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Waste cooking oil (WCO) from various sources were evaluated as low-cost carbon feedstock to produce poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] copolymer, which is a type of polyhydroxyalkanoate (PHA). Different Cupriavidus necator transformants harbouring phaCBP−M−CPF4 were employed as PHA-producing organisms in one-stage cultivation in shake flasks. Biological and chemical recovery of PHA were evaluated. C. necator Re2058/pHT1-CBP−M−CPF4 was able to achieve a cell dry weight (CDW) of 8.5 g/L, PHA content of 75.5 wt% and PHA concentration of 6.4 g/L from 1% (v/v) WCO. The weight-average molecular weight (Mw) of the synthesized PHA obtained from the same strain was 1440 kDa. The CDW, PHA content and concentration acquired using WCO are comparable to those using fresh oils as carbon substrates. The copolymer produced from distinct sources of WCO and different extraction methods did not have significant differences in molecular weights and thermal properties. Mealworms can perform biological recovery of PHA from cells fed with WCO from distinct sources. This study suggests that WCO from any source can be used as carbon source for bacteria to produce PHA. WCO can be a sustainable alternative for the more expensive fresh oils, and the synthesized PHA can be biologically recovered by mealworms. A combination approach of utilising low-cost substrate such as WCO, high-performance PHA producer including C. necator transformants and biological recovery to extract PHA is beneficial in biosynthesis of PHA copolymer, since this can make PHA production more economical and can improve the ability of PHA to compete with conventional petrochemical plastics. [ABSTRACT FROM AUTHOR]

Objectives: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. Results: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD+-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose –nitrogen mineral salt medium (FN). Conclusion: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80. [ABSTRACT FROM AUTHOR]

Purpose: The main objective of the present study is to assess and improve fermentation conditions to produce polyhydroxyalkanoates (PHAs), such as the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from renewable and inexpensive industrial waste by wild type Cupriavidus necator. Methods: Untreated residual glycerol, a byproduct generated by biodiesel industry, was used as the main carbon source by wild type C. necator. The bacterial adaptation stage, the glycerol concentration and other medium components were adjusted to enhance the PHB production. Fed batch fermentation with a pulse feeding strategy of propionic acid solution was employed for PHBV production. Results: C. necator, previously adapted to glycerol utilization, was able to accumulate PHB up to 4.1 g/l under optimal substrate concentration conditions. The bacterium could also synthesize up to 3.4 g/l PHBV, with a mole fraction of 3-hydroxyvalerate (3HV) up to 12% by fed-batch fermentation with a pulsed propionic acid feed strategy. Conclusion: This is the first report on the use of residual glycerol as ideal feedstock for PHA production by wild type C. necator strain. [ABSTRACT FROM AUTHOR]

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C0) and the specific growth rate (µ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C0 = 2.9–23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C0 = 19.1 g/L) saponified SCG oil. Peak µ-values of 0.139 h−1 and 0.145 h−1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates (µ = 0.35 h−1) than on the crude SCG oil (µ = 0.23 h−1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA. Key points: • The bioavailability of plant oil substrates can be improved via saponification. • Cell growth and inhibition were accurately described by the Han-Levenpsiel model. • Mixing crude and saponified oils enable variation of free fatty acid content. [ABSTRACT FROM AUTHOR]

