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.

1. Prepare Starting Materials and Equipment for Assays
<ol>
	<li>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...

<ol>
	<li>Perlack, R. D., Stokes, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. , (2011).</li><li>Prior, B. A., Kilian, S. G., duPreez, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentation+of+D-xylose+by+the+yeasts+Candida+shehatae+and+Pichia+stipitis.">Fermentation of D-xylose by the yeasts Candida shehatae and Pichia stipitis.</a> Process Biochem. 24 (1), 21-32 (1989).</li><li>Kurtzman, C. P., Suzuki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+ascomycete+yeasts+that+form+coenzyme+Q-9+and+the+proposal+of+the+new+genera+Babjeviella,+Meyerozyma,+Millerozyma,+Priceomyces+and+Scheffersomyces.">Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces and Scheffersomyces.</a> Mcoscience. 51 (1), 2-14 (2010).</li><li>Slininger, P. J., Bothast, R. J., Okos, M. R., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+ethanol+production+by+xylose-fermenting+yeasts+presented+high+xylose+concentrations.">Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations.</a> Biotechnol. Lett. 7 (6), 431-436 (1985).</li><li>Slininger, P. J., Bothast, R. J., Ladisch, M. R., Okos, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimum+pH+and+temperature+conditions+for+xylose+fermentation+by+Pichia+stipitis.">Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis.</a> Biotechnol. Bioeng. 35 (7), 727-731 (1990).</li><li>Slininger, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+kinetics+of+xylose+fermentation+by+Pichia+stipitis.">Stoichiometry and kinetics of xylose fermentation by Pichia stipitis.</a> Annals NY Acad. Sci. 589, 25-40 (1990).</li><li>Slininger, P. J., Dien, B. S., Gorsich, S. W., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+source+and+mineral+optimization+enhance+D-xylose+conversion+to+ethanol+by+the+yeast+Pichia+stipitis+NRRL+Y-7124.">Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.</a> Appl. Microbiol. Biotechnol. 72 (6), 1285-1296 (2006).</li><li>Slininger, P. J., Thompson, S. R., Weber, S., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+xylose-specific+enzymes+by+ethanol+in+Scheffersomyces+(Pichia)+stipitis+and+utility+of+repitching+xylose-grown+populations+to+eliminat+diauxic+lag.">Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminat diauxic lag.</a> Biotechnol. Bioeng. 108 (8), 1801-1815 (2011).</li><li>Slininger, P. J., Gorsich, S. W., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+nutrition+and+physiology+impact+inhibitor+tolerance+of+the+yeast+Pichia+stipitis+NRRL+Y-7124.">Culture nutrition and physiology impact inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124.</a> Biotechnol. Bioeng. 102 (3), 778-790 (2009).</li><li>Agbogbo, F. K., Coward-Kelly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+ethanol+production+using+the+naturally+occurring+xylose-fermenting+yeast,+Pichia+stipitis.">Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis.</a> Biotechnol. Lett. 30 (9), 1515-1524 (2008).</li><li>Balan, V., Bals, B., Chundawat, S., Marshall, D., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignocellulosic+pretreatment+using+AFEX.">Lignocellulosic pretreatment using AFEX.</a> Biofuels: Methods and protocols, Methods in Molecular Biology. 581, 61-77 (2009).</li><li>Jin, M., Gunawan, C., Uppugundla, N., Balan, V., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+integrated+biological+process+for+cellulosic+ethanol+production+featuring+high+ethanol+productivity,+enzyme+recycling,+and+yeast+cells+reuse.">A novel integrated biological process for cellulosic ethanol production featuring high ethanol productivity, enzyme recycling, and yeast cells reuse.</a> Energ. Environ. Sci. 5 (5), 7168-7175 (2012).</li><li>Nigam, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+xylose-fermenting+yeast+Pichia+stipitis+for+ethanol+production+through+adaptation+on+hardwood+hemicellulose+acid+prehydrolysate.">Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate.</a> J. Appl. Microbiol. 90 (2), 208-215 (2001).</li><li>Nigam, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+production+from+wheat+straw+hemicellulose+hydrolysate+by+Pichia+stipitis.">Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis.</a> J. Biotechnol. 87 (1), 17-27 (2001).