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

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

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

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.

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]

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

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

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]

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

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

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

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

Tuna condensate, an organic-rich by-product from the tuna canning industry, was assessed as a substrate for polyhydroxyalkanoate (PHA) production using Cupriavidus necator TISTR 1095. The effect of cultivation parameters on PHA accumulation was studied, including substrate concentration, carbon to nitrogen (C/N) ratio, initial pH-value control and fermentation strategies. For the bacterium, a biomass of 3.8 ± 0.1 g/L, PHA of 1.64 ± 0.1 g/L with PHA productivity of 0.027 g/L.h were obtained under batch cultivation using 100% tuna condensate with a C/N ratio of 88:1 and no control of pH. However, the PHA production was increased 1.3-fold when repeated-batch cultivation was applied. The highest biomass (7.5 ± 0.1 g/L) and PHA (3.8 ± 0.1 g/L) with 0.063 g/L.h of PHA productivity were achieved after the third cycle of repeated-batch cultivation. High chemical oxygen demand (COD) removal efficiency of 70% under the optimal condition was also demonstrated. The polymers generated by C. necator TISTR 1095 were characterised. The size of polymer granules was in the range of 0.7-0.8 μm. The polymer produced in the optimal medium under batch and repeated-batch cultivation was identified as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 20 mol% of 3-hydroxyvalerate. The molecular mass (Mn) and polydispersity of the polymer were 2 × 106 Da and 2.5, respectively. The results demonstrated that tuna condensate could be used as a cheap substrate for PHA production on an industrial scale. [ABSTRACT FROM AUTHOR]

Photovoltaic cells have considerable potential to satisfy future renewable-energy needs, but efficient and scalable methods of storing the intermittent electricity they produce are required for the large-scale implementation of solar energy. Current solar-to-fuels storage cycles based on water splitting produce hydrogen and oxygen, which are attractive fuels in principle but confront practical limitations from the current energy infrastructure that is based on liquid fuels. In this work, we report the development of a scalable, integrated bioelectrochemical system in which the bacterium Ralstonia eutropha is used to efficiently convert CO2, along with H2 and O2 produced from water splitting, into biomass and fusel alcohols. Water-splitting catalysis was performed using catalysts that are made of earth-abundant metals and enable low overpotential water splitting. In this integrated setup, equivalent solar-to-biomass yields of up to 3.2% of the thermodynamic maximum exceed that of most terrestrial plants. Moreover, engineering of R. eutropha enabled production of the fusel alcohol isopropanol at up to 216 mg/L, the highest bioelectrochemical fuel yield yet reported by >300%. This work demonstrates that catalysts of biotic and abiotic origin can be interfaced to achieve challenging chemical energy-to-fuels transformations.

[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]

Abstract Background 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO2 and H2. Results When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism’s ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co–metabolise 3-HP alongside other carbon and energy sources such as fructose and CO2/H2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of β-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3). Conclusions Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain represents an ideal chassis for autotrophic 3-HP production.

The current study shows that Cupriavidus necator has the ability to grow on waste glycerol as carbon source, and can synthesize a highly thermostable copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Batch cultivation on waste glycerol showed accumulation of 6.76 g L–1 biomass containing 4.84 g L–1 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer with 3-hydroxyvalerate content of 24.6 mol%. A novel recovery strategy was developed for the extraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from Cupriavidus necator using recyclable solvents, i.e., propylene carbonate, butyl acetate, isoamyl alcohol, and ethyl acetate. Propylene carbonate demonstrated the recovery efficiency of 90 % and polymer purity of 95 %, at 120 °C after 30 min. Ethyl acetate exhibited a higher efficiency than butyl acetate in terms of recovering the copolymer from cells. Ethyl acetate extraction demonstrated a recovery yield of 96 % and purity of 93 % at 100 °C. Efficacy of an anionic surfactant, linear alkylbenzene sulfonic acid (LAS), was also tested for extraction, and it showed maximum yield of 84 % and purity of 90 % at 80 °C and pH 5.0. Extraction of copolymer using these solvents could help in replacing generally used chlorinated toxic solvents, such as 1,2 dichloroethane and chloroform. Further, GPC, TGA and DSC analysis revealed that the thermo-physical properties were not significantly affected by the extraction method. However, the molecular weight distribution of the polymer showed a variation depending on the type of solvent used for extraction. Subsequently, endotoxins were removed efficiently to less than 5 EU g–1 of copolymer using alkali at optimized conditions of 6 h digestion time and 2.5 N NaOH concentration for medical applications.

