Castro, C. C.

Martins, Rui C .

Oliveira, J. M.

Silva, Fernando F. Macieira da

Silva, J. S.

Silva, Sílvia S.

Teixeira, J. A.

Universidade do Minho: RepositoriUM

Escola de EngenhariaSemana da Escola de Engenharia October 20 - 26, 2011 FERMENTATIVE METABOLISM OF YEAST STRAINS UNDER INDUCED STRESS CONDITIONSCristiana C. Castro, João S. Silva, Sílvia S. Silva, Fernando F. Macieira-Silva, José M. Oliveira, Rui C. Martins and José A. TeixeiraDepartment of Biological EngineeringE-mail: cristianacastro@deb.uminho.ptKEYWORDSSaccharomyces cerevisiae, stress response, morphology, HPLC, GC-FID .ABSTRACTSaccharomyces  cerevisiae can  undergo  changes  in  its replicative  pattern  and  morphology  according  to  the environmental  conditions.  In  the  present  work,  three different yeast strains – S288c, CA11 and PE-2 - were grown in YPD agar and broth, where different  stress-inducing compounds were added, including ethanol, 1-butanol,  isopropanol,  2-methyl-2-butanol,   2-furaldehyde,  5-hydroxymethyl-furfural  and  cyclic-adenosine-monophosphate.  The  three  strains  showed different  fermentation  rates,  under  the  same fermentation  media,  being  higher  for  PE-2  strain. Different  fermentation  condition  also  led  to  different fermentation rates, using the same strain. The presence of 1-butanol and amylic alcohols, as well as furans in the  growth  media  showed  to  be  the  most  stressful environments for yeasts growth. This was confirmed by the low fermentation  rates  under these conditions,  by the  chemical  information  and  in  certain  cases  by morphology  changes  mainly  by  hyphae  or  filaments formation. INTRODUCTIONThe yeast  is  one of the most studied microorganisms, due  to  the  large  ability  to  adapt,  grow  and  be manipulated  under  laboratory  conditions  and  to  its essential  role  in  fermentation  processes  in  different industrial  activities.  The fermentation  process,  despite its massive use in the food industry, cannot be totally controlled. The means of control available are external to the process and are centered primarily in the selection of strains and optimization and control  of the process conditions.  Saccharomyces  cerevisiae is  a  dimorphic yeast  with  the  ability  to  change  its  morphology according to the environment. Under stress conditions it can  modify  from a  unicellular  to  a  filamentous  form (Casalone  et  al.  2005),  and  different  strains  and/or growth conditions lead to different responses (Palková 2004; Casalone et al. 2005). In  this  work,  we  used  three  different  S.  cerevisiae strains - S288c, a standard laboratory strain unable to form pseudohyphae (Liu et al. 1996); PE-2, a strain with a high tolerance to ethanol widely used in the industry (Pereira  et  al.  2011);  and  Ca-11,  a  flocculant  strain isolated from Brazilian  cachaça fermentations (Pereira et al. 2010). The three strains were grown in YPD agar (YPDa)  and  YPD broth (YPDb),  in  the presence  and absence  of stress inducers, and compared in terms of fermentation  profiles,  chemical  composition  and morphological changes.MATERIAL AND METHODSS.  cerevisiae  strains  -  S288c,  CA11 and PE-2 -  were obtained from the microbiological collection of the IBB at  the  University  of  Minho.  The  incubation  was performed in YPDb medium (Sigma) during 12 hours at 30 ºC under constant agitation (150 rpm). Fermentations were performed in both solid and liquid medium, where different  growth  conditions  were  induced.  The  three strains  were  inoculated  in  the  surface  of  YPD  agar media  (20  μl),  during  96h,  and  in  50  ml  YPDb (106 cells/ml),  during  24h,  without  any  stress  condition (control) and with: 1 % (v/v) ethanol (Riedel-de-Han), 1 % (v/v) isoamyl alcohol (SAFC), 1 % (v/v) 1-propanol (Sigma Aldrich), 1 % (v/v) 2-methyl-2-butanol (Sigma Aldrich)  under  30  ºC.  