Waste paper holds great potential as a substrate for the microbial production of bioplastic (Poly-3-hydroxybutyrate (PHB)). This study aimed to produce PHB by utilizing office paper as a substrate using Cupriavidus necator through batch and fed-batch simultaneous saccharification and fermentation (SSF) approach. For the batch experiment, different loadings of shredded office paper (3, 5 and 10%) with two different pretreatments H2O2 (OPH) and H2O2 and Triton X-100 (OPTH) were carried out. For the fed-batch experiment, paper loading started with 3% and two more additions were made at 36 and 84 h. Both experiments were conducted at 30 °C, 200 rpm and pH 7 using 55.5 FPU/g of cellulase and 37.5 CBU/g of β-glucosidase with a fixed amount of nitrogen source. High PHB yield was observed with OPH in all loadings, though the OPHT showed a better hydrolysis. Maximum PHB yield (4.27 g/L) was achieved with 10% OP on the sixth day of fermentation in batch SSF. Whereas, maximum PHB yield (4.19 g/L) was obtained within a shorter time (66 h)with OPH in the fed-batch experiment. The extracted PHB showed well-matched characteristic features to the standard PHB. Finally, this study proves the feasibility of employing the SSF process for PHB production using waste paper as an alternative approach to overcome the shortcoming of the separate hydrolysis and fermentation (SHF) process. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are biopolymer synthesized as storage granules by microbes in the cell's cytoplasm under excess carbon and limited nitrogen conditions. This study aims to examine the production and optimization of PHA from Ceiba pentandra oil, a non-edible feedstock, by Cupriavidus necator. Response surface methodology was used to optimize the concentrations of carbon, nitrogen, and inoculum for the production of PHA. To improve the physical, mechanical, and thermal properties of PHA, it was blended with polylactic acid (PLA) which possesses high bio-compatibility and bio-absorbability. Furthermore, batch mode cultivation of PHA in a 3-L bioreactor was performed under the optimized conditions (Ceiba pentandra oil—17.13 g/L, urea—1.34 g/L, and inoculum—53.42 mL/L), and 8.51 g/L (0.47 g PHA/g of Ceiba pentandra oil) of PHA product was obtained. The PHA was characterized by FTIR and the presence of functional groups such as C=O, OH, and CH were observed. 1H and 13C NMR spectroscopy revealed the existence of hydroxy valerate (HV) and hydroxybutyrate (HB), and maximal decomposition temperature was identified to be 217.7 °C from TGA analysis. Therefore, Ceiba pendantra oil appears to be an attractive alternate carbon source for PHA production. [ABSTRACT FROM AUTHOR]

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active β-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important β-ketothiolases for C6 metabolism in β-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. Key points: • Role of six β-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter. [ABSTRACT FROM AUTHOR]

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. Key points: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization. [ABSTRACT FROM AUTHOR]

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

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]

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]

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]

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]

Purpose: Sludge palm oil (SPO), a difficult-to-be-used solid byproduct of the palm oil milling industry, was evaluated as potential carbon source for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production using recombinant Cupriavidus necator Re2058/pCB113.Methods: The biosynthesis of polyhydroxyalkanoate (PHA) was conducted using single stage shake flask growth to study different parameters affecting the bacterial growth. Fed-batch fermentations were conducted using SPO to increase the PHA productivity and to reach high density culture which is deemed necessary for large scale production.Results: Initial shake flask studies showed that SPO can be utilized by the bacteria for growth and PHA accumulation. However, the yield of SPO conversion into cell biomass and PHA was low due to the difficulty in using the solid oil in liquid medium. With the aid of surfactant and mixing strategy, SPO which consisted mainly of free fatty acids was successfully emulsified in the mineral medium and was used for cell growth and PHA accumulation whereby 10 g/L of SPO supplied as emulsion solution produced 9.7 g/L of CDW containing 74 wt% P(3HB-co-22 mol% 3HHx). The high yield of biomass obtained indicates that SPO is an excellent feedstock for this strain. Fed-batch fermentation was conducted to increase the yield and productivity whereby a biomass productivity of 1.9 g/L/h and PHA productivity of 1.1 g/L/h were achieved.Conclusion: These results suggest that SPO, an inexpensive waste material, can be used to produce PHA in large scale for commercialization purpose with reduced production cost.Graphical Abstract: [ABSTRACT FROM AUTHOR]

