SECOND-GENERATION ETHANOL PRODUCTION by Wickerhamomyces Anomalus Hydrolysates (Da Cunha-Pereira Et Al
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An Acad Bras Cienc (2020) 92(Suppl. 2): e20181030 DOI 10.1590/0001-3765202020181030 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal BIOLOGICAL SCIENCES Second-generation ethanol production Running title: SECOND- by Wickerhamomyces anomalus strain GENERATION ETHANOL adapted to furfural, 5-hydroxymethylfurfural PRODUCTION BY Wickerhamomyces anomalus (HMF), and high osmotic pressure Academy Section: BIOLOGICAL NICOLE T. SEHNEM, ÂNGELA S. MACHADO, CARLA R. MATTE, SCIENCES MARCOS ANTONIO DE MORAIS JR & MARCO ANTÔNIO Z. AYUB Abstract: The aims of this work were to improve cell tolerance towards high e20181030 concentrations of furfural and 5-hydroxymethylfurfural (HMF) of an osmotolerant strain of Wickerhamomyces anomalus by means of evolutionary engineering, and to determine its ethanol production under stress conditions. Cells were grown in the presence of 92 furfural, HMF, either isolated or in combination, and under high osmotic pressure (Suppl. 2) conditions. The most toxic condition for the parental strain was the combination of 92(Suppl. 2) both furans, under which it was unable to grow and to produce ethanol. However, the tolerant adapted strain achieved a yield of ethanol of 0.43 g g-1glucose in the presence of furfural and HMF, showing an alcohol dehydrogenase activity of 0.68 mU mg protein-1. For this strain, osmotic pressure, did not affect its growth rate. These results suggest that W. anomalus WA-HF5.5strain shows potential to be used in second-generation ethanol production systems. Key words: Second-generation ethanol, Wickerhamomyces anomalus, furaldehydes tol- erance, osmotic pressure, lignocellulosic hydrolysates. INTRODUCTION process that could produce inhibitory compounds of yeast metabolism. Therefore, There is a worldwide effort to ensure the biomass hydrolysates pose a challenging to production of energy from various sources of their use in bioprocess, since the operational renewable raw materials. Second-generation costs of medium detoxifi cation are, at present, ethanol, sometimes called bioethanol, is prohibitive if second-generation ethanol must obtained from lignocellulosic biomass, especially compete with first-generation sugarcane or from agriculture residues, which are abundant, starch-based ethanol. inexpensive, and a desirable feedstock for the In the research for second-generation sustainable production of biofuels, among other ethanol production it is fundamentally important things because these residues do not directly to consider the presence of pentoses and hexoses compete with food production for arable land, in the hydrolysates, which requires specifi c cell as is the case of ethanol from sugarcane and metabolisms for the total conversion of these maize (Hasunuma et al. 2013). The production of sugars. S. cerevisiae, the most efficient yeast ethanol from lignocellulosic feedstock requires for conventional ethanol production, cannot pretreatments of biomass and hydrolyses steps assimilate pentoses, including xylose, usually in order to release sugars for yeast fermentation, present in great amounts in lignocellulosic An Acad Bras Cienc (2020) 92(Suppl. 2) NICOLE T. SEHNEM et al. SECOND-GENERATION ETHANOL PRODUCTION BY Wickerhamomyces anomalus hydrolysates (da Cunha-Pereira et al. 2011, Cortivo 2-furaldehyde (furfural), which is formed by et al. 2018, Sehnem et al. 2017). Therefore, it has dehydration of pentoses, and 5-hydroxymethyl- been of great concern the research for isolating 2-furaldehyde (5-hydroxymethylfurfural - HMF), or engineering pentose-fermenting yeasts, with which is formed by dehydration of hexoses (Liu some reports showing pentose bioconversions et al. 2009, 2008, Lenihan et al. 2010, Mansilla et by several non-recombinant strains of Candida al. 1998, Kim et al. 2015). Furaldehydes interfere guilliermondii, Pichia stipitis, Pachysolen with microbial growth by hampering glycolytic tannophylus, Spathaspora arborariae and enzymes activities, as well as by impairing Wickerhamomyces anomalus (da Cunha-Pereira protein and RNA synthesis (Almeida et al. 2007, et al. 2011, Schirmer-Michel et al. 2008, Yadav et Luo et al. 2002, Modig et al. 2002, Kim et al. al. 2011, Zhao et al. 2008, Tao et al. 2011). 2015), and negative synergistic effects of HMF The ascomycetous yeast W. anomalus and furfural have been demonstrated for S. (formerly Pichia anomala and Hansenula cerevisiae and S. arborariae (Taherzadeh et al. anomala) has been isolated from many different 2000, da Cunha-Pereira et al. 2011). A common habitats and shows a remarkable physiological furaldehyde detoxification metabolism in yeasts robustness to cultivation stresses, such as is the reduction of the aldehydes into the less extreme pH and low water activity. Compared toxic corresponding alcohols. This reaction to other yeast strains, W. anomalus is highly is catalyzed by alcohol dehydrogenases. competitive in terms of growth and can inhibit These enzymes are NAD(P)H-dependent cell growth of other microorganisms (Passoth et oxidoreductases that catalyze the reversible al. 2011). This species has been tested for several oxidation of alcohols to aldehydes or ketones biotechnological applications, including food and (De Smidt et al. 2008, Liu et al. 2004, Almeida et beverage applications (as probiotics, sourdough al. 2007, Guarnieri et al. 2017). fermentations, volatile aromas in wine), One approach extensively used to select environmental bioremediation (sophorolipis as tolerant yeast strains to adverse environmental biosurfactants), biopharmaceuticals (production stresses, such as high temperature, ethanol of aminobutiric acid), and biofuels (ethanol and inhibition, osmotic pressure, and inhibitory isobutanol productions) (Walker 2011, Sehnem furaldehydesis the simple experiment of et al. 2017). evolutionary engineering (Zhao & Bai 2009, Li During ethanol fermentation, metabolism et al. 2017). inhibition by substrate and product, besides In the present study, a strain of W. anomalus medium osmotic pressure, are the most was submitted to evolutionary engineering important adverse conditions (Zhao & Bai in order to increase its resistance to furfural 2009, Sehnem et al. 2017, Hickert et al. 2014). and HMF present in the culture medium. Both In second-generation ethanol production, the parental and its tolerant-derived strain there is a further important problem, which is were evaluated in their abilities to grow and the degradation of sugars during hydrolyses to produce ethanol, which were correlated to of biomass and the consequent formation of alcohol dehydrogenase enzymatic activity and to toxic compounds (Almeida et al. 2008, Margeot the reduction of the toxic furan concentrations et al. 2009, da Cunha-Pereira et al. 2011, Li et in the medium. The ability of these strains to al. 2017, Guarnieri et al. 2017, Ling et al. 2014). resist high osmotic pressures was also evaluated The main inhibitors of yeast metabolism are and compared. An Acad Bras Cienc (2020) 92(Suppl. 2) e20181030 2 | 12 NICOLE T. SEHNEM et al. SECOND-GENERATION ETHANOL PRODUCTION BY Wickerhamomyces anomalus MATERIALS AND METHODS flasks filled with 60 mL of medium at 28 °C and 150 rpm on a rotatory shaker, which corresponds Microorganisms, cell maintenance and to an oxygen limitation condition. Inoculum was chemicals set as a cell suspension of 1.0 OD (600 nm). The strain used in this work is part of the yeast Strains were cultivated in absence (control) collection of Bioteclab, named W. anomalus WA- -1 -1 and presence of furfural (3 g.L ), HMF (3 g.L ), 001. This strain was isolated by this research -1 or the combination of both (1.5 g.L of each). group from piles of decomposing rice hulls Samples were taken at determined times during deposited in the environment and it was 48 h of cultivation for determination of glucose, identified comparing ITS1 and ITS4 amplicon ethanol, glycerol, furfural, HMF concentration, sequences with GenBank database (www.ncbi. and for biomass formation. nlm.nih.gov/BLASTn). Stock cultures were kept frozen at -20 °C in medium composed of 20 Enzyme activities assays % glycerol and 80 % of mid-exponential cell To determine furfural and HMF reducing suspensions. All chemicals used in this research metabolic activities, samples were taken at 24 h were of analytical grade and purchased from of cultivation under conditions mentioned in the Sigma-Aldrich (St. Louis, USA). next item. Crude protein extracts were prepared by lysing cells with glass beads attrition. Cells Evolutionary engineering for HMF and furfural were resuspended in 400 μL of 100 mM K HPO resistance 2 4 buffer, pH 7.0, and 2 μL of a solution of 100 mM The evolutionary engineering approach was used phenylmethylsulfonyl fluoride (PMSF) in a 2 mL aiming to increase furaldehydes resistance of W. Eppendorf tube, and added of an equal volume anomalus WA-001. Cultivations were performed of glass beads (diameter of 500 μm). Cells were using YPD medium (composed of, in g L-1: disrupted by six cycles of vortexing for 60 s, glucose, 20; yeast extract, 10; and peptone, 20), with samples cooled on ice for 60 s between and were carried out in 125 mL Erlenmeyer flasks each cycle. Protein extracts were collected after containing 30 mL of medium, at 28 °C and 150 centrifugation at 13000 g (5 min at 4 °C) and its rpm on a rotatory shaker. Cells were inoculated concentration was determined using the Lowry (10 % volume fraction of a cell suspension of OD assay method. Alcohol dehydrogenase activity 1.0, 600 nm) into YPD medium added of HMF and was performed by recording the decrease in furfural (0.25 g L-1 each chemical), and incubated absorbance at 340 nm using NADH as cofactor. for 24 h. From this mother culture, successive NADPH was used as cofactor when HMF was used batches were run by transferring 10 % (volume as substrate. The reaction mixture consisted of fraction) of cells (OD 1.0) to re-inoculate YPD 10 mM HMF substrate, final concentration, and medium with increasing concentrations of 100 μM of NADPH prepared in 100 mM potassium furaldehydes, up to 1 g L-1 each. phosphate buffer, pH 7.0. All reagents were kept at 25 °C prior to use.