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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.

Objectives: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. Results: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD+-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose –nitrogen mineral salt medium (FN). Conclusion: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator , thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 g PHA /g Cells. For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations. • Polyhydroxyalkanoates (PHA) are the most promising alternatives to conventional plastics. • We developed a Raman-based soft sensor for in-line monitoring of PHA biomanufacturing. • An optimized multivariate data analysis allows to extract quantitative information. • Metabolite concentrations and cell dry weight can be reliably tracked. • For the first time, the molecular weight distribution of PHA can be measured in-line. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoate (PHA)-producing bacteria represent a powerful synthetic biology chassis for waste bioconversion and bio-upcycling where PHAs can be produced as the final products. In this study, we present a seamless plasmid construction for orthogonal expression of recombinant PET hydrolase (PETase) in model PHA-producing bacteria P. putida and C. necator. To this end, this study described seamless cloning and expression methods utilizing SureVector (SV) system for generating pSV-Ortho-PHA (pSVOP) expression platform in bioengineered P. putida and C. necator. Genetic parts specifically Trc promoter, pBBR1 origin of replication, anchoring proteins and signal sequences were utilized for the transformation of pSVOP-based plasmid in electrocompetent cells and orthogonal expression of PETase in both P. putida and C. necator. Validation steps in confirming functional expression of PETase activity in corresponding PETase-expressing strains were also described to demonstrate seamless and detailed methods in establishing bioengineered P. putida and C. necator as whole-cell biocatalysts tailored for plastic bio-upcycling. • Seamless plasmid construction for orthogonal expression in PHA-producing bacteria. • Step-by-step guide for high-efficiency generation of electrotransformants of P. putida and C. necator. • Adaptable methods for rapid strain development (Design, Build, Test and Learn) for whole-cell biocatalysis.

Bacterial flagella are assembled with ∼30 different proteins in a defined order via diverse regulatory systems. In gram-negative bacteria from the Gammaproteobacteria and Betaproteobacteria classes, the transcription of flagellar genes is strictly controlled by the master regulator FlhDC. In Gammaproteobacteria species, the FlhDC complex has been shown to activate flagellar expression by directly interacting with the promoter region in flagellar genes. To obtain the DNA-binding mechanism of FlhDC and determine the conserved and distinct structural features of Betaproteobacteria and Gammaproteobacteria FlhDCs that are necessary for their functions, we determined the crystal structure of Betaproteobacteria Cupriavidus necator FlhDC (cnFlhDC) and biochemically analyzed its DNA-binding capacity. cnFlhDC specifically recognized the promoter DNA of the class II flagellar genes flgB and flhB. cnFlhDC adopts a ring-like heterohexameric structure (cnFlhD 4 C 2) and harbors two Zn-Cys clusters, as observed for Gammaproteobacteria Escherichia coli FlhDC (ecFlhDC). The cnFlhDC structure exhibits positively charged surfaces across two FlhDC subunits as a putative DNA-binding site. Noticeably, the positive patch of cnFlhDC is continuous, in contrast to the separated patches of ecFlhDC. Moreover, the ternary intersection of cnFlhD 4 C 2 behind the Zn-Cys cluster forms a unique protruding neutral structure, which is replaced with a charged cavity in the ecFlhDC structure. • Cupriavidus necator FlhDC complex (cnFlhDC) adopts a heterohexameric structure. • CnFlhDC interacted with the promoter sequences of the flagellar flgB and flhB genes. • CnFlhDC contains Zn-Cys clusters to stabilize the complex structure. • CnFlhDC harbors positively charged, continuous patch for DNA recognition. [ABSTRACT FROM AUTHOR]

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO 2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse β-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical. [Display omitted] • Combination of process and metabolic engineering strategies for the biological upgrading of formate into a platform chemical. • Formate toxicity is assessed in C. necator under both formatotrophic and mixotrophic growth modes. • A small-scale, pH-stat system with addition of formic acid is developed to obtain formatotrophic growth in the g/L range. • Engineering a crotonate biosynthetic pathway results in product formation under heterotrophic and formatotrophic conditions. • An ATP-dependent, thermodynamically favorable malonyl-CoA bypass increases crotonate titers, rates and yields. [ABSTRACT FROM AUTHOR]

