Cunha, M. R. M.

Domingues, Lucília

Guimarães, Pedro M. R.

Teixeira, J. A.

Universidade do Minho: RepositoriUM

Book  of  Abstracts  of  the  10th  International  Chemical  and Biological Engineering Conference – CHEMPOR 2008 Integration of life sciences& engineering  855 High ethanol productivity by fermentation of concentrated industrial substrates using ethanol-tolerant Saccharomyces cerevisiae strains  Marisa R.M. Cunha, Pedro M.R. Guimarães, José A. Teixeira, Lucília Domingues IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal  Keywords: bioethanol, ethanol tolerance, ethanol titre, industrial media, corn steep liquor.   Growing industrial development makes imperative to find environmentally sustainable energy sources. Biofuels have gained popularity in the last decades, especially due to the fossil fuels depletion and the increasing environmental concern. Of the many fossil fuels’ alternatives, ethanol has been highly used and studied, since it can be added to fuels for combustion engines to levels of up to 20% without any modification to the engine (MDA, 2007). In order to make ethanol production more economical, a high productivity bioprocess is necessary. Ethanol productivity can be improved by using highly concentrated substrates. However, ethanol is well known as a toxic metabolite for yeast cells, which raises a serious problem. Thus, strains that can grow well under high ethanol stress condition are highly desirable. This work aims to select and characterize Saccharomyces cerevisiae strains with improved ethanol tolerance. Moreover, it aims to evaluate the feasibility of industrial residues as fermentation media and to optimize the composition of such media. Screening for strains or mutants with high ethanol tolerance has been often performed by viability observation on ethanol-containing agar plates (Carrasco et al., 2001) and by growth rates determination on ethanol-containing medium (Hirasawa et al., 2007). Although these and other similar techniques are easy and fast, they screen for the ability to grow under high ethanol stress rather then for fermentation ability in these conditions. Thus, selection must be conducted under conditions similar to those faced by yeasts in industrial processes. In the present work, strains were selected for ethanol tolerance in batch fermentations of high-glucose media, which causes osmotic (sugar) stress in the beginning and increasingly ethanol stress towards the end of the fermentation.  Three Saccharomyces cerevisae strains were tested for ethanol production and tolerance, from which CA 116 and RL 11 are industrial strains isolated from Brazilian “cachaça” fermentation and BY 4741 is a laboratorial strain. The most ethanol-tolerant strain was able to ferment 300 g/L glucose, producing up to 17.4 % (v/v) of ethanol in trials carried out in anaerobic shake-flasks. Aiming to develop an inexpensive and highly fermentable industrial based medium, corn steep liquor (CSL) has been added to high glucose solutions, as sole nutrients source in replacement of both peptone and yeast extract. CSL), a major by-product of corn starch processing, is a cheap and readily available source of proteins, amino acids, minerals, vitamins, reducing sugars (such as dextrose), organic acids (in particular, lactic acid), enzymes, and elemental nutrients (such as nitrogen) (Aktar et al., 1997; Amartley et al., 2000; Keller and Heckman, 2006). Although its primary uses are as feed supplement for ruminants and nutrient source for poultry (Akhtar et al. 1997; Keller and Heckman, 2006), CSL has been used in many works as a rich and effective nutrient supplement, including for ethanol production (Lawford, 1997; Amartley et al., 2000; Carvalheiro et al., 2006). Likewise, the results obtained have confirmed the feasibility of CSL as peptone and yeast extract substitute, since supplementation of 300 g/L glucose medium with CSL concentrations around 90 - 110                                                  Corresponding author. Tel + 351‐253‐605406. E‐mail:marisa.cunha@deb.uminho.pt Book  of  Abstracts  of  the  10th  International  Chemical  and Biological Engineering Conference – CHEMPOR 2008 Integration of life sciences& engineering  856 g/L has resulted in fermentation performance similar to that observed in YP medium with the same glucose concentration. Recent studies have reported ethanol titres as high as 19 % v/v (Alfenore et al., 2002; Cot et al., 2007). However, these values have been achieved in fed batch processes with exponential vitamins feeding, besides other controls such as pH regulation and constant glucose concentration. In the current work, fermentations were carried out in simple anaerobic shake-flasks without process control; thus, higher performance may be achieved by process improvement. In order to design sustainable bioethanol production processes it is important to understand which factors contribute for a higher microbial ethanol tolerance, as well as to design fermentation media totally based in industrial by-products or other cheap resources.   References Akhtar, M; Lentz, JL; Blanchette, RA and Kirk, TK (1997). Corn steep liquor lowers the amount of inoculum for biopulping. Tappi Journal, Vol. 80, pp 161. Alfenore, S; Molina-Jouve, C; Guillouet, SE; Uribelarrea, J-L; Goma, G; Benbadis, L (2002). Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. Appl Microbiol Biotechnol, Vol. 60, pp 67. Amartley, SA and Leungl, JPC (2000). Corn steep liquor as a source of nutrients for ethanologic fermentation by Bacillus Stearothermophilus T-13. Bulletin of the Chemists and Technologists of Macedonia, Vol. 19, pp. 65. Carrasco, P; Querol, A and del Olmo, M (2001). Analysis of the stress resistance of commercial wine yeast strains. Arch Microbiol, Vol. 175, pp 450. Carvalheiro, F; Duarte, LC; Lopes, S; Parajó, JC; Pereira, H; Gírio, FM (2006). Supplementation requirements of brewery’s spent grain hydrolysate for biomass and xylitol production by Debaryomyces hansenii CCMI 941. J Ind Microbiol Biotechnol, Vol. 33, pp 646. Cot, M; Loret, MO; François, J and Benbadis, L (2007). Physiological behaviour of Saccharomyces cerevisiae in aerated fed-batch fermentation for high level production of bioethanol. FEMS Yeast Res, Vol. 7, pp 22. Hirasawa, T; Yoshikawa, K; Nakakura, Y; Nagahisa, K; Furusawa, C; Katakura, Y; Shimizu, H and Shioya, S (2007). Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis. J Biotechnol, Vol. 131, pp 34. Keller and Heckman LLP (2006). Assessment plan for corn steep liquor (CAS #66071-94-1) in accordance with the USEPA high production volume chemical challenge program. Available at www.epa.gov/hpv/pubs/summaries/cornstlq/c16469.pdf, 10 March 2008. Lawford, HG (1997). Corn steep liquor as a cost-effective nutrition adjunct in high-performance Zymomonas ethanol fermentations. Appl Biochem Biotechnol, Vol. 63-65, pp 287. Minnesota Department of Agriculture (2007). E20: The Feasibility of 20 Percent Ethanol Blends by Volume as a Motor Fuel. Available at http://www.mda.state.mn.us/renewable/ethanol/default.htm, 12 March 2008. 

High ethanol productivity by fermentation of concentrated industrial substrates using ethanol-tolerant Saccharomyces cerevisiae strains

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High ethanol productivity by fermentation of concentrated industrial substrates using ethanol-tolerant Saccharomyces cerevisiae strains

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