CELLS IMMOBILIZED WITH CALCIUM ALGINATE IN THE PRESENCE OF MAGNETIC NANOPARTICLES ORTIZ, S.1

Transcription

1 EVALUATION OF Saccharomyces cerevisiae CELLS IMMOBILIZED WITH CALCIUM ALGINATE IN THE PRESENCE OF MAGNETIC NANOPARTICLES ORTIZ, S. 1 ; BORK, J. A. 1 ; HENRIQUES, R. O. 1 ; FURIGO Jr., A. 1 1 Department of Chemical Engineering, Federal University of Santa Catarina, Florianópolis-SC, Brazil, ( samaraeq@gmail.com, j.bork@ymail.com, rosanahrq@gmail.com, agenor@enq.ufsc.br)

2 EVALUATION OF Saccharomyces cerevisiae CELLS IMMOBILIZED WITH CALCIUM ALGINATE IN THE PRESENCE OF MAGNETIC NANOPARTICLES ORTIZ, S. 1 ; BORK, J. A. 1 ; HENRIQUES, R. O. 1 ; FURIGO Jr., A. 1 1 Department of Chemical Engineering, Federal University of Santa Catarina, Florianópolis-SC, Brazil, ( samaraeq@gmail.com, j.bork@ymail.com, rosanahrq@gmail.com, agenor@enq.ufsc.br) Abstract The alcoholic fermentation with immobilized cell is extensively studied, and one of the research goals is to test different immobilization supports. The main yeast used in research is Saccharomyces cerevisiae, which is used in industrial scale. Currently, the magnetic particles are being applied as support for the enzymes immobilization. In this study, the magnetite was added in the Saccharomyces cerevisiae yeast immobilization in calcium alginate. The influence of the magnetic nanoparticle presence was analyzed in comparison to a fermentation process with cell immobilized in calcium alginate without the presence of the magnetite. After the visual and substrate consumption analysis, it was concluded that the magnetic particles presence on the cell immobilization does not present significant changes in the fermentation process. Keywords Alcoholic fermentation; immobilized cell; Saccharomyces cerevisiae; magnetite. INTRODUCTON Saccharomyces cerevisiae yeast is belonging to the family of Saccharomycetaceae, being a genre of Fungi kingdom. The genome of Saccharomyces yeasts have 6,000 genes, of which over 2,000 have their unknown functions (Fonseca and Teixeira, 2007). Sodium alginate is the compound most widely used in various industries. This is because it is soluble in water and become insoluble in the presence of divalent cations (Ca++ or Mg++, for example). These cations are attached forming a gelatinous three-dimensional structure, held together by calcium or magnesium ions, known by egg-box model (Souza et al, 2008; Cargill, 2014). The magnetite (Fe3O4) is an important ferromagnetic mineral containing Fe II and Fe III. The magnetite is black, and its density is 5.18 g.cm -3. The magnetite, which is studied mostly in enzymes immobilization, can be also known as black iron oxide, magnetic iron ore, natural magnet, among others (Cornell and Schwertmann, 2003). This work aims to analyze the influence of the magnetite nanoparticles presence in cell immobilization efficiency of Saccharomyces cerevisiae yeast in calcium alginate, qualitatively (visual observations) and quantitative (the substrate consumption during the fermentation process). MATERIALS AND METHODS Magnetite The magnetite synthesis was according to the methodology proposed by Bork et al (2014).