Microscopy  analysis  were performed  for  morphological  changes  observation  for both  solid  and  liquid  medium  and  YPDb  medium fermentations were controlled by optical  density, cells concentration,  pH  and  CO2 production  and  major fermentation  metabolites  were  measured  using  HPLC and GC-FID techniques.Escola de EngenhariaSemana da Escola de Engenharia October 20 - 26, 2011 DISCUSSIONMorphological changesThe  presence  of  1-butanol  and  the  amylic  alcohols induced  cells  morphology  changes  by  pseudo-hyphae and filaments production. Morphological changes were more evident in solid media when compared to liquid. Fermentation profilesThe three studied S. cerevisiae strains showed different fermentation  rates  under  the  same  fermentation condition  (Figure  1a),  being  higher  for  PE-2  (1.17 g/L.h), followed by CA11 (0.98 g/L.h) and S288c (0.93 g/L.h), i.e., the industrial strain is more adapted to the used conditions, comparing to the others. The presence of 1-butanol, 2-furaldehyde and 5-HMF showed distinct profiles with lower fermentation rates (Figure 1b),: 0.59, 0.31 and 0.31 g/L.h, respectively, comparing to the rest (≈ 0.9 g/L.h), using S288c. Figure 1: CO2 production during fermentation in: (a) YPDb control, using: (♦) S288c, (■) CA11 and (▲) PE-2; and (b) YPDb using S288c in: (♦) control, (■) ethanol, (▲) 1-butanol, (×) isopropanol, (+) 2-methyl-2-butanol, (*) 2-furaldehyde, and (●) 5-HMF.Chemical informationSingular value decomposition methodology of chemical information obtained by HPLC and GC-FID, confirmed that  the  presence  of  1-butanol,  2-furaldehyde  and  5-HMF leads to very distinct behavior. Samples in time 0h of  fermentations  under  stress  seem  to  have  similar chemical  composition,  and different  from the  control. Fermentation with ethanol seems to approach, (after 10 hours) to the control, throughout fermentation time.REFERENCESCasalone, E.; Barberio C.; Cappellini L.; M. Polsinelli. 2005. “Characterization  of  Saccharomyces  cerevisiae natural populations  for  pseudohyphal  growth  and  colony morphology.”  Research  in  Microbiology,  No.156,  191-200.Liu,  X; Kim, C.N.; Yang, J.; Jemmerson,  R; and X. Wang. 1996.  “Induction  of  apoptotic  program  in  cell-free extracts: requirement for dATP and cytochrome c.”  Cell, Vol.86(1), 147-57.Palková,  Z. 2004.  “Multicellular microorganisms: laboratory versus nature – review.” EMBO reports, Vol.5(5).Pereira,  F.B.;  Guimarães,  P.M.;  Teixeira,  J.A.;  and  L. Domingues.  2010.  “Selection  of  Saccharomyces  cerevisiae strains  for  efficient  very  high  gravity  bio-ethanol  fermentation  processes.”  Biotechnology  Letters,  No. 32, 1655–1661.Pereira,  F.B.;  Guimarães,  P.M.;  Teixeira,  J.A.;  and  L. Domingues.  2011.  “Robust  industrial  Saccharomyces cerevisiae  strains  for  very  high  gravity  bio-ethanol fermentations.” Journal of Bioscience and Bioengineering. Vol.112(2), 130-6.AUTHORS’ BIOGRAPHIESCRISTIANA C. CASTRO was born in  Miranda  do  Douro,  Portugal  and went  to  University  of Minho, where she studied Chemical  and Biological Engineering  and  obtained  her  MSc degree in 2007. She worked for two years  in  the  project  PTDC/BIO/ 69310/2006  before  start  the  PhD  in CEB  in  2009.  Her  email  address  is: cristianacastro@deb.uminho.pt and her web-page can be found in http://www.ceb.uminho.pt/pessoas/pid.aspx?id=328.ACKNOWLEDGEMENTSThe author Castro C.C. gratefully acknowledges herDoctoral  grant  to  the  Fundação  para  a  Ciência  e Tecnologia (FCT). This research was funded by FEDER through  the  Programa  Operacional  Factores  de Competitividade  –  COMPETE  and  national  funds through  FCT  under  PTDC/BIO/69310/2006  and PDTC/AGR-ALI/103392 projects. 

Fermentative metabolism of yeast strains under induced stress conditions

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