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

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

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

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

Cupriavidus necator, a facultative hydrogen-oxidizing bacterium, was grown on carbon dioxide, hydrogen, and oxygen for value-added products. High cell density and productivity were the goal of gas fermentation, but limited by gas substrates because of their low solubility in the aqueous medium solution. Enhancement of gas fermentation was investigated by (i) adding n-hexadecane as a gas vector to increase the volumetric mass transfer coefficient (kLa) and gas solubility, (ii) growing C. necator under a raised gas pressure, and (iii) using cell mass hydrolysates as the nutrients of chemolithotrophic growth. In contrast to previous studies, little positive but negative effects of the gas vector were observed on gas mass transfer and cell growth. The gas fermentation could be significantly enhanced under a raised pressure, resulting in a higher growth rate (0.12 h-1), cell density (18 g L-1), and gas uptake rate (200 mmole L-1 h-1) than a fermentation under atmospheric pressure. The gain, however, was not proportional to the pressure increase aspredicted by Henry's law. The hydrolysates of cell mass were found a good source of nutrients and the organic nitrogen was equivalent to or better than ammonium nitrogen for chemolithotrophic growth of C. necator on carbon dioxide. [ABSTRACT FROM AUTHOR]

Featured Application: production of bioplastics with tailored composition and properties from the feedstock CO2, in view of maximal CO2 fixation and minimal H2 consumption. White biotechnology is promising to transform CO2 emissions into a valuable commodity chemical such as the biopolymer polyhydroxyalkanaotes (PHA). Our calculations indicated that the indirect conversion of acetic acid from CO2 into PHA is an interesting alternative for the direct production of PHA from CO2 in terms of CO2 fixation, H2 consumption, substrate cost, safety and process performance. An alternative cultivation method using acetic acid as an indirect sink of CO2 was therefore developed and a proof-of-concept provided for the synthesis of both the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The aim was to compare key performance parameters with those of existing cultivation methods for direct conversion of CO2 to PHA. Fed-batch cultivations for PHA production were performed using a pH-stat fed-batch feeding strategy in combination with an additional Dissolved Oxygen (DO)-dependent feed. After 118 h of fermentation, 60 g/L cell dry matter (CDM) containing 72% of PHB was obtained, which are the highest result values reported so far. Fed-batch cultivations for PHBV production resulted in 65 g/L CDM and 48 g/L PHBV concentration with a 3HV fraction of 27 mol %. Further research should be oriented towards process optimisation, whole process integration and design, and techno-economic assessment. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Poly(hydroxyalkanoates) (PHAs) are biodegradable polymers that can replace conventional plastics, but microbial production of PHAs must be optimized for commercial success. The aim of this study is to provide a simple mathematical model based on previous studies in the literature to represent the production of PHAs in chemostat and batch cultures by Pseudomonas putida GPo1 (ATCC 29347) and Cupriavidus necator (DSM 545) grown on octanoate and glucose, respectively. RESULTS: Kinetic and stoichiometric equations, dependent on the specific growth rate of residual biomass (µR), were developed for carbon‐ and nitrogen‐limiting growth conditions. The parameters have biochemical significance and are independent of the biomass concentration. The results revealed that the Luedeking–Piret model is growth‐associated for both fermentations under carbon limitation. In addition, the PHA content increases with µR. Meanwhile, the PHA content is amplified under nitrogen limitation but decreases as μR increases. Also, the Luedeking–Piret model exhibits a profile that depends on the synthesized polyhydroxyalkanoate. CONCLUSION: This study demonstrates that strategies for the production of PHAs require an in‐depth understanding of the process kinetics. This model gives satisfactory predictions, may be extended to fed batch cultures and may be adapted to other fermentations. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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

Biodegradable polymer polyhydroxyalkanoates are one of the promising alternatives for conventional plastics. The present article focuses on a modified and novel method for the synthesis of poly (3‐hydroxybutyrate) (PHB) by two microorganisms, viz. Bacillus megaterium and Cupriavidus necator. These microbial cells were grown over fructose as a carbon source, and the produced PHB was recovered using ultrasound as well as solvent assisted extraction. The extracted PHB was characterized using FTIR, 1H, and 13C NMR to observe the functional groups in the PHB molecule. The XRD characterization confirmed the partial crystalline nature of PHB, and the results of TGA, DTG, and DSC analysis attributed to the thermal stability of PHB. The major step of weight loss of PHB derived by B. megaterium and C. necator in TGA analysis was found to be 415°C and 289°C, respectively. These values were comparatively higher than standard PHB, for which it is 260°C. Similarly, the maximum degradation temperature for standard PHB is 236°C, whereas the maximum degradation temperature of PHB synthesized by B. megaterium and C. necator are 248°C and 277°C, respectively. This ascertains that the produced PHB has greater resistance to thermal degradation as compared with PHB standard. The melting point of synthesized PHBs were found to be 175°C to 176°C, which is similar to standard PHB. The glass transition temperature of the synthesized PHBs varies from –8°C to 6°C. The plausible reason behind the variances could be due to difference in crystallinity and molecular weight of polymer matrix. Nevertheless, thermal properties of PHB produced by B. megaterium and C. necator are found to be similar or much better than commercial PHB. The degree of crystallinity of synthesized PHBs are lower than previously reported literatures, which extends its range of applications. [ABSTRACT FROM AUTHOR]