Polyhydroxyalkanoates (PHA) are polymers produced by microorganisms with increasing commercialization potential; Cupriavidus necator has been the model microorganism to research PHA production. Despite many contributions concerning the formation and degradation of PHA granules, as well as the morphological changes in cells, these phenomena have not been univocally explained yet. Thus, this study aims to integrate the microscopic and analytical analysis to characterize changes in bacterial cell/PHA granules morphology, PHA content, and yield coefficients under different cultivation strategies of C. necator ATCC 17697. The cell size and morphology, granule size and amount, residual biomass, and PHA concentration along the fermentation and degradation depend greatly on nutritional conditions and cultivation time of C. necator. It was proposed to calculate a yield coefficient for the residual biomass production in the PHA utilization stage, related to the bacteria's ability to survive without a carbon source in the culture medium by utilizing the accumulated PHA previously. Maximum granule length reached 1.07 µm after 72 h of PHA accumulation stage under optimum nutritional conditions. This value is twice the values previously reported for C. necator. It is important since the larger PHA granules facilitate the recovery of PHA and different application development. [ABSTRACT FROM AUTHOR]

Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram‐scale production of the sesquiterpene α‐humulene in final aqueous concentrations of 2 g L−1 with the recombinant strain Cupriavidus necator pKR‐hum in a fed‐batch mode on fructose as carbon source and n‐dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost‐effectiveness of both processes based on a production of 10 t α‐humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3‐hydroxymethylglutaryl‐CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR‐hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant‐based variant to increase α‐humulene production titers in the future. [ABSTRACT FROM AUTHOR]

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaCA–04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA–04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA–04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA–04 protein with high YP/S and PHB productivity. Soluble phaCA–04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA–04 from pColdTF-phaCABA–04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCABA–04 enhanced soluble protein formation to approximately 3.09−4.1 times higher than that from pColdI-phaCABA–04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCABA–04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Pollution resulting from the persistence of plastics in the environment has driven the development of substitutes for these materials through fermentation processes using agro-industrial wastes. Polyhydroxybutyrate (PHB) is a rapidly biodegradable material with chemical and mechanical properties comparable to those of some petroleum-derived plastics. PHB accumulates intracellularly as an energy reserve in a wide variety of microorganisms exposed to nutritionally imbalanced media. The objective of this study was to evaluate the use of a banana waste product as a carbon source for PHB production. PHB was extracted by acid methanolysis and detected by gas chromatography-mass spectrometry. Eleven bacterial strains with potential for PHB production were evaluated by in vitro fermentation in a culture broth containing fructose as the carbon source and limited nitrogen. A 22 central composite rotational design was applied to optimize the concentrations of banana juice and ammonium chloride needed to maximize the PHB-producing biomass concentration. The process was then carried out in a 3 L fed-batch fermentation system that included an initial stage of biomass growth. Banana juice was used as the carbon source and fructose pulses were added to maintain the test sugar concentrations of 30, 40, and 50 g/L. The control strain, Cupriavidus necator (ATCC 17699), produced 2.816 g/L of PHB, while productivity of the most promising isolate, C. necator (CR-12), was 0.495 g/L. Maximum biomass production was obtained using 5% banana juice and 2 g/L ammonium chloride. PHB production was not detected in fed-batch fermentations supplemented with 30 or 40 g/L of fructose, while the mean PHB production in fermentations with 50 g/L of fructose was 1.3 g/L.

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Nonionic surfactant-based cloud point extraction of polyhydroxyalkanoate from fermentation broth was implemented in a modified rotating disc contactor. The effect of rotor speed and phase flowrates on dispersed micellar phase holdup, mass transfer coefficient, yield, and purity were studied with micellar system contains TX114 (4.5 wt %)+ TMN6 (0.5 wt %) and 0.1 M ammonium chloride at broth pH of 3. The rotor speed and the micellar phase flowrate had a significant effect. A maximum PHA recovery of 85.48% and purity of 86.01% were achieved. The correlation was developed to predict the dispersed-phase holdup. [ABSTRACT FROM AUTHOR]

The article presents the astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses. Topics include the polyP kinases will critically assess of the underlying data on the multiple functions of polyP, and the most other functions of polyP are not relevant for survival of Ralstonia eutropha important beta-proteobacterial species.

