Robust industrial Saccharomyces cerevisiae strains for very high gravity bio-ethanol fermentations

The application and physiological background of two industrial Saccharomyces cerevisiae strains, isolated from harsh industrial environments, were studied in Very High Gravity (VHG) bio-ethanol fermentations. VHG laboratory fermentations, mimicking industrially relevant conditions, were performed with PE-2 and CA1185 industrial strains and the CEN.PK113-7D laboratory strain. The industrial isolates produced remarkable high ethanol titres (>19%, v/v) and accumulated an increased content of sterols (2 to 5-fold), glycogen (2 to 4-fold) and trehalose (1.1-fold), relatively to laboratory strain. For laboratory and industrial strains, a sharp decrease in the viability and trehalose concentration was observed above 90 g l-1 and 140 g l-1 ethanol, respectively. PE-2 and CA1185 industrial strains presented important physiological differences relatively to CEN.PK113-7D strain and showed to be more prepared to cope with VHG stresses. The identification of a critical ethanol concentration above which viability and trehalose concentration decrease significantly is of great importance to guide VHG process engineering strategies. This study contributes to the improvement of VHG processes by identifying yeast isolates and gathering yeast physiological information during the intensified fermentation process, which, besides elucidating important differences between these industrial and laboratory strains, can drive further process optimization.The authors thank Daniel Gomes for performing some of the fermentation samples analyses, COPAM - Companhia Portuguesa de Amidos S.A. (Portugal) for kindly providing the CSL, and Rosane Schwan (Federal University of Lavras, Brazil) for kindly providing the yeast strains PE-2 and CA1185. The financial support of Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, is acknowledged: project ProBioethanol PTDC/BIO/66151/2006, grant SFRH/BD/64776/2009 to F.B. Pereira and grant SFRH/BPD/44328/2008 to P.M.R. Guimaraes

Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs

Statistical experimental designs were used to develop a medium based on corn steep liquor (CSL) and other low-cost nutrient sources for high-performance very high gravity (VHG) ethanol fermentations by Saccharomyces cerevisiae. The critical nutrients were initially selected according to a Plackett–Burman design and the optimized medium composition (44.3 g/L CSL; 2.3 g/L urea; 3.8 g/L MgSO4·7H2O; 0.03 g/L CuSO4·5H2O) for maximum ethanol production by the laboratory strain CEN.PK 113-7D was obtained by response surface methodology, based on a three-level four-factor Box-Behnken design. The optimization process resulted in significantly enhanced final ethanol titre, productivity and yeast viability in batch VHG fermentations (up to 330 g/L glucose) with CEN.PK113-7D and with industrial strain PE-2, which is used for bio-ethanol production in Brazil. Strain PE-2 was able to produce 18.6 ± 0.5% (v/v) ethanol with a corresponding productivity of 2.4 ± 0.1 g/L/h. This study provides valuable insights into cost-effective nutritional supplementation of industrial fuel ethanol VHG fermentations.The authors thank Marisa Cunha for performing the preliminary fermentations with varying CSL concentrations, COPAM - Companhia Portuguesa de Amidos, S.A. (Portugal) for kindly providing glucose syrup and CSL, and Rosane Schwan (University of Lavras, Brazil) for kindly providing the yeast strain PE-2. The financial support of Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, is acknowledged: Project ProBioethanol PTDC/BIO/66151/2006, Grant SFRH/BD/64776/2009 to F.B. Pereira and Grant SFRH/BPD/44328/2008 to P.M.R. Guimaraes

Optimization of glycerol-organosolv pretreatment for improving enzymatic saccharification of Eucalyptus wood

This work contributes to the improvement of biomass pretreatment technologies and shows an efficient pretreatment by glycerol -water with a good lignocellulose biomass fractionation and an enhanced enzymatic susceptibility of pretreated solid. The results show that solubilized wood fraction after glycerol treatment is composed by 15.9 and 13.2 g of lignin and xylose / 100 g of raw material in the liquid phase, respectively, whereas the solid phase was hydrolyzed by enzymes (achieving up to 90 % conversion of cellulose to glucose). So, the use of glycerol in organosolv treatment is a suitable alternative for the use of an industrial by-product and the structural modification of biomass for second generation bioethanol production

Integrated approach for effective bioethanol production using whole slurry from autohydrolyzed Eucalyptus globulus wood at high-solid loadings