The industrial waste generated in the extraction of carrageenan from red seaweed, Eucheuma spinosum, was tested in this study to produce fermentable sugars that could be used for the production of high‐value‐bioproducts with a biorefinery approach. A sequential process was used: thermochemical pretreatment with HCl and enzymatic hydrolysis. Hydrogen chloride concentrations in the range from 0 mol L−1 to 0.5 mol L−1 and pretreatment times from 15 to 100 min were assayed. The best conditions found for pretreatment were HCl 0.3 mol L−1 for 60 min, leading to reducing sugar concentrations of 21.4 g L−1 (274 mg of reducing sugars per gram of algal residue). The hydrolysates coming from the sequential process under the pretreatment conditions of HCl 0.3 mol L−1 for 60 and 80 min have been used successfully for the production of polyhydroxyalkanoates by Cupriavidus necator. The yields of polyhydroxybutyrate were 0.21–0.26 g PHB g−1 reducing sugar consumed and the accumulation of the biopolymer was of the order of 58% dry cell weight. © 2023 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Third-generation (3G) biorefineries harnessing industrial off-gases have received significant attention in the transition towards a sustainable circular economy. However, uncertainties surrounding their techno-economic feasibility are hampering widespread commercialization to date. This study investigates the production of single-cell protein (SCP), a sustainable alternative food and feed protein, from steel mill off-gas through an efficient coupled fermentation approach utilizing acetate as an intermediate. A comprehensive model that comprises both the gas-to-acetate and the acetate-to-SCP fermentation processes, as well as gas pretreatment and downstream processing (DSP) operations, was developed and used to perform a techno-economic analysis (TEA). Sensitivity analyses demonstrated that significant cost reductions can be achieved by the process intensification of the gas-to-acetate fermentation. As such, an increase in the acetate concentration to 45 g/L and productivity to 4 g/L/h could lead to a potential cost reduction from 4.15 to 2.78 USD/kg. In addition, the influence of the production scale and other economic considerations towards the commercialization of off-gas-based SCPs are discussed. Conclusively, this research sheds light on the practical viability of a coupled fermentation process for SCP production by identifying key cost-influencing factors and providing targets for further optimization of the acetate platform, fostering sustainable and economically feasible bio-based innovations. [ABSTRACT FROM AUTHOR]

Because of its application potential and biodegradability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate;PHBV), a member of the polyhydroxyalkanoates (PHA) biopolymer family, is one of the most extensively studied PHA. High PHBV productivity with a significant amount of hydroxyvalerate (HV) content is very appealing for commercial scale production. The goal of this study was to investigate the efficiency of various defined limitation strategies, namely nitrogen, phosphorus, and oxygen-limitation, for high yield PHBV production by Cupriavidus necator H16 with increased HV unit using waste frying vegetable oil (WFO) and propionic acid (PA) in a high cell density culture (5 L bioreactor). With optimized WFO and PA feeding, highest PHBV harvest (121.7 ± 2.59 g/L; HV 13.9 ± 0.44% (w/w)) and volumetric productivity (2.03 ± 0.04 gPHBV/L·h) were obtained in oxygen-limited operation, while highest HV content (19.8 ± 0.28 wt%) and yield coefficient (0.43 ± 0.017 gHV/gPA) were observed during phosphorus-limited cultivation. Although nitrogen limitation is widely applied in the production of PHA, nitrogen-limited cultivation had the lowest cell dry matter, PHBV production, volumetric productivity, oil-to-HB and PA-to-HV yield coefficients for the given conditions. The results of the present study demonstrate the highest PHBV yield together with the highest HV content using WFO as main carbon source and PA as the HV precursor ever reported in the literature. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate;PHBV), a member of the polyhydroxyalkanoates (PHA) biopolymer family, is one of the most extensively studied PHA due to its high application potential and biodegradability properties. High PHBV productivity with substantial amount of hydroxyvalerate (HV) content is of great interest for commercial scale PHA production to compete with conventional plastic costs. In this study, we investigated the effectiveness of different nutrient limitation strategies in optimizing the production of PHBV from waste frying vegetable oil and propionic acid. Although there are individual studies investigating the performance of operational PHBV production strategies such as nitrogen, phosphorus, or oxygen limitation, none of them have comprehensively compared the effect of different limitation strategies on PHA production parameters using waste frying vegetable oil as the main carbon source and propionic acid as the cheapest HV precursor. To our knowledge, this is the first study that evaluates the impact of these three limitation strategies on the efficient production of PHBV from waste frying oil and propionic acid in high cell density fermentations of Cupriavidus necator H16. The results demonstrate the highest PHBV yield together with the highest HV content using waste frying oil as main carbon source and propionic acid as the cheapest HV precursor ever reported in the literature. In addition, it is shown that the PHBV yield and its HV content could be intercorrelated by switching between oxygen and phosphorus-limited strategies for desired material specifications. [ABSTRACT FROM AUTHOR]

