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Poly-(3-hydroxybutyrate) [P(3HB)] is a polyester synthesized as a carbon and energy reserve material by a wide number of bacteria. This polymer is characterized by its thermo-plastic properties similar to plastics derived from petrochemical industry, such as polyethylene and polypropylene. Furthermore, P(3HB) is an inert, biocompatible and biodegradable material which has been proposed for several uses in medical and biomedical areas. Currently, only few bacterial species such as Cupriavidus necator, Azohydromonas lata and recombinant Escherichia coli have been successfully used for P(3HB) production at industrial level. Nevertheless, in recent years, several fermentation strategies using other microbial models such as Azotobacter vinelandii, A. chroococcum, as well as some methane-utilizing species, have been developed in order to improve the P(3HB) production and also its mean molecular weight.

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The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA Ntr , previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM=1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.

BackgroundAzotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P)+ ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions.ResultsThe results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L−1 h−1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L−1 h−1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L−1 h−1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH).ConclusionsWe have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.

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BACKGROUND Poly‐3‐hydroxybutyrate P(3HB) is a bioplastic that is characterized for its biodegradability and biocompatibility and can be used in the biomedical field. The aim of this study was to evaluate the scaling‐up process at pilot level, for the production and recovery of P(3HB) synthesized by Azotobacter vinelandii OPNA strain, based on the use of fed‐batch fermentation coupled to a recovery method using nonhalogenated solvents (ethanol and acetone). RESULTS In fed‐batch cultivations at the 3‐L scale, using yeast extract and sucrose of industrial grade with a carbon:nitrogen ratio of 14 and manipulating the agitation rate from 500 to 700 rpm to give a maximal biomass concentration of 33.8 ± 1.5 g L−1, P(3HB) concentrations of 29 ± 4.5 g L−1 were reached. By simulating the gassed volumetric power input observed in the 3‐L bioreactors at the different agitation rates (500 and 700 rpm), a scaling‐up to 30‐L bioreactors was performed. At this scale, the P(3HB) concentration, and productivity were similar or even better than those values obtained at the smaller scale. The production process was coupled to a P(3HB) separation process, in which the polymer was precipitated with ethanol and washed with acetone, reaching a purity close to 95% and a yield of 85%. The P(3HB) obtained was of ultra‐high molecular mass, with values of 5693 ± 615 kDa. CONCLUSION In this study a successful scaling‐up process for the production of P(3HB) of ultra‐high molecular weight at pilot scale was developed. © 2019 Society of Chemical Industry

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