</li><li>Hughes, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+UV-C+mutagenesis+of+Scheffersomyces+(formerly+Pichia)+stipitis+NRRL+Y-7124+to+improve+anaerobic+growth+on+lignocellulosic+sugars.">Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars.</a> J. Ind. Microbiol. Biotechnol. 39 (1), 163-173 (2012).</li><li>Bajwa, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutants+of+the+pentose-fermenting+yeast+Pichia+stipitis+with+improved+tolerance+to+inhibitors+in+hardwood+spent+sulfite+liquor.">Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor.</a> Biotechnol. Bioeng. 104 (5), 892-900 (2009).</li><li>Bajwa, P. K., Pinel, D., Martin, V. J. J., Trevors, J. T., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+improvement+of+the+pentose-fermenting+yeast+Pichia+stipitis+by+genome+shuffling.">Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling.</a> J. Microbiol. Methods. 81 (2), 179-186 (2010).</li><li>Slininger, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolved+strains+of+Scheffersomyces+stipitis+achieving+high+ethanol+productivity+on+acid-+and+base-pretreated+biomass+hydrolyzate+at+high+solids+loading.">Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.</a> Biotechnol. Biofuels. 8:60, 1-27 (2015).</li><li>Jeffries, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+lignocellulosic-bioconverting+and+xylose-fermenting+yeast+Pichia+stipitis.">Genome sequence of the lignocellulosic-bioconverting and xylose-fermenting yeast Pichia stipitis.</a> Nature Biotechnol. 25 (3), 319-326 (2007).</li><li>Zabriski, D. W., Armiger, W. B., Phillips, D. H., Albano, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentation+media+formulation.">Fermentation media formulation.</a> Trader's Guide to Fermentation Media Formulation. , 1-39 (1980).</li><li>Syzbalski, W., Bryson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+studies+on+microbial+cross+resistance+to+toxic+agents.+I.+Cross+resistance+of+Escherichia+coli+to+fifteen+antibiotics.">Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics.</a> J. Biotechnol. 64 (4), 489-499 (1952).</li><li>Klinke, H. B., Thomsen, A. B., Ahring, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ethanol-producing+yeast+and+bacteria+by+degradation+products+produced+during+pre-treatment+of+biomass.">Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass.</a> Appl. Microbiol. Biotechnol. 66, 10-26 (2004).</li><li>Almeida, J. R. M., Bertilsson, M., Gorwa-Grauslund, M. F., Gorsich, S., Liden, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+effects+of+furaldehydes+and+impacts+on+biotechnological+processes.">Metabolic effects of furaldehydes and impacts on biotechnological processes.</a> Appl. Microbiol. Biotechnol. 82, 625-638 (2009).</li><li>Allen, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Furfural+induces+reactive+oxygen+species+accumulation+and+cellular+damage+in+Saccharomyces+cerevisiae.">Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae.</a> Biotechnol. Biofuels. 3, (2010).</li><li>Weisburger, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenic,+carcinogenic,+and+chemopreventive+effects+of+phenols+and+catechols:+the+underlying+mechanisms.">Mutagenic, carcinogenic, and chemopreventive effects of phenols and catechols: the underlying mechanisms.</a> ACS Symposium Series. 507, 35-47 (2009).</li><li>Slininger, P. J., Dien, B. S., Lomont, J. M., Bothast, R. J., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+kinetic+model+for+computer+simulation+of+growth+and+fermentation+by+Scheffersomyces+(Pichia)+stipitis+fed+D-xylose.">Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.</a> Biotechnol. Bioeng. 111 (8), 1532-1540 (2014).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+metabolic+profiling+revealed+limitations+in+xylose-fermenting+yeast+during+co-fermentation+of+glucose+and+xylose+in+the+presence+of+inhibitors.">Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.</a> Biotechnol. Bioeng. 111 (1), 152-164 (2014).</li><li>Slininger, P. J., Branstrator, L. E., Bothast, R. J., Okos, M. R., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth,+death,+and+oxygen+uptake+kinetics+of+Pichia+stipitis+on+xylose.">Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose.</a> Biotechnol. Bioeng. 37 (10), 973-980 (1991).</li></ol>

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...

