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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.

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.

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.

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

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 260.75µAcm -2 and a coulombic efficiency of 64.1 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Poly(3-hydroxybutyrate) (PHB) belongs to the family of polyhydroxyalkanoates, biopolymers used for agricultural, industrial, or even medical applications. However, scaling up the production is still an issue due to the myriad of parameters involved in the fermentation processes. The present work seeks, firstly, to scale up poly(3-hydroxybutyrate) (PHB) production by wild type C. necator ATCC 17697 from shaken flasks to a stirred-tank bioreactor with the optimized media and fructose as carbon source. The second purpose is to improve the production of PHB by applying both the batch and fed-batch fermentation strategies in comparison with previous works of wild type C. necator with fructose. Furthermore, thinking of biomedical applications, physicochemical, and cytotoxicity analyses of the produced biopolymer, are presented.Fed-batch fermentation with an exponential feeding strategy enabled us to achieve the highest values of PHB concentration and productivity, 25.7 g/l and 0.43 g/(l h), respectively. The PHB productivity was 3.3 and 7.2 times higher than the one in batch strategy and shaken flask cultures, respectively. DSC, FTIR, 1H, and 13C NMR analysis led to determine that the biopolymer produced by C. necator ATCC 17697 has a molecular structure and characteristics in agreement with the commercial PHB. Additionally, the biopolymer does not induce cytotoxic effects on the NIH/3T3 cell culture.Due to the improved fermentation strategies, PHB concentration resulted in 40 % higher of the already reported one for wild type C. necator using other fed-batch modes and fructose as a carbon source. Thus the produced PHB could be attractive for biomedical applications, which generate a rising interest in polyhydroxyalkanoates during recent years.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490–520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dependency on plastic commodities has led to a recurrent increase in their global production every year. Conventionally, plastic products are derived from fossil fuels, leading to severe environmental concerns. The recent coronavirus disease 2019 pandemic has triggered an increase in medical waste. Conversely, it has disrupted the supply chain of personal protective equipment (PPE). Valorisation of food waste was performed to cultivate C. necator for fermentative production of biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The increase in biomass, PHBV yield and molar 3-hydroxy valerate (3HV) content was estimated after feeding volatile fatty acids. The fed-batch fermentation strategy reported in this study produced 15.65 ± 0.14 g/L of biomass with 5.32 g/L of PHBV with 50% molar 3HV content. This is a crucial finding, as molar concentration of 3HV can be modulated to suit the specification of biopolymer (film or fabric). The strategy applied in this study addresses the issue of global food waste burden and subsequently generates biopolymer PHBV, turning waste to wealth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered., Competing Interests: Competing interests The authors declare that there are no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Broken rice, a low-cost starchy residue of the rice industry, can be an interesting substrate to reduce the polyhydroxyalkanoates (PHAs) production cost. However, since the most common PHAs-producing strains lack amylases, this waste must be firstly hydrolysed by additional commercial enzymes. In this work, the acidogenesis phase of the anaerobic digestion was exploited as efficient hydrolysis step to convert broken rice into volatile fatty acids (VFAs) to be used as PHAs carbon source by Cupriavidus necator DSM 545, one of the most promising PHAs-producing microbes. Broken rice, both non-hydrolysed and enzymatically hydrolysed, was processed in two continuous stirred tank reactors, at hydraulic retention times (HRT) of 5, 4 and, 3 days, to produce VFAs. The highest VFAs levels were obtained from non-hydrolysed broken rice which was efficiently exploited for PHAs accumulation by C. necator DSM 545. PHAs contents were higher after 96 h of incubation and, noteworthy, reached the highest value of 0.95 g/L in the case of 4 days HRT without any chemicals supplementation, except vitamins. Moreover, in view of a biorefinery approach, the residual solid fraction was used for methane production resulting in promising CH 4 levels. Methane yields were very promising again for 4 days HRT. As such, this HRT resulted to be the most suitable to obtain effluents with high promise in terms of both PHAs accumulation and CH 4 production. In addition, these results demonstrate that broken rice could be efficiently processed into two valuable products without any costly enzymatic pre-treatment and pave the way for future biorefining approaches where this by-product can be converted in a cluster of added-value compounds. Techno-economical estimations are