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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]

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 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]

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]

[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]

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]

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

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

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

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

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

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

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

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

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

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

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]

• Oil (16.8 %, w/w) was extracted from spent coffee grounds (SCG) with n -hexane. • Cupriavidus necator DSM-545 was cultivated on SCG and sunflower oils. • Cells accumulated 89.6 % (w/w) P(3HB- co -3 HV) (6.8 mol% 3 HV) when grown on SCG oil. • Synthesis of 3 HV likely occurs via the succinate-propionate metabolic pathway. Oil extracted from spent coffee grounds (SCG) [yield 16.8 % (w/w)] was discovered to be a highly suitable carbon substrate for the biosynthesis of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [P(3HB- co -3 HV)] copolymers by Cupriavidus necator DSM 545 in the absence of any traditional 3 HV precursors. Cells cultivated in a 3 L bioreactor (batch) reached a total biomass concentration of 8.9 g L−1 with a P(3HB- co -3 HV) (6.8 mol% 3 HV) content of 89.6 % (w/w). In contrast, cells grown on sunflower oil reached a total biomass concentration of 9.4 gL−1 with a P(3HB- co -3 HV) (0.2 mol% 3 HV) content of 88.1 % (w/w). It is proposed that the organism could synthesize 3 HV monomers from succinyl CoA, an intermediate of the tricarboxylic acid (TCA) cycle, via the succinate-propionate metabolic pathway. [ABSTRACT FROM AUTHOR]

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-P BAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadE E.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1 P.p , at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co -polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes. [ABSTRACT FROM AUTHOR]

The valorization of agricultural waste is a sustainable way of how to utilize renewable resources wisely. Grape pomace is a by-product of the manufacturing of the must. The potential of Cupriavidus necator, Halomonas halophile and Halomonas organivorans to produce poly(3-hydroxybutyrate) (PHB) by using oil and fermentable sugars derived from grape pomace has been studied. C. necator proved to be the best producer of PHB when utilizing grape pomace extract as a carbon source. After 29.5 h of fermentation of C. necator in 2-L bioreactor, PHB titers reached 8.3 g l-1 and 63 % PHB content in bacteria cell dry mass. The average molecular weight and the polydispersity of accumulated polymer was 512.2 kDa and 1.2, respectively. In addition to fermentable sugars, this work indicates that oil, pigments, phenolics, lignin and cellulose can be isolated from seeds and the rest of the grape pomace to support the cascading utilization of agricultural waste and natural valuable resources recovery. © 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. [ABSTRACT FROM AUTHOR]

In order to exploit an abundant and cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, the rice husk was pretreated with dilute sulfuric acid and steam flash-explosion to enhance the enzymatic saccharification. The yield of reducing sugars of pretreated rice husk hydrolysate came up to 266.5 mg/g rice husk, much higher than that of untreated rice husk hydrolysate (72.67 mg/g rice husk). This result indicated that the pretreatment can significantly enhance the yield of reducing sugars. Then the hydrolysate was used as a sole carbon source for PHB production by using Cupriavidus necator. Response surface method was used to optimize the fermentation conditions. Under optimum fermentation conditions (carbon source, 31.81 gL−1; nitrogen source, 1.8 gL−1; pH-value, 7.0; temperature, 27.1 °C), the PHB yield was 5.0 gL−1, which was in good agreement with the predicted value. • It is the first time to pretreat rice hull with dilute sulfuric acid and SFE. • Dilute sulfuric acid and SFE is an effective pretreatment method for rice hull. • A new and abundant carbon source for PHB production has been found. • Usage of rice hull hydrolysate for PHB production can protect environment. [ABSTRACT FROM AUTHOR]

Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate- co -(R)-3-hydroxyhexanoate] [P(3HB- co -3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of C upriavidus necator transformants harbouring the phaC BP-M-CPF4 gene (including PHB¯4/pBBR1-C BP-M-CPF4) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa , along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of β-ketothiolase (PhaA Cn) and NADPH-dependent acetoacetyl-CoA reductase (PhaB Cn) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB- co -3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 × 106 to 1.8 × 106 Da. PhaC BP-M-CPF4 displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study. [ABSTRACT FROM AUTHOR]

