution rate. Conversely, the nucleic acid and polyhydroxyalkanoate contents decreased with the dilution rate from 8.8 %CDW to 4.8 %CDW and 9.8 %CDW to 0.6 %CDW respectively. Working at a low dilution rate seems to be a good way to maximize protein content while minimizing unwanted nucleic acids and polyhydroxyalkanoates. [Display omitted] • C. necator continuous cultivations led to total consumption of the carbon sources. • Biomass composition changes with dilution rate regardless carbon source. • Bioplastics were produced even under carbon limitation. [ABSTRACT FROM AUTHOR]

The "knallgas" bacterium Cupriavidus necator is attracting interest due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO2 via the Calvin-Benson-Bassham (CBB) cycle, and grows on organic acids and sugars. Its tripartite genome is notable for its size and duplications of key genes (CBB cycle, hydrogenases, and nitrate reductases). Little is known about which of these isoenzymes and their cofactors are actually utilized for growth on different substrates. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on succinate, fructose, hydrogen (H2/CO2), and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that (i) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. (ii) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. (iii) For hydrogenases, both soluble and membrane-bound enzymes were utilized for lithoautotrophic growth. (iv) Of the six terminal respiratory complexes in C. necator H16, only some are utilized, and utilization depends on the energy source. (v) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necator's genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium and inspire future engineering strategies. IMPORTANCE The soil bacterium Cupriavidus necator can grow on gas mixtures of CO2, H2, and O2. It also consumes formic acid as carbon and energy source and various other substrates. This metabolic flexibility comes at a price, for example, a comparatively large genome (6.6 Mb) and a significant background expression of lowly utilized genes. In this study, we mutated every non-essential gene in C. necator using barcoded transposons in order to determine their effect on fitness. We grew the mutant library in various trophic conditions including hydrogen and formate as the sole energy source. Fitness analysis revealed which of the various energy-generating iso-enzymes are actually utilized in which condition. For example, only a few of the six terminal respiratory complexes are used, and utilization depends on the substrate. We also show that the protein cost for the various lowly utilized enzymes represents a significant growth disadvantage in s pecific conditions, offering a route to rational engineering of the genome. All fitness data are available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/. [ABSTRACT FROM AUTHOR]

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

Waste cooking oil (WCO) from various sources were evaluated as low-cost carbon feedstock to produce poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] copolymer, which is a type of polyhydroxyalkanoate (PHA). Different Cupriavidus necator transformants harbouring phaCBP−M−CPF4 were employed as PHA-producing organisms in one-stage cultivation in shake flasks. Biological and chemical recovery of PHA were evaluated. C. necator Re2058/pHT1-CBP−M−CPF4 was able to achieve a cell dry weight (CDW) of 8.5 g/L, PHA content of 75.5 wt% and PHA concentration of 6.4 g/L from 1% (v/v) WCO. The weight-average molecular weight (Mw) of the synthesized PHA obtained from the same strain was 1440 kDa. The CDW, PHA content and concentration acquired using WCO are comparable to those using fresh oils as carbon substrates. The copolymer produced from distinct sources of WCO and different extraction methods did not have significant differences in molecular weights and thermal properties. Mealworms can perform biological recovery of PHA from cells fed with WCO from distinct sources. This study suggests that WCO from any source can be used as carbon source for bacteria to produce PHA. WCO can be a sustainable alternative for the more expensive fresh oils, and the synthesized PHA can be biologically recovered by mealworms. A combination approach of utilising low-cost substrate such as WCO, high-performance PHA producer including C. necator transformants and biological recovery to extract PHA is beneficial in biosynthesis of PHA copolymer, since this can make PHA production more economical and can improve the ability of PHA to compete with conventional petrochemical plastics. [ABSTRACT FROM AUTHOR]

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.

Important improvements have been achieved in developing the coupling of electrochemical CO2 reduction to formate with its subsequent microbial conversion to polyhydroxybutyrate (PHB) by Cupriavidus necator. The CO2 based formate electrosynthesis was optimised by electrolysis parameter adjustment and application of Sn based gas diffusion electrodes reaching almost 80 % Faradaic efficiency at 150 mA cm−2. Thereby, catholyte with the high formate concentration of 441±9 mmol L−1 was generated as feedstock without intermediate downstream processing for semi‐automated formate feeding into a fed‐batch reactor system. Moreover, microbial formate conversion to PHB was studied further, optimised, and successfully scaled from shake flasks to semi‐automated bioreactors. Therein, a PHB per formate ratio of 16.5±4.0 mg g−1 and a PHB synthesis rate of 8.4±2.1 mg L−1 OD−1 h−1 were achieved. By this process combination, an almost doubled overall process yield of 22.3±5.5 % was achieved compared to previous reports. The findings allow a detailed evaluation of the overall CO2 to PHB conversion, providing the basis for potential technical exploitation. [ABSTRACT FROM AUTHOR]

