Name: techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol
Uploaded: 2016-10-24T13:00:00
Description: Watch this Scientific Journal Video about Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol at JoVE.com

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 ...

Research

Education

JoVE Journal

JoVE Core

Biology

Bioengineering

Evolution

Natural Selection

SCIENTISTS IN ACTION

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...