This work presents the dielectric characterization of rare earth elements (REEs) biosorption by Cupriavidus necator using dielectrophoretic crossover frequency measurements. Traditional means of characterizing biomass for biosorption is limited and time consuming. In this research we are presenting, for the first time, an electrokinetic method termed as dielectrophoresis (DEP) for the characterization of biosorption (uptake) of rare earth elements (REEs) by gram negative bacteria - Cupriavidus necator. To characterize, a 3mm-diameter point and planar microwell device platform is used to measure the DEP crossover frequency that yields the dielectric properties of the targeted biosorbents. Quantified dielectric properties of native Cupriavidus necator (REE−) and those exposed to rare earth elements (REE+): europium, neodymium, and samarium revealed a substantial change in the surface characteristics of the Cupriavidus necator after exposure to the REE solution. The response of C. necator to changes in REE exposure is substantially different for europium but similar between neodymium and samarium. Statistically both the REE+ and REE− groups dielectric signatures were significantly different proving that the REEs were absorbed by the bacteria. This research will revolutionize and impact the researchers and industrialists in the field of biosorption seeking for economical, greener, and sustainable means to recover REEs. Image 1 • Traditional means of characterizing biomass for biosorption is time consuming. • Dielectrophoresis for biosorption by Cupriavidus necator shows promise. • Point and planar microwell to quantify the dielectric properties of the biosorbents. • Quantified dielectric properties of native and REE exposed bacteria revealed changes. • This research will revolutionize and impact the field of biosorption to recover REEs. [ABSTRACT FROM AUTHOR]

• A bioreactor was designed to cultivate C.necator under a safe environment and high pressure. • Cultivations led to growth and production of C. necator up to 3 g DCW L-1 and 3.5 g L-1 isopropanol. • First demonstration of g L-1-scale molecule production from autotrophic engineered bacteria. A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L-1 level from CO 2 for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO 2 , H 2 and air inputs and the ability to operate at high pressures. The bioreactor configuration and cultivation strategy enabled growth of Cupriavidus necator strains for long periods, to reach over 3 g L-1 dry cell weight. No negative impact of the high pressure was observed on viability of the strains up to more than 4 bar overpressure. The cultivation was then carried out using an engineered isopropanol producing strain; in this case, 3.5 g L-1 isopropanol was obtained from CO 2 as the sole carbon source. This is the first reported demonstration of a successful production from engineered bacteria of product in the g L-1 range on CO 2 , raising the prospect of future development of CO 2 -based bioprocesses. [ABSTRACT FROM AUTHOR]

• 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 cost of polyhydroxyalkanoates can be reduced through biological recovery method. • Freeze-dried cells contained a high amount of mineral salts from the culture medium. • Washing the residual mineral salts improved cell consumption by the mealworms. • Mealworms on washed cells recovered PHA granules of higher purity. • The resulting mealworm showed a significant increase in protein content. The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68–70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value <90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Four PHB extraction routes were economically evaluated. • Pretreatment equipment and extraction efficiency defined the best production route. • Extraction efficiency alone is not decisive to define the lower production cost. • Biomass concentration and production capacity are key economic elements. • A production cost of US$ 2.71 per kg can be achieved in a high production scenario. Abstract The aim of this study was to conduct an economic assessment of the entire polyhydroxybutyrate (PHB) production process by Cupriavidus necator , using citric molasses as carbon source and employing propylene carbonate as solvent coupled with different extraction pretreatment methods. Four extraction alternatives were assayed. The alternatives that uses high pressure and heat to pretreat the biomass showed equally lower production costs (US$ 4.28 per kg) and better economic indicators among evaluated scenarios. It was observed that method's extraction capacity is not decisive in selecting the best alternative, but there must be the simultaneous analysis of extractability capacity and expenditures in equipment and utilities. Through five times increase in production capacity, the production cost significantly decreased, reaching US$ 2.71 per kg. Furthermore, achieving high cellular concentration can be responsible for a reduction of up to 18% in the production cost. [ABSTRACT FROM AUTHOR]