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 capable of surviving and efficiently functioning to use both hexose and pentose sugars in the presence of such inhibitory compounds are needed. A significant additional weakness of traditional industrial yeast strains, such as Saccharomyces cerevisiae, is the inability to efficiently ferment the xylose available in hydrolyzates of plant biomass.
Pichia stipitis type strain NRRL Y-7124 (CBS 5773), recently renamed Scheffersomyces stipitis, is a native pentose fermenting yeast that is well known to ferment xylose to ethanol.2,3 The evolution of strain NRRL Y-7124 was pursued here because it has been documented to have the greatest potential of native yeast strains to accumulate economically recoverable ethanol exceeding 40 g/L with little xylitol byproduct.4,5,6 In optimal media, S. stipitis strain NRRL Y-7124 produces 70 g/L ethanol in 40 hr (1.75 g/L/hr) at a yield of 0.41 &#177; 0.06 g/g in high cell density cultures (6 g/L cells).7,8 Resistance to fermentation inhibitors ethanol, furfural, and HMF has also been reported,9 and S. stipitis has been ranked among most promising native pentose-fermenting yeasts available for commercial scale ethanol production from lignocellulose.10 Our objective was to apply diverse undetoxified lignocellulosic hydrolyzates and ethanol selection pressures to force evolution toward a more robust derivative of strain NRRL Y-7124 suitable for industrial applications. Key among improved features sought were faster sugar uptake rates in concentrated hydrolyzates, reduced diauxy for more efficient mixed sugar utilization, and higher tolerances of ethanol and inhibitors. The application of S. stipitis to undetoxified hydrolyzates was a key focus of the research to eliminate the added operating expense associated with hydrolyzate detoxification processes, such as overliming.
Two industrially promising hydrolyzates were applied to force evolution: enzyme saccharified ammonia fiber expansion-pretreated corn stover hydrolyzate (AFEX CSH) and dilute acid-pretreated switchgrass hydrolyzate liquor (PSGHL).11,12 AFEX pretreatment technology is being developed to minimize the production of fermentation inhibitors, while dilute acid pretreatment represents the current lowest cost technology most commonly practiced to expose cellulosic biomass for enzymatic saccharification. PSGHL is separable from the cellulose remaining after pretreatment and is characteristically rich in xylose from the hydrolyzed hemicellulose, but low in glucose. AFEX CSH and PSGHL compositions differ from one another in key aspects which were exploited to manage the evolution process. AFEX CSH is lower in furan aldehydes and acetic acid inhibitors but higher in amino acids and ammonia nitrogen sources compared with PSGHL (Table 1). PSGHL presents the additional challenge of xylose being the predominant sugar available. Thus PSGHL is appropriate to specifically enrich for improved xylose utilization in hydrolyzates, a weakness preventing commercial use of available yeast. Even among native pentose fermenting yeasts, the reliance on the suboptimal sugar xylose to support cell growth and repair becomes even more challenging in hydrolyzates because of a variety of reasons: nutrient deficiencies, inhibitors causing widespread damage to cell structural integrity, and disruption to metabolism due to redox imbalances.9 Nitrogen supplementation, especially in the form of amino acids, can represent a significant operating cost for fermentations. The impact of nitrogen supplementation on isolate screening and ranking was explored with switchgrass hydrolyzates.
Improved individuals were 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. Selection pressures were applied in parallel and in series to explore the evolution progress of S. stipitis toward desired derivatives able to grow and ferment efficiently in hydrolyzates (Figure 1). The repetitive culturing of functional populations in increasingly challenging hydrolyzates was accomplished in microplates employing a dilution series of either 12% glucan AFEX CSH or else PGSHL prepared at 20% solids loading. The application of ethanol-challenged growth on xylose in continuous culture further improved AFEX CSH adapted populations by enriching for phenotypes demonstrating less susceptibility to ethanol repression of xylose utilization. The latter feature was recently shown problematic to pentose utilization by strain NRRL Y-7124 following glucose fermentation.8 Enrichment on PSGHL was next explored to broaden hydrolyzate functionality.
