BIOETHANOL PRODUCTION: OPTIMIZATION OF REGIONAL CIDER WASTE PRE-TREATMENT AND SELECTION OF NATIVE Saccharomyces cerevisiae STRAINS

JOSEFINA FONTANINI; ANDREA C. ORIGONE; MARÍA F. GORORDO ; MARCELA P. SANGORRÍN; MARIA E. RODRIGUEZ

Rosario

Congreso; XVI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2021

Asociación Civil de Microbiología General SAMIGE

The use of lignocellulosic biomass (LB), in addition to the potential of yeasts to ferment reducing sugars, has proved to be a robust feature for bioethanol production that could replace the use of limited, environmentally damaging petroleum-derived resources. However, it is necessary to apply suitable pre-treatments of the LB in order to ensure both availability of fermentable sugars and the absence of compounds that can inhibit fermentation. The aim of the present work was to study the behaviour of a pool of Saccharomyces cerevisiae yeast strains from different origins (including wine, apple chicha and Toddy beverages) under stress conditions encountered during the bioethanol production. Furthermore, the optimization of apple bagasse (AB) pre-treatment was evaluated for its possible application in the production of bioethanol as well as to reduce its availability as a waste from the cider industry. A total of 60 S. cerevisiae stains were assayed in microplates containing 0-15% (v/v) ethanol. OD growth data were fitted to Gompertz function and kinetic parameters (μmax and λ) were obtained. Twelve yeast strains were selected for their ethanol tolerance (higher than 12% v/v ethanol). The selected strains showed the shortest λ (media of 11.83±0.89 h) and the highest μmax (media of 0.15±0.01 h-1). Later analysis for their tolerance to temperature (25-45°C), pH (2-5), glucose (2-300 g/L), Na2SO4 (0-50 g/L) and acetic acid (0-8 g/L) concentrations evidenced that glucose and Na2SO4 did not affect the growth. However, all the strains were able to grow at temperatures below 40°C and at pH 3, 4 and 5, as well as at 3g/L of acetic acid. The AB pre-treatment involved an initial screening, using a fractional factorial design, to establish the significant variables for optimization. For the phosphoric acid (PA) pre-treatment, a Central Compound Design (CCD) was assayed with 16 runs and 3 factors: solid:liquid ratio (1:5-1:7), temperature (121-131°C) and PA concentration (0.2-1% w/v). Experiments were carried out in 100 mL Erlenmeyers, incubated during 40 min, and both total reducing sugars and glucose concentrations (g/100 g dry weight) were determined. The optimization conditions suggested by the model were: 1:5 of S:L, 121°C, PA 0.2% w/v. On the other hand, the alkali pre-treatment showed optimal conditions of NH4OH 6%, at room temperature for 24 h. The subsequent hydrolysis of the pre-treated AB was optimized using a cocktail of enzymes (endo-β-glucanase, pectinase and cellulose) at equal concentrations, with 10 runs and 2 factors: cocktail concentration (1.88-6.12%) and time (190-530 min). The optimal conditions obtained were 6.12% cocktail for 360 min. The pre-treated and hydrolyzed AB evidenced an increase of 20 and 22% of glucose and reducing sugar content. In addition, the calculation of bias and precision factors close to 1 indicated a good fit of the models. This results and the tolerance to stress factors of selected strains suggest the possibility of producing bioethanol using regional industry wastes.