Highlights • PHA rich cells of non-halophilic bacterium are resistant to hypotonic lysis. • PHA also helps halophilic bacterium to survive hypotonic shock. • The protective effect of PHA against hypotonic shock may have practical applications. Abstract Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator , the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila ; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity. [ABSTRACT FROM AUTHOR]

[Display omitted] • First reported evidence of PHA autotrophic accumulation from biogenic H 2 and CO 2. • Under autotrophic conditions, C. necator DSM 545 increased 3HB accumulation of 42%. • Two reactors: (i) acetogenesis of melon waste; (ii) H 2 and CO 2 fermentation in PHA. • To boost H 2 and CO 2 , methanogenesis was stopped and OLR increased to 20 gCOD/L/d. • 13 gCOD/L of VFAs and alcohols were also produced and converted into 3HB and 3HV. The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator , able to convert CO 2 and H 2 into PHAs, offer an additional strategy. Typically, the preferred source for CO 2 and H 2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H 2 and CO 2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H 2 (26.7 %) and CO 2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB -co- 3HV). This is the first proof-of-concept of using acidogenic-derived H 2 and CO 2 from fruit waste to produce PHAs. [ABSTRACT FROM AUTHOR]

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator 's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO 2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future. • Taxonomy of Cupriavidus necator strains is examined by comparative genomics. • The metabolism of C. necator is suited to diverse aerobic and anaerobic lifestyles. • C. necator can convert many waste products to polyhydroxyalkanoates. • Metabolic modeling of C. necator is a powerful tool for bioprocess optimization. [ABSTRACT FROM AUTHOR]

[Display omitted] • 84.9 % of VFA was recovered from landfill leachate by pilot-scale MVR. • 91.3 % of ammonia was removed from landfill leachate by pilot-scale MVR. • The recovered VFA was converted to PHBV in a yield of 96% (g PHBV/g DCW). • The ratio of 3HV to 3HB of PHBV produced from landfill leachate was 45%. Serious global problems faced due to many petroleum-based materials in the last century, which is called the plastic age, constitute the main motivation of this research. Considering wastewater treatment from this perspective, both the recovery of organic acids from wastewater and their conversion into bioplastics are extremely important in terms of reducing petroleum dependency. In this study, while the treatment of landfill leachate was provided with biological process integrated into Mechanical Vapor Recompression (MVR), simultaneously PHBV production was carried out with 84.9% recovered VFA as carbon source. The effects of C/N/P ratio and feeding regime on PHBV storage were investigated by Cupriavidus necator. PHBV storage of 96% (g PHBV/g DCW) was maximized by 2-stage feeding and nitrogen restriction. The ratio of 3HV to 3HB of PHBV was 45%. In addition, extracted PHBV was compared with standard PHA in terms of thermal and chemical properties with FTIR, XRD, TGA and DSC analyses. [ABSTRACT FROM AUTHOR]

