Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading

Biotechnology for Biofuels

Patricia J Slininger¹  Maureen A Shea-Andersh¹  Stephanie R Thompson¹  Bruce S Dien¹  Cletus P Kurtzman²  Venkatesh Balan³  Leonardo da Costa Sousa³  Nirmal Uppugundla³  Bruce E Dale³  Michael A Cotta¹ 

[1] Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, 1815 N. University, Peoria 61604, IL, USA

[2] Bacterial Foodborne Pathogens and Mycology Research, National Center for Agricultural Utilization Research, USDA-ARS, 1815 N. University, Peoria 61604, IL, USA

[3] DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing 48824, MI, USA

关键词: Fermentation;    Pichia stipitis;    Yeast;    Adaptation;    Biofuel;    Lignocellulose;   

Others  :  1177294
DOI  :  10.1186/s13068-015-0239-6

 received in 2014-07-29, accepted in 2015-03-13,  发布年份 2015

【 摘 要 】

Background

Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment.

Results

Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions.

Conclusions

Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

【 授权许可】

   
2015 Slininger et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

【 参考文献 】

• [1]U.S. Department of Energy: U.S. billion-ton update: biomass supply for a bioenergy and bioproducts industry (R.D. Perlack and B.J. Stokes (Leads)) In Book U.S. billion-ton update: biomass supply for a bioenergy and bioproducts industry (R.D. Perlack and B.J. Stokes (Leads)). U.S. Department of Energy, Oak Ridge National Laboratory, Oak Ridge, TN; 2011:227.
• [2]Ladisch MR, Dyck K: Dehydration of ethanol: new approach gives positive energy balance. Science 1979, 205:898-900.
• [3]Cardona Alzate CA, Sanchez Toroa OJ: Energy consumption analysis of integrated flowsheets for production of fuel ethanol from lignocellulosic biomass. Energy 2006, 31:2447-59.
• [4]Prior BA, Kilian SG, DuPreez JC: Fermentation of D-xylose by the yeasts Candida shehatae and Pichia stipitis. Process Biochem 1989, 24:21-32.
• [5]Kurtzman CP, Suzuki M: Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces and Scheffersomyces. Mycoscience 2010, 5:2-14.
• [6]Bruinenberg PM, De Bot PHM, VanDijken JP, Scheffers WA: NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeasts. Appl Microbiol Biotechnol 1984, 19:256-60.
• [7]Slininger PJ, Bothast RJ, Okos MR, Ladisch MR: Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations. Biotechnol Lett 1985, 7:431-6.
• [8]Slininger PJ, Bothast RJ, Ladisch MR, Okos MR: Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis. Biotechnol Bioeng 1990, 35:727-31.
• [9]Slininger PJ, Branstrator LE, Lomont JM, Dien BS, Okos MR, Ladisch MR, et al.: Stoichiometry and kinetics of xylose fermentation by Pichia stipitis. Annals New York Acad Sci 1990, 589:25-40.
• [10]Slininger PJ, Dien BS, Gorsich SW, Liu ZL: Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124. Appl Microbiol Biotechnol 2006, 72:1285-96.
• [11]Slininger PJ, Thompson SR, Weber S, Liu ZL: Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag. Biotechnol Bioeng 2011, 108:1801-15.
• [12]Slininger PJ, Gorsich SW, Liu ZL: Culture nutrition and physiology impact inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124. Biotechnol Bioeng 2009, 102:778-90.
• [13]Agbogbo FK, Coward-Kelly G: Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis. Biotechnol Lett 2008, 30:1515-24.
• [14]Balan V, Bals B, Chundawat S, Marshall D, Dale BE: Lignocellulosic biomass pretreatment using AFEX. In Biofuels: Methods and protocols, Methods in Molecular Biology. Volume 581. Edited by Mielenz JR. Humana Press, a part of Springer Science+Business Media, LLC, Springer, New York, NY; 2009:61-77.
• [15]Jin M, Gunawan C, Uppugundla N, Balan V, Dale BE: A novel integrated biological process for cellulosic ethanol production featuring high ethanol productivity, enzyme recycling, and yeast cells reuse. Energy Environ Sci 2012, 5:7168-75.
• [16]Nigam JN: Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate. J Appl Microbiol 2001, 90:208-15.
• [17]Nigam JN. Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis. In: Book Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis (Editor ed.^eds.), vol. 87. 2001. p. 17–27.
• [18]Hughes SR, Gibbons WR, Bang SS, Pinkelman R, Bischoff KM, Slininger PJ, et al.: Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars. J Indus Microbiol Biotechnol 2012, 39:163-73.
• [19]Bajwa PK, Shireen T, D’Aoust F, Pinel D, Martin VJJ, Trevors JT, et al.: Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor. Biotechnol Bioeng 2009, 104:892-900.
• [20]Bajwa PK, Pinel D, Martin VJJ, Trevors JT, Lee H: Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling. J Microbiol Methods 2010, 81:179-86.
• [21]Jeffries TW, Grigoriev I, Grimwood J, Laplaza JM, Aerts A, Salamov A, et al.: Genome sequence of the lignocellulosic-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat Biotechnol 2007, 25:319-26.
• [22]Kurtzman CP, Robnett CJ: Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 1998, 73:331-71.
• [23]Casey E, Sedlak M, Ho NWY, Mosier NS: Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae. FEMS Yeast Res 2010, 10:385-93.
• [24]Wang X, Jin M, Balan V, Jones AD, Li X, Li B-Z, Dale BE, Yuan Y-J. Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors. Biotechnology and Bioengineering 2013:n/a-n/a.
• [25]Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW: Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Indus Microbiol Biotechnol 2004, 31:345-52.
• [26]Liu ZL, Slininger PJ, Gorsich SW: Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl Biochem Biotechnol 2005, 121–124:451-60.
• [27]Wiegert B, Klein C, Rizzi M, Lauterbach C, Dellweg H: Xylose fermentation by yeasts. 8. Influence of furfural on the aerobic growth of the yeast Pichia stipitis. Biotechnolgy Letters 1988, 10:895-900.
• [28]Hahn-Hagerdal B, Jeppsson H, Skoog K, Prior BA: Biochemistry and physiology of xylose fermentation by yeasts. Enzyme Microb Technol 1994, 16:933-43.
• [29]Syzbalski W, Bryson Y: Genetic studies on microbial cross resistance to toxic agents, I. Cross resistance of Escherichia coli to fifteen antibiotics. J Biotechnol 1952, 64:489-99.