Putative improved derivatives of S. stipitis NRRL Y-7124 were isolated from each phase of the evolution process using targeted enrichment under stress conditions and dilution plating to pick colonies from the most prevalent populations. Dimensionless relative performance indices (RPIs) were used to rank strains based on overall performance, where kinetic behavior was evaluated on the different hydrolyzate types and nutrient supplements applied. Although the successes of various adaptation procedures to improve the functionality of S. stipitis in lignocellulosic hydrolyzates have been previously documented, strains demonstrating economical ethanol production on undetoxified hydrolyzates have not been previously reported.13-17 Using the evolution procedures to be visualized in more detail here, Slininger et al.18 developed strains that are significantly improved over the parent strain NRRL Y-7124 and are able to produce &#62;40 g/L ethanol in AFEX CSH and enzyme saccharified switchgrass hydrolyzate (SGH) appropriately supplemented with nitrogen sources. These novel strains are of future interest to the developing lignocellulose to ethanol industry and as subjects of additional genomics studies building on those of previously sequenced strain NRRL Y-11545.19 A genomics study of top strains produced during various phases of evolution diagramed in Figure 1 would elucidate the history of genetic changes that occurred during development as a prelude to further strain improvement research.

<table border="0" cellpadding="0" cellspacing="0" width="1252">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cellic Ctec, Contains Xylanase (endo-1,4-)</td>
			<td style="width:251px;">Novozymes</td>
			<td style="width:180px;">No product number</td>
			<td style="width:467px;">www.novozymes.com, 1-919-494-3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cellic Htec, Contains Cellulase and Xyalanase</td>
			<td style="width:251px;">Novozymes</td>
			<td style="width:180px;">No product number</td>
			<td>www.novozymes.com, 1-919-494-3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Toasted Nutrisoy Flour</td>
			<td style="width:251px;">Archer Daniels Midland Co. (ADM)</td>
			<td style="width:180px;">63160</td>
			<td>ADM, 4666 Faries Parkway, Decatur, IL&#160; 1800-37-5843</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Pluronic F-68 (Surfactant)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">P1300</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Difco Vitamin Assay Casamino Acids</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">228830</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D,L-tryptophan&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:180px;">T3300</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-cysteine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:180px;">C7352</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Becton Dickinson and Company</td>
			<td style="width:180px;">214010</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Malt Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">218630</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Yeast Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">212750</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Peptone Type IV from soybean</td>
			<td style="width:251px;">Fluka</td>
			<td style="width:180px;">P0521-500g</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Adenine, &#62; 99% powder</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">A8626</td>
			<td>CAS 73-24-5,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cytosine, &#62; 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">C3506</td>
			<td>CAS 71-30-7,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Guanine, SigmaUltra</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">G6779</td>
			<td>CAS 73-40-5,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Thymine, 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">T0376</td>
			<td>CAS 65-71-4,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Uracil, 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">U0750</td>
			<td>CAS 66-22-8,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dextrose (D-Glucose), Anhydrous, Certified ACS</td>
			<td style="width:251px;">Fisher Chemical</td>
			<td style="width:180px;">D16-500</td>
			<td>CAS 50-99-7, Could use other brands.&#160; Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">D-Xylose, assay &#62; 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">X1500</td>
			<td>CAS 58-86-6, Could use other brands.&#160; Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">96-well, flat bottom plates</td>
			<td style="width:251px;">Becton Dickinson Falcon</td>
			<td style="width:180px;">351172</td>
			<td>multiple suppliers:&#160; e.g. Thermo-Fisher, VWR, Daigger</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Wypall L40 Wiper</td>
			<td style="width:251px;">Kimberly-Clark</td>