The effective fermentation of organic waste for polyhydroxyalkanoate (PHA) production is crucial for waste pollution prevention and comprehensive utilization. Glycerol, a common component of organic waste and a by-product of biodiesel production, can serve as a valuable raw material for PHA synthesis. This study explored the potential of metal-organic frameworks (MOFs), specifically MOF-808, to enhance the PHA production by Cupriavidus necator (C. necator) and Bacillus cereus (B. cereus) using glycerol as the substrate. Our results demonstrated that the addition of MOF-808 significantly promoted PHA synthesis without altering the PHA composition. Notably, the proportion of PHA to cell dry weight (CDW) increased significantly (P < 0.05) in both C. necator (from 17.17% to 30.43%) and B. cereus (from 11.30% to 37.93%), with a shortened growth period observed in the latter. The enhanced superproton transport properties of MOF-808 elevated the ATP energy supply level, regulated coenzyme A metabolism, and modulated the PHA synthesis pathway in the bacterial strains. Additionally, MOF-808 improved the utilization of the PHA precursor acetyl-CoA by C. necator , and enhanced the PHA production in B. cereus by facilitating cell growth and PHA synthesis in two distinct stages. Importantly, the recovered MOF-808 structure remained stable, enabling potential recycling. This study provides compelling evidence for the enhancement of PHA production and offers valuable insights into the utilization of organic waste as a valuable resource. [Display omitted] • Enhancement of MOF-808 on PHA production by C. necator and B. cereus was studied. • MOF-808 forms hydrogen bond network to increase ATP energy supply for PHA synthesis. • The proton gradient is heightened, and coenzyme A is activated by the abundant H+. • MOF-808 accelerates the B. cereus growth and diverts the carbon flow of C. necator. • This study provides new idea for improving PHA production from organic waste. [ABSTRACT FROM AUTHOR]

Highlights • Novel two-stage fermentation process for PHB production from grass silage. • Various carbon sources in grass silage used for bioconversion to PHB. • Nutrient-rich PJ as a suitable ready-to-use medium for bacterial growth. • Upstream LAB fermentation of PC hydrolyzate prevents repression effects. • PHB accumulation on consumption of organic acids reached 8.5 g L−1 in 32 h. Abstract Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L−1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L−1 contained 42% PHB. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHAs) are diverse biopolyesters produced by numerous microorganisms and have attracted much attention as a substitute for petroleum-based polymers. Despite several decades of study, the detailed molecular mechanisms of PHA biosynthesis have remained unknown due to the lack of structural information on the key PHA biosynthetic enzyme PHA synthase. The recently determined crystal structure of PHA synthase, together with the structures of acetyl-coenzyme A (CoA) acetyltransferase and reductase, have changed this situation. Structural and biochemical studies provided important clues for the molecular mechanisms of each enzyme as well as the overall mechanism of PHA biosynthesis from acetyl-CoA. This new information and knowledge is expected to facilitate production of designed novel PHAs and also enhanced production of PHAs. Highlights Polyhydroxyalkanoates (PHAs) are natural polyesters consisting of various hydroxyalkanoates (HAs) and have attracted much attention as a feasible substitute to conventional petroleum-based plastics. The PHA biosynthetic pathway consists of three enzymes: acetyl-CoA acetyltransferase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaA catalyzes the two-step condensation reaction of acetyl-CoA to acetoacetyl-CoA, and the size of the binding pocket for acyl-S-enzyme intermediate determines the substrate specificity of the enzyme. Acetoacetyl-CoA is reduced to (R)-3-hydroxybutyryl-CoA by PhaB using NADPH as a cofactor. PHA polymerization reaction takes place at the C-terminal domain of PhaC via non-processive ping-pong mechanism, and the N-terminal domain of PhaC plays an important role in the binding and localization of PhaC to the PHA granule. PhaM binds to the N-terminal domain of PhaC and activates PhaC by reinforcing the binding capacity of PhaC to the growing PHB polymer. The amphipathic surfactant properties of the PhaP are crucial for its interactions with polymer and the other granule-associated proteins PhaR and PhaZ. [ABSTRACT FROM AUTHOR]

A combination of crude glycerol and rapeseed meal from biodiesel production can be utilised to produce a complete microbial feedstock for value-added chemicals production. In this study, rapeseed meal was transformed into a solution rich in free amino nitrogen and was used as a nitrogen source, in combination with crude glycerol as carbon source, to produce poly(3-hydroxybutyrate) (PHB). Using Cupriavidus necator in fed-batch fermentation, PHB concentration at 24.75 g/L with a productivity of 0.21 g·L −1  h −1 , and a yield of 0.32 g-PHB/g-glycerol were obtained. Based on these results, a process for bioconversion of biodiesel by-products to PHB is proposed. In this process, 3.41 L of crude glycerol and 0.72 kg of rapeseed meal are all that is required for the production of 1 kg of PHB. The study demonstrates clearly that a complete microbial feedstock with no requirement for further nutrient supplements can be derived directly from the principal by-products of a conventional biodiesel process. [ABSTRACT FROM AUTHOR]

