Aloia Romaní

Bioethanol production from hydrothermally pretreated Eucalyptus globulus wood

Eucalyptus globulus wood samples were pretreated in aqueous media under non-isothermal conditions to reach maximal temperatures (T(MAX)) in the range 195-250 degrees C, in order to assess the effects of the pre-treatment severity on the fractionation of wood and on the susceptibility of processed samples toward enzymatic hydrolysis. Both the fraction of cellulose susceptible to hydrolysis and the hydrolysis rate increased with the severity of the pre-treatments, but the overall glucose yield decreased for substrates pretreated at T(MAX) above 220 degrees C owing to cellulose losses. Using substrates pretreated at T(MAX)=220 degrees C, up to 94% of polysaccharides were recovered in the hydrolysis media as mono- or oligo-saccharides. High glucose to ethanol conversions were obtained operating at low enzyme charges in Simultaneous Saccharification and Fermentation mode.

Eucalyptus globulus wood fractionation by autohydrolysis and organosolv delignification.

This work provides an assessment on the fractionation of Eucalyptus globulus wood by sequential stages of autohydrolysis (to cause the solubilization of hemicelluloses) and organosolv pulping (to dissolve lignin, leaving solids enriched in cellulose). With this approach, valuable products (hemicellulose-derived saccharides, sulphur-free lignin fragments and cellulosic substrates with low contents of residual hemicelluloses) are obtained in separate streams, according to the biomass refinery approach. Autohydrolysis was carried out under optimized operational conditions, and organosolv pulping was performed using uncatalyzed ethanol-water solutions. The effects of the most influential operational variables (autohydrolysis severity, delignification temperature and ethanol concentration in the organosolv stage) on solid yield, solid composition, cellulose susceptibility and recovery of the various fractions was assessed using statistical methods, which enabled the identification of the most favourable operational conditions.

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 0.8g ethanol/Lh corresponding to 94% of the theoretical yield.

Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield.

Extracting value-added products before pulping: Hemicellulosic ethanol from Eucalyptus globulus wood

Abstract Xylose solutions have been produced from Eucalyptus globulus wood by autohydrolysis (with hot, compressed water) and post-hydrolysis (in presence of sulfuric acid). This two-stage process led to solids enriched in cellulose and lignin (suitable as a substrate for pulping) and liquors containing xylose as the major component. The liquid phase from post-hydrolysis also contained other sugars (glucose, arabinose) and acetic acid. Neutralized liquors (as obtained, or after membrane concentration), were employed (directly or after detoxification by ion exchange) as fermentation media for the production of hemicelluosic bioethanol with the yeast Pichia stipitis CECT 1922T. Under the best conditions assayed (fermentation of neutralized, concentrated and detoxified two-stage hydrolysis liquors), bioconversion took place at nearly stoichiometric yield, with a volumetric productivity of 0.37 g l-1·h-1.

Contribution of PRS3, RPB4 and ZWF1 to the resistance of industrial Saccharomyces cerevisiae CCUG53310 and PE-2 strains to lignocellulosic hydrolysate-derived inhibitors

PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes.

Combined alkali and hydrothermal pretreatments for oat straw valorization within a biorefinery concept

The aim of this work was the evaluation of lime pretreatment combined or not with previous step of autohydrolysis for oat straw valorization. Under selected conditions of lime pretreatment, 96% of glucan and 77% of xylan were recovered and 42% of delignification was achieved. Xylose fermentation to ethanol by metabolic engineered Saccharomyces cerevisiae (MEC1133) strain improved the ethanol production by 22% achieving 41g/L. Alternatively, first step of autohydrolysis (S0=4.22) allowed a high oligosaccharides recovery (68%) and subsequent lime pretreatment attained a 57% of delignification and 99% of glucan to glucose conversion. Oat straw processed by autohydrolysis and lime pretreatment reached the maximal ethanol concentration (50g/L). Both strategies led to oat straw valorization into bioethanol, oligosaccharides and lignin indicating that these pretreatments are adequate as a first stage within an oat straw biorefinery.

Integrated approach for selecting efficient Saccharomyces cerevisiae for industrial lignocellulosic fermentations: Importance of yeast chassis linked to process conditions

In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods (Eucalyptus and Paulownia) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process.

Comparison of microwave and conduction-convection heating autohydrolysis pretreatment for bioethanol production

This work describes the application of two forms of heating for autohydrolysis pretreatment on isothermal regimen: conduction-convection heating and microwave heating processing using corn stover as raw material for bioethanol production. Pretreatments were performed using different operational conditions: residence time (10-50 min) and temperature (160-200°C) for both pretreatments. Subsequently, the susceptibility of pretreated solids was studied using low enzyme loads, and high substrate loads. The highest conversion was 95.1% for microwave pretreated solids. Also solids pretreated by microwave heating processing showed better ethanol conversion in simultaneous saccharification and fermentation process (92% corresponding to 33.8g/L). Therefore, microwave heating processing is a promising technology in the pretreatment of lignocellulosic materials.

A biorefinery approach based on fractionation with a cheap industrial by-product for getting value from an invasive woody species.

Acacia dealbata wood (an invasive species) was subjected to fractionation with glycerol (a cheap industrial by-product), and the resulting solid phase was used as a substrate for enzymatic hydrolysis. Glycerol fractionation allowed an extensive delignification while preserving cellulose in solid phase. The solids from the fractionation stage showed high susceptibility to enzymatic hydrolysis. Solids obtained under selected fractionation conditions (glycerol content of media, 80 wt%; duration, 1h; liquid to solid ratio, 6 g/g; alkaline and neutral washing stages) were subjected to enzymatic saccharification to achieve glucose concentrations up to 85.40 g/L, with almost complete cellulose conversion into glucose. The results confirmed the potential of glycerol as a fractionation agent for biorefineries.