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 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.

Aims:  To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results:  Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l−1 at 1 bar to 533·5 U l−1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions:  OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study:  Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.

In this work, an empirical correlation is proposed to describe k L a 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 k L a 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 ␥-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 k L a on the production of the aroma. It was demonstrated that k L a 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 k L a value of 70 h −1 was necessary to attain the maximal aroma production.

Yarrowia lipolytica converts methyl ricinoleate to c-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-c-decalactone produced by the yeast (263 mg l -1 ) occurred by increasing the k L a up to 120 h )1 at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O 2 solubility, but, although growth was accelerated, c-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec)2-en-4-olide and dec-3-en-4-olide (70 mg l )1 ).

Improvement of microbial cell cultures oxygenation can be achieved by the increase of total air pressure, which increases oxygen solubility in the medium. In this work, a pressurized bioreactor was used for Yarrowia lipolytica batch cultivation under increased air pressure from 1 to 6 bar. Cell growth was strongly enhanced by the pressure rise. Fivefold and 3.4-fold increases in the biomass production and in specific growth rate, respectively, were observed under 6 bar. The increase of oxygen availability caused the induction of the antioxidant enzyme superoxide dismutase, which indicates that the defensive mechanisms of the cells against oxidative stress were effective and cells could cope with increased pressure. The pregrowth of Y. lipolytica under increased pressure conditions did not affect the lipase production ability of the cells. Moreover, the extracellular lipase activity increased 96% using a 5-bar air pressure instead of air at 1-bar pressure during the enzyme production phase. Thus, air pressure increase in bioreactors is an effective mean of cell mass and enzyme productivity enhancement in bioprocess based in Y. lipolytica cultures.

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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.