Cupriachelin is a photoreactive lipopeptide siderophore produced by the freshwater bacterium Cupriavidus necator H16. In the presence of sunlight, the iron-loaded siderophore undergoes photolytic cleavage, thereby releasing solubilized iron into the environment. This iron is not only available to the siderophore producer, but also to the surrounding microbial community. In this study, the cupriachelin-based interaction between C. necator H16 and the freshwater diatom Navicula pelliculosa was investigated. A reporter strain of the bacterium was constructed to study differential expression levels of the cupriachelin biosynthesis gene cucJ in response to varying environmental conditions. Not only iron starvation, but also culture supernatants of N. pelliculosa were found to induce cupriachelin biosynthesis. The transcription factors involved in this differential gene expression were identified using DNA-protein pulldown assays. Besides the well-characterized ferric uptake regulator, a two-component system was found to tune the expression of cupriachelin biosynthesis genes in the presence of diatom supernatants. [ABSTRACT FROM AUTHOR]

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization ( csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene ( cscA) and sucrose permease gene ( cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene ( ccr), ethylmalonyl-CoA decarboxylase gene ( emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution. [ABSTRACT FROM AUTHOR]

Statistical distribution of cell and poly[3-( R)-hydroxybutyrate] (PHB) granule size and number of granules per cell are investigated for PHB production in a five-stage cascade (5CSTR). Electron microscopic pictures of cells from individual cascade stages (R1-R5) were converted to binary pictures to visualize footprint areas for polyhydroxyalkanoate (PHA) and non-PHA biomass. Results for each stage were correlated to the corresponding experimentally determined kinetics (specific growth rate μ and specific productivity π). Log-normal distribution describes PHA granule size dissimilarity, whereas for R1 and R4, gamma distribution best reflects the situation. R1, devoted to balanced biomass synthesis, predominately contains cells with rather small granules, whereas with increasing residence time τ, maximum and average granule sizes by trend increase, approaching an upper limit determined by the cell's geometry. Generally, an increase of intracellular PHA content and ratio of granule to cell area slow down along the cascade. Further, the number of granules per cell decreases with increasing τ. Data for μ and π obtained by binary picture analysis correlate well with the experimental results. The work describes long-term continuous PHA production under balanced, transient, and nutrient-deficient conditions, as well as their reflection on the granules size, granule number, and cell structure on the microscopic level. [ABSTRACT FROM AUTHOR]

Being biodegradable and renewable, polyhydroxyalkanoates (PHAs), a green polymer, attract much attentions as potential alternative for conventional plastics due to increased concern towards environmental issue and resource depletion. However, PHAs not only have suffered some economic disadvantages on the market, and its environmental-friendliness has also been questioned as well. Therefore, there is a growing demand to improve both economic and environmental performances of PHAs production, especially at earlier stage of the process where there are plenty of opportunities and the modification cost is cheap. Therefore, a preliminary integrated assessment is introduced to provide a rapid evaluation for PHAs biosynthesis at R&D stage by coupling material cost analysis together with life-cycle assessment. Using fuzzy approach multi-objective optimization, crude glycerol is the most optimum substrate for biopolymer productions from Cupriavidus necator. The insight from sensitivity analysis has showed that the integrated assessment is sensitive to fluctuation in price and yield of substrate, while maintaining its robustness as similar result is obtained when using different multi-objective optimization tools. Providing some novel insights on PHAs biosynthesis like performance and site selection influencing factor, the integrated assessment can be used to facilitate screening for large-scale production of PHAs. [ABSTRACT FROM AUTHOR]

Bacterial synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymer [P(3HB- co-3HV)] using the hydrolysate of rice straw waste as a carbon source was affected by the composition of the hydrolysate, which depends highly on the rice straw pretreatment condition. Acid digestion with 2 % sulfuric acid generated larger production of P(3HB- co-3HV) than 6 % sulfuric acid, but 3HV concentration in the copolymer produced with 2 % acid hydrolysate was only 8.8 % compared to 18.1 % with 6 % acid hydrolysate. To obtain a higher 3HV mole fraction for enhanced flexibility of the copolymer, an additional heating was conducted with the 2 % acid hydrolysate after removal of residual rice straw. As the additional heating time increased a higher concentration of levulinic acid was generated, and consequently, the mole fraction of 3HV in P(3HB- co-3HV) increased. Among the conditions tested (i.e., 20-, 40-, 60-min), 60-min additional heating following 2 % sulfuric acid digestion achieved the highest 3HV mole fraction of 22.9 %. However, a longer heating time decreased the P(3HB- co-3HV) productivity, probably due to the increased intermediates concentrations acting as inhibitors in the hydrolysates. Therefore, the use of additional heating needs to consider both the increase in the 3HV mole fraction and the decrease in the P(3HB- co-3HV) productivity. [ABSTRACT FROM AUTHOR]

