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.

Approximately 1 million tons of waste cooking oils (WCO) are generated in European Union per year. Oftentimes, these oily wastes are discharged through public sewerage systems, increasing the water treatment costs in wastewater treatment plants. The oleaginous yeast Yarrowia lipolytica W29 is used to simultaneously degrade WCO and to produce lipase (market demand is increasing due to its application in the field of bioenergy). A statistical experimental design based on Taguchi method is employed to assess the effect of initial medium pH, WCO, and arabic gum concentration on lipase production. Initial medium pH is found to be the most significant parameter and the interaction between WCO and arabic gum concentration had the highest influence for lipase production. Additionally, the effect of oxygen mass transfer is studied in batch cultures in a stirred tank bioreactor and the maximum lipase activity (12 000 U · L−1) is obtained at kLa of 16 h−1. Simultaneously with lipase production, also lipid‐rich biomass (48% of lipids mass per dry cellular mass), enriched in unsaturated fatty acids (oleic and linoleic acids) is obtained. Practical Applications: In this work it was demonstrated the possibility of replacing an expensive edible oil (olive oil) by a non‐edible oil waste (WCO) for lipase production. Moreover, no more wastes are produced, since whole cells of Yarrowia lipolytica W29 (lipid‐rich biomass), are a potential source for food supplements. Thus, the bioprocess described fulfill the requisites of circular economy: a low cost substrate is used as raw material, added‐value products (lipase and lipid‐rich biomass) are obtained and no further wastes are generated (take‐make‐dispose concept). Waste cooking oils (WCO) are generated from vegetable oils used at high temperatures in food frying. The biological degradation of WCO by the oleaginous yeast Yarrowia lipolytica W29, with concomitant production of added‐value compounds (lipase and lipid‐rich biomass), allows the reduction of pollutant burden of these oily wastes.

Yarrowia lipolytica is a suitable microbial platform to convert low-value hydrophobic substrates into microbial oils and other important metabolites. In this work, this yeast species was used to simultaneously synthetize ex novo lipids and produce citric acid and lipase from animal fat (pork lard) adding higher value to the low-cost fatty substrate. The effect of pH, lard concentration, arabic gum concentration and oxygen mass transfer rate (OTR) on lipids accumulation on Y. lipolytica batch cultures was assessed by an experimental design based on Taguchi method. OTR was by far the most influential parameter in the range of 96mgLh-480mgLh. A bio-modification of initial lipidic substrate was observed and, depending on the nutritional and operational conditions, specialty lipids with specific composition and high added-value were obtained. The unsaturated-to-saturated fatty acids ratio of these microbial lipids is higher than in initial substrate, which indicates that they are more suitable than animal fat for food additives. Moreover, the simultaneous induction of lipase and citric acid by Y. lipolytica growing in animal fat demonstrates that a biorefinery approach may be designed based on animal fat raw material.

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L(-1) to 200 g L(-1) were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.

In recent years, the interest to find alternative extraction methods has increased. Extraction of natural phenolics by enzymes produced during solid-state fermentation (SSF) is a useful and novel technique environmentally friendly. On the other hand, agro-industrial wastes are an excellent source of natural antioxidants as phenolics. The aim of the work was to evaluate the increase of antioxidant compounds extraction from agro-industrial wastes after SSF and to relate it to the production of lignocellulolytic enzymes. Several filamentous fungi were evaluated as candidates to increase the extraction of antioxidant compounds by solid-state fermentation of wineries, olive mill and brewery wastes. Fermented and unfermented agro-industrial wastes were extracted with water and lignocellulolytic enzymes, total phenolic compounds and antioxidant activity were determined in the extract. The maximum xylanase and cellulase activities were achieved by A. ibericus strains using brewer´s spent grain (BSG) as substrate and ranged from 300 to 313 U xylanase/g and 51-62 U cellulase/g. The best producer of β-glucosidase was A. niger CECT2088 using BSG as substrate (94 ± 4 U/g). The results of extraction of phenolic compounds revealed a higher extraction by SSF in olive mill wastes followed by winery wastes. In the same form, the maximum increase of antioxidant activity was achieved by SSF of exhausted olive pomace by Rhizopus oryzae MUM 10.260, increasing 12.9-fold in relation to unfermented waste. In addition, the production of enzymes, the extraction of phenolic compounds and the increase of antioxidant activity were related by principal component analysis. The first component grouped the BSG along with maximum enzymes production, the second component related positively the production of enzymes and the extraction of phenolic compounds and the increase of antioxidant activity. SSF proved to be an innovative environmentally friendly process that can improve the extraction of antioxidant compounds and simultaneously to produce lignocellulolytic enzymes from different agro-industrial wastes.

Olive mills generate a large amount of waste that can be revaluated. This work aim to improve the production lignocellulolytic enzymes by solid-state fermentation using ultrasounds pretreated olive mill wastes. The composition of olive mill wastes (crude and exhausted olive pomace) was compared and several physicochemical characteristics were significantly different. The use of both wastes in SSF was evaluated and a screening of fungi for xylanase and cellulase production was carried out. After screening, the use of exhausted olive pomace and Aspergillus niger led to the highest enzyme activities, so that they were used in the study of ultrasounds pre-treatment. The results showed that the sonication led to a 3-fold increase of xylanase activity and a decrease of cellulase activity. Moreover, the liquid fraction obtained from ultrasounds treatment was used to adjust the moisture of solid and a positive effect on xylanase (3.6-fold increase) and cellulase (1.2-fold increase) production was obtained.

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO2.