Yeast biomass production: a new approach in glucose-limited feeding strategy

Brazilian Journal of Microbiology 44, 2, 551-558 (2013) ISSN 1678-4405 Copyright © 2013, Sociedade Brasileira de Microbiologia www.sbmicrobiologia.org.br Research Paper Yeast biomass production: a new approach in glucose-limited feeding strategy Érika Durão Vieira1, Maria da Graça Stupiello Andrietta2, Silvio Roberto Andrietta2 1 Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, SP, Brazil. 2 Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, Paulínia, SP, Brazil. Submitted: June 27, 2011; Approved: September 10, 2012. Abstract The aim of this work was to implement experimentally a simple glucose-limited feeding strategy for yeast biomass production in a bubble column reactor based on a spreadsheet simulator suitable for industrial application. In biomass production process using Saccharomyces cerevisiae strains, one of the constraints is the strong tendency of these species to metabolize sugars anaerobically due to catabolite repression, leading to low values of biomass yield on substrate. The usual strategy to control this metabolic tendency is the use of a fed-batch process in which where the sugar source is fed incrementally and total sugar concentration in broth is maintained below a determined value. The simulator presented in this work was developed to control molasses feeding on the basis of a simple theoretical model in which has taken into account the nutritional growth needs of yeast cell and two input data: the theoretical specific growth rate and initial cell biomass. In experimental assay, a commercial baker’s yeast strain and molasses as sugar source were used. Experimental results showed an overall biomass yield on substrate of 0.33, a biomass increase of 6.4 fold and a specific growth rate of 0.165 h-1 in contrast to the predicted value of 0.180 h-1 in the second stage simulation. Key words: yeast, biomass, fed-batch, process simulation, Saccharomyces cerevisiae. Introduction Microbial biomass is a suitable supplemental protein source obtained from processes in which bacteria, yeasts, other fungi or algae are cultivated in large quantities. Yeast biomass is extensively used as human or animal protein supplement in animal feed or in human nutrition (Halasz and Lasztity, 1991). Common strains used as single cell protein source includes Saccharomyces cerevisiae and Candida utilis strains. Typical microbial biomass products include bakers’ yeast and yeast extracts where Saccharomyces cerevisiae strains are the host for heterologous-protein production (Beudeker et al., 1990; Hensing et al., 1995). Basically, the industrial process concept relies on propagating cells from pure culture agar slants to large bioreactors increasing volume in each propagation stage till the final bioreactor volume (Rose, 1979; Burrows, 1979; EPA, 1995; Rendez-Gil, Sanz and Priteo, 1999; Di Serio et al., 2001; Di Serio, Tesser and Santacesaria, 2001). How- ever, besides this simple concept, industrial production aims at efficient conversion of sugar feedstock into yeast biomass mainly in the later stages where biomass volume is high. An efficient transformation of sugar in yeast protein requires that anaerobic metabolites production such as ethanol and acetaldehyde (Bauer et al., 1999) is minimized, i.e., that sugar metabolism is deviated to the oxidative pathway to achieving maximum ATP energy yield and biomass formation (Van Hoek, Van Dijken and Pronk, 1998). Moreover, fermentation products such as ethanol and, in particular, acetaldehyde are toxic. This problem becomes especially relevant during cultivation at high biomass densities (Hensing et al., 1995; van Dijken, Weusthuis and Pronk, 1993). It’s generally known that Saccharomyces cerevisiae species tends to metabolize glucose glycolytically under glucose excess even in fully aerobic conditions producing ethanol, a phenomenon known as the Crabtree effect (Käppeli, 1986; Sonnleitner and Käppeli, 1986; Verduyn, Send correspondence to E.D Vieira. School of Chemical Engineering, University of Campinas, Cidade Universitária “Zeferino Vaz”, C.P. 6066, 13083-970 Campinas, SP, Brazil. E-mail: . 552 1991; Dynesen, 1998). Additionally, not only glucose but also fructose has shown to triggers catabolite repression on Saccharomyces cerevisiae strains (Dynesen, 1998). This catabolite repression renders low biomass yield when cultivating Saccharomyces cerevisiae in batch cultures and negatively affects biomass yields due to the low ATP yield from alcoholic fermentation. Despite this, biomass formation can be achieved by innumerous metabolic pathways (Frick and Whitmann, 2005) and biomass yield on substrate can reach values up to 50% in pure oxidative growth (Akinyemi, Betiku and Solomon, 2005). To overcome these constraints on yeast biomass production two important variables are of major importance: oxygen transfer rate and glucose concentration in the broth. In heterogeneous gas-liquid reactions, e.g., in aerobic fermentation, the liquid phase controls mass transfer processes due to the relative insolubility of gases. This limitation is minimized by the use of bubble column reactors due to its good oxygen transfer with low cost operation when compared to stirred tank reactors (Kantarcia, Borakb and Ulgen, 2005). The minimum sugar concentration in broth can be reached by the use of a fed-batch process. This process concept is the current one in industrial scale and renders good biomass yields when appropriate process control strategy is used. Traditionally, in industrial production, molasses or another feedstock feeding follows a strategy built on the basis of factory historic data and so it is peculiar to a determined strain and other process conditions. Nowadays, not only for economic reasons but also because of environmental policy, some industries are investing in new strategies of process control to avoid emission of toxic pollutants (EPA, 1995). In scientific literature, many articles can be found about baker’s yeast production dealing with yeast growth modeling and aiming at different goals such as productivity, yield and yeast quality as well as new and robust online sensors (Reyman, 1992; Rigbom, Rothberg and Saxen, 1996; Rendez-Gil, Sanz and Priteo, 1999; Jones and Kompala, 1999; Di Serio et al., 2001; DiSerio, Tesser and Santacesaria, 2001; Soley, 2005). However, there are few simple theoretical models for yeast biomass production that could fit to any strain and process and could be applicable to industrial scale. The objective of the present work was, therefore, to develop and implement experimentally a simple theoretical model for yeast growth based on few parameters aiming at achieving a glucose-limited feeding strategy applicable to industrial scale. For this purpose, a spreadsheet was developed in which the theoretical model could predict at each time interval the nutritional growth needs of yeast cell based on two major input d (...truncated)