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

Recent studies of the impact and dimension of plastic pollution have drawn the attention to finding more sustainable alternatives to fossil-based plastics. Microbially produced polyhydroxyalkanoates (PHAs) biopolymers are strong candidates to replace conventional plastic materials, due to their true biodegradability and versatile properties. However, widespread use of these polymers is still hindered by their high cost of production. In the present study, we target high yields of the PHA copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] using a substrate-flexible two-stage fed-batch approach for the cultivation of the recombinant Cupriavidus necator strain Re2058/pCB113. A more substrate-flexible process allows to cope with constant price fluctuations and discontinuous supply of feedstocks on the market. Utilizing fructose for biomass accumulation and rapeseed oil for polymer production resulted in a final biomass concentration of 124 g L–1 with a polymer content of 86 wt% holding 17 mol% of HHx. Productivities were further optimized by operating the biomass accumulation stage in a “drain and fill” modus where 10% of the culture broth was recycled for semi-continuous biomass accumulation, after transferring 90% to a second bioreactor for PHA production. This strategy succeeded in shortening process times rising productivity yields to ∼1.45 g L–1 h–1.

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.

Abstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. Results An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.

Microalgae biomass has been recently used as an inexpensive substrate for the industrial production of polyhydroxyalkanoates (PHAs). In this work, a dilute acid pretreatment using 0.3 N of hydrochloric acid (HCl) was performed to extract reducing sugars from 10% (w/v) of defatted Chlorella biomass (DCB). The resulting HCl DCB hydrolysate was used as a renewable substrate to assess the ability of three bacterial strains, namely Bacillus megaterium ALA2, Cupriavidus necator KCTC 2649, and Haloferax mediterranei DSM 1411, to produce PHA in shake flasks. The results show that under 20 g/L of DCB hydrolysate derived sugar supplementation, the cultivated strains successfully accumulated PHA up to 29.7–75.4% of their dry cell weight (DCW). Among the cultivated strains, C. necator KCTC 2649 exhibited the highest PHA production (7.51 ± 0.20 g/L, 75.4% of DCW) followed by H. mediterranei DSM 1411 and B. megaterium ALA2, for which a PHA content of 3.79 ± 0.03 g/L (55.5% of DCW) and 0.84 ± 0.06 g/L (29.7% of DCW) was recorded, respectively. Along with PHA, a maximum carotenoid content of 1.80 ± 0.16 mg/L was produced by H. mediterranei DSM 1411 at 120 h of cultivation in shake flasks. PHA and carotenoid production increased by 1.45- and 1.37-fold, respectively, when HCl DCB hydrolysate biotransformation was upscaled to a 1 L of working volume fermenter. Based on FTIR and 1H NMR analysis, PHA polymers accumulated by B. megaterium ALA2 and C. necator KCTC 2649 were identified as homopolymers of poly(3-hydroxybutyrate). However, a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 3-hydroxyvalerate fraction of 10.5 mol% was accumulated by H. mediterranei DSM 1411. [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 present study focuses on sustainable production of biodegradable polymers by Cupriavidus necator DSMZ 545 using glycerol as substrate. The batch growth and biopolymer production kinetics were established in a 7-L bioreactor, which resulted in a total biomass of 8.88 g L-1 and poly(3-hydroxybutyrate) (PHB) accumulation of 6.76 g L-1. The batch kinetic and independently acquired substrate inhibition data were then used to develop a mathematical model for PHB production process. This was eventually used to design different nutrient feeding strategies under constant feed rate, decreasing feed rate, and pseudo steady state of substrate (glycerol) to optimize the PHB production during fed-batch cultivation. Among all the fed-batch cultivation strategies, the highest PHB accumulation and productivity of 13.12 g L-1 and 0.27 g L-1 h-1, respectively, was achieved in fed-batch bioreactor cultivation where a pseudo steady state with respect to glycerol was maintained. [ABSTRACT FROM AUTHOR]

