Search

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.

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.

Green waste, e.g., grass clippings, is currently insufficiently recycled and has untapped potential as a valuable resource. Our aim was to use juice from grass clippings as a growth medium for microorganisms. Herein, we demonstrate the production of the sesquiterpene α-humulene with the versatile organism Cupriavidus necator pKR-hum on a growth medium from grass clippings. The medium was compared with established media in terms of microbial growth and terpene production. C. necator pKR-hum shows a maximum growth rate of 0.43 h−1 in the grass medium and 0.50 h−1 in a lysogeny broth (LB) medium. With the grass medium, 2 mg/L of α-humulene were produced compared to 10 mg/L with the LB medium. By concentrating the grass medium and using a controlled bioreactor in combination with an optimized in situ product removal, comparable product concentrations could likely be achieved. To the best of our knowledge, this is the first time that juice from grass clippings has been used as a growth medium without any further additives for microbial product synthesis. This use of green waste as a material represents a new bioeconomic utilization option of waste materials and could contribute to improving the economics of grass biorefineries.

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]

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

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

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

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

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.

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.

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.

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.

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.

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.

To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a high conversion of sucrose (88.9%) to hexoses. We showed the necessity to adjust the chemical composition of molasses hydrolysate to balance with the physiological needs of C. necator B-10646 and reduce excess sugars and nitrogen and eliminate phosphorus deficiency. The modes of cultivation of bacteria on diluted hydrolyzed molasses with the controlled feeding of phosphorus and glucose were implemented. Depending on the ratio of sugars introduced into the bacterial culture due to the molasses hydrolysate and glucose additions, the bacterial biomass concentration was obtained from 20–25 to 80–85 g/L with a polymer content up to 80%. The hydrolysates of molasses containing trace amounts of propionate and valerate were used to synthesize a P(3HB-co-3HV) copolymer with minor inclusions of 3-hydroxyvlaerate monomers. The introduction of precursors into the medium ensured the synthesis of copolymers with reduced values of the degree of crystallinity, containing, in addition to 3HB, monomers 3HB, 4HB, or 3HHx in an amount of 12–16 mol.%.

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]

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

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.

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]

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]

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]

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]

Qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance are assessed. Safety concerns identified for a taxonomic unit (TU) are where possible to be confirmed at strain or product level, reflected by 'qualifications'. No new information was found that would change the previously recommended QPS TUs and their qualifications. The list of microorganisms notified to EFSA was updated with 54 biological agents, received between April and September 2019; 23 already had QPS status, 14 were excluded from the QPS exercise (7 filamentous fungi, 6 Escherichia coli, Sphingomonas paucimobilis which was already evaluated). Seventeen, corresponding to 16 TUs, were evaluated for possible QPS status, fourteen of these for the first time, and Protaminobacter rubrum, evaluated previously, was excluded because it is not a valid species. Eight TUs are recommended for QPS status. Lactobacillus parafarraginis and Zygosaccharomyces rouxii are recommended to be included in the QPS list. Parageobacillus thermoglucosidasius and Paenibacillus illinoisensis can be recommended for the QPS list with the qualification 'for production purposes only' and absence of toxigenic potential. Bacillus velezensis can be recommended for the QPS list with the qualifications; the absence of toxigenic potential and the absence of aminoglycoside production, including the genes encoding this. Cupriavidus necator, Aurantiochytrium limacinum and Tetraselmis chuii can be recommended for the QPS list with the qualification; for production purposes only. Pantoea ananatis is not recommended for the QPS list due to lack of body of knowledge in relation to its pathogenicity potential for plants. Corynebacterium stationis, Hamamotoa singularis, Rhodococcus aetherivorans and Rhodococcus ruber cannot be recommended for the QPS list due to lack of body of knowledge. Kodamaea ohmeri cannot be recommended for the QPS list due to safety concerns. [ABSTRACT FROM AUTHOR]

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

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

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

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