Fed-batch versus batch cultures of Yarrowia lipolytica for γ-decalactone production from methyl ricinoleate

Constant medium feeding rate and intermittent fed-batch fermentation strategies were investigated aiming to increase the yields of γ-decalactone production by Yarrowia lipolytica, using methyl ricinoleate as substrate and ricinoleic acid source. The accumulation of another compound, 3-hydroxy-γ-decalactone, was also analyzed since it derives from the direct precursor of γ-decalactone thereby providing information about the enzymatic activities of the pathway. Both strategies were compared with the traditional batch mode in terms of overall productivity and yield in respect to the substrate. Although the productivity of γ-decalactone was considerably higher in the batch mode (168 mg l−1 h−1), substrate conversion to lactone (73 mg γ-decalactone g−1) was greater in the intermittent fed-batch giving 6.8 g γ-decalactone l−1. This last strategy therefore has potential for γ-decalactone production at an industrial level.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/28039/2006)

Empirical modelling as an experimental approach to optimize lactone production

The biotransformation of ricinoleic acid, carried out by Yarrowia lipolytica, leads to the formation of gama-decalactone, a well-known peach-like aroma compound, interesting to produce and to use in the flavouring industry, reason why it is imperative to define the most appropriate conditions for its production. Thus, the aim of this work is the optimization of operating conditions for this lactone. However, as the accumulation of another compound, namely 3-hydroxy-g-decalactone (the precursor of two other aromatic compounds, dec-2-enolide and dec-3-enolide), may also occur simultaneously in the biotransformation medium, and since this compound may as well be of interest for the flavouring industry, the operating conditions for its production were also a focus of attention. Therefore, a 3^2 level full-factorial design was used to determine the effect of pH and dissolved oxygen concentration (DO) on the production of gama-decalactone and 3-hydroxy-gama-decalactone. Since both factors were found to influence the two lactones production, a response surface methodology (RSM) analysis was also applied to identify the optimal conditions for the production of those two compounds. The statistical model pointed out pH = 6.17 and DO = 44.4% as the best conditions optimizing gama-decalactone production. Using these optimalconditions, the maximal gama-decalactone concentration achieved was 680.9 mg/L, which was quite similar to the predicted value of 718.7 mg gama-decalactone per liter. Among the range of operating conditions tested, no optimization was possible for 3-hydroxy-gama-decalactone production, since all possible solutions corresponded to operating conditions not analyzed.The authors aknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/28039/2006) and Hector Ruiz for the help provided with MATLAB

Oxygen effect in γ-decalactone production through biotransformation of ricinoleic acid

γ-Decalactone can be produced biotechnologically from the degradation of ricinoleic acid by yeasts, namely Yarrowia lipolytica. Preliminary studies using two different ricinoleic acid sources (methyl ricinoleate and castor oil) at different concentrations were tested as substrates, in flask experiments. Although high amounts of γ-decalactone (up to 2 g L-1) were achieved with oil concentrations of 3% and 5% (v/v) for each substrate, the productivity of the process was small (10 to 14.5 mg L-1 h-1). In order to increase these values, essays were conducted in a 2-L bioreactor, with 3% (v/v) methyl ricinoleate at different aeration and agitation rates, in the herein presented work. Under these conditions, the highest amount of γ-decalactone achieved was lower (approximately 1 g L-1). However, the productivity of the process was much higher: 87 mg L-1 h-1. Furthermore, another compound derived from the direct precursor of γ-decalactone, 3-hydroxy-γ-decalactone, was detected in large amounts (up to 8 g L-1), which implies a change in the metabolic pathway control

Oxygen mass transfer in a biphasic medium: influence on the biotransformation of methyl ricinoleate into γ-decalactone by the yeast Yarrowia lipolytica

In this work, an empirical correlation is proposed to describe kLα as a function of operating conditions (agitation and aeration rates) and of oil and surfactant volumetric fractions in a biotransformation medium, an oil-in-water dispersion. An interaction effect between the oil and the surfactant effects was found, since oil presence increased kLα in the absence of the surfactant but had an opposite effect when Tween 80 was available in the medium. The biotransformation of methyl ricinoleate (MR) into γ-decalactone (an aroma compound of industrial interest), by the yeast Yarrowia lipolytica, was carried out at different conditions of operation, to evaluate the influence of kLα on the production of the aroma. It was demonstrated that kLα had an influence on the aroma production; however, for the low hydrophobic substrate concentration used (1.08% v/v) and cellular density of 2.0 × 10 7 cells mL−1, a minimal kLα value of 70 h−1 was necessary to attain the maximal aroma production

Gas-liquid interfacial area in oxygen absorption into oil-in-water emulsions

Present work includes an exhaustive study about gas-liquid interfacial area between gas phase and liquid heterogeneous medium generated in an airlift bioreactor. The model system studied is composed by water, methyl ricinoleate and Tween 80, since it is the base of the medium used for the production of γ-decalactone through the biotransformation of ricinoleic acid, by the yeast Yarrowia lipolytica. Experimental results allow describing the hydrodynamic behavior of the gas phase into the biphasic medium. In the oil-in-water system used, a decrease of the gas-liquid interfacial area with the oil concentration increase was found, due to the redistribution of surfactant between interface and bulk phases

Freeze/thawing and sonication of Escherichia coli TB1 cells for cytochrome b5 recovery

The influence of sonication power, suspension volume and cell concentration on the kinetics of cytochrome b5 and intracellular protein release by sonication of Escherichia coli TB1 cells was studied. The influence of freezing and thawing of the cell suspension was also evaluated. Freezing and thawing increased the recovery yield of cytochrome b5. The sonication efficiency increased with the increase of sonication power and with the decrease of the suspension volume and cell concentration

Optimization study of Escherichia coli TB1 cell disruption for cytochrome b5 recovery in a small-scale bead mill

The recovery of a recombinant intracellular protein, cytochrome b5, from Escherichia coli TB1 cells was carried out by bead mill disintegration in a discontinuous smallscale instrument. This process was optimized by the use of experimental factorial design. Several parameters were studied: operating time, amount and size of beads, cellular suspension concentration, and presence of toluene and lysozyme. For the experimental conditions used, only the time of treatment and bead load had significant effects. The optimal values of these variables were found by applying the response surface methodology.Junta Nacional de Investigação Científica e Tecnológica (JNICT