In this study volatile fatty acids (VFAs), which are by-products from anaerobic fermentations, have been used as the carbon source for polyhydroxyalkanoates (PHA) production by pure culture bacteria, Cupriavidus necator. A number of factors influence the conversion efficiencies of VFAs to PHAs including the bacterial feeding regimes. When VFA was supplied as a single feed, it was found that concentrations higher than 2% v/v VFA led to substrate inhibition and only 18% acetic acid and 12% of butyric acid was converted into PHA. This resulted in less than 65% (w/w) of PHA content within the microbial cells. Hence, the single VFA feeding strategy was found to provide low conversion rates of VFA into polymer. An improved feeding strategy was found to be the use of an automatic VFA feed based on the pH control of the medium, which led to a more continuous feeding regime. The conversion of VFA to PHA was increased by almost 2-fold to 33 and 22% for acetic acid and butyric acid respectively, with up to 75% (w/w) of PHA resultant within the microbial cells. [ABSTRACT FROM AUTHOR]

This work describes potential opportunities for utilization of agro-industrial residues to produce green biodegradable plastics of poly(3-hydroxybutyrate) (PHB). Wheat straws were examined with good efficacy of carbon substrates using Cupriavidus necator. Production was examined in separate hydrolysis and fermentation (SHF) in the presence and absence of WS hydrolysis enzymes, and in simultaneous saccharification and fermentation (SSF) with enzymes. Results showed that production of PHB in SSF was more efficient in terms of viable cell count, cell dry weight, and PHB production and yield compared to those of SHF and glucose-control cultures. While glucose control experiment produced 4.6 g/L PHB; SSF produced 10.0 g/L compared to 7.1 g/L in SHF when utilizing enzymes during WS hydrolysis. Results showed that most of sugars produced during the hydrolysis were consumed in SHF (~98 %) compared to 89.2 % in SSF. Results also demonstrated that a combination of glucose and xylose can compensate for the excess carbon required for enhancing PHB production by C. necator. However, higher concentration of sugars at the beginning of fermentation in SHF can lead to cell inhibition and consequently catabolite repressions. Accordingly, results demonstrated that the gradual release of sugars in SSF enhanced PHB production. Moreover, the presence of sugars other than glucose and xylose can eliminate PHB degradation in medium of low carbon substrate concentrations in SSF. [ABSTRACT FROM AUTHOR]

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R = 0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process. [ABSTRACT FROM AUTHOR]

The utilization of captured CO as a part of the CO capture and storage system to produce biopolymers could address current environmental issues such as global warming and depletion of resources. In this study, the effect of feeding strategies of CO and valeric acid on cell growth and synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] in Cupriavidus necator was investigated to determine the optimal conditions for microbial growth and biopolymer accumulation. Among the studied CO concentrations (1-20 %), microbial growth and poly(3-hydroxybutyrate) accumulation were optimal at 1 % CO using a gas mixture at H:O:N = 7:1:91 % (v/v). When valeric acid was fed together with 1 % CO, (R)-3-hydroxyvalerate synthesis increased with increasing valeric acid concentration up to 0.1 %, but (R)-3-hydroxybutyrate synthesis was inhibited at >0.05 % valeric acid. Sequential addition of valeric acid (0.05 % at Day 0 followed by 0.025 % at Day 2) showed an increase in 3HV fraction without inhibitory effects on 3HB synthesis during 4 d accumulation period. The resulting P(3HB-co-3HV) with 17-32 mol % of 3HV is likely to be biocompatible. The optimal concentrations and feeding strategies of CO and valeric acid determined in this study for microbial P(3HB-co-3HV) synthesis can be used to produce biocompatible P(3HB-co-3HV). [ABSTRACT FROM AUTHOR]

Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a β-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g h, yield 0.32 Cmole Cmole) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole) and the overall specific productivity (0.044 g g h) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures. [ABSTRACT FROM AUTHOR]

The aim of this study was to increase the density of wild type Cupriavidus necator H16 biomass grown on fructose in order to produce sufficient copolymer of short-chain-length (scl) and medium-chain-length (mcl) polyhydroxyalkanoate (PHA) from canola oil for mechanical testing of the PHA. Initial batch cultivation on fructose was followed by exponential feeding of fructose at a predetermined μ to achieve 44.4 g biomass/l containing only 20 % w/w of polyhydroxybutyrate (PHB) with a Y of 0.44 g/g. In a third stage, canola oil was added under N-limited conditions to produce 92 g/l of biomass with 48 % w/w scl-mcl PHA. Using known standards, the PHA composition was confirmed by GC-MS analysis as 99.81 % 3-hydroxybutyrate, 0.06 % 3-hydroxyvalerate, 0.09 % 3-hydroxyhexanoate and 0.04 % 3-hydroxyoctanoate. The melting temperature (179 °C), crystallinity (54 %), tensile stress (25.1 Mpa) and Young's modulus (698 Mpa) for a PHB standard decreased to 176 °C, 52 %, 19.1 and 443 Mpa respectively for C. necator PHA produced in the 3-stage process. [ABSTRACT FROM AUTHOR]

Whole whey hydrolyzed by Alcalase (WWH) was tested as a complex nitrogen source for the production of poly(3-hydroxybutyrate) (PHB) from waste frying oils by Cupriavidus necator H16. Addition of WWH (10 % (v/v) of cultivation media) supported the growth and PHB accumulation; PHB yields in Erlenmeyer flasks were more than 3.5-fold higher than in control cultivations. The positive influence of WWH on PHB production was confirmed in experiments performed in laboratory fermentor. C. necator cultivated with WWH produced 28.1 g PHB l resulting in a very high product yield coefficient of 0.94 g PHB per g oil. Since PHB yields were ~40 % higher than in the control cultivation, WWH can be considered as an excellent inexpensive nitrogen source for PHB production by C. necator. [ABSTRACT FROM AUTHOR]

Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP + ratio. It is likely that as a side effect of the increase of NADPH/NADP + ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate- co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria. [ABSTRACT FROM AUTHOR]

A multistage system for poly(hydroxyalkanoate) (PHA) production consisting of five continuous stirred tank reactors in series (5-CSTR) with Cupriavidus necator DSM 545 as production strain was modelled using formal kinetic relations. Partially growth-associated production of PHA under nitrogen limited growth was chosen as modelling strategy, thus the Luedeking-Piret's model of partial growth-associated product synthesis was applied as working hypothesis. Specific growth rate relations adjusted for double substrate (C and N source) limited growth according to Megee et al. and Mankad-Bungay relation were tested. The first stage of the reactor cascade was modelled according to the principle of nutrient balanced continuous biomass production system, the second one as two substrate controlled process, while the three subsequent reactors were adjusted to produce PHB under continuous C source fed and nitrogen deficiency. Simulated results of production obtained by the applied mathematical models and computational optimization indicate that PHB productivity of the whole system could be significantly increased (from experimentally achieved 2.14 g L h to simulated 9.95 g L h) if certain experimental conditions would have been applied (overall dilution rate, C and N source feed concentration). Additionally, supplemental feeding strategy for switching from batch to continuous mode of cultivation was proposed to avoid substrate inhibition. [ABSTRACT FROM AUTHOR]

