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The production of high-purity fructooligosaccharides (FOS), known as prebiotics, by sucrose fermentation using whole microbial cells has been recently explored. At the end of the fermentation process, FOS are present in mixture with small saccharides that are known to have an inhibitory effect of transfructosylating enzymes and to decrease the prebiotic activity of the mixture. This issue can be overcome by reducing the small saccharides present in FOS broth, which can be done using a combined microbial treatment, among others, improving as well the further purification of FOS by Simulated Moving Bed (SMB) chromatography. The main goal of this work was the use of combined microbial treatment approaches to improve FOS production and enhance a high purity FOS content. Aureobasidium pullulans and Saccharomyces cerevisiae were used combined to produce FOS and reduce the small sugars in the culture, respectively. FOS-producing microorganism was used free, immobilized to a non-conventional carrier or encapsulated in Ca-alginate beads, in mixture with the non-oligosaccharides consuming microorganism, free or encapsulated in Ca-alginate beads. A factorial design, considering three different variables, was performed, to select the microbial treatment approach through which increased FOS levels and yields can be obtained. The 38 assays were performed in shaken-flasks and the most suitable one was scaled-up to a 3L bioreactor. The inoculation time of S. cerevisiae showed to be the most relevant variable for FOS production, and the use of immobilized A. pullulans, mixed with encapsulated S. cerevisiae inoculated after 20h of fermentation, was the best combination, with statistical relevance (p

The interest on oligosaccharides such as fructo-oligosaccharides (FOS) has strongly increased in recent years for food and pharmaceutical applications, mainly due to their improved technological and functional properties. FOS can be produced by fermentative processes from sucrose, and can be found in mixture with other mono- and di-saccharides and salts, at the end of the process [1]. Unlike FOS, the small saccharides (SGF), namely fructose, glucose and sucrose in the mixture, are known to be cariogenic, caloric and do not present prebiotic activity. The purification of FOS from the other sugars can represent and important increment on the economic value of the final product, which can be further used in diabetic and dietetic food [2]. Different strategies have been developed for this purpose, including microbial treatment [3], ultra and nano-filtration, activated charcoal systems [4], or ion-exchange chromatography [5]. Ion exchange resins may be then used in batch or continuous chromatographic processes, as Simulated Moving Bed (SMB) chromatography, to purify sugars. A screening of different commercial resins was previously done in order to select the most suitable to separate the oligosaccharides [5]. The resin Diaion 535Ca showed an increased recovery yield and purity of FOS (92 and 90%, respectively). In the present work, the separation process was implemented in the SMB, using the selected resin, namely. Equilibrium adsorption isotherms were determined by the Retention Time Method (RTM), for each single sugar. The resin was afterwards packed in eight SMB columns, and tested in the pilot plant. Different operation parameters, including switching time, extra time, internal flow-rates and operating pump flow-rates for feed, raffinate, desorbent, eluent and recycling streams, were tested in the plant. The separation of fructose from glucose and FOS from the SGF was evaluated. Firstly, the separation of a binary sugar mixture of fructose/sucrose (~ 50/50%) was performed followed by the separation of FOS from a fermentative broth. Fructose was purified from 53 to 76% and sucrose from 47 to 77%. FOS and SGF were purified from 50 to 67%. The implementation of UV detectors between the SMB columns allowed following the sugar concentration profile online during the separation process. The accurate selection of the operating parameters was made using the concentration signal obtained and showed to be a crucial step for an improved separation.

Study of culture strategies of Dunaliella tertiolecta combining high cell density and accumulation of lipids to produce biodiesel. Microalgae are photosynthetic organisms using light to capture CO2. Some species can accumulate, under specific growth conditions, carbon as lipids (triglycerides). This characteristic led the scientists to think about cultivating this microorganism to produce biodiesel. The following study is based on the cultivation of a 5 to 10 µm length green biflagellate microalgae, Dunaliella tertiolecta. Two objectives will be presented in parallel: first, the growth rate and then the oil content. An optimal design of experiment has been used to determine the influence of the concentration of different components in the medium as sodium chloride, nitrate and phosphate on the two responses. The fluorescence technique allows measurements of oil level within the microalgae. The experimental results show that increasing the growth leads to an oil level reduction. The sudden depletion (stress) of an essential nutrient stops the growth but increase the lipids' storage. A nitrate stress allows lipid dry mass percentage of around 19%.

Fructo-oligosaccharides (FOS) are produced from sucrose by Aureobasidium pullulans in yields between 40-60% (w/w). To increase the amount of FOS it is necessary to remove the small, non-prebiotic sugars, present. Two methods for producing high-purity FOS have been developed: the use of microorganisms able to consume small saccharides; and the use of continuous chromatography to separate sugars: simulated moving bed (SMB). It is herein proposed the combination of both methods. The aim of this study is to optimize the composition of the fermentative broth (in terms of salts and sugars) that will be further purified by SMB. A yield of 0.63 gFOS.gSucrose-1 was obtained with A. pullulans using low amounts of salts in the initial fermentative broth. By removing the small sugars, Saccharomyces cerevisiae and Zymomonas mobilis increased the percentage of FOS from around 56.0% to 83% (w/w) in average, losing only 10% (w/w) of FOS during the recovery process.