There is considerable interest in the development of simple methods for quantifying production of the biodegradable polyester poly(3-hydroxybutyrate) [P(3HB)] by bacteria. Cells of Cupriavidus necator were grown on agar medium containing different concentrations of glucose (10–25 g/L) as a sole carbon source. Trace amounts (100±5 μg) of dried C. necator cells were directly subjected to thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of tetramethylammonium hydroxide (TMAH). On the resulting chromatograms, a series of characteristic peaks, attributed to the THM products from poly(3-hydroxybutylate) accumulated in the bacterial cells, were clearly observed without any interfering component. Based on the peak intensities, the contents of P(3HB) in C. necator were determined precisely and rapidly without using any cumbersome sample pretreatment. Furthermore, the values of the P(3HB) contents coincided overall with those obtained by the conventional method involving solvent extraction followed by gravimetric determination. [ABSTRACT FROM AUTHOR]

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

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

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

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

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

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor ) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55 wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that – purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome. [ABSTRACT FROM AUTHOR]

[Display omitted] • The improvement of PHA metabolic pathways in C. necator and B. cereus were studied. • C. necator has better PHA synthesis ability and substrate versatility than B. cereus. • Lactate has active amino acid pathway and aminobutyric acid pathway in PHA synthesis. • PHA was inhibited by the carbon competition or limited by the amount of acetyl-CoA. • This study enhances the understanding of PHA synthesis of organic solid waste. The production of polyhydroxyalkanoate (PHA) from organic solid waste is a research hotspot. However, the allocation and conversion mechanism of different carbon resources between precursor routes and PHA metabolism remains unclear. We compared the PHA synthesis of dominant functional bacteria, Cupriavidus necator (C. necator) and Bacillus cereus (B. cereus), and replenished the metabolic pathways of primitive components (glucose, glycerin, lactic acid and propionic acid). The results showed that the PHA accumulation ability of C. necator (11.8%∼36.5%) was better than that of B. cereus (10.1%∼24.5%). C. necator had a faster rate of cell growth and PHA synthesis than B. cereus , which showed advantages in production efficiency. Most of the carbon of glucose flowed to the direct poly-3-hydroxybutyrate synthesis pathway. The carbon competition inhibited the PHA production from glycerin in C. necator, and cysteine metabolism was an effective pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in B. cereus. There were active amino acid pathway and aminobutyric acid pathway in the utilization of lactic acid in C. necator and B. cereus , respectively. The yield of copolymer was limited by acetyl-CoA in propionic acid system. This study laid the foundation for exploring the PHA synthesis from the components of organic solid waste. [ABSTRACT FROM AUTHOR]

[Display omitted] • Conversion of CO 2 to GlcNAc was achieved for the first time by engineered C. necator. • GlcNAc biosynthetic pathway was constructed by expression of Cegna1 *. • PHB synthesis pathway and ED pathway disruption increased the production of GlcNAc. • The maximum GlcNAc titer from CO 2 reached 75.3 mg/L. The conversion of CO 2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N -acetylglucosamine (GlcNAc) from CO 2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF , nagE , nagC , nagA and nagB genes. Second, the GlcNAc-6-phosphate N -acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner–Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO 2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO 2 under normal conditions.. [ABSTRACT FROM AUTHOR]

Fatty acids produced by H2-metabolizing bacteria are sometimes observed to be more D-depleted than those of photoautotrophic organisms, a trait that has been suggested as diagnostic for chemoautotrophic bacteria. The biochemical reasons for such a depletion are not known, but are often assumed to involve the strong D-depletion of H2. Here, we cultivated the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha H16) under aerobic, H2-consuming, chemoautotrophic conditions and measured the isotopic compositions of its fatty acids. In parallel with the wild type, two mutants of this strain, each lacking one of two key hydrogenase enzymes, were also grown and measured. In all three strains, fractionations between fatty acids and water ranged from -173‰ to -235‰, and averaged -217‰, -196‰, and -226‰, respectively, for the wild type, SH- mutant, and MBH- mutant. There was a modest increase in δD as a result of loss of the soluble hydrogenase enzyme. Fractionation curves for all three strains were constructed by growing parallel cultures in waters with δDwater values of approximately -25‰, 520‰, and 1100‰. These curves indicate that at least 90% of the hydrogen in fatty acids is derived from water, not H2. Published details of the biochemistry of the soluble and membrane-bound hydrogenases confirm that these enzymes transfer electrons rather than intact hydride (H-) ions, providing no direct mechanism to connect the isotopic composition of H2 to that of lipids. Multiple lines of evidence thus agree that in this organism, and presumably others like it, environmental H2 plays little or no direct role in controlling lipid δD values. The observed fractionations must instead result from isotope effects in the reduction of NAD(P)H by reductases with flavin prosthetic groups, which transfer two electrons and acquire H+ (or D+) from solution. Parallels to NADPH reduction in photosynthesis may explain why D/H fractionations in C. necator are nearly identical to those in many photoautotrophic algae and bacteria. We conclude that strong D-depletion is not a diagnostic feature of chemoautotrophy.

The aim of this study was to evaluate the production of polyhydroxyalkanoates (PHAs) by fermentation of Crude Glycerin, a byproduct of the biodiesel industry, by Cupriavidus necator IPT 026, 027 and 028. The influence of fermentation time and temperature in shake flasks were evaluated. The highest PHA production (2.82 g L-1) occurred at 35 ºC for 72 h of fermentation. The melting and initial thermal degradation temperatures of this PHA were 177.9 ºC and 306.33 ºC, respectively, with 55% crystallinity. FTIR spectrum was similar to those reported in literature. The polymer obtained presented three different methyl esters of hydroxyalkanoates in its composition, with molecular weight of 630 kDa. Bacteria can use Crude Glycerin as an inexpensive substrate to produce value-added biodegradable products, such as PHA.

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs’ and the absorption coefficient µa at 638 nm. A correlation of µs’ with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L−1·h−1 and a final biomass of 106 g·L−1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.