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaC) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaC was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1STQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, ). The LA-polymerizing activity of the PhaCA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaC(A510X) mutants, 15 synthesized P (LA- co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The C and H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA- co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10 Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaC mutant may not be a random copolymer, but presumably had a block sequence. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l) was achieved using soybean oil at 40 g l and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g lh, and the calculated Y value was 0.85 g g. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe. [ABSTRACT FROM AUTHOR]

The feasibility of utilizing non edible rice (broken rice) for production of fine materials such as poly(3-hydroxybutyrate) (PHB) was considered as one of the alternative ways of keeping the environment clean for sustainable development. Thus, production of PHB from broken rice by simultaneous saccharification and fermentation (SSF) was investigated. During the SSF process, the rice (15% w/v) material was hydrolyzed to glucose, which was utilized by Cupriavidus necator for growth and production of PHB. The PHB content reached 38% at 58 h fermentation. The PHB had weight average molar mass (Mw) and polydipersity index of 3.82 × 10 (g/mol) and 4.15, respectively. Differential calorimetric scan of the PHB showed a melting temperature (Tm) of 176 °C. Given that the PHB was a homopolymer (which consisted of ( R)-3-hydroxybutyric acid monomers), it was thought that broken rice could be a raw material for production of both PHB and ( R)-3-hydroxybutyric acid. This SSF process would not only help in the utilization of broken rice or non edible rice, but would also serve as a model for utilization of other raw materials that contain starch for production of PHB. [ABSTRACT FROM AUTHOR]

A new halogen-free and environmental-friendly method using water and ethanol is developed as an alternative for the recovery of polyhydroxyalkanoates (PHA) from recombinant Cupriavidus necator in comparison to the established chloroform extraction method. After optimisation, our results showed that the halogen-free method produced a PHA with 81% purity and 96% recovery yield, in comparison to the chloroform extraction system which resulted in a highly pure PHA with 95% yield. Although the purity of the PHA using the new method is lower, the molecular weight of the extracted PHA is not compromised. This new method can be further developed as an alternative and more environmental-friendly method for industrial application. [ABSTRACT FROM AUTHOR]

Poly(hydroxyalkanoates) (PHAs) constitute biodegradable polyesters and are considered among the most promising candidates to replace common petrochemical plastics in various applications. To date, all commercial processes for PHA production employ microbial discontinuous fed-batch fermentations. These processes feature drawbacks such as varying product quality and the inevitable periods of downtime for preparation and post-treatment of the bioreactor equipment. An unprecedented approach to PHA production was chosen in the presented work using a multistage system consisting of five continuous stirred tank reactors in series (5-SCR), which can be considered as a process engineering substitute of a continuous tubular plug flow reactor. The first stage of the reactor cascade is the site of balanced bacterial growth; thereafter, the fermentation broth is continuously fed from the first into the subsequent reactors, where PHA accumulation takes place under nitrogen-limiting conditions. Cupriavidus necator was used as production strain. The focus of the experimental work was devoted to the development of a PHA production process characterized by high productivity and high intracellular polymer content. The results of the experimental work with the reactor cascade demonstrated its potential in terms of volumetric and specific productivity (1.85 g L h and 0.100 g g h, respectively), polymer content (77%, w/ w) and polymer properties ( M = 665 kg/mol, PDI = 2.6). Thus, implementing the technology for 5-SCR production of PHB results in an economically viable process. The study compares the outcome of the work with literature data from continuous two-stage PHA production and industrial PHA production in fed-batch mode. [ABSTRACT FROM AUTHOR]

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P, P, or P derived from Escherichia coli, or promoter regions of phaC1 ( P) or phaP1 ( P) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P, P, P, and P mediated constitutive gene expression, among which P was the strongest promoter. lacI- P was not thoroughly functional even after addition of isopropyl-β- d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC- P could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR- P exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR- P for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator. [ABSTRACT FROM AUTHOR]

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l, respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l h, respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate- co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1% which resulted in 8% content of 3-hydroxyvalerate in copolymer. [ABSTRACT FROM AUTHOR]

