The authors would like to thank intern Evelyn Von Neida who provided paramount insights regarding the preparation of biomass samples for both of the high-throughput pipelines discussed in this manuscript. Support for the development of this work and manuscript was provided by the BioEnergy Science Center. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The National Renewable Energy Laboratory (NREL) is a national laboratory of the US DOE Office of Energy Efficiency and Renewable Energy, operated for DOE by the Alliance for Sustainable Energy, LLC. This work was supported by the U.S. Department of Energy under Contract No. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory.

1.高通量测定葡萄糖和木糖产量继酶糖化9,29 <ol><li>样品制备（研磨，去上浆，提取，预处理） <ol><li>磨至少300毫克用Wiley磨，使得颗粒通过20目（850微米）筛每个生物质的样品。转移到防静电顶部拉链袋（通常条形码）和记录样本的信息的条形码数据库。 </li><li>加入约250毫克或更多的地面生物量从防静电袋一个编号（用铅笔，不使用墨水或标记）茶叶袋，仔细地卷起的茶包，是一定会随着生物质折端，以防止在去上浆和提取的损失。 </li><li>包裹使用镀锡铜线关闭袋泡茶。记录每个条码样本袋泡茶数量。 </li><li>准备从商业酶（典型地，0.25％（体积/体积）葡糖淀粉酶（〜1600 AGU / L）和脱上浆酶溶液1.5％（体积/体积）的α-淀粉酶（〜2900 KNU-S / L）在0.1M乙酸钠（pH 5.0））。准备每克散装生物质16毫升或每120袋泡茶样品500毫升。注:荷载能够从地面生物量去除淀粉应由消化后检测淀粉与一系列酶的负荷和比率的经验来确定。 </li><li>在塑料容器中，每1克的批量地上生物量添加加入16毫升去上浆酶溶液。对于间歇脱上浆协议，添加120茶袋至500ml缓冲 - 酶溶液。 </li><li>孵育摇床中以55℃，24（±4）小时，以120rpm以除去可能的淀粉。 </li><li>解上浆孵育后，冲洗并浸泡生物质中几升的去离子水30分钟。重复此过程，附加2次，以除去缓冲盐可以导致碰撞（形成气泡的在溶液中，可能导致突然和剧烈上升，溶液的水平，从而引起热液体乙醇级联出提取期间反应容器）的。 </li><li>在脱浆生物质的详尽漂洗，将茶袋放入索氏柱进行萃取。没有顶针需要此步骤。 </li><li>设置索氏回流，用95％的乙醇和提取的样品24小时。 </li><li>从索氏反应器取出茶袋和摊开在单个层上的平板状的托盘。 </li><li>允许将样品干燥过夜，在室温下在通风橱中。 </li><li>展开的茶包，并返回干燥的生物质原条形码防静电袋。 注意:防静电袋是有效减少在生物量静电，改善了操作特性。如果其他的存储选项时，附加的生物质可能需要由于生物质抱住存储容器的侧面。 </li><li>传送至少50毫克的干燥的生物量的一个条码料斗（使用更多的材料是用于精确分配更好）。小号可以防静电袋和所述接纳料斗条形码以确保准确的抽样跟踪。 </li><li>装载斗到固体称重机器人，密切关注在机架中的样品的顺序。加载足够反应器板以包含所有的样品，并选择基于所用板的数量分配的协议。 </li><li>机器人称量5毫克的干燥样品（±0.3毫克）成耐酸不锈钢96孔板中。称出该板周围分布各样品的3个重复，以尽量减少任何局部的变化。 </li><li>为了跟踪测定性能包括良好表征的生物材料标准的8控制生物质样品。对多块板，使用多个标准生物质料斗将最小化引起的过重复分配循环筛分在料斗粒径漂移。 