One of the most important targets and challenges in the second generation bioethanol is the development of a cost-effective process on large-scale. In this context, the high solid loading on saccharification and fermentation and the use of whole-slurry from pretreatment could be promising alternatives to obtain high ethanol concentrations and to decrease operational costs and wastewater. In this work, Eucalyptus globulus wood was submitted to non-isothermal autohydrolysis treatment (Tmax = 210 °C) and the whole-slurry obtained was assayed for the optimization of enzymatic saccharification at different solid and enzymes (CTec2 and HTec2) loadings using a Box–Behnken experimental design. Under the optimized conditions (liquid solid ratio 6.4 g/g, cellulase to substrate ratio 22.5 FPU/g and hemicellulase to substrate ratio 500 UI/g), two strategies were evaluated for ethanol production (Simultaneous Saccharification and Fermentation, SSF and Presaccharification and Simultaneous Saccharification and Fermentation, PSSF), using an industrial and robust Saccharomyces cerevisiae strain. High concentrations of ethanol (50.2 and 48.8 g/L) and productivities (0.63 and 0.55 g/L h) were obtained by SSF and PSSF, respectively. The SSF process proved to be an advantageous strategy to obtain concentrations >6% (v/v) of ethanol with elevated conversion (95%) even employing high solid loading and non-detoxified hydrolysate. Following an integrated optimization process, cost-effective bioethanol production conditions from whole-slurry E. globulus wood were determined and validated experimentally, representing a step-forward towards its industrial implementation.The authors A. Roman and F.B. Pereira thank to the "Fundacao para a Ciencia e a Techologia" (FCT, Portugal) for their fellowships (grant number, SFRH/BPD/77995/2011 and SFRH/BD/64776/2009, respectively) and Gil Garrote (University of Vigo, Spain) for assistance in the pre-treatment of EGW. Research described in this article was financially supported by FEDER and FCT: Strategic Project PEst-OE/EQB/LA0023/2013, Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028'' Co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte) QREN, FEDER

Industrial robust yeast isolates with great potential for fermentation of lignocellulosic biomass

The search of robust microorganisms is essential to design sustainable processes of second generation bioethanol. Yeast strains isolated from industrial environments are generally recognised to present an increased stress tolerance but no specific information is available on their tolerance towards inhibitors that come from the pretreatment of lignocellulosic materials. In this work, a strategy for the selection of different yeasts using hydrothermal hydrolysate from Eucalyptus globulus wood, containing different concentrations of inhibitors, was developed. Ten Saccharomyces cerevisiae and four Kluyveromyces marxianus strains isolated from industrial environments and four laboratory background strains were evaluated. Interestingly, a correlation between final ethanol titer and percentage of furfural detoxification was observed. The results presented here highlight industrial distillery environments as a remarkable source of efficient yeast strains for lignocellulosic fermentation processes. Selected strains were able to resourcefully degrade furfural and HMF inhibitors, producing 0.8 g ethanol/Lh corresponding to 94% of the theoretical yield.The authors thank Rosane Schwan (Federal University of Lavras, Brazil) for kindly providing the yeast strains (isolated from Brazilian ''cachaca'' and ''bio-ethanol'' fermentations) and Juan Carlos Parajo and Gil Garrote (University of Vigo, Spain) for assistance in the pre-treatment of lignocellulose biomass. Research described in this article was financially supported by FEDER funds of EU and ''Fundacao para a Ciencia e a Tecnologia'' (FCT), Portugal: Contract PTDC/BIO/66151/2006, Strategic Project PEst-OE/EQB/LA0023/2013, Project ''BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124FEDER-000028'' Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER and the PhD grant to FP and Pos-Doc grant to AR

Use of whole-slurry from autohydrolyzed Eucalyptus wood for bioethanol production

The development of a cost-effective process on large-scale is one of the most important targets in the second generation bioethanol. The use of pretreated whole-slurry allows savings in washing-steps and water consumption. In this work the whole-slurry from pretreated Eucalyptus wood (EW) was used for the bioethanol production by saccharification and fermentation process. Firstly, EW was submitted to autohydrolysis treatment and the slurry obtained was employed for the optimization of enzymatic saccharification using an experimental design. The optimized conditions were employed for bioethanol production using a robust industrial Saccharomyces cerevisiae strain. The highest ethanol concentration obtained was 50 g/L corresponding to an ethanol conversion of 95 %

Effect of hemicellulose liquid phase on the enzymatic hydrolysis of autohydrolyzed eucalyptus globulus wood

In this work, Eucalyptus globulus wood (EGW) was pretreated under autohydrolysis process at 210 and 220 ºC, obtaining a pretreated solid with high cellulose content. Moreover, the effect of the hemicellulosic liquid phase (HLP) addition on the enzymatic hydrolysis was studied. When the enzymatic hydrolysis was performed without addition of HLP, 87.38 and 107.46 g/L of glucose was obtained for 210 ºC and 220 ºC, respectively, showing that the unwashed pretreated solids are susceptible to the enzymatic hydrolysis contributing to reduce operational cost. Additionally, the impact of the inhibitory compounds in the HLP was shown to affect the enzymatic hydrolysis. However, a positive effect was shown on the enzymatic hydrolysis when the xylanases were added using 100 % of HLP, increasing to 35 % and 29 % in the glucose production respect to whole-slurry without addition of xylanases

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates

The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.The authors thank Juan Carlos Parajo and Hector Ruiz for assistance in the pre-treatment of lignocellulose biomass. Research described in this article was financially supported by FEDER and "Fundacao para a Ciencia e a Tecnologia" (FCT) (Contracts PEst-OE/EQB/LA0023/2011, PTDC/BIO/66151/2006, PTDC/AGR-ALI/102608/2008 and ERA-IB/0002/2010 and PhD grant (SFRH/BD/64776/2009) to FP)