Improved Sugarcane-Based Fermentation Processes by an Industrial Fuel-Ethanol Yeast Strain

Abstract

In Brazil, sucrose-rich broths (cane juice and/or molasses) are used to produce billions of liters of both fuel ethanol and cachaça per year using selected Saccharomyces cerevisiae industrial strains. Considering the important role of feedstock (sugar) prices in the overall process economics, to improve sucrose fermentation the genetic characteristics of a group of eight fuel-ethanol and five cachaça industrial yeasts that tend to dominate the fermentors during the production season were determined by array comparative genomic hybridization. The widespread presence of genes encoding invertase at multiple telomeres has been shown to be a common feature of both baker's and distillers' yeast strains, and is postulated to be an adaptation to sucrose-rich broths. Our results show that only two strains (one fuel-ethanol and one cachaça yeast) have amplification of genes encoding invertase, with high specific activity. The other industrial yeast strains had a single locus (SUC2) in their genome, with different patterns of invertase activity. These results indicate that invertase activity probably does not limit sucrose fermentation during fuel-ethanol and cachaça production by these industrial strains. Using this knowledge, we changed the mode of sucrose metabolism of an industrial strain by avoiding extracellular invertase activity, overexpressing the intracellular invertase, and increasing its transport through the AGT1 permease. This approach allowed the direct consumption of the disaccharide by the cells, without releasing glucose or fructose into the medium, and a 11% higher ethanol production from sucrose by the modified industrial yeast, when compared to its parental strain.

Keywords: bioethanol; fermentation; sugarcane; yeast.

Figure 1

Copy number differences and similarities among industrial sugarcane yeast strains for genes involved in the metabolism and transport of sugar, and of vitamins B1 and B6: (A) array-CGH data showing the copy number of genes involved in sugar fermentation among the different strains; (B) array-CGH data showing the copy number of genes involved in thiamine (vitamin B1) and pyridoxine (vitamin B6) biosynthesis and transport among the different strains. A scale of relative gene copy number is shown at the bottom.

Figure 2

Detection of SUC genes in the industrial sugarcane yeasts. (a) PFGE separation of yeast chromosomes (ethidium-bromide stained). (b) Southern blot of gel shown in panel (a), hybridized with a probe for SUC2 to detect which chromosomes carry SUC genes. (Lane 1) Reference laboratory strain S288C, which contains SUC2 on chromosome IX (indicated to the left of panel (a)); (lanes 2–6) strains CAT-1, PE-2, BG-1, SA-1, and UFPE-136, respectively. This last strain has both the SUC2 gene in chromosome IX and the SUC1 gene in chromosome VII.

Figure 3

Invertase activity of the sugarcane industrial yeast strains. The extracellular invertase activity was determined after the growth of the cells in rich medium containing 20 g/L sucrose, 20 g/L ethanol plus 30 g/L glycerol (derepressed conditions), or in these derepressed conditions with media supplemented with 1 g/L glucose.

Figure 4

Sucrose batch fermentation by selected sugarcane industrial yeast strains. Panel (a) shows the concentrations of sucrose (circles) and biomass (squares), panel (b) shows the concentrations of glucose (triangles) and fructose (diamonds), while panel (c) shows the concentrations of ethanol (inverted triangles) during the fermentation by strain PE-2 (white symbols), UFPE-179 (light gray symbols), BG-1 (gray symbols), and UFMG-1007 (black symbols).

Figure 5

Sucrose batch fermentation by sugarcane industrial yeast strains with amplification of the SUC genes. Panel (a) shows the concentrations of sucrose (circles) and biomass (squares), panel (b) shows the concentrations of glucose (triangles) and fructose (diamonds), while panel (c) shows the concentrations of ethanol (inverted triangles) during the fermentation by strain UFMG-829 (white symbols) and UFPE-135 (black symbols).

Figure 6

Sucrose batch fermentation by strains CAT-1 and GMY08. Panel (a) shows the concentrations of sucrose (circles) and biomass (squares), panel (b) shows the concentrations of glucose (triangles) and fructose (diamonds), while panel (c) shows the concentrations of ethanol (inverted triangles) during the fermentation by strain CAT-1 (white symbols) or GMY08 (black symbols).