注:在每个板，应该有24个样品用3个重复，4坯料包括去离子水和酶，和8 CONTROLS，利用先前表征的标准生物质。每4个角的板的3角井的糖标准品的保留。 </li><li>检查空白和糖的标准井错误的生物质颗粒，如果存在删除。 </li><li>加入250微升去离子水到各孔中，并密封用硅氧烷粘合剂背衬聚四氟乙烯（PTFE）胶带将板。 </li><li>用1/8"烙铁头，刺破聚四氟乙烯带在每个蒸汽口（117个）的每块板。使用一个空盘子堆放在密封反应器板为指导，​​限制PTFE薄膜的运动。 </li><li>夹紧板紧密0.031"厚玻璃纤维增​​强聚四氟乙烯板和空板之间的垫片（预穿孔与孔蒸汽端口）的顶部和堆叠的底部。预处理用蒸汽反应器设定为180℃17.5分钟，或基于期望严重性其他温度/时间组合的样本。 </li><li>凉爽反应器板至50°下通过用水浸去离子水。 </li></ol></li><li>酶糖化<ol><li>准备由8％（体积/体积）的酶溶液中的1.0M柠檬酸钠，pH值5.0的酶糖化溶液。准备每个反应器板5毫升注意:需要应根据特定股票酶溶液的活性和蛋白质含量来确定稀释。 </li><li>当板是冷，离心它们在吊桶式转子中以1500×g离心20分钟。除去封口膜。注意:这些板块都很重，离心机规格应检查兼容性。 </li><li>添加40微升8％酶母液的向每个孔（每克生物量70毫克酶）。 </li><li>重新封装新的PTFE带。将密封板进入磁板钳。 </li><li>轻轻地混合样品进行反演（至少15次），和孵化在50℃，70小时。 </li><li>当糖化的结论，颠倒混合和离心板在1500 x克20分钟。 </li></ol></li><li>糖含量<ol><li>准备一组6个葡萄糖和0.014柠檬酸缓冲木糖合成校准标准（pH5.0）中从0到0.750毫克/毫升（0，0.2％，0.3，0.45，0.65，和0.75毫克/毫升推荐）和0至0.600毫克/毫升（0，0.1，0.2，0.35，0.45，和0.6毫克/毫升推荐）为葡萄糖和木糖，分别。 </li><li>根据试剂盒说明书制备葡萄糖氧化酶/过氧化物酶（GOPOD）和木糖脱氢酶（XDH）试剂。 </li><li>使用移液管，在4角孔和边缘孔邻近于反应器板的角部的孔（12总孔）除去200微升的液体。 </li><li>使用移液管，取180微升去离子水至96孔聚苯乙烯平底板稀释板的每孔中。不要加水至12糖标准和角井（如果使用的是96通道的头删除这些提示）。 </li><li>使用移液管，取180微升GOPOD试剂的葡萄糖测定的每个孔复吃了。 </li><li>使用移液管，取180微升的XDH试剂木糖测定板的各孔中。 </li><li>使用移液管，转移20微升的水解产物的等分试样从96孔反应器板固定到稀释板。吸管从孔中的上段，以避免生物质固体和在角落孔残余液体。通过研磨混合至少10个周期。 </li><li>使用移液管，转移110微升糖标准，一式两份，向稀释板的角落孔中。 </li><li>使用移液管，从稀释板转移20微升等分试样的葡萄糖和木糖的测定板。通过研磨混合。 </li><li>孵育葡萄糖和木糖的测定板在室温下30分钟。阅读前仔细破坏任何表面的气泡。简要地通过在板的表面上的热枪效果很好。 </li><li>使用紫外/可见的96孔读板器，将测得的波长至510纳米，记录对试剂空白的吸光度。该测定监视的形成醌亚胺的，它正比于葡萄糖浓度。中的葡萄糖浓度从基于在1.3.1中制备的校准标准的校准曲线计算。 </li><li>使用紫外/可见的96孔读板器，将测得的波长至340纳米，记录吸光度。这种测量监测NAD +的还原成NADH，其正比于木糖浓 ​​度。木糖浓度从基于1.3.1制备的校准标准校准曲线计算。 </li></ol></li></ol> 2.高通量测定的总木质素和木质素单体含量使用pyMBMS 28 <ol><li>样品制备<ol><li>研磨并提取利用在步骤1.1.1，和1.1.6-1.1.8描述的方法的生物质。这包括研磨和提取一组标准的使用作为实验对照。 注:pyMBMS测量电对此语句不是颗粒尺寸依赖，因此，如果只使用pyMBMS协议，生物质制剂应进行，以使样品适合于样品架，&lt;4毫米。 </li><li>使用小刮勺分配大约4毫克（3至5mg优选）所制备的生物量成80μl的不锈钢杯设计的自动采样器。 </li><li>确保被分析的样品至少10％的控制标准，如那些可从标准和技术研究所（甘蔗渣-8491;杨-8492;松-8493;或麦秸-8494）。