Name: techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol
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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 Começando Materiais e Equipamentos para Ensaios <ol><li> Prepare hidrolisados ​​usando 18 a 20% de biomassa inicial de peso seco na reacção de pré-tratamento para utilização na evolução, o isolamento e a procedimentos de classificação. Veja Slininger et al. 2015 18 para os métodos detalhados para preparar AFEX CSH, PSGHL e SGH com suplementos de nitrogênio N1 ou N2 utilizados em evolução, isoladamente ou ranking. Ver Tabela 1 para composição de cada t...

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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. 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Vários passos eram críticos para o sucesso do processo de evolução. Em primeiro lugar, é fundamental para escolher pressões de seleção apropriadas para impulsionar a evolução da população para com os fenótipos desejados que são necessários para a aplicação bem-sucedida. As seguintes tensões seletivos foram escolhidos para S. stipitis desenvolvimento e aplicados em alturas apropriadas para guiar o enriquecimento para os fenótipos desejados: forças crescentes de 12% glucano AFEX CSH (o que obr...

Estima-se que 1,3 bilhões de toneladas secas anuais de biomassa lignocelulósica poderia apoiar a produção de etanol e permitir que os EUA a reduzir o seu consumo de petróleo em 30%. 1 Embora biomassa vegetal misturas de açúcar rendimentos de hidrólise ricos em glicose e xilose, inibidores da fermentação são gerados pelo pré-tratamento químico necessário para quebrar hemicelulose e celulose para expor ataque enzimático. ácido acético, furfural e hidroximetilfurfural (HMF) são considerados os principais componentes entre muitos inibidores que se formam durante o pré-tratamento. A fim de mover a indústria de etanol lignocelulósico para a frente, a investigação e procedimentos para permitir a evolução de estirpes de leveduras capazes de sobreviver e funcionar eficazmente para usar tanto hexoses e pentoses, na presença de tais compostos inibidores são necessários. Um ponto fraco adicional significativo de estirpes de leveduras industriais tradicionais, tais como Saccharomyces cerevisiae, é a incapacidade de fermen eficientementet a xilose disponíveis em hidrolisados ​​de biomassa vegetal. Pichia stipitis estirpe NRRL Y-7124 (CBS 5773), recentemente renomeado stipitis Scheffersomyces, é uma levedura de fermentação pentose nativa que é bem conhecido para fermentar a xilose em etanol. 2,3 A evolução da estirpe NRRL Y-7124 foi prosseguido aqui porque tem sido documentada a têm o maior potencial de linhagens de leveduras nativas para acumular etanol economicamente recuperável superior a 40 g / L com pouco subproduto xilitol. 4,5,6 na mídia ideal, S. stipitis estirpe NRRL Y-7124 produz 70 g / L de etanol, em 40 h (1,75 g / L / h), com um rendimento de 0,41 ± 0,06 g / g em culturas de elevada densidade celular (células de 6 g / l). 7,8 Resistência para inibidores da fermentação etanol, furfural, e HMF também tem sido relatado, 9 e S. stipitis foi classificado entre mais promissores pentoses-fermentação de leveduras nativas disponíveis para productio etanol em escala comercialn de lignocelulose. 10 O nosso objectivo era aplicar diversas hidrolisados lignocelulósicos undetoxified e pressões de seleção de etanol para forçar evolução em direção a um derivado mais robusto da estirpe NRRL Y-7124 adequada para aplicações industriais. Chave entre características melhoradas procurados eram taxas mais rápidas de açúcar de absorção em hidrolisados ​​concentrados, reduzida diauxy para uma utilização mais eficiente de açúcar misturado, e tolerâncias mais elevadas de etanol e inibidores. A aplicação de S. stipitis para hidrolisados undetoxified foi um dos principais focos da pesquisa para eliminar a despesa operacional adicional associado com processos de desintoxicação hidrolisado, como calagem excessiva. Dois hidrolisados industrialmente promissores foram aplicadas para forçar evolução:. Enzima Sacarificada fibra de amônia pré-tratados com expansão hidrolisado de palha de milho (AFEX CSH) e diluir o licor switchgrass hidrolisado pré-tratadas com ácido (PSGHL) 11,12 tecnologia de pré-tratamento AFEX está sendo desenvolvido paraminimizar a produção de inibidores de fermentação, enquanto que o pré-tratamento com ácido diluído representa o menor custo actual tecnologia mais comumente praticado para expor biomassa celulósica para sacarificação enzimática. PSGHL é separável a partir da celulose remanescente após pré-tratamento e é tipicamente rica em xilose a partir do hidrolisado hemicelulose, mas pobre em glucose. AFEX CSH e composições PSGHL diferem uns dos outros em aspectos-chave que foram exploradas para gerenciar o processo de evolução. AFEX CSH é menor em aldeídos furano e inibidores do ácido acético, mas superior em aminoácidos e as fontes de azoto, quando comparada com amoníaco PSGHL (Tabela 1). PSGHL apresenta o desafio adicional de xilose sendo o açúcar predominante disponível. Assim PSGHL é apropriado para enriquecer