2.2.3. Bioethanol production

Wood samples from trees of each species, crop density, and site were evaluated. A wood physical treatment method was chosen for this study (Balan, 2014), and the samples were milled to 12 mm in a continuous flow mill (Marconi MA 600, Brazil). Bioethanol production was performed using a pre-hydrolysis simultaneous saccharification and fermentation (PSSF) process that ensures the optimal temperature for cellulase enzyme activity, which is an advantage over the traditional simultaneous saccharification and fermentation (SSF) process (McIntosh et al., 2016; Trevorah et al., 2018). For this purpose, 15 g of wood in 300 mL (5% w/v) of pH 4.8 acetate buffer was sterilized separately for 15 min at 121 °C, and 1 atm, and then pre-hydrolysed with a commercial cellulase complex (36.6 Filter Paper Units per gram of dry biomass; Sunson, China), at 50 °C at 150 rpm for 72 h in a shaker incubator.

Fermentation was undertaken with using the yeast strain SacSV-10, which was obtained from the culture collection of the Biochemistry and Biotechnology Laboratory (CIN, UdelaR, Uruguay). This strain was obtained by -irradiation of the M522 Saccharomyces cerevisiae strain (Vázquez et al., 2012), and was maintained at –20°C in 10% v/v low-fat milk. The inoculum of SacSV–10 was prepared in liquid YPD medium (yeast extract, peptone and dextrose), at a concentration of 106 cells mL⁻¹ and incubated at 37 °C for 72 h, without shaking. After 72 h of wood pre-hydrolysis, the temperature was cooled to 37 °C to optimize the yeast performance, and the inoculum of 106 cell mL⁻¹ (10% v/v inoculum) was added to begin the fermentation to a 4.5% w/v solids concentration in a final volume) of 330 mL of medium. The PSSF process was maintained for 24 h at 37 °C in a shaker incubator at 130 rpm.

Quantification of total reducing sugars was performed using the dinitrosalicylic acid technique (Bonifacino-Buttiglione, 2012; Chaplin and Kennedy, 1986) at the end of the PSSF. The ethanol concentration at the end was measured using the potassium dichromate technique (Bonifacino-Buttiglione, 2012; Isarankura-Na-Ayudhya et al., 2007) after simple distillation of the samples. All analyses were conducted in duplicate. The yield of the PSSF process for each Eucalyptus species at each crop density and site was calculated as the percentage of the mass of ethanol obtained relative to its theoretical mass (Eq. 2) according to Dowe and McMillan (2008). The bioethanol productivity for each species, planting density, and site was calculated relative to the megagrams (Mg) of biomass (Eqs. 3 and 4) and per hectare of crop (Eq. 5).