			<td style="width:180px;"></td>
			<td style="width:467px;">towel in microplate boxes to absorb water for humidification;&#160; multiple suppliers:&#160; e.g. Thermo-Fisher, uline, Daigger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Corning graduated pyrex flask, 125-mL, narrow opening (stopper #5)</td>
			<td style="width:251px;">Corning Life Science Glass</td>
			<td style="width:180px;">4980-125</td>
			<td>multiple suppliers:&#160; e.g. Thermo-Fisher, VWR, Daigger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Innova 42R shaker/incubator, 2.5 cm (1&#34;) rotation</td>
			<td style="width:251px;">New Brunswick Scientific (1-800-631-5417)</td>
			<td style="width:180px;">M1335-0016</td>
			<td>multiple suppliers:&#160; e.g. Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1&#34;) throw could be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Duetz Cover clamp for 4 deepwell MTP plates</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">Z365001700</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Duetz System sandwich cover for 96 deepwell plates</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">Z365001296</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Duetz System silicone seal (0.8mm black low evap) for 96 deep well plate cover</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">V0W1040027</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Blue microfiber layer for Duetz system sandwich cover</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">V0W1040001</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">96 well, 2 mL square well pyramid bottom plates, natural popypropylene</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">ZC3DXP0240</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Bellco 32mm silicon sponge plug closures, pk of 25 for 125-mL flasks</td>
			<td style="width:251px;">Bellco</td>
			<td style="width:180px;">1924-00032</td>
			<td>Thomas Scientific, their Catalog number is 1203K27</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:355px;">Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100-mL&#160; working volume.&#160; This design no longer manufactured.</td>
			<td style="width:251px;">Bellco</td>
			<td style="width:180px;">1968-00100 (original Cat. No.)</td>
			<td style="width:467px;">Jacketed vessels have lower inlet &#38; 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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Mathis Labomat IR Dryer Oven</td>
			<td style="width:251px;">MathisAg</td>
			<td style="width:180px;">Typ-Nbr BFA12 215307</td>
			<td>Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dual Channel Biochemistry Analyzer</td>
			<td>YSI Life Sciences</td>
			<td>2900D-UP</td>
			<td style="width:467px;">www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PowerWave XS Microplate Spectrophotometer</td>
			<td>Bio-Tek Instruments, Inc</td>
			<td>MQX200R</td>
			<td><a href="http://www.biotek.com/">www.biotek.com</a></td>
		</tr>
	</tbody>
</table>

techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to 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.

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...

Watch this Scientific Journal Video about Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol at JoVE.com

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

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 ...

The overall goal of this adaptive evolution procedure is to derive robust strains of the native xylose fermenting yeast scheffersomyces stipitis NRRL Y-7124 that can more rapidly ferment the hexose and pentose sugars in undetoxified hydrolysates to economically recoverable ethanol. This method moves the lignocellulose to ethanol industry forward by providing a road map to strains with complex modifications, allowing fermentation of sugars in hydrolysates which contain microbial inhibitors. Here, the main advantage of targeted evolution is that robust strains are molded from a native yeast able to ferment xylose, eliminating the need to engineer industrial saccharomyces strains.We realized that any single yeast strain was unlikely to be robust in different types of hydrolysates, such as AFEX pretreated corn stover or dilute acid pretreated switchgrass. The ability of scheffersomyces stipitis to undergo a sexual reproductive cycle is beneficial to further exploitation in the present work because the various strains developed can be combined into a single, more robust strain through mating of strains having these various traits. Demonstrating the procedure will be coauthors Stephanie Thomson and Maureen Shea-Andersh who are biological science technicians in my laboratory.Prior to starting this enrichment procedure, prepare a preculture of strain NRRL Y-7124 in a 125 milliliter flask and a dilution series of the enzyme saccharified ammonia fiber expansion pretreated corn stover hydrolysate, or AFEX CSH. Use a repeating pipette to