especificamente para melhor utilização xilose na hidrolisados, uma fraqueza prevenção do uso comercial de levedura disponíveis. Mesmo entre leveduras de fermentação de pentoses nativas, a dependência em relação à xilo açúcar abaixo do idealse a apoiar o crescimento celular e reparação torna-se ainda mais desafiador na hidrolisados por causa de uma variedade de razões:. deficiências de nutrientes, inibidores causando danos generalizados para a célula integridade estrutural, e as perturbações do metabolismo devido a desequilíbrios redox 9 suplementação de nitrogênio, especialmente na forma de aminoácidos, pode representar um custo operacional significativa para as fermentações. O impacto da suplementação de nitrogênio sobre o rastreio isolado e ranking foi explorada com hidrolisados ​​switchgrass. Indivíduos melhoradas foram enriquecidos em uma população evoluindo usando várias pressões de seleção dependente de diversidade genética natural do S. população e as mutações stipitis induzida pela exposição a dois hidrolisados diversas, etanol ou radiação UV. Pressões de selecção foram aplicados em paralelo e em série para explorar o progresso evolução de S. stipitis para derivados desejados capazes de crescer e fermentar eficientemente hidrolisados em(Figura 1). A cultura repetitivo de populações funcionais hidrolisados ​​em cada vez mais difíceis foi realizado em microplacas utilizando uma série de diluições de 12%, quer CSH AFEX glucano ou então PGSHL preparado em 20% de carga de sólidos. A aplicação de crescimento desafiou-etanol em xilose em cultura contínua melhorou ainda mais CSH adaptada populações AFEX, através do enriquecimento de fenótipos que demonstram menor suscetibilidade ao etanol repressão da utilização de xilose. A última característica foi recentemente mostrado problemática à utilização das pentoses pela estirpe NRRL Y-7124 após fermentação da glicose. 8 Enriquecimento em PSGHL foi próxima explorado para ampliar a funcionalidade hidrolisado. Derivados melhorados putativos de S. stipitis NRRL Y-7124 foram isolados de cada fase do processo de evolução usando enriquecimento alvo sob condições de estresse e à diluição em placas para escolher colónias a partir das populações mais prevalentes. relativa adimensionalíndices de desempenho (RPIS) foram usadas para classificar estirpes com base no desempenho global, em que o comportamento cinético foi avaliada sobre os diferentes tipos de hidrolisados ​​e suplementos nutrientes aplicados. Embora os sucessos de vários procedimentos de adaptação para melhorar a funcionalidade do S. stipitis na hidrolisados lignocelulósicos foram documentados anteriormente, as estirpes que demonstram a produção de etanol econômica na hidrolisados undetoxified não tenham sido previamente relatado. 13-17 Utilizando os processos de evolução para ser visualizado em mais detalhes aqui, Slininger et al. 18 cepas que são significativamente aprimorados em relação desenvolvido a estirpe-mãe NRRL Y-7124 e são capazes de produzir&gt; 40 g / L de etanol em AFEX CSH e enzima hidrolisado virgatum sacarificado (SGH) adequadamente complementada com fontes de azoto. Estas novas estirpes são de futuro interesse para a lignocelulose em desenvolvimento para a indústria de etanol e como sujeitos de genômica adicionais estudos edifícionaqueles de tensão previamente sequenciado NRRL Y-11545 19. Um estudo da genómica das principais linhagens produzidas durante as várias fases de evolução diagramado na Figura 1 poderia elucidar a história de mudanças genéticas que ocorreram durante o desenvolvimento como um prelúdio para novas pesquisas de melhoria estirpe.

<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Becton Dickinson and Company</td>
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			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Malt Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
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			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Yeast Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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 foi desenvolvido usando combinações de três culturas de selecção, que incluíram AFEX CSH, PSGHL, e cultura contínua xilose-Fed desafiou-etanol. A Figura 1 mostra o diagrama esquemático da evolução das experiências realizadas juntamente com os isolados encontrados tanto para executar mais eficazmente em geral, ou de forma mais eficaz em um dos hidrolisados testadas. a Tabela 3 mostra os números de acesso NRRL destes isolados superiores e resume as tensõe...

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

Evolução e isolamento de técnicas adaptativas são descritos e demonstrados para se obter derivados de Scheffersomyces stipitis estirpe NRRL Y-7124 que é capaz de consumir rapidamente de hexoses e pentoses em açúcares mistos enzima sacarificado hidrolisados undetoxified e a acumular-se sobre 40 g / L de etanol.

Técnicas para a Evolução da levedura Pentose-fermentação robusta para bioconversão de transformação de lignocelulose em etanol

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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