Polyhydroxyalkanoates (PHA), of which polyhydroxybutyrate (PHB) is the most abundant, are polymers of bacterial origin used for various applications in the medical, industrial and agricultural fields. In the present study we worked on the selection, evaluation and improvement of the significant variables of the medium for the production of PHB by Cupriavidus necator ATCC 17697. In order to address the selection of the main factors and optimize the culture medium, a complete factorial experimental design based on the coupled response surface methodology, was presented. The model with the best adjustment of the variables turned out to be quadratic in fructose (C), linear in ammonium sulphate (N) and pH, with interaction in pH and phosphate solution (P), where the pH was the most significant (p < 0.0001) while the micro-elements solution could be neglected. Thus, optimum carbon concentration, adequate nitrogen limitation and interaction between initial pH and phosphate solution concentration are important factors to ensure a high production of PHB. The optimal values of the selected variables were C = 20 g/l, N = 1.5 g/l, P = 8.75 g/l and pH 7.5. A maximum PHB production of 4.6 g/l, obtained under these conditions, increased almost 2.5 times. The polymer accumulated in the cytoplasm of C. necator ATCC 17697 in the form of granules showed an FTIR spectrum corresponding to that of commercial PHB.

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, 1 H, and 13 C 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.

One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.

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.

Purpose:The main objective of the present study is to assess and improve fermentation conditions to produce polyhydroxyalkanoates (PHAs), such as the homopolymer polyhydroxybutyrate (PHB) and the copolymer polyhydroxybutyrate-co-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 (HV) 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 native C. necator strain.

Purpose : The main objective of the present study is to assess and improve fermentation conditions to produce polyhydroxyalkanoates (PHAs), such as the homopolymer polyhydroxybutyrate (PHB) and the copolymer polyhydroxybutyrate-co-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 (HV) 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 native C. necator strain.

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