fill a 96 well microplate with 15 microliters per well, and eight wells per hydrolysate dilution. Inoculate with a few microliters of preculture per well to allow an initial absorbance at 620 nanometers, or A620 of greater than or equal to 0.1.Place the microplate in a plastic box with a wet paper towel for humidity and incubate statically for 24 to 28 hours at 25 degrees Celsius. Next, use a microplate reading spectrophotometer to identify the cultures that physically grew to an A620 greater than 1.0 in the most concentrated hydrolysate dilution. Pool the identified wells in a microfuge tube, and distribute to a fill plate.Transfer one to five microliters from the fill plate to each well of a new hydrolysate dilution series to achieve an initial A620 of greater than or equal to 0.1 and incubate the plate as before. Monitor the growth of the cultures in the microplate with a microplate reading spectrophotometer. Prepare glycerol stocks of adaptation cultures regularly.Pool four wells of the greatest hydrolysate concentration colonized in a cryofile, and mix one to one with 20%sterile glycerol. Freeze cells at minus 80 degrees Celsius. Repeat this enrichment procedure until growth in 12%glucan AFEX CSH is consistently visible at 24 hours.To begin this procedure, streak selected glycerol stocks of adaptation cultures to yeast malt peptone dextrose, or YM agar. Then after growth, use streaks to inoculate 50 microliters of 3%or 6%glucan AFEX CSH in each of three microplate wells. Incubate the microplate for 24 hours at 25 degrees Celsius.Pool replicate colonized wells at the highest hydrolysate strength with strong growth. And dilution plate to YM or 6%glucan AFEX CSH agar. Incubate the plates in a 25 degree Celsius incubator for 24 to 48 hours.Pick five single colonies from the highest dilution plate showing growth to freeze as glycerol stock cultures. Streak each colony to a YM plate and incubate for 24 hours. With a sterile loop, transfer a developed streak of cells to a small volume of 10%glycerol mixed to suspend the cells and distribute to two to three cryofiles for freezing at minus 80 degrees Celsius.To test isolates in simple 6%glucan AFEX CSH batch cultures, transfer cells from 48 hour plates streaked from glycerol stocks to pH 7 potassium phosphate buffer to prepare cell suspensions. After measuring the optical density of each cell suspension, adjust the volume with additional buffer to reach an A620 of ten. Use one microliter of each suspension to inoculate each of four wells of 50 microliters of 3%glucan hydrolysate to an initial A620 of 0.2.Incubate the microplate for 24 hours. Next, transfer two 24 hour microplate wells to inoculate 25 milliliter precultures of 6%glucan AFEX CSH at pH 5. Close the flasks with silicon sponge closures and incubate for 24 hours at 25 degrees Celsius and 150 RPM.Use the 6%glucan AFEX CSH precultures to inoculate similar 25 milliliter growth cultures in duplicate to an initial A620 of 0.1. Incubate the growth cultures for 24 hours at 25 degrees Celsius and 150 RPM. To test dioxic lack of isolates in batch cultures of optimal defined medium, or ODM, with mixed sugars, inoculate precultures of 75 milliliters ODM with 150 grams per liter of xylose in 125 milliliter flasks with silicone sponge closures.Incubate for 24 hours at 25 degrees Celsius and 150 RPM. Use the precultures to inoculate to an A620 of 0.1 similar duplicate 75 milliliter test cultures with ODM containing 75 grams per liter of glucose and 75 grams per liter of xylose at pH 6.5. Shake flasks at 25 degrees Celsius and 150 RPM.24 hours prior to inoculating a continuous culture, prepare a preculture of a representative AFEX CSH tolerant colony in ethanol-free ODM in a 125 milliliter flask. Use five to 10 milliliters of the AFEX CSH tolerant isolate preculture to inoculate 100 milliliters of ODM in a jacketed 100 milliliter spinner flask to obtain an initial A620 of 0.5. Maintain the culture holding volume at 25 degrees Celsius.Stir it at 200 RPM and outfit it with a sterilizable pH electrode, a pH controller, and a temperature controlled circulating water bath. Initially, since cell growth will be slow due to the ethanol exposure, set up a pH actuated pump so that when the culture fermentation decreases the pH to 5.4, the pump will feed pH 6.3 ODM with 100 grams per liter of xylose and 50 grams per liter of ethanol to stop further pH decreases. Maintain the volume at 100 milliliters with an effluent pump continuously skimming the culture surface.Measure the effluent volume collected and draw samples from continuous cultures every 48 to 72 hours for analysis of viable cell concentration, products, and substrates as described in the manuscript. Save glycerol stocks regularly by isolating colonies from viability plates of buffered sample dilutions forming 30 to 100 colonies, representing the most prevalent robust colonies at that time in enrichment. Flood the plates with five milliliters of 