Abstract Background Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Background: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry. [ABSTRACT FROM AUTHOR]

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner–Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden–Meyerhof–Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD600 of the phaC1AB1-knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD600 of phaC1AB1-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator, an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden–Meyerhof–Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator. [ABSTRACT FROM AUTHOR]

The aim of this work was to produce the biopolymer polyhydroxybutyrate (PHB) from industrial flue gas as CO2 source and electrolysis originating hydrogen via microbial electrosynthesis. Besides the laboratory experiments, the experiments were carried out under industrial conditions directly on-site in a cogeneration plant. We were able to demonstrate that the use of flue gas as a CO2 source has no detectable negative effect on bacterial growth and PHB production in comparison to a pure gas mixture. In an electrochemical H-cell 333 ± 44 mg L−1 PHB were obtained, which corresponds to a PHB content of 43 ± 3 % of the cell dry weight. By using flue gas for the production of the biopolymer PHB, not only CO2 emissions are reduced, but also possible environmental pollution from non-biodegradable plastics. To the best of our knowledge, this is the first time that the production of PHB from flue gas has been demonstrated using Cupriavidus necator.

Bioelectrochemical systems with Cupriavidus necator present a viable solution for harnessing H2/CO2 mixtures as substrates, employing mediated electron transfer to an infinite electron acceptor in the form of an anode instead of O2. Fourteen redox mediators were spectroelectrochemically characterized, and their efficiency was evaluated through screening with C. necator in common cuvettes with screen printed electrodes (e-Cuvettes). Key performance indicators, including total turnover number, reduction rate, and growth, were analyzed. Ferricyanide emerged as highly effective for anodic respiration, reaching a total turnover number of 8.38 over 120 h of cultivation. On the other hand, phenazine methosulfate exhibited the highest reduction rate at 2.49 mM h−1 with a total of 5.16 turnovers. Contrary, growth impairment is reported for menadione, possibly leading to deficient anodic electron transfer. The utilization of a broad spectrum of these shuttle molecules highlights the potential for optimizing bioelectrochemical applications involving C. necator.

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

[Display omitted] • Sudan black absorption, optical density monitoring of intracellular PHA is proposed. • PLS model on autoscaled data can reliably predict polymer content. • Model is resilient to random error perturbation. • Intracellular protein remains the most important predictor in PLS regression. • Method limit of detection for intracellular PHA is quantified. Rapid quantitation of intracellular polyhydroxyalkanoate (PHA) is essential for monitoring and possibly intervening in ongoing bioprocesses. The traditional multistep protocol involving cell separation, lysis, organic solvent extraction, and purification of intracellular polymers is time intensive. In this study, indirect Sudan black (ISB) adsorption data combined with culture optical density and intracellular protein measurements were used to develop partial least squares (PLS) regression models to predict the intracellular PHA concentration in Cupriavidus necator. ISB analysis supported the hypothesized partitioning of Sudan black into residual biomass, PHA, and abiotic compartments. The PLS regression model developed with autoscaled data offered excellent fit on cross-validation (R2 CV of 0.987) and independent prediction (R2 P of 0.994) sets. The method limit of detection for the PLS model (0.036 g l−1) was reasonably low to track biopolymer accumulation in microbial culture, even in early growth phase. The conservative estimate suggests a significantly shorter processing time with the machine learning-based approach (190 min) than with the traditional protocol (940 min) while retaining analytical interchangeability. [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.)

[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]

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.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB- co -17 mol% 3 HV- co -44 mol% 4 HV) and Poly (25 mol% 3HB- co -75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB−4 harboring the gene phaC BP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB- co -11 mol% 3 HV- co -25 mol% 4 HV- co -32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB- co -3 HV- co -4 HV) is a rubber-like amorphous polymer and Poly (3HB- co -5 HV) has a high tensile strength and elongation at break. Poly (3HB- co -3 HV- co -4 HV- co -5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV. [ABSTRACT FROM AUTHOR]

