F. Garcia-Ochoa

Oxygen transfer and uptake rates during xanthan gum production

Oxygen uptake rate and oxygen mass transfer rate have been studied during xanthan gum production process in stirred tank bioreactor. Empirical equations for the oxygen mass transfer coefficient have been obtained taking into account several variables such as air flow rate, stirrer speed and apparent viscosity. Oxygen uptake rate evolution in the course fermentation has been measured, obtaining an equation as a function of biomass concentration, including overall growth and non growth-associated oxygen uptake. A metabolic kinetic model has been employed for xanthan gum production description including oxygen mass transfer and uptake rates. The results point out that this model is able to describe adequately not only oxygen dissolved evolution, but also of the production of xanthan and substrate consumption. Also, the influence of several parameters (k(L)a, air flow rate and dissolved oxygen) in the evolution of the key compounds of the system have been studied. The results of the simulation shown that an increasing of dissolved oxygen concentration favor the xanthan gum production.

Studies on the activity and the stability of β-galactosidases from Thermus sp strain T2 and from Kluyveromyces fragilis

Abstract The activity and the stability of the β-galactosidases from Thermus sp strain T2 and Kluyveromyces fragilis have been compared. Both enzymes have been partially purified by gel permeation chromatography, determining their molecular weights too. The influence of several metal cations and some buffers on the activity of the enzymes has been tested. The specificity of the enzymes for galactosyl moieties and β-bonds has been established by testing their activity on several synthetic chromogenic substrates and disaccharides. Also, it has been determined that both enzymes showed a remarkable hydrolytic activity and a weak transgalactosilation activity, even in the presence of high concentrations of lactose. The stability of both enzymes in soft and extreme conditions of pH and temperature and in the presence of aggressive chemicals (organic miscible solvents, oxygen peroxide, surfactants and urea) was studied. The thermophilic enzyme showed a higher resistance to hydrophobic agents and a higher stability at different temperatures, pHs and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, showing that some residues important for its stability were affected by oxidation. Kinetic studies on the ONPG hydrolysis with both enzymes were carried out in a wide range of temperatures and substrate and product concentrations. The data obtained at all the temperatures were fitted by a nonlinear technique to different kinetic models and two of them were selected to describe the reaction catalysed by the enzymes. The enzyme from K. fragilis was strongly inhibited by o-nitrophenol in a acompetitive way but it was weakly and competitively inhibited by galactose. The thermophilic enzyme was competitively inhibited by galactose much strongly than its mesophilic counterpart but the inhibition did not change with the temperature.

Activity over lactose and ONPG of a genetically engineered β-galactosidase from Escherichia coli in solution and immobilized: kinetic modelling

The kinetic study of the hydrolysis of lactose and o-nitrophenol-β-D-galactoside (ONPG) with a β-galactosidase from Escherichia coli, both in solution and covalently immobilized on a silica-alumina, is presented. The enzyme employed in this work had been modified previously by genetic engineering and purified to homogeneity by affinity chromatography. Firstly, the influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. Secondly, hydrolysis runs of lactose and ONPG with both forms of the enzyme were carried out in a wide experimental range of temperature and concentrations of substrates, products and enzyme. Data obtained were fitted to several kinetic models based on the Michaelis-Menten mechanism by non-linear regression. Finally, the models and their parameters were compared to determine the influence of the immobilization process and the substrate on the activity of the enzyme. In the hydrolysis of lactose and with both forms of the enzyme, acompetitive inhibition due to glucose was observed while the most common inhibition by galactose (which is usually a competitive inhibitor of β-galactosidases) was not observed. Curiously, when the immobilized enzyme was the catalyst employed, lactose was an acompetitive inhibitor of the hydrolysis. When the substrate hydrolysed was the o-nitrophenol-β-D-galactoside (ONPG), the galactose acted as a competitive inhibitor and the o-nitrophenol (ONP) was an acompetitive inhibitor for the free enzyme, being the immobilization process able to avoid the interaction between the ONP and the enzyme.

Estimation of oxygen mass transfer coefficient in stirred tank reactors using artificial neural networks.

The estimation of volumetric mass transfer coefficient, k(L)a, in stirred tank reactors using artificial neural networks has been studied. Several operational conditions (N and V(s)), properties of fluid (µ(a)) and geometrical parameters (D and T) have been taken into account. Learning sets of input-output patterns were obtained by k(L)a experimental data in stirred tank reactors of different volumes. The inclusion of prior knowledge as an approach which improves the neural network prediction has been considered. The hybrid model combining a neural network together with an empirical equation provides a better representation of the estimated parameter values. The outputs predicted by the hybrid neural network are compared with experimental data and some correlations previously proposed in the literature for tanks of different sizes.

Stirred Tank Bioreactors

Stirred tank bioreactors (STBRs) are the reactors most widely employed for culturing of biological agents such as cells, enzymes, or antibodies. They are contactors where the well-mixed among phases is obtained mainly by internal mechanical agitation. The impeller must provide sufficiently rapid agitation to disperse all compounds and achieve an effectively homogeneous concentration inside the bioreactor. The technical design of an STBR for a bioprocess must determine the required volume, which depends on the production. Therefore, it is necessary to know the rates of the different phenomena involved (mass transport, heat transfer, complex network of reactions, etc.) and combine the characteristic variables and parameters of each one of these phenomena into a kinetic model that adequately describes the complex evolution of the biological system. For this purpose, the balances of mass and energy have to be formulated. This work is focused on design of STBR in batch and continuous operation. As a means of illustrating typical design equations for both way operations, the xanthan gum production has been used. The simulation and discussion of results with different kinetics model proposed in the literature are presented.