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Introduction

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Acknowledgements

Materials

References

Bioengineering

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Published: October 24th, 2016

DOI:

10.3791/54227

1Bioenergy Research Unit, National Center for Agricultural Utilization Research, 2Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, 3Chemical Engineering and Material Science, Great Lakes Bioenergy Center, Michigan State University

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

An estimated annual 1.3 billion dry tons of lignocellulosic biomass could support ethanol production and allow the U.S. to reduce its petroleum consumption by 30%.1 Although plant biomass hydrolysis yields sugar mixtures rich in glucose and xylose, fermentation inhibitors are generated by the chemical pretreatment necessary to break down hemicellulose and expose cellulose for enzymatic attack. Acetic acid, furfural, and hydroxymethylfurfural (HMF) are thought to be key components among many inhibitors that form during pretreatment. In order to move the lignocellulosic ethanol industry forward, research and procedures to allow the evolution of yeast strains ....

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1. Prepare Starting Materials and Equipment for Assays

  1. Prepare hydrolyzates using 18 to 20% initial biomass dry weight in the pretreatment reaction for use in the evolution, isolation and ranking procedures. See Slininger et al. 201518 for the detailed methods to prepare AFEX CSH, PSGHL, and SGH with nitrogen supplements N1 or N2 used in evolution, isolation or ranking. See Table 1 for composition of each hydrolyzate type.
    NOTE: Nitrogen fortifications of SGH were d.......

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S. stipitis was evolved using combinations of three selection cultures, which included AFEX CSH, PSGHL, and ethanol-challenged xylose-fed continuous culture. Figure 1 shows the schematic diagram of the evolution experiments performed along with the isolates found either to perform most effectively overall, or most effectively on one of the hydrolyzates tested. Table 3 shows the NRRL accession numbers of these superior isolates and summarizes the adaptation stresses applied in th.......

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Several steps were critical to the success of the evolution process. First, it is key to choose appropriate selection pressures to drive the population evolution toward the desired phenotypes that are needed for successful application. The following selective stresses were chosen for S. stipitis development and applied at appropriate times to guide enrichment for the desired phenotypes: increasing strengths of 12% glucan AFEX CSH (which forces growth and fermentation of diverse sugars in the presence of acetic a.......

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We would like to express our sincere appreciation to Drs. Kenneth Vogel, Robert Mitchell and Gautam Sarath, Grain, Forage, and Bioenergy Research Unit, Agricultural Research Service, Lincoln, NE for their kind supply of switchgrass for this project. We also thank U.S. Department of Energy for funding to VB through the DOE Great Lakes Bioenergy Research Center (GLBRC) Grant DE-FC02-07ER64494.

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Name Company Catalog Number Comments
Cellic Ctec, Contains Xylanase (endo-1,4-) Novozymes No product number www.novozymes.com, 1-919-494-3000
Cellic Htec, Contains Cellulase and Xyalanase Novozymes No product number www.novozymes.com, 1-919-494-3000
Toasted Nutrisoy Flour Archer Daniels Midland Co. (ADM) 63160 ADM, 4666 Faries Parkway, Decatur, IL  1800-37-5843
Pluronic F-68 (Surfactant) Sigma-Aldrich P1300 Sigma-Aldrich
Difco Vitamin Assay Casamino Acids Becton Dickinson and Company 228830 multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
D,L-tryptophan  Sigma-Aldrich T3300 multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
L-cysteine  Sigma-Aldrich C7352 multiple suppliers:  e.g. Fisher Scientific, Sigma-Aldrich
Bacto Agar Becton Dickinson and Company 214010 multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
Bacto Malt Extract Becton Dickinson and Company 218630 multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
Bacto Yeast Extract Becton Dickinson and Company 212750 multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
Peptone Type IV from soybean Fluka P0521-500g multiple suppliers:  e.g. Fisher Scientific, VWR, Daigger
Adenine, > 99% powder Sigma-Aldrich A8626 CAS 73-24-5,  Could use other brands.  Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC
Cytosine, > 99% Sigma-Aldrich C3506 CAS 71-30-7,  Could use other brands.  Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC
Guanine, SigmaUltra Sigma-Aldrich G6779 CAS 73-40-5,  Could use other brands.  Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC
Thymine, 99% Sigma-Aldrich T0376 CAS 65-71-4,  Could use other brands.  Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC
Uracil, 99% Sigma-Aldrich U0750 CAS 66-22-8,  Could use other brands.  Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC
Dextrose (D-Glucose), Anhydrous, Certified ACS Fisher Chemical D16-500 CAS 50-99-7, Could use other brands.  Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich
D-Xylose, assay > 99% Sigma-Aldrich X1500 CAS 58-86-6, Could use other brands.  Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich
96-well, flat bottom plates Becton Dickinson Falcon 351172 multiple suppliers:  e.g. Thermo-Fisher, VWR, Daigger
Wypall L40 Wiper Kimberly-Clark towel in microplate boxes to absorb water for humidification;  multiple suppliers:  e.g. Thermo-Fisher, uline, Daigger
Corning graduated pyrex flask, 125-mL, narrow opening (stopper #5) Corning Life Science Glass 4980-125 multiple suppliers:  e.g. Thermo-Fisher, VWR, Daigger
Innova 42R shaker/incubator, 2.5 cm (1") rotation New Brunswick Scientific (1-800-631-5417) M1335-0016 multiple suppliers:  e.g. Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1") throw could be used. 
Duetz Cover clamp for 4 deepwell MTP plates Applikon Biotechnology Z365001700 applikon-biotechnology.com (U.S.), 1-650-578-1396
Duetz System sandwich cover for 96 deepwell plates Applikon Biotechnology Z365001296 applikon-biotechnology.com (U.S.), 1-650-578-1396
Duetz System silicone seal (0.8mm black low evap) for 96 deep well plate cover Applikon Biotechnology V0W1040027 applikon-biotechnology.com (U.S.), 1-650-578-1396
Blue microfiber layer for Duetz system sandwich cover Applikon Biotechnology V0W1040001 applikon-biotechnology.com (U.S.), 1-650-578-1396
96 well, 2 mL square well pyramid bottom plates, natural popypropylene Applikon Biotechnology ZC3DXP0240 applikon-biotechnology.com (U.S.), 1-650-578-1396
Bellco 32mm silicon sponge plug closures, pk of 25 for 125-mL flasks Bellco 1924-00032 Thomas Scientific, their Catalog number is 1203K27
Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100-mL  working volume.  This design no longer manufactured. Bellco 1968-00100 (original Cat. No.) Jacketed vessels have lower inlet & upper outlet ports for temp. control with circulating water bath. Vessels are 75mm in outer diam and 200mm in height. There are four side ports at ~45o angles and one top port. Port openings appropriate size for size 0 neoprene stoppers (21-22mm inner diameters on ports).
Mathis Labomat IR Dryer Oven MathisAg Typ-Nbr BFA12 215307 Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157
Dual Channel Biochemistry Analyzer YSI Life Sciences 2900D-UP www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format
PowerWave XS Microplate Spectrophotometer Bio-Tek Instruments, Inc MQX200R www.biotek.com

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