[Display omitted] • Spectroelectrochemical analysis of redox mediators. • Database of mediators for anodic respiration with versatile C. necator. • Phenazine metho- and ethosulfate achieve high reduction rates and turnovers. • Highest turnover numbers can be achieved with ferricyanide. Bioelectrochemical systems with Cupriavidus necator present a viable solution for harnessing H 2 /CO 2 mixtures as substrates, employing mediated electron transfer to an infinite electron acceptor in the form of an anode instead of O 2. Fourteen redox mediators were spectroelectrochemically characterized, and their efficiency was evaluated through screening with C. necator in common cuvettes with screen printed electrodes (e-Cuvettes). Key performance indicators, including total turnover number, reduction rate, and growth, were analyzed. Ferricyanide emerged as highly effective for anodic respiration, reaching a total turnover number of 8.38 over 120 h of cultivation. On the other hand, phenazine methosulfate exhibited the highest reduction rate at 2.49 mM h−1 with a total of 5.16 turnovers. Contrary, growth impairment is reported for menadione, possibly leading to deficient anodic electron transfer. The utilization of a broad spectrum of these shuttle molecules highlights the potential for optimizing bioelectrochemical applications involving C. necator. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cupriavidus necator can grow by consuming CO 2 derived from raw biogas streams. • The choice of growth conditions is crucial for the setup of autotrophic cultivation. • Biogas upgrading is achieved through the effective CO 2 assimilation by C. necator. • Transcriptomics prove the relevance of inoculum adaptation for autotrophic growth. • C. necator adapts to CO 2 changing the respiratory chain and central carbon metabolism. Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator , known for converting carbon dioxide (CO 2) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO 2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H 2 and O 2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO 2 -adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications. [ABSTRACT FROM AUTHOR]

The aim of this work was to produce the biopolymer polyhydroxybutyrate (PHB) from industrial flue gas as CO 2 source and electrolysis originating hydrogen via microbial electrosynthesis. Besides the laboratory experiments, the experiments were carried out under industrial conditions directly on-site in a cogeneration plant. We were able to demonstrate that the use of flue gas as a CO 2 source has no detectable negative effect on bacterial growth and PHB production in comparison to a pure gas mixture. In an electrochemical H-cell 333 ± 44 mg L−1 PHB were obtained, which corresponds to a PHB content of 43 ± 3 % of the cell dry weight. By using flue gas for the production of the biopolymer PHB, not only CO 2 emissions are reduced, but also possible environmental pollution from non-biodegradable plastics. To the best of our knowledge, this is the first time that the production of PHB from flue gas has been demonstrated using Cupriavidus necator. • First-ever production of polyhydroxybutyrate from flue gas with Cupriavidus necator. • Microbial growth and product formation not affected by flue gas components. • Using CO 2 as a resource while reducing greenhouse gas emissions. [ABSTRACT FROM AUTHOR]

Important improvements have been achieved in developing the coupling of electrochemical CO 2 reduction to formate with its subsequent microbial conversion to polyhydroxybutyrate (PHB) by Cupriavidus necator. The CO 2 based formate electrosynthesis was optimised by electrolysis parameter adjustment and application of Sn based gas diffusion electrodes reaching almost 80 % Faradaic efficiency at 150 mA cm -2 . Thereby, catholyte with the high formate concentration of 441±9 mmol L -1 was generated as feedstock without intermediate downstream processing for semi-automated formate feeding into a fed-batch reactor system. Moreover, microbial formate conversion to PHB was studied further, optimised, and successfully scaled from shake flasks to semi-automated bioreactors. Therein, a PHB per formate ratio of 16.5±4.0 mg g -1 and a PHB synthesis rate of 8.4±2.1 mg L -1  OD -1  h -1 were achieved. By this process combination, an almost doubled overall process yield of 22.3±5.5 % was achieved compared to previous reports. The findings allow a detailed evaluation of the overall CO 2 to PHB conversion, providing the basis for potential technical exploitation., (© 2024 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