in progress to assess the feasibility of the entire process, in view of supporting the low-cost conversion of organic waste into valuable products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal views that have appeared to influence the work described in this manuscript., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The nature of air-inactivation of the formate dehydrogenase FdsDABG from Cupriavidus necator has been investigated. It is found that superoxide, generated in the reaction of reduced enzyme with oxygen, is responsible for the loss of activity and that superoxide dismutase protects the enzyme from air-inactivation. Inhibition appears to be due to the reaction of superoxide with the catalytically essential MoS group of the enzyme's molybdenum center in such a way that generates sulfite. SYNOPSIS: Superoxide generated in the reaction of reduced formate dehydrogenase FdsDABG from Cupriavidus necator with O 2 is found to be responsible for the loss of activity. Catalytic amounts of superoxide dismutase are found to protect FdsDABG just as well as more generally used stabilizing inhibitors such as nitrate., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Cu 2+ 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 × 10 6  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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 10 7 CFU/μg 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Slaughterhouse residues are greatly available and can pose a threat to the environment if not disposed of correctly. Such by-products can be proficiently processed into polyhydroxyalkanoates by accurately selected and developed bacterial strains. Cupriavidus necator DSM 545, one of the most efficient polyhydroxyalkanoates-producing strain, cannot grow well on fatty substrates. In this work, a recombinant lipolytic C. necator microbe was developed for the efficient conversion of slaughtering by-products into polyhydroxyalkanoates. Two lipase sequences, lipC and lipH of Pseudomonas stutzeri BT3, were effectively expressed in C. necator DSM 545. The engineered strain C. necator DSM 545 JR11, selected for the outstanding extracellular lipolytic activity, produced high levels of polyhydroxyalkanoates (nearly 65% of cell dry mass) from udder, jowl and membrane caul fat. This research is crucial to the cost-effective one-step processing of slaughterhouse waste into polyhydroxyalkanoates with useful applications in several industrial and medical sectors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal views that have appeared to influence the work described in this manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

One of the major problems with pesticides is linked to the non-negligible proportion of the sprayed active ingredient that does not reach its intended target and contaminates environmental compartments. Here, we have implemented and provided new insights to the preventive bioremediation process based on the simultaneous application of the pesticide with pesticide-degrading microorganisms to reduce the risk of leaching into the environment. This study pioneers such a practice, in an actual farming context. The 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) and one of its bacterial mineralizing-strains (Cupriavidus necator JMP134) were used as models. The 2,4-D biodegradation was studied in soil microcosms planted with sensitive (mustard) and insensitive (wheat) plants. Simultaneous application of a 2,4-D commercial formulation (DAM®) at agricultural recommended doses with 10 5 cells.g -1 dw of soil of the JMP134 strain considerably accelerated mineralization of the herbicide since its persistence was reduced threefold for soil supplemented with the mineralizing bacterium without reducing the herbicide efficiency. Furthermore, the inoculation of the Cupriavidus necator strain did not significantly affect the α- and β-diversity of the bacterial community. By tackling the contamination immediately at source, the preventive bioremediation process proves to be an effective and promising way to reduce environmental contamination by agricultural pesticides., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Particulate photocatalysts developed for the solar energy-driven reduction of the greenhouse gas CO 2 have a small product range and low specificity. Hybrid photosynthesis expands the number of products with photocatalysts harvesting sunlight and transferring charges to microbes harboring versatile metabolisms for bioproduction. Besides CO 2 , abiotic photocatalysts have been employed to increase microbial production yields of reduced compounds from organic carbon substrates. Most single-reactor hybrid photosynthesis systems comprise CdS assembled in situ by microbial activity. This approach limits optimization of the morphology, crystal structure, and crystallinity of CdS for higher performance, which is usually done via synthesis methods incompatible with life. Here, shape and activity optimized CdS nanorods were hydrothermally produced and subsequently applied to Cupriavidus necator for the heterotrophic and autotrophic production of the bioplastic polyhydroxybutyrate (PHB). C. necator with CdS NR under light produced 1.5 times more PHB when compared to the same bacterium with suboptimal commercially-available CdS. Illuminated