Microorganisms have the potential to produce nutrient‐rich products that can be consumed as food or feed. The protein‐rich powder derived from heat treatment of the whole‐cell biomass of polyhydroxybutyrate‐deficient Cupriavidus necator, a metabolically versatile organism that uses elements found in the air, is an example of such a product. To assess the safety of the protein powder for use as a nutritional ingredient in human food, in accordance with internationally accepted standards, its genotoxic potential and repeated‐dose oral toxicity were investigated. A bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test were performed. No evidence of mutagenicity or genotoxicity was found. Additionally, a 90‐day repeated‐dose oral toxicity study in rats was completed, in which a total of 100 male and female Wistar rats were exposed by gavage to daily doses of 1000, 2000, or 3000 mg/kg bw/day of the test material. Following 90 days of continuous exposure, no mortality or treatment‐related adverse effects were observed and no target organs were identified. Therefore, a no observed adverse effect level was determined at 3000 mg/kg bw/day, the highest dose tested. The whole‐cell biomass of polyhydroxybutyrate‐deficient Cupriavidus necator is protein rich. The derived protein concentrate has the potential for use as an environmentally sustainable nutrient‐dense food ingredient. Therefore, we assessed the genotoxic potential and oral toxicity of the heat‐treated and powdered product, in preclinical safety studies. No evidence of genotoxicity or mutagenicity was found. In a 90‐day repeated‐dose oral toxicity study, a no observed adverse effect level in rats of 3000 mg/kg bw/day, the highest feasible dose, was determined. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The 3HV content of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), a biobased and biodegradable polyester, must be controlled for improved thermomechanical properties. Propionic acid (PA) is the main acid allowing 3HV monomer biosynthesis which is present in volatile fatty acid mixes produced from agro-industrial coproducts acidogenesis. With the strain Cupriavidus necator, we investigated the impact of carbon-limiting vs. carbon-overflow conditions thanks to fed-batch cultures, with sequential or continuous feeding of propionic acid as the sole carbon source, and phosphorus deficiency conditions. Regardless of the C-limiting continuous specific feeding rate imposed (i.e., below the determined critical specific feeding rate (q PAcrit) of 0.24 Cmol.Cmol−1.h−1), a regular incorporation of the 3HV in the copolymer at a content of 23 ± 4 mol% was observed with a high molecular weight (≈ 1000 kDa) and a remarkably low polydispersity index (≈ 1.5). This feeding strategy maximized both the productivity (up to 0.78 g.L−1.h−1) and conversion yield of propionic acid into P(3HB-co-3HV) (up to 0.59 Cmol.Cmol−1) if operated close to q PAcrit. With C-overflow sequential feeding (i.e., above q PAcrit), the residual propionic acid concentration has been highlighted as a key parameter in 3HV content improvement (≥ 44 mol%) even though it occurred at the expense of the copolymer yield and productivity. [Display omitted] • C-overflow conditions vs C-limiting conditions were studied. • Propionic acid (PA) feeding rate investigated to tailor P(3HB-co-3HV) synthesis. • Critical PA specific uptake rate (q PAcrit) determined at 0.24 Cmole.Cmole−1.h−1. • Below q PAcrit , a regular random copolymer produced with 23 ± 4 mol% 3HV. • Above q PAcrit , PA accumulation in the medium lead to increased 3HV content. [ABSTRACT FROM AUTHOR]

