PHA granule formation and degradation by Cupriavidus necator under different nutritional conditions.

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.

Use of enzymes in extraction of polyhydroxyalkanoates produced by Cupriavidus necator.

Poly(3-hydroxybutyrate) (P(3HB)) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV), are biodegradable thermoplastic polymers. They are members of the polyhydroxyalkanoate (PHA) family, synthesized and accumulated as a carbon and energy reserve by a variety of microorganisms. The aim of this study was to evaluate the use of the proteases Corolase® L10, Alcalase® 2.4L, Corolase® 7089 and Protemax® FC and glycosidases Celumax® BC, Rohament® CL and Rohalase® Barley for the recovery of P(3HB) and P(3HB-co-3HV) synthesized by Cupriavidus necator. The enzyme Celumax® BC provided better lysis of the bacterial cell membrane and the results for the optimization of the operating conditions showed that this enzyme is most stable in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). The optimization of the operating conditions showed that the enzyme Celumax® BC provided better lysis of the bacterial cell in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). These conditions resulted in lysis of the membrane of the bacteria with a recovery of 93.2% P(3HB-co-3HV) with 94% purity. The results showed that the use of enzymes for the polymer extraction is an efficient process that assists in the cell disruption of Cupriavidus necator.

A suitable procedure to choose antimicrobials as controlling agents in fermentations performed by bacteria

This work evaluated the influence of nitrofurantoin, erythromycin and streptomycin at 50, 25 and 12,5 (per cent) of the minimal inhibitory concentration (MIC) on maximum specific growth rate (µmax) and specific polymer accumulation rate (µPHB) of "Alcaligenes eutrophus", considered resistant to those antimicrobials. Nitrofurantoin strongly affected µmax even at 50(per cent) MIC. Streptomycin moderately affected µmax only at 50(per cent) MIC. Nitrofurantoin showed the most harmful effect on µPHB when 50(per cent) MIC was applied and erythromycin was not harmful.