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

Because of availability and inexpensiveness, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by Cupriavidus necator was found to produce lower PHA with low bacterial growth rates. According to our study, C. necator was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the Cupriavidus necator H16 experiment give rise to the break down the bonds of α,α-1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (C6H14O6), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs. [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]

Polyhydroxyalkanoates are polyesters of R-hydroxyalkonic acids, prominently used as bioplastics on grounds of their complete biodegradable and environment-friendly characteristics. There is an upsurge in need of an alternative low-cost, renewable carbon source for the production of PHA for enhanced economic and to exert a positive impact on the industries. In the present work, cashew apple juice (CAJ) was supplemented as a carbon source for Cupriavidus necator to produce PHA. (NH4)2SO4, NH4Cl, NH4NO3 and CO(NH2)2, and NaNO3 were tested and urea was found to be the best nitrogen source that supports optimal growth of the microorganism. The production process was then optimized using response surface methodology by incorporating the effects of total reducing sugar concentration, urea concentration, and inoculum size. Under optimized condition, the resulting PHA yield was found to be 15.78 g/L with total reducing sugar concentration of 50 g/L, inoculum size of 50 mL/L, and urea concentration of 3 g/L. FT-IR, NMR, TGA, and DSC analysis revealed the product to be a copolymer of hydroxybutyrate and hydroxyvalerate. [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]

CO2 has been electrochemically reduced to the intermediate formate, which was subsequently used as sole substrate for the production of the polymer polyhydroxybutyrate (PHB) by the microorganism Cupriavidus necator. Faradaic efficiencies (FE) up to 54 % have been reached with Sn‐based gas‐diffusion electrodes in physiological electrolyte. The formate containing electrolyte can be used directly as drop‐in solution in the following biological polymer production by resting cells. 56 mg PHB L−1 and a ratio of 34 % PHB per cell dry weight were achieved. The calculated overall FE for the process was as high as 4 %. The direct use of the electrolyte as drop‐in media in the bioconversion enables simplified processes with a minimum of intermediate purification effort. Thus, an optimal coupling between electrochemical and biotechnological processes can be realized. [ABSTRACT FROM AUTHOR]

Alligator weed (Alternanthera philoxeroides) is a stoloniferous, amphibious and perennial herb which has invaded many parts of the world and led to serious environmental and ecological problems. In order to exploit cheap carbon source for poly(3‐hydroxybutyrate) (PHB) production, alligator weed hydrolysates were prepared by acid and enzyme treatment and used for PHB production via Cupriavidus necator. The bacterium utilized alligator weed enzymatic hydrolysate and produced the PHB concentration of 3.8 ± 0.2 g/L at the conditions of pH 7.0, 27.5°C, 1.5 g/L of nitrogen source, and 25 g/L of carbon source, this exceeded the value of 2.1 ± 0.1 g/L from acid hydrolysate media at the same conditions. In order to obtain the optimum conditions of PHB production, response surface methodology was employed which improved PHB content. The optimum conditions for PHB production are as follows: carbon source, 34 g/L; nitrogen source, 2 g/L; pH, 7; temperature, 28°C. After 72 hr of incubation, the bacterium produced 8.5 g/L of dry cell weight and 4.8 g/L of PHB. The PHB was subjected to Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and Molecular weight analysis and found the melting temperature, number average molecular mass, and polydispersity were 168.20°C, 185 kDa, and 2.1, respectively. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA), polyesters accumulated by numerous prokaryotes in the form of intracellular granules, have been for decades considered being predominantly storage molecules. However, numerous recent discoveries revealed and emphasized their complex biological role for microbial cells. Most of all, it was repeatedly reported and confirmed that the presence of PHA granules in prokaryotic cells enhances stress resistance and robustness of microbes against various environmental stress factors such as high or low temperature, freezing, oxidative, and osmotic pressure. It seems that protective mechanisms of PHA granules are associated with their extraordinary architecture and biophysical properties as well as with the complex and deeply interconnected nature of PHA metabolism. Therefore, this review aims at describing novel and unexpected properties of PHA granules with respect to their contribution to stress tolerance of various prokaryotes including common mesophilic heterotrophic bacteria, but also extremophiles or photo-autotrophic cyanobacteria. Key points: • PHA granules present in bacterial cells reveal unique properties and functions. • PHA enhances stress robustness of bacterial cells. [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]

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