The environmental impact caused by the disposal of plastics has motivated the development of biodegradable materials. Recent studies showed that supplementation with oleic acid (OA) in cultures producing poly(3-hydroxybutyrate), P(3HB), increased the polymer productivity. However only few studies have shown the properties and biodegradation profile of the polymer obtained. This research investigated the influence of OA concentration on the biodegradation of the P(3HB) obtained from cultures of Cupriavidus necator. The crystallinity of the casting films determined by differential scanning calorimetry (DSC) was reduced from 70% (0 g L of OA) to 52% (3.0 g L of OA). A reduction of 11 °C in the melting temperature was observed with 3.0 g L of OA. The kinetic of biodegradation was: 3.0 > 1.5 > 0.9 > 0.3 > 0 g L of OA. [ABSTRACT FROM AUTHOR]

Polychlorobiphenyls (PCBs) are classified as “high-priority pollutants.” Diverse microorganisms are able to degrade PCBs. However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA, and degraded 3,5-CBA in the presence of m-toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4′-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of 2,4′-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the development of improved biocatalysts for remediation of PCB-contaminated environments. [ABSTRACT FROM AUTHOR]

Exposition of Cupriavidus necator to ethanol or hydrogen peroxide at the beginning of the stationary phase increases poly(3-hydroxybutyrate) (PHB) yields about 30%. Hydrogen peroxide enhances activity of pentose phosphate pathway that probably consequently increases intracellular ratio NADPH/NADP+. This effect leads to stimulation of the flux of acetyl-CoA into PHB biosynthetic pathway and to an increase of enzymatic activities of β-ketothiolase and acetoacetyl-CoA reductase while activity of PHB synthase remains uninfluenced. During ethanol metabolisation, in which alcohol dehydrogenase is involved, acetyl-CoA and reduced coenzymes NAD(P)H are formed. These metabolites could again slightly inhibit TCA cycle while flux of acetyl-CoA into PHB biosynthetic pathway is likely to be supported. As a consequence of TCA cycle inhibition also less free CoA is formed. Similarly with hydrogen peroxide, activities of β-ketothiolase and acetoacetyl-CoA reductase are increased which results in over-production of PHB. Molecular weight of PHB produced under stress conditions was significantly higher as compared to control cultivation. Particular molecular weight values were dependent on stress factor concentrations. This could indicate some interconnection among activities of β-ketothiolase, acetoacetyl-CoA reductase and PHB molecular weight control in vivo. [ABSTRACT FROM AUTHOR]

Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdCIDIEIFI and tfdDIICIIEIIFII. The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in vivo studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR1 and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to Ic1R-type transcriptional regulator members and located near the tfdII gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families. [ABSTRACT FROM AUTHOR]

Autohydrogenotrophic batch growth of Ralstonia eutropha H16 was studied in a stirred-tank reactor with nitrate and nitrite as terminal electron acceptors and the sole limiting substrates. Assuming product inhibition by nitrite, saturation kinetics with the two limiting substrates and a simple switching function, which allows growth on nitrite only at low nitrate concentrations, resulted in a kinetic growth model with nine model parameters. The data of two batch experiments were used to identify the kinetic model. The kinetic model was validated with two additional batch experiments. The model predictions are in very good agreement with the experimental data. The maximum nitrite concentration was estimated to be 30.7 mM (total inhibition of growth). After complete reduction of nitrate, the growth rate decreases almost to zero before it increases again because of the following nitrite respiration. The maximum autohydrogenotrophic growth rate of Ralstonia eutropha with nitrate as a final electron acceptor (0.509 d−1) was found to be reduced by 90–95% compared to the so far reported autohydrogenotrophic growth rates with oxygen. [ABSTRACT FROM AUTHOR]

