Evaluation of divergent yeast genera for fermentation-associated stresses and identification of a robust sugarcane distillery waste isolate Saccharomyces cerevisiae NGY10 for lignocellulosic ethanol production in SHF and SSF

Abstract

Background: Lignocellulosic hydrolysates contain a mixture of hexose (C6)/pentose (C5) sugars and pretreatment-generated inhibitors (furans, weak acids and phenolics). Therefore, robust yeast isolates with characteristics of C6/C5 fermentation and tolerance to pretreatment-derived inhibitors are pre-requisite for efficient lignocellulosic material based biorefineries. Moreover, use of thermotolerant yeast isolates will further reduce cooling cost, contamination during fermentation, and required for developing simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SScF), and consolidated bio-processing (CBP) strategies.

Results: In this study, we evaluated thirty-five yeast isolates (belonging to six genera including Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Ogatea and Wickerhamomyces) for pretreatment-generated inhibitors {furfural, 5-hydroxymethyl furfural (5-HMF) and acetic acid} and thermotolerant phenotypes along with the fermentation performances at 40 °C. Among them, a sugarcane distillery waste isolate, Saccharomyces cerevisiae NGY10 produced maximum 49.77 ± 0.34 g/l and 46.81 ± 21.98 g/l ethanol with the efficiency of 97.39% and 93.54% at 30 °C and 40 °C, respectively, in 24 h using glucose as a carbon source. Furthermore, isolate NGY10 produced 12.25 ± 0.09 g/l and 7.18 ± 0.14 g/l of ethanol with 92.81% and 91.58% efficiency via SHF, and 30.22 g/l and 25.77 g/l ethanol with 86.43% and 73.29% efficiency via SSF using acid- and alkali-pretreated rice straw as carbon sources, respectively, at 40 °C. In addition, isolate NGY10 also produced 92.31 ± 3.39 g/l (11.7% v/v) and 33.66 ± 1.04 g/l (4.26% v/v) ethanol at 40 °C with the yields of 81.49% and 73.87% in the presence of 30% w/v glucose or 4× concentrated acid-pretreated rice straw hydrolysate, respectively. Moreover, isolate NGY10 displayed furfural- (1.5 g/l), 5-HMF (3.0 g/l), acetic acid- (0.2% v/v) and ethanol-(10.0% v/v) tolerant phenotypes.

Conclusion: A sugarcane distillery waste isolate NGY10 demonstrated high potential for ethanol production, C5 metabolic engineering and developing strategies for SSF, SScF and CBP.

Keywords: Ethanol; Fermentation; Inhibitors; SHF; SSF; Thermo-tolerance.

Fig. 1

Phylogenetic tree displaying evolutionary relationship among yeast isolates based on ITS sequences. ITS sequences were aligned by ClustalW (a multiple sequence alignment tool) and phylogenetic analysis was performed by MEGA 6.0 software using maximum likelihood method with bootstrap value 1000 and Tamura-Nei model. Cluster 1: C. tropicalis isolates (NGY21, NGY22, NGY19, NGY18, NGY17, NGY9, NGY6, NGY5, NGY4, NGY3, NGY23, NGY24 and NGY25) and C. albicans isolate SC5314; Cluster 2: C. lusitaniae isolate NCIM3484 and P. kudriavzevii isolates (NGY12, NGY13, NGY15, NGY16 and NGY20); Cluster 3: C. sehatae isolate NCIM3500, S. stipitis isolates (NCIM3507 and NCIM3498) and O. thermophilla isolate NGY11; Cluster 4: C. glabrata isolates (NGY7, NGY14 and CBS138); Cluster 5: S. cerevisiae isolates (CEN.PK-122, NGY1, NGY10 and NCIM3570) and Cluster 6: Kluyveromyces sp. isolates (NGY8, NCIM3465 and NCIM3551). W. anomalus isolate NGY2 did not cluster with any other yeast sp

