Nelma Gomes

Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.

Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims:  To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures.

Adaptation of dinitrosalicylic acid method to microtiter plates

A microtiter plate adaptation of the well-known dinitrosalicylic acid (DNS) colorimetric method, for measurement of reducing sugars, is described. This method allows a substantial volume reduction of the reagents and a rapid analysis of a large number of samples, having an economic cost benefit and a positive impact on the environment. Therefore, it is well suited as an high-throughput technique for reducing sugars determination.

The use of methyl ricinoleate in lactone production by Yarrowia lipolytica: Aspects of bioprocess operation that influence the overall performance

Abstract Yarrowia lipolytica was used to produce γ-decalactone by the degradation of methyl ricinoleate (MR). A new method for inoculating the biotransformation medium was tested, which avoided the laborious step of washing cells from the growth medium. The consequent cell hydrophobicity increase led to an enhancement of aroma production. In a study of MR concentration in shake flasks, the highest productivity (15 mg L−1 h−1) was achieved using 30 g MR L−1. Lipase and protease activities were induced but no correlation between lipase induction and aroma production was found. The effects of different aeration and agitation rates were studied in bioreactor assays. Productivity was improved to 87 mg L−1 h−1, and another compound, 3-hydroxy-γ-decalactone, was detected in large amounts. Dehydration of this lactone produced two decenolides with aroma characteristics. The direct influence of oxygen on the production of both lactones was demonstrated.

Application of a novel oscillatory flow micro-bioreactor to the production of γ-decalactone in a two immiscible liquid phase medium

A novel micro-bioreactor based on the oscillatory flow technology was applied to the scale-down of the biotechnological production of γ-decalactone. A decrease up to 50% of the time required to obtain the maximum concentration of the compound was observed, when compared with other scaled-down platforms (stirred tank bioreactor or shake flask). A three-fold increase in γ-decalactone productivity was obtained by increasing oscillatory mixing intensity from Reo ~482 to Reo ~1447. This was presumably related to the effective contribution of the reactor geometry to enhanced mass transfer rates between the two immiscible liquid phases involved in the process by increasing the interfacial area.

Oil-in-water emulsions characterization by laser granulometry and impact on γ-decalactone production in Yarrowia lipolytica

Oil-in-water emulsions composed of methyl ricinoleate (MR) or castor oil (CO) as the organic phase, stabilized by Tween 80, are in the basis of the biotechnological production of γ-decalactone. Yarrowia lipolytica was used due to its ability to grow on hydrophobic substrates and to carry out the biotransformation. The characterization of oil droplets size distribution by laser granulometry was performed under different oil concentrations. The impact of the presence of cells on droplets size was also analyzed as well as the relevance of washing inoculum cells. Furthermore, the granulometric characterization of the emulsions was related with γ-decalactone production and it was observed that, in the presence of non-washed cells, the smaller droplets disappeared, using both oils, which increased γ-decalactone concentration. This suggests that the access of cells to the substrate occurs by their adhesion around larger oil droplets.