Purpose: The main objective of the present study is to assess and improve fermentation conditions to produce polyhydroxyalkanoates (PHAs), such as the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from renewable and inexpensive industrial waste by wild type Cupriavidus necator. Methods: Untreated residual glycerol, a byproduct generated by biodiesel industry, was used as the main carbon source by wild type C. necator. The bacterial adaptation stage, the glycerol concentration and other medium components were adjusted to enhance the PHB production. Fed batch fermentation with a pulse feeding strategy of propionic acid solution was employed for PHBV production. Results: C. necator, previously adapted to glycerol utilization, was able to accumulate PHB up to 4.1 g/l under optimal substrate concentration conditions. The bacterium could also synthesize up to 3.4 g/l PHBV, with a mole fraction of 3-hydroxyvalerate (3HV) up to 12% by fed-batch fermentation with a pulsed propionic acid feed strategy. Conclusion: This is the first report on the use of residual glycerol as ideal feedstock for PHA production by wild type C. necator strain. [ABSTRACT FROM AUTHOR]

Cupriavidus necator H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear β-hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from C. necator B-4383 grown under iron limitation. The characterization of B-4383 cupriachelins is based on MS/MS fragmentation analysis, which was confirmed by 1D- and 2D-NMR for the most abundant analog (1). The cupriachelin congeners distinguish these two strains with differences in the preferred lipid tail; however, our rigorous metabolomic investigation also revealed minor analogs with changes in the peptide core, hinting at a potential mechanism by which these siderophores may reduce biologically unavailable ferric iron (4–6). Antifungal screening of the C. necator B-4383 supernatant extract and the isolated cupriachelin analog (1) revealed inhibitory activity against Cryptococcus neoformans, with IC50 values of 16.6 and 3.2 μg/mL, respectively. This antifungal activity could be explained by the critical role of the iron acquisition pathway in the growth and pathogenesis of the C. neoformans fungal pathogen.

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution.Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions.Conclusion:C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L−1 could be achieved. When the isopropanol concentration has reached about 8 g. L−1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L−1 of IPA compared to the reference strain (8 g. L−1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L−1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L− 1. • GroESL improved cellular tolerance to isopropanol. • PSK hok/sok expression improve plasmid stability up to a threshold IPA concentration. • Metabolic load due to GroESL and PSK hok/sok expression deleterious to overall isopropanol production compared to the reference strain. [ABSTRACT FROM AUTHOR]

A promising strategy to alleviate the plastic pollution from traditional petroleum‐based plastics is the application of biodegradable plastics, in which polyhydroxyalkanoates (PHAs) have received increasing interest owing to their considerable biodegradability. In the PHAs family, poly(3‐hydroxybutyrate‐co‐3‐hydroxvalerate) (PHBV) has better mechanical properties, which possesses broader application prospects. With this purpose, the present study adopted Cupriavidus necator to synthesize PHBV utilizing volatile fatty acids (VFAs) as sole carbon sources. Results showed that the concentration and composition of VFAs significantly influenced the production of PHAs. Especially, even carbon VFAs (acetate and butyrate) synthesized only poly(3‐hydroxybutyrate) (PHB), while the addition of odd carbon VFAs (propionate and valerate) resulted in PHBV production. The 3‐hydroxyvalerate (3HV) contents in PHBV were directly determined by the specific VFAs compositions, in which valerate was the preferred substrate for 3HV accumulation. After optimization by response surface methodology, the highest PHBV accumulation achieved 79.47% in dry cells, and the conversion efficiency of VFAs to PHBV reached 40%, with the PHBV production of 1.20 ± 0.05 g/L. This study revealed the metabolic rule of VFAs converting into PHAs by C. necator and figured out the optimal VFAs condition for PHBV accumulation, which provides a valuable reference for developing downstream strategies of PHBV production in industrial applications in future. [ABSTRACT FROM AUTHOR]