Cupriavidus necator H16 Re2058/pCB113 could effectively utilize oil sources to synthesize P(3HB- co -3HHx). In this study, P(3HB- co -3HHx) biosynthesis procedure with optimally controlled (between 2–28) 3HHx mol% was established for C. necator H16 Re2058/pCB113 by regulating the amounts of dual carbon sources supplied; date molasses and date seed oil. Alone, date molasses supplied at 20 g/L total sugar equivalents to recombinant C. necator resulted in pure P(3HB) biosynthesis, with accumulation up to 28 wt% of cell dry weight (CDW), and effective total biomass of 4 g/L. Whereas, date seed oil supply alone or in combination with date molasses resulted in P(3HB- co -3HHx) biosynthesis. P(3HB- co -3HHx) with as high as 28 mol% 3HHx, up to 80 wt% of CDW and total biomass of 7.6 g/L were produced using 10 g/L date seed oil as the sole carbon source. The results of PHA production in the presence of date seed oil as a sole carbon source was obtained with a modest yield at a range of 0.38–0.62 g-PHA per g-date oil used. Gel permeation chromatography (GPC) analyses of these P(3HB- co -3HHx) copolymers exhibited weight average molecular weights ( M w ) between 500 − 690 kDa. Differential scanning calorimetry (DSC) analyses showed T m values of 157 °C and 85 °C for P(3HB) and P(3HB- co -28 mol% 3HHx), respectively. NMR analyses ( 1 H and 13 C) of P(3HB- co -3HHx) copolymers confirmed the incorporation of 3HHx monomers between 2–28 mol% depending on the supplied dual carbon source composition. [ABSTRACT FROM AUTHOR]

Industrial manufacturing of polyhydroxyalkanoates (PHAs) requires purification of PHAs granules from high-cell-density cultures. Cells are broken by homogenization and PHAs granules are cleansed and treated to obtain PHAs latexes. However, cell lysis releases large amounts of DNA which results in an increasing viscosity of the medium, hampering the following downstream steps. Drop in viscosity is generally achieved by costly procedures such as heat treatment or the supplementation of hypochlorite and commercially available nucleases. Searching for a cost-effective solution to this issue, a nuclease gene from Staphylococcus aureus has been integrated into two efficient PHAs-producing bacteria: Cupriavidus necator DSM 545 and Delftia acidovorans DSM 39. Staphylococcal nuclease has been proficiently expressed in both microbial hosts without affecting PHAs production. Moreover, the viscosity of the lysates of recombinant C. necator cells was greatly reduced, indicating that the engineered strain is expected to yield large reduction cost in PHAs downstream processing. [ABSTRACT FROM AUTHOR]