• C. necator H16 produces P(3HB)/PLA (40/60) using a lactate-enriched fermented wastewater from a candy industry. • Lactate content in P(3HB)/PLA can be tailored in a defined medium using C. necator H16 resting cells. • Proteomic analysis provides new insights into P(3HB)/PLA production by native enzymes in C. necator H16. The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC–MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • More archaea, bacteria, microalgae, and yeasts show potentials as PHA producers. • Feeding strategies and C/N ratio are essential for Cupriavidus necator to produce PHA. • Biomass waste and C1 compounds are the alternatives and economical raw materials. • PHA manufacturing from bench to industrial scale is demonstrated in this review. • Integrative waste utilization and carbon capture is the new opportunity of PHA industry. The pursuit of carbon neutrality goals has sparked considerable interest in expanding bioplastics production from microbial cell factories. One prominent class of bioplastics, polyhydroxyalkanoates (PHA), is generated by specific microorganisms, serving as carbon and energy storage materials. To begin with, a native PHA producer, Cupriavidus necator (formerly Ralstonia eutropha) is extensively studied, covering essential topics such as carbon source selection, cultivation techniques, and accumulation enhancement strategies. Recently, various hosts including archaea, bacteria, cyanobacteria, yeast, and plants have been explored, stretching the limit of microbial PHA production. This review provides a comprehensive overview of current advancements in PHA bioproduction, spanning from the native to diversified cell factories. Recovery and purification techniques are discussed, and the current status of industrial applications is assessed as a critical milestone for startups. Ultimately, it concludes by addressing contemporary challenges and future prospects, offering insights into the path towards reduced carbon emissions and sustainable development goals. [ABSTRACT FROM AUTHOR]

[Display omitted] • Industrial scale PHB production model using various carbon sources was developed. • Techno-economic analysis was used to assess the effect of raw materials on cost. • Cheapest PHB unit production cost was 3.63 $/kg when fructose was consumed. • Sensitivity analysis show that the price of H 2 effects PHB price the most. • Using CO 2 and formate for producing PHB can be feasible in the bioplastic market. Polyhydroxybutyrate (PHB) is a biodegradable polymer that has potential to replace petroleum-derived plastics. However, the commercialisation of PHB is hindered by high production costs. In this study, the material flow and economics of an industrial scale PHB production process using fructose, formic acid and carbon dioxide (CO 2) as carbon sources were simulated and analysed. The lowest breakeven price of 3.64 $/kg PHB was obtained when fructose was utilized as carbon source. When formic acid and CO 2 were used, the breakeven price was 10.30 and 10.24 $/kg PHB due to raw material cost, respectively. Although using formic acid and CO 2 is more expensive, they meet the emerging sustainable needs for plastic production and contribute to the circular economy via CO 2 fixation. This study suggests that the use of formic acid and CO 2 as feedstock for PHB production has potential to become competitive in the bioplastic market with further research. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are biodegradable polyesters that can be produced from renewable resources. However, PHA biomanufacturing is costly compared to petrochemical-based plastics. A promising solution consists of using cassava (Manhiot esculenta) waste, abundant biomass in developing countries, as a carbon source for PHA production. This study involved characterising untreated and acid-hydrolysed cassava peel (CP) to confirm the degradation of polysaccharides into fermentable sugars after pre-treatment. A chemical and biological integrated process was developed, optimising the pre-treatment using a central composite design. The highest conversion of CP into reducing sugars was 97% (w/w) using 3 M H 2 SO 4 , 120 min and 90 ºC. The ability of Cupriavidus necator to grow on CP hydrolysate and produce PHA was screened resulting in up to OD 600 15.8 and 1.5 g/L of PHA (31% (g PHA /g DCW)). Flow cytometry allowed rapid, simple, and high-throughput assessment of PHA content. These findings pave the way for developing a biorefinery platform for PHA production from cassava waste. [Display omitted] • Cassava peel (CP) is a renewable resource with great valorisation potential. • CP was converted into polyhydroxyalkanoates (PHA) via chemical and microbial routes. • Optimisation of CP acid hydrolysis resulted in 97% conversion into reducing sugars. • The integration of chemical and biological processes resulted in 31% of PHA. • Flow cytometry allowed rapid, simple and high-throughput assessment of PHA content. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate) (PHB) is one of the most well-known biodegradable and biocompatible biopolymers produced by prokaryotic microorganisms. It belongs to the family of polyhydroxyalkanoates (PHAs), and it has gained significant attention in recent years due to its potential as a sustainable alternative to traditional petroleum-based plastics. Cupriavidus necator has been identified as a potential producer of PHB for industrial applications due to its ability to produce high amounts of the polymer under controlled conditions, using a wide range of waste substrates. In this study, the ability of Cupriavidus necator DSM 545 strain to produce PHB was tested in a fed-batch strategy providing two different organic substrates. The first is a sugar-based syrup (SBS), derived from cereal waste. The second is an acetate-rich medium obtained through CO 2 -H 2 fermentation by the acetogenic bacterium Acetobacterium woodii. The carbon sources were tested to improve the accumulation of PHB in the strain. C. necator DSM 545 proved to be able to grow and to perform high accumulation of biopolymer on waste substrates containing glucose, fructose, and acetate, reaching about 10 g/L of PHB, 83% of biopolymer accumulation in cell dry mass, in 48 h of fed-batch fermentation in 0.6 L working volume in a bioreactor. Moreover, a Life Cycle Assessment analysis was performed to evaluate the environmental impact of the process converting the sugar syrup alone and the integrated one. It demonstrated that the integrated process is more sustainable and that the most impactful step is the PHB production, followed by the polymer extraction. • Cupriavidus necator DSM 545 growth and poly(3-hydroxybutyrate) biosynthesis using a sugar-based syrup from cereal waste. • C. necator DSM 545 growth and PHB biosynthesis providing a cereal waste substrate and acetate from gas fermentation. • Environmental evaluation of the integrated bioprocess for PHB production through the Life Cycle Assessment. [ABSTRACT FROM AUTHOR]