The production of poly(hydroxylalkanoates) (PHA) is limited by the high cost of the feedstock since various biomass wastes look attractive as possible sources for polymer production. The originality of this present study is in the biotransformation of mixed Chlorella-based substrates into PHAs. The synthetic potential of Cupriavidus necator B8619 cells was studied during the bioconversion of algae biomass in mixtures with spent immobilized mycelium of different fungi (genus Rhizopus and Aspergillus) into PHAs. The biomass of both microalgae Chlorella and fungus cells was accumulated due to the use of the microorganisms in the processes of food wastewater treatment. The biosorption of Chlorella cells by fungal mycelium was carried out to obtain mixed biomass samples (the best ratio of "microalgae:fungi" was 2:1) to convert them by C. necator B8619 into the PHA. The influence of conditions used for the pretreatment of microalgae and mixed types of biomass on their conversion to PHA was estimated. It was found that the maximum yield of reducing sugars (39.4 ± 1.8 g/L) can be obtained from the mechanical destruction of cells by using further enzymatic hydrolysis. The effective use of the enzymatic complex was revealed for the hydrolytic disintegration of treated biomass. The rate of the conversion of mixed substrates into the biopolymer (440 ± 13 mg/L/h) appeared significantly higher compared to similar known examples of complex substrates used for C. necator cells. [ABSTRACT FROM AUTHOR]

The number of genes encoding β-oxidation enzymes in Cupriavidus necator H16 (synonym, Ralstonia eutropha H16) is high, but only the operons A0459-A0464 and A1526-A1531, each encoding four genes for β-oxidation enzymes, were expressed during growth with long-chain-length fatty acids (LCFAs). However, we observed that C. necator ΔA0459-A0464 ΔA1526-A1531 and C. necator H16 showed the same growth behavior during growth with decanoic acid and shorter FAs. The negative effect of the deletion of these two operons increased with an increasing chain length of the utilized FAs. Transcriptome sequencing (RNA-Seq) revealed the expression profiles of genes involved in the catabolism of medium-chain-length fatty acids (MCFAs) in C. necator H16. Operon A0459-A0464 was expressed only during growth with nonanoic acid, whereas operon A1526-A1531 was highly expressed during growth with octanoic and nonanoic acid. The gene clusters B1187-B1192 and B0751-B0759 showed a log2 fold change in expression of up to 4.29 and 4.02, respectively, during growth with octanoic acid and up to 8.82 and 5.50, respectively, with nonanoic acid compared to sodium gluconate-grown cells. Several acyl-CoA ligases catalyze the activation of MCFAs with coenzyme A (CoA), but fadD3 (A3288), involved in activation of LCFAs, was not detected. The expression profiles of C. necator strain ΔA0459-A0464 ΔA1526-A1531 showed that the growth with nonanoic acid resulted in the expression of further β-oxidation enzyme-encoding genes. Additional insights into the transport of FAs in C. necator H16 revealed the complexity and putative involvement of the DegV-like protein encoded by A0463 in the transport of odd-chain-length FAs and of siderophore biosynthesis in the transport mechanism. [ABSTRACT FROM AUTHOR]

[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]

Membrane biofilm reactors are a growing trend in wastewater treatment whereby gas-transfer membranes provide efficient bubbleless aeration. Recently, there has been a growing interest in using these bioreactors for industrial biotechnology using microorganisms that can metabolise gaseous substrates. Since gas fermentation is limited by the low solubilities of gaseous substrates in liquid media, it is critical to characterise mass transfer rates of gaseous substrates to enable the design of membrane biofilm reactors. The objective of this study is to measure and analyse mass transfer rates and reaction engineering characteristics for a single tube membrane biofilm reactor using Cupriavidus necator H16. At elevated Reynolds numbers, the dominant resistance for gas diffusion shifts from the liquid boundary layer to the membrane. The biofilm growth rate was observed to decrease after 260 μm at 96 h. After 144 h, some sloughing of the biofilm occurred. Oxygen uptake rate and substrate utilisation rate for the biofilm developed showed that the biofilm changes from a single-substrate limited regime to a dual-substrate-limited regime after 72 h which alters the localisation of the microbial activity within the biofilm. This study shows that this platform technology has potential applications for industrial biotechnology.

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]

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]