We aimed to develop a non-toxic and simple recovery procedure for poly(3-hydroxybutyrate) based on the use of green solvent. In this study, 2-methyltetrahydrofuran, 1,3-propanediol, 1,3-dioxolane and ethyl lactate were investigated and compared with chloroform in the extraction of poly(3-hydroxybutyrate) from Cupriavidus necator strain A-04. A water-miscible system was developed using 1,3-dioxolane and water. PHB was extracted from dried cells at 80 °C for 6 h in 1,3-dioxolane then water was used to cause phase separation of PHB film from 1,3-dioxolane solution resulted in the recovery yield 92.7% ± 1.4% and purity up to 97.9% ± 1.8%. The physical properties and molecular weight distribution of the PHB recovered were not significantly affected by the 1,3-dioxolane extraction. The PHB obtained from wet cells by 1,3-dioxolane extraction at 80 °C for 6 h had a M W of 4.86 × 10 5 , M N of 3.74 × 10 5 , polydispersity index of 1.30. The recovery process is non-toxic and very simple, thus reducing purification costs. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are biodegradable polymers found in the cellular masses of a wide range of bacterial species and the demand for PHA is steadily growing. In this work we have produced PHA from a low-cost substrate, Calophyllum inophyllum oil, using Cupriavidus necator . Effects of various process parameters such as Oil concentration, Nitrogen source and inoculum size on the production of PHA were studied using Response Surface Methodology. A quadratic equation was used in the model to fit the experimental data. It was found that the model could satisfactorily predict the PHA yield (R 2 = 99.17%). Linear, quadratic and interaction terms used in the model were found to be statistically significant. Maximum PHA yield of 10.6 g L −1 was obtained under the optimized conditions of oil concentration - 17.5%, inoculum concentration - 50 mL/L and nitrogen content - 1.125 g L −1 , respectively. The product obtained was characterized using FTIR and NMR to confirm that it was PHA. The results demonstrate that C. inophyllum oil, a non-edible oil, can be potentially used as a low-cost substrate for the production of PHA. [ABSTRACT FROM AUTHOR]

The present work aimed to verify the feasibility of producing polyhydroxyalkanoates (PHAs) at high concentrations from an alternative and cheap carbon source such as the carboxylic acids (VFAs) obtained by the anaerobic fermentation of cheese whey (CW). An electrodialysis (ED) step was proposed for the obtainment of a concentrated acidic stream, suitable for being employed as feeding solution in the consecutive fed-batch culture system for PHAs production. Experiments conducted in duplicate shown that a packed bed anaerobic bioreactor resulted a robust and high repeatable culture system for a high performing continuous production of VFAs from CW. The acidogenic effluent contained 12.55 ± 1.10 g L −1 of carboxylic acids ( ca. 90% of the effluent soluble COD), including the hexanoic (4.13 ± 0.56 g L −1 ) and octanoic (3.12 ± 0.94 g L −1 ) acids. The application of 7 sequential batch ED processes allowed to obtain a carboxylic acids concentrated stream ( ca. 63 g L −1 ); by the achievement of high molar flow. Finally, this stream was utilized as the feeding solution in a fed-batch fermentation aimed to produce PHAs. The attained PHAs yield ( ca. 0.60 g PHAs  g VFAs −1 ) was comparable to that obtained in a parallel test accomplished with a VFAs-water simulating solution and also to those previously reported from pure VFAs; i.e. no inhibition effects due to the employment of an actual biowaste as the feedstock were detected. As a whole, the results allow to conclude that the proposed integrated valorisation scheme fed with CW for the production of PHAs by an ED-concentrated carboxylic acids solution is technically feasible and robust. [ABSTRACT FROM AUTHOR]

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates. [ABSTRACT FROM AUTHOR]

Waste paper, a major fraction of municipal solid waste, has a potential to serve as renewable feedstock for the biorefineries of fuels, chemicals and materials due to rich in cellulose and abundant at low cost. This study evaluates the possibility of waste office paper (WOP) to serve as a potential feedstock for the biorefinery production of poly (3-hydroxybutyrate). In this study, the WOP was pretreated, enzymatically saccharified and the hydrolysate was used for PHB production. The hydrolysate mainly consists of glucose (22.70 g/L) and xylose (1.78 g/L) and the corresponding sugar yield was about 816 mg/g. Ammonium sulphate and C/N ratio 20 were identified as most favorable for high yield of PHB. The batch fermentation of Cupriavidus necator using the pretreated WOP hydrolysate resulted in cell biomass, PHB production and PHB content of 7.74 g/L, 4.45 g/L and 57.52%, respectively. The volumetric productivity and yield achieved were 0.061 g/L/h and 0.210 g/g sugar, respectively. The results suggested that WOP could be a potential alternative feedstock for the biorefinery production of bioplastics. [ABSTRACT FROM AUTHOR]