C. necator with CdS NR synthesized 1.41 g PHB from fructose over 120 h and 28 mg PHB from CO 2 over 48 h. Interestingly, the beneficial effect of CdS NR was specific to C. necator as the metabolism of other microbes often employed for bioproduction including yeast and bacteria was negatively impacted. These results demonstrate that hybrid photosynthesis is more productive when the photocatalyst characteristics are optimized via a separated synthesis process prior to being coupled with microbes. Furthermore, bioproduction improvement by CdS-based photocatalyst requires specific microbial species highlighting the importance of screening efforts for the development of performant hybrid photosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest Authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A Chlorella sp.-Cupriavidus necator (C. necator) microcosm was artificially established for phenol degradation. The cooperation relationship between Chlorella sp. and C. necator was initially demonstrated, and then the effects of Chlorella sp./C. necator inoculation ratio, light intensity, temperature and pH on the performance of this microcosm were systematically evaluated and optimized. The optimal conditions for phenol degradation were as follows: a Chlorella sp./C. necator inoculation ratio of 1:1, a light intensity of 110 μmol m -2  s -1 , a temperature in the range of 25-32 °C and a pH in the range of 5.5-7.5. Under optimal conditions, this microcosm could degrade phenol with a maximum concentration of 1200 mg L -1 within 60 h. It was found that only when the phenol concentration was reduced to the tolerance concentration of microalgae, that is, the last stage of phenol degradation, the cooperation effect could be generated, indicating that the tolerance of microalgae to phenol may be more important than its degradation performance. Comparative transcriptomic analysis was conducted to discuss the cooperation mechanism of this microcosm subject to high phenol concentrations. The up-regulation of genes involved in photosynthesis and carbon fixation of Chlorella sp. demonstrated the CO 2 and O 2 exchange between Chlorella sp. and C. necator and their cooperation relationship. This study suggests that this microcosm has great potential for the bioremediation of phenol contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 Cupriavidusnecator 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 × 10 6 to 1.8 × 10 6  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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 10.6gL -1 was obtained under the optimized conditions of oil concentration - 17.5%, inoculum concentration - 50mL/L and nitrogen content - 1.125gL -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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A combination of palm olein (POl) and fructose was used as carbon source for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by recombinant Cupriavidus necator Re2058/pCB113. Cultures grown using 5g/L PO alone as carbon source produced cell dry weight (CDW) of 5.13g/L, 67% PHA/CDW and accumulated a copolymer containing 27mol% 3HHx in shake flask cultures. When cultures were grown in 5g/L fructose alone as the carbon source they produced CDW of 2.32g/L, 11% PHA/CDW and accumulated only poly(3-hydroxybutyrate) [P(3HB)] homopolymer. When the cells were cultured in 5g/L POl in combination with 7g/L fructose, CDW of 7.41g/L and 80% PHA/CDW was obtained with 17mol% 3HHx monomer fraction. Biosynthesis was carried out using a 13L fermenter to study the accumulation of 3HHx monomer fraction in the bacterial cells at different time point. The molecular weights of P(3HB-co-3HHx) with 4-15mol% 3HHx monomer were in the range between 5.47-6.85×10 5 Da, which were at least two fold higher than previously reported values. Interestingly, the increase in M w of the copolymer along with the increase in 3HHx molar fraction was observed. The viscoelastic property of the copolymer further confirmed the increase in M w ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Low frequency sonic waves, less than 10kHz were introduced to assist cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator present within the crude broth. Process parameters including surfactant system variables and sonication parameters were studied for their effect on extraction efficiency. Introduction of low frequency sonic waves assists in the dissolution of microbial cell wall by the surfactant micelles and release of cellular content, polyhydroxyalkanoate granules released were encapsulated by the micelle core which was confirmed by crotonic acid assay. In addition, sonic waves resulted in the separation of homogeneous surfactant and broth mixture into two distinct phases, top aqueous phase and polyhydroxyalkanoate enriched bottom surfactant rich phase. Mixed surfactant systems showed higher extraction efficiency compared to that of individual Triton X-100 concentrations, owing to increase in the hydrophobicity of the micellar core and its interaction with polyhydroxyalkanoate. Addition of salts to the mixed surfactant system induces screening of charged surfactant head groups and reduces inter-micellar repulsion, presence of ammonium ions lead to electrostatic repulsion and weaker cation sodium enhances the formation of micellar