The exploitation of agri-food wastes is of great importance for environmental and economic reasons. Date wastes are attractive biomasses that could be used as a carbon source for the growth of microorganisms to obtain added-value products. In this work, spoilage date syrup, containing 102.01 and 101.00 g/L of glucose and fructose, respectively, was assessed as a feedstock for the production of bioethanol and polyhydroxyalkanoates (PHAs) by Saccharomyces cerevisiae MEL2 and Cupriavidus necator DSM 545, respectively. The waste date syrup was first evaluated as a carbon source for microbial growth and resulted to sustain the growth of both strains. 47.95 g/L of ethanol, corresponding to the 93.52% of the theoretical yield, were obtained from the fermentation of date syrup by S. cerevisia e MEL2, here adopted as a proficient bioethanol yeast strain. Furthermore, C. necator DSM 545, a well-known PHAs-producer, was able to accumulate up to 79.20 % (w/w on dry mass) of PHAs. This study demonstrates that bioethanol and PHAs can be obtained from date wastes, contributing to developing cost-effective exploitation of these residues with economic and environmental advantages. [Display omitted] • Spoilage dates are suitable substrate for bioethanol and PHAs production. • A simple hot water extraction allows to obtain a date syrup containing simple sugars. • Simple sugars in syrup were converted by Saccharomyces cerevisiae MEL2 into ethanol. • A one step fermentation mode appears efficient in terms of sugars to PHAs conversion. • Cupriavidus necator DSM 545 is an effective PHAs‐ accumulator from date syrup. [ABSTRACT FROM AUTHOR]

The trend in bioplastic application has increased over the years where polyhydroxyalkanoates (PHAs) have emerged as a potential candidate with the advantage of being bio-origin, biodegradable, and biocompatible. The present study aims to understand the effect of acetic acid concentration (in combination with sucrose) as a mixture variable and its time of addition (process variable) on PHA production by Cupriavidus necator. The addition of acetic acid at a concentration of 1 g l -1 showed a positive influence on biomass and PHA yield; however, the further increase had a reversal effect. The addition of acetic acid at the time of incubation showed a higher PHA yield, whereas maximum biomass was achieved when acetic acid was added after 48 h. Genetic algorithm (GA) optimized artificial neural network (ANN) was used to model PHA concentration from mixture-process design data. Fitness of the GA-ANN model (R 2 : 0.935) was superior when compared to the polynomial model (R 2 : 0.301) from mixture design. Optimization of the ANN model projected 2.691 g l -1 PHA from 7.245 g l -1 acetic acid, 12.756 g l -1 sucrose, and the addition of acetic acid at the time of incubation. Sensitivity analysis indicates the inhibitory effect of all the predictors at higher levels. ANN model can be further used to optimize the variables while extending the bioprocess to fed-batch operation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The discovery of formate dehydrogenase (Me-FDH1) from Methylorubrum extorquens has provided an avenue for sustainable CO 2 fixation and utilization. However, the mass production of Me-FDH1 is challenging due to the presence of its unique tungsto-bis-metalopterin guanine dinucleotide (W-bis-MGD) cofactor, limiting its practical applications. In this study, C. necator H16 is proposed as a host for the large-scale production of Me-FDH1, utilizing fructose as a carbon source and its inherent machinery for cofactor synthesis. In a minimal salt medium, C. necator H16 could produce active Me-FDH1, which exhibited a specific activity of 80 to 100 U/mg for CO 2 conversion to formate. In fed batch bioreactor experiments, approximately 50 g CDW /L (cell dry weight/L) and 10,000 U/L Me-FDH1 were achieved within 50 h. This study highlights C. necator H16 as the recombinant host for Me-FDH1, paving the way for the future development of efficient mass-production methods for this crucial enzyme., Competing Interests: Declaration of competing interest 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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single-stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/g inulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of M w , and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization., (© 2023. The Author(s).)