3 Cell Immobilization The cell immobilization evaluated in this study was entrapment. Initially, there was prepared 10 ml of sodium alginate solution (1.5%). This solution was maintained under agitation for the complete granules dissolution. Then the magnetic particles (1%) were added. After brief manual stirring, the suspension of sodium alginate and magnetic particles was subjected to ultrasonic treatment (25 khz), with agitation of 600 rpm for 30 minutes. In parallel, it was prepared 100 ml of calcium chloride solution (CaCl2) 0.1M. In the sodium alginate and magnetite mixture, it was added 1.5g of Saccharomyces cerevisiae. Then, this mixture was placed in a burette and dropped in the calcium chloride solution, with a flow rate of 3 ml/min and dropping height of 24 cm. The formed spheres remained for at least 2 hours immersed in the calcium chloride solution at 4 C. Then, they were washed with sterile distilled water, being ready for their utilization in fermentation processes. For comparative purposes, the immobilization was performed in parallel without the magnetic particles presence, maintaining all other variables equal, and used in a similar fermentation process. Alcoholic Fermentation The medium for the alcoholic fermentation was composed of glucose (100 g/l), yeast extract (1.0 g/l), ammonium sulfate (10.3 g/l), potassium phosphate monobasic (0.5 g/l) and magnesium sulfate heptahydrate (0.5 g/l). The Erlenmeyer flask containing 10 ml of medium was previously sterilized in an autoclave for 15 minutes at 121 C. After that, the spheres were added and the system was maintained in an incubator at 30 C and agitation of 150 rpm for 24 hours, with samples collection every 2 hours for analysis. The total reducing sugars concentration (TRSs) was analyzed by the DNS method, according to the methodology proposed by Gonçalves et al (2010). RESULTS AND DISCUSSION Magnetite Synthesis In the Figure 1 we can observe the magnetite particles being attracted by the magnet. Before using the particles in the immobilization step, it was necessary to grind them to facilitate its mix to the alginate. Figure 1. Magnetite particles being attracted by the magnet.

4 TRS concentration (g/l) TRS concentration (g/l) Alcoholic Fermentation The graphic of the total reducing sugars consumption versus fermentation time for the process using immobilized cell with the magnetic particle is presented in Figure 2. In Figure 3 is presented the graphic for the alcoholic fermentation using immobilized cell without the magnetic particle. In both, it is possible to observe similar behavior, resulting in final sugars concentrations of approximately zero, in the proposed time Time (h) Figure 2. Graphic of substrate consumption during fermentation time using cell immobilization with the magnetic particle Time (h) Figure 3. Graphic of substrate consumption during fermentation time using cell immobilization without the magnetic particle. In the substrate consumption analysis, we can realize that the magnetic nanoparticle presence does not have significant influence on the fermentation process. In the visual analysis, no change in both culture media was observed. After 24 hours, the immobilized cells with the magnetic particles showed cracks and fissures, but maintained the attraction by the magnet, according to the Figure 4. The spheres did not crumbled with the action of the agitation during the fermentation process, deforming only due to cell growth into the sphere.

5 CONCLUSIONS Figure 4. Immobilized cells with magnetic particles after 24 hours of fermentation. This study is innovative in the alcoholic fermentation area with cell immobilized, and preliminary studies are required to demonstrate the availability of scale-up studies and obtain reliable parameters. This is particularly necessary because of the cost of the magnetic particles synthesis and the sodium alginate. We can conclude that it was possible to perform the cell immobilization with calcium alginate and the magnetic particles. Analyzing the substrate consumption during the fermentation process, we can conclude that the presence of the particle had no influence. The immobilization, in the studied conditions, resisted the stipulated time, being viable for future studies. ACKNOWLEDGEMENTS Authors thanks financial support from CAPES. REFERENCES Bork, J. A.; Henriques, R. O.; Weinsberger, G. M. N.; Furigo Jr, A. Síntese e Imobilização da Lipase de Rhizomucor miehei em Nanopartículas Magnéticas Modificadas com Moléculas de Lauril Sulfato. COBEQ Florianópolis/SC, Cargill. Alginatos. Available in: index.jsp. Accessed on 04/09/2014. Cornell, R. M.; Schwertmann, U. The Iron Oxides: Structure, Properties, Reactions, Occurences and Uses. Second, Completely Revised and Extended Edition. WILEY-VCH GmbH & Co. KGaA, Germany, ISBN , 664 p., Fonseca, M. Manuela da; Teixeira, José A. Reactores biológicos: Fundamentos e Aplicações. Lisboa: Lidel, xxxii, 483 p, il. (Coleção biotec) Gonçalvez, Cristiana; Rodriguez-Jasso, Rosa Maria; Gomes, Nelma; Teixeira, José A.; Belo, Isabel. Adaptation of dinitrosalicylic acid method to microtiter plates. Analytical Methods, v. 2, p , Souza, Kely Viviane de; Peralta-Zamora, Patricio; Zawadzki, Sônia Faria. Immobilization of iron (II) in alginate matrix and its use in textile dye degradation by Fenton processes. Química Nova, v. 31, n.5 (Print version ISSN ), 2008.