Fig. 2

Growth phenotypes in the presence of pretreatment-generated inhibitors and fermentation stresses. a The cells were grown in SD medium containing 2.0% glucose with varying concentration of pretreatment-generated inhibitors such as furfural (0.5 g/l, 1.0 g/l and 1.5 g/l), 5-HMF (1.0 g/l, 2.0 g/l and 3.0 g/l), acetic acid (0.2%, 0.3% and 0.4% v/v) and ethanol (6.0%, 8.0% and 10% v/v) at 40 °C. Relative growth in the presence of inhibitors was calculated by considering 100% growth in the absence of inhibitors. b Chemogenetic network profile: In silico Chemogenetic network profile was generated using Cytoscape 3.6.0 software using 1.5 g/l of furfural, 3.0 g/l of 5-HMF, 0.3% v/v of acetic acid and 10.0% v/v ethanol individually as well as in combinations. c Isolate NGY10 growth phenotypes in the presence of inhibitor: % growth reduction in presence of 1.0 g/l furfural, 3.0 g/l 5-HMF, 0.3% v/v acetic acid, 10% v/v ethanol, cocktail A (1.0 g/l furfural, 3.0 g/l 5-HMF, 0.3% v/v acetic acid and 10% v/v ethanol) and cocktail B (furfural: 0.618 g/l, 5-HMF: 0.748 g/l, acetic acid: 0.18% v/v and ethanol 5.0% v/v) as compared to without inhibitors in SD medium containing 2.0% glucose at 40 °C

Fig. 3

Ploidy determination, thermotolerant phenotypes and pretreatment-generated inhibitors tolerance profile of isolate NGY10. a Ploidy determination: Total DNA content of the cells was analyzed by flow cytometry followed by propidium iodide (PI) staining. Ploidy was determined by comparing the FACS spectra of isolate NGY10 with the spectra of reference haploid (CEN.PK-1137D) and diploid (CEN.PK-122) strains. Unstained and stained cells are represented by purple and green colors. b Thermotolerant phenotype: isolate NGY10 cells were grown in YEPD broth at 30 °C (filled black diamonds) and 40 °C (clear black diamonds), respectively, followed by OD₆₀₀ measurement after every 30 min interval. c Pre-treatment inhibitors-tolerant phenotypes: Serial tenfold dilution of isolate NGY10 cells (OD₆₀₀ = 0.1) was spotted on SD agar plates containing 2.0% glucose and inhibitors (Furfural 1.5 g/l, 5-HMF 3.0 g/l, acetic acid 0.2% v/v and ethanol 10% v/v), and incubated at 40 °C for 24 h

Fig. 4

Fermentation kinetics: Isolate NGY10 cells were grown in YEPD broth and 5% v/v inoculum of overnight grown cells was diluted in different fermentation media and fermentation was performed for 24 h. Glucose (circle), ethanol (triangle) and cell biomass (star) were estimated at 30 °C (filled symbols) and 40 °C (clear symbols) after every 3.0 h. a Fermentation kinetics in synthetic media containing 100 g/l glucose. b Fermentation kinetics in acid-pretreated rice straw enzymatic hydrolysate (containing glucose: 26.38 g/l; xylose: 7.38 g/l; furfural: 0.618 g/l; HMF: 0.748 g/l and acetic acid: 1.91 g/l) and c Fermentation kinetics in alkali pre-treated rice straw enzymatic hydrolysate (containing glucose: 17.15 g/l; xylose: 5.63 g/l; furfural: 0.142 g/l; HMF:0.148 g/l and acetic acid:0.51 g/l)

Fig. 5

Fermentation profile in the presence of high sugar concentration. a Fermentation kinetics of isolate NGY10 (circle), CEN.PK-122 (triangle) and Angel yeast (square) in presence of 30% w/v glucose at 30 °C (filled symbols) and 40 °C (clear symbols), respectively. Ethanol and glucose concentrations are represented by black and red edges symbols, respectively. b Comparative ethanol yield with 30% w/v glucose. c Comparative ethanol yield with ×1 (black coloured bar), ×2 (light grey coloured bar) and ×4 (dark grey coloured bar) concentrated acid-pretreated rice straw hydrolysate. Statistical Student t-test for ethanol yield was performed for isolate NGY10 with reference strains CEN-PK-122 and Angel yeast, and showed significance (p < 0.05)

Fig. 6

Acid- and alkali-pretreated RS fermentation via SSF without pre-saccharification (blue coloured bar) and with pre-saccharification (orange coloured bar), using isolate NGY10. SSF was performed at 40 °C for 72 h employing 15 FPU cellulase/g of RS and 5.0% v/v inoculums of overnight YEPD grown isolate NGY10. a With 5.0% w/v solid loading and b with 10.0% w/v solid loading. For SSF without pre-saccharification both cellulase and inoculums were added simultaneously; however, in case of pre-saccharification, cellulases were added to RS and incubated at 50 °C for 6 h before adding the yeast inoculums