Production of bioethanol from red macroalgae by galactose-adapted yeasts or recombinant yeast

Abstract

Seaweeds are a promising biomass to produce bioethanol due to several advantages including no require for arable land and fresh water. Seaweeds are classified into three group;green, brown and red seaweed. The red seaweed composed of high carbohydrate and the main monosaccharide is galactose along with glucose. The seaweeds are hydrolyzed to monosaccharides and then convert to ethanol using three main steps including pretreatment, hydrolysis and fermentation. The popular pretreatment method is acid hydrolysis due to its efficiency. Particularly, hyper thermal acid hydrolysis can produce high monosaccharides with low inhibitors as by products. Enzymatic hydrolysis hydrolyzes remained cellulose from pretreatment in order to increase monosaccharides. The final step is fermentation using yeasts. Pichia stipitis, Kluyveromyces marxianus and Saccharomyces cerevisiae are the most efficient yeast in bioethanol. However, the efficiency of fermentation from red seaweed hydrolysate is low due to various monosaccharides composition. To overcome, the adaptation to high galactose and genetically engineered techniques are applied to improve galactose consumption. This study employed a statistical method to obtain optimal hyper thermal acid hydrolysis conditions using Gelidium amansii (red seaweed) as a source of biomass. The optimal hyper thermal acid hydrolysis using G. amansii as biomass was determined as 12 % (w/v) slurry content, 358.3 mM H2SO4, and temperature of 142.6°C for 11 min. After hyper thermal acid hydrolysis, enzymatic saccharification was carried out. The total monosaccharide concentration was 45.1 g/L, 72.2% of the theoretical value of the total fermentable monosaccharides of 62.4 g/L based on 120 g dry weight/L in the G. amansii slurry. To increase ethanol production, 3.8 g/L 5-hydroxymethylfurfural (HMF) in the hydrolysate was removed by treatment with 3.5% (w/v) activated carbon for 2 min and fermented with Pichia stipitis adapted to high galactose concentrations via separate hydrolysis and fermentation. With complete HMF removal and the use of P. stipitis adapted to high galactose concentrations, 22 g/L ethanol was produced (yield = 0.50). Fermentation with total HMF removal and yeast adapted to high galactose concentrations increased the fermentation performance and decreased the fermentation time from 96 h to 36 h compared to traditional fermentation. Gracilaria verrucosa, red seaweed, is a promising biomass for bioethanol production due to its high carbohydrate content. The optimal hyper thermal (HT) acid hydrolysis conditions are 12% (w/v) G. verrucosa with 0.2 M H2SO4 at 130°C for 15 min, with a severity factor of 1.66. This HT acid hydrolysis produces 50.7 g/L monosaccharides. The maximum monosaccharide concentration of 58.0 g/L was achieved with 96.6% of the theoretical monosaccharide production from 120 g dry weight/L G. verrucosa slurry after HT acid hydrolysis and enzymatic saccharification. Fermentation was carried out by removing an inhibitory compound and via yeast adaptation to galactose. Both Pichia stipitis and Kluyveromyces marxianus adapted to galactose were excellent producers, with the ethanol yield (YEtOH) of 0.50 and 29.0 g/L ethanol production. However, the bioethanol productivity with Pichia stipitis adapted to galactose is higher than that with Kluyveromyces marxianus adapted to galactose, being 0.81 and 0.35 g/L/h, respectively. The results from this study can be applied to industrial scale bioethanol production from seaweed. A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.