network. Addition of polyethylene glycol 8000 resulted in increasing interaction with the surfactant tails of the micelle core there by reducing the purity of polyhydroxyalkanoate., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The brown seaweed Sargassum sp. was used as a feedstock to produce polyhydroxybutyarte (PHB) using Cupriavidus necator PTCC 1615. In order to release monomeric sugars, dilute acid hydrolysis of Sargassum sp. biomass was followed by enzymatic saccharification. In addition, the effect of different nitrogen sources was evaluated for PHB production. The fermentation of hydrolysate with the ammonium sulfate as selected nitrogen source resulted PHB yield of 0.54±0.01g/g reducing sugar. Then, NaCl was used as external stress factor which was added to the media. Addition of 8g/L NaCl had a positive impact on high PHB yield of 0.74±0.01g/g reducing sugar. Increasing trend of NaCl concentration to 16g/L was found to inhibit the production of PHB. Based on obtained results using 20g/L of reducing sugar, at desired condition the highest cell dry weight and PHB concentrations were 5.36±0.22 and 3.93±0.24g/L, respectively. The findings of this study reveal that Sargassum sp. is a promising feedstock for biopolymer production. The characteristics of produced PHB were analyzed by FTIR, differential scanning calorimetry and 1 H NMR., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Agro-industrial residues with a low carbon content, such as whey, stillage or wastewater from plant oil mills are abundant and cheap. However, they cannot be used directly in highly productive industrial poly(3-hydroxybutyrate) (P3HB) production, as the classical fed-batch fermentation strategy requires highly concentrated feed streams. This problem has been circumvented in this report by retaining the cells during the fermentation in the bioreactor using an external microfiltration module. Synthetic medium containing a glucose concentration of 50g/L was continuously fed to Cupriavidus necator, which converted the sugar to P3HB. With this setup we were able to achieve high productivities (3.10g P3HB/(Lh)) and reach high cell densities (148g/L) containing 76% P3HB, and obtained good yields (0.33g P3HB/g added glucose). The added sugar from the feed was instantly consumed by the bacteria, resulting in a negligible loss of sugar to the permeate. This approach creates the possibility of polyhydroxyalkanoate production from a range of cheap and easily available substrates, for which only waste water treatment or biogas production has been cost-competitive until now., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study investigated the effects of using a combination of substrates (fructose, propanol, citric acid, acetic acid, propionic acid and beef extract) on the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate), which is also known as PHBV. The results of batch PHBV optimization by RSM, indicated that PHB was synthesized only when fructose was used as the sole carbon source. Fructose in combination with propanol and beef extract, exhibited better PHBV production performance, as compared to that of propionic acid which was used in previous studies. GC, (1)H and (13)C NMR spectroscopy revealed that the best copolymer composition was 54.1 mol% of 3-hydroxyvalerate (3HV) which was obtained in a basal medium combined with 0.3% (v/v) propanol and 0.0172% (w/v) beef extract. This is the first report on the production of PHBV with HV content of 54.1% by a wild-type Cupriavidus necator. The Fourier Transform Infrared Spectroscopy (FTIR) spectra of the PHAs indicated absorption bands at 1723, 1274, 1373, 1453, 2932cm(-1) corresponding to CO, CO stretching, CH3, -CH2 and -CH groups, respectively. This study showed that the recovered PHBV biopolymer from the optimized culture to have a melting temperature of 103.6°C, melting enthalpy of 55.67J/g and crystallization temperature of 76.1°C., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A fat-containing waste produced from the margarine manufacturing process was tested as a low cost carbon source for cultivation of different polyhydroxyalkanoates (PHAs) producing bacterial strains, including Cupriavidus necator, Comamonas testosteroni and several Pseudomonas strains. The margarine waste was mainly composed of free fatty acids (76wt.%), namely mystiric, oleic, linoleic and stearic acids. In preliminary shake flask experiments, several strains were able to grow on the margarine waste, but C. necator reached the highest PHA content in the biomass (69wt.%). This strain was selected for batch bioreactor experiments, wherein it reached a cell dry weight of 11.2g/L with a polymer content of 56wt.%. The culture produced 6.4g/L of polyhydroxybutyrate, P3(HB), within 20h of cultivation, which corresponds to a volumetric productivity of 0.33gPHA/Lh. The P3(HB) polymer produced by C. necator from the margarine waste had a melting point of 173.4°C, a glass transition temperature of 7.9°C and a crystallinity of 56.6%. Although the bioprocess needs to be optimized, the margarine waste was shown to be a promising substrate for P(3HB) production by C. necator, resulting in a polymer with physical and chemical properties similar to bacterial P(3HB) synthesized from other feedstocks., (Copyright © 2014 Elsevier B.V. All rights reserved.)