Producción de polihidroxialcanoatos (PHA) a partir de ácidos grasos en Pseudomonas putida KT2440: optimización del proceso y cambio de escala en biorreactor tipo tanque agitado

Abstract

The work carried out focuses on the optimization of the culture medium of Pseudomonas putida KT2440 with the aim of increasing its growth, as well as the production of medium-chain polyhydroxyalcanoates (mcl-PHAs). For this purpose, the initial concentrations of the carbon source (sodium octanoate) and phosphates (monopotassium phosphate) that showed better results during the optimization of the medium were analysed and selected separately. It was performed by studying a moderate range of concentrations of these nutrients in the culture medium in agitated flask. The stirring speed in the flasks has been optimized in order to establish the best growing conditions and thus facilitate the scaling to laboratory bioreactor (2 litres of workload). The fermenting culture has been carried out with the optimized growth medium and under different stirring conditions to select the one that provided optimal growth and production of PHA. In addition, tests were also performed under semi-continuous modes of operation (fed-batch) in flask, to determine the possibility of prolonging the cultivation over time, to increase cell growth and PHA production in comparison to previous discontinuous culture (batch). For the study and selection of the best parameters of all the tests carried out, a multitude of analysis techniques were used. High Performance Liquid Chromatography (HPLC) was used for the detection of carbon sources consumption, The consumption of phosphates and ammonium, as well as for the quantification of the biomass produced in the broth were examined by spectrophotometry techniques. The production of biopolymers was analyzed by gas chromatography coupled to a mass spectrometer, showing the monomer composition of the biopolymers produced. Finally, the bioreactor culture features auxiliary probes coupled to the fermenter for pH monitoring and temperature control. In this way it was possible to study the best production strategy for PHA production by P. putida KT2440 at laboratory scale, which will provide the key parameters for the scale of the process to industrial-sized equipment or pilot plant type.