Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

Abstract

Background: Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2.

Results: With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth.

Conclusions: Laboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.

Figure 1

The two xylose assimilation pathways. XDH, xylitol dehydrogenase; XI, xylose isomerase; XR, xylose reductase.

Figure 2

Time courses of mixed glucose-xylose fermentation in 5% hydrolyzate_X. Depicted are the first 50 h of fermentation using strains (A) BP10001, (B) IBB10A02 and (C) IBB10B05. Glucose (approximately 14 g/L) was depleted within the first 5 h and the ‘glucose phase’ is shown in Additional file 3. Data points are mean values of two independent fermentation experiments. Full diamonds, xylose; empty triangles, glycerol; empty squares, xylitol; empty circles, ethanol.

Figure 3

Time courses of mixed glucose-xylose fermentation in 15% hydrolyzate. Depicted are the first 50 h of fermentation utilizing strains (A) BP10001, (B) IBB10A02 and (C) IBB10B05. Data points are mean values of two independent fermentation experiments. Crosses, glucose; full diamonds, xylose; empty triangles, glycerol; empty squares, xylitol; empty circles, ethanol.