This study aimed to enhance production of polyhydroxybutyrate P(3HB) by a newly engineered strain of Cupriavidus necator NSDG-GG by applying response surface methodology (RSM). From initial experiment of one-factor-at-a-time (OFAT), glucose and urea were found to be the most significant substrates as carbon and nitrogen sources, respectively, for the production of P(3HB). OFAT experiment results showed that the maximum biomass, P(3HB) content, and P(3HB) concentration of 8.95 g/L, 76 wt%, and 6.80 g/L were achieved at 25 g/L glucose and 0.54 g/L urea with an agitation rate of 200 rpm at 30 °C after 48 h. In this study, RSM was applied to optimize the three key variables (glucose concentration, urea concentration, and agitation speed) at a time to obtain optimal conditions in a multivariable system. Fermentation experiments were conducted in shaking flask by cultivation of C. necator NSDG-GG using various glucose concentrations (10-50 g/L), urea concentrations (0.27-0.73 g/L), and agitation speeds (150-250 rpm). The interaction between the variables studied was analyzed by ANOVA analysis. The RSM results indicated that the optimum cultivation conditions were 37.70 g/L glucose, 0.73 g/L urea, and 200 rpm agitation speed. The validation experiments under optimum conditions produced the highest biomass of 12.84 g/L, P(3HB) content of 92.16 wt%, and P(3HB) concentration of 11.83 g/L. RSM was found to be an efficient method in enhancing the production of biomass, P(3HB) content, and P(3HB) concentration by 43, 21, and 74%, respectively. [ABSTRACT FROM AUTHOR]

With rapid technology progress and cost reduction, clean hydrogen from water electrolysis driven by renewable powers becomes a potential feedstock for CO2 fixation by hydrogen-oxidizing bacteria. Cupriavidus necator (formally Ralstonia eutropha), a representative member of the lithoautotrophic prokaryotes, is a promising producer of polyhydroxyalkanoates and single cell proteins. This paper reviews the fundamental properties of the hydrogen-oxidizing bacterium, the metabolic activities under limitation of individual gases and nutrients, and the value-added products from CO2, including the products with large potential markets. Gas fermentation and bioreactor safety are discussed for achieving high cell density and high productivity of desired products under chemolithotrophic conditions. The review also updates the recent research activities in metabolic engineering of C. necator to produce novel metabolites from CO2. [ABSTRACT FROM AUTHOR]

Different concentrations of palm oil and urea were used in a preliminary experiment conducted in a shake-flask to determine the optimum conditions for the biosynthesis of polyhydroxyalkanoates (PHA). The best concentration was found to be 10 g/L palm oil and 0.54 g/L urea which produced the highest concentration of PHA (8.11 g/L). A 13 L bioreactor was employed using a fed-batch fermentation with the derived optimum conditions and yielded a PHA concentration of 97 g/L. The melting temperature and thermal degradation temperature of PHA were determined to be 173 °C and 254 °C, respectively. The PHA was applied as a binder to make particleboard from para-rubber-wood flakes which were pretreated with microwaves and steam before being mixed with PHA, and control panels were also manufactured from un-pretreated flakes and without the addition of PHA. The mechanical and physical properties of the panels were determined including the modulus of rupture (MOR), internal bond strength (IB), water absorption, and thickness swelling according to the Japanese Industrial Standard for particleboard (JIS A 5908, 2003). The result showed that none of the panels produced met all the minimum requirements for Type 8 Particleboard of Japanese Industrial Standard JIS A 5908. The MOR of the control panel passed the JIS A 5908 standard for MOR (> 8.00 MPa) but did not meet the IB requirement (>0.15 MPa). The addition of PHA as a binder in proportions of 15, 20 and 30 wt% in the control panel enhanced the IB to 0.18, 0.19. 0.22 MPa, respectively. However none of the other parameters of these boards met the relevant JIS A 5908 standard. The pretreatment process was also unable to enhance the properties of the particleboard but that using PHA as a binder instead of urea formaldehyde was able to meet the relevant internal bonding standard for particleboard manufactured from para-rubber-wood flakes which are a waste product in the furniture industry. [ABSTRACT FROM AUTHOR]