10%glycerol to allow preparation of duplicate cryofiles.If cultures can grow steadily at specific rates of greater than 0.01 per hour on xylose in the presence of more than 25 grams per liter of ethanol, start the continuous culture feed at a low dilution rate of about 0.01 per hour to the 100 milliliter holding volume. Over time, raise the ethanol in the feed toward 50 grams per liter. Ultraviolet irradiation of inocula for restarting continuous cultures is an option to hasten the mutation rate.Capture advanced populations in glycerol stocks. Prior to isolating single cell colonies, glycerol stock populations with improved xylose fermentation in the presence of ethanol, are identified as described in the text protocol. Use these superior populations to streak plates.The streaked plates are used to prepare dense buffered cell suspensions of an A620 equal to five. Then cell suspensions are used to inoculate precultures in 96 deep well plates. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit.Next, enrich tolerant colonies by using each preculture to inoculate 16 one milliliter replicate cultures to an initial A620 of 0.5 in acid pretreated switchgrass hydrolysate liquor, mixed one to one with ODM plus 50 grams per liter of xylose, and enough ethanol to provide a 20, 30 or 40 grams per liter of ethanol challenge. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit. For each different glycerol stock, harvest the culture wells with the highest ethanol concentration allowing growth and xylose use.Plate each cell line to YM agar to obtain prevalent single colonies to preserve in glycerol. Pick 10 colonies per cell line and streak each to a YM agar plate for glycerol stock preparation. After adaptation on AFEX CSH, a comparison of fermentation performances in ODM between the parent strain and adapted populations reveal that the adapted populations use xylose more efficiently to make ethanol more rapidly and in higher amounts.A hydrolysate tolerant colony was further developed for improved ethanol tolerance by continuous culture selection on ODM containing xylose and high levels of ethanol. All adapted populations surpassed the parent in the ability to induce xylose metabolism in the presence of 40 grams per liter of ethanol. These graphs summarize the performances of superior tolerant isolates assessed in terms of xylose uptake rates and ethanol yields relative to the parent strain for each formulation of PSGHL.The best strains selected from the primary screen on xylose rich PSGHL were subsequently screened on three complete hydrolysates and the relative performance index, or RPI, was calculated for the fermentation of each isolate within each hydrolysate. Next, the combined overall performance indices for each isolate were calculated. Five isolates, three, 14, 27, 28 and 33, had overall RPIs above 60, which ranked them as the most robust to all of the variations of hydrolysate and nutrient conditions combined.After watching this video, you should know how to challenge a xylose fermenting yeast like scheffersomyces stipitis NRRL Y-7124 in a targeted evolution process, enrich, isolate, and evaluate more robust derivatives which more effectively ferment mixed sugars in undetoxified hydrolysates. Once mastered, this technique can be used with a daily time investment averaging about an hour per day. Significant changes in the evolution process can be documented in as little as four months.While attempting this procedure, it's important to save advancing populations regularly by preparing glycerol stocks to freeze at minus 80 degrees Celsius. This allows population recovery, periodic evaluation, isolation or restarting the evolution process. To manage various mutations, remember to regularly screen the yeast under process specific conditions for an array of phenotypes.Calculation of overall dimensionless relative performance indices, or RPIs, based on kinetic parameters, allows assessment of strain rank and robustness to variations in fermentation conditions like hydrolysate type and nutrient supplementation. Superior yeasts arising from each phase of the evolution process are candidates for genome sequencing so that the nature of the improvements such as inhibitor tolerance are reduced and dioxic lag can be understood and used for design of next generation biocatalytic strains.

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11.0K visualizações.  National Center for Agricultural Utilization Research. The overall goal of this adaptive evolution procedure is to derive robust strains of the native xylose fermenting yeast scheffersomyces stipitis NRRL Y-7124 that can more rapidly ferment the hexose and pentose sugars in undetoxified hydrolysates to economically recoverable ethanol. This method moves the lignocellulose to ethanol industry forward by providing a road map to strains with complex modifications, allowing fermentation of sugars in hydrolysates which contain microbial inhibitors. He...