该标准还可以是类似于待分析的样本已经使用标准的方法，其特征的任何物种。 </li><li>随机装载样品到使用镊子，以避免偏见的自动进样杯，由于可能的光谱漂移随着时间的推移。注:样品的典型随机化将包括一旦所有检体的测定，随后重新随机化的样本进行重复测量的顺序。随机程序可在网上。 </li><li>使用标准打孔，手动型A / D玻璃纤维片材生产玻璃过滤盘，无粘合剂。持圆形玻璃纤维过滤器，镊子，居中过样品杯，并推入样品用3.5毫米的内六角扳手在实验过程中，以限制在每个杯中的材料。 </li></ol></li><li>器乐协议<ol><li>校准使用公知的标准，有峰强度超过可能存在的实验样品的化合物的整个范围质谱仪。对于典型的生物样品，使用全氟三丁胺（PFTBA）。 </li><li>置使用气体流量计的氦载气流速以0.9升/分。 </li><li>设定自动采样器炉以500℃的热解温度，并使用自动取样软件接口温度为350℃。注:1/8"不锈钢加热包裹在该自动取样连接到质谱仪热带传输线是用在250℃下的热控制器控制。 </li><li>开始质谱仪的数据采集，并等待至少60秒，以获得足够的数据背景光谱采集。 </li><li>开始自动自动取样方法与来自2.2.2的规格。注意:自动取样单独地滴每个样品放入自动取样炉。总的数据采集时间是约1.5分钟;然而，一种典型的4毫克样品的热解是30秒之后完成。 </li><li>使用质谱仪软件在0.5秒的扫描速度每个样品的记录总离子含量（TIC）。 M / Z 30至450之间的记录强度。 注:典型的生物质化合物，使用软电离为17伏特。该仪器可以记录从M / Z 1大间隔1000;然而，扫描速度将通过电脑CPU功率的限制。 </li><li>用马从光谱去除背景NUAL增强特征在软件中。注:在数据采集开始时的基准的60扫描一部分被用来计算平均背景值。这个平均的背景光谱被从质谱仪中软件的实验样品自动光谱。 </li><li>导入由含各样品的光谱数据质谱仪软件创建的单个列的文本文件，数据库程序，所有的样品合并为一个数据库。添加任何适用的元数据到电子表格。导入格式的数据（电子表格/ CSV文件）到一个统计软件包，平均规范化的频谱来解释变化的热解样品群众。 </li><li>使用统计软件包使用所述光谱数据来分析在测量中使用的复制的标准样品的分组来执行主成分分析（PCA），以及评估其峰是不可或缺的化学品的分类在负载化合物积28。 注意:PCA组根据它们的光谱的相似性的样品中，并且允许标准的检查，以了解实验误差由于在运行期间仪器漂移。 </li><li>为了计算木质素紫丁香（S）/愈创木（G）的比例，总结在S峰的面积（M / Z = 154，167，168，182，194，208和210）和除以G的峰的总和在124，137，138，150，164，和178。 </li><li>计算总木质素含量，总结木质素峰M / Z = 120，124，137，138，150，152，154，164，167，178，180，182，194，和210。 </li><li>计算用于缩放pyMBMS测量到的标准方法来估计总木质素，如Klason木质素校正系数。通过测量使用pyMBMS该标准样品的总木质素含量除以个人本位的Klason木质素值。 </li><li>将此校正因子的所有像物种中的数据集。重复每种类型的生物质进行分析。 ñOTE:校正因子的变化在显著基于所分析的生物的S / G。 </li></ol></li></ol>

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Technol. 17 (4), 399-417 (1997).</li><li>Ona, T., Sonoda, T., Ohshima, J., Yokota, S., Yoshizawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+quantitative+method+to+assess+eucalyptus+wood+properties+for+kraft+pulp+production+by+FT-Raman+spectroscopy.">A rapid quantitative method to assess eucalyptus wood properties for kraft pulp production by FT-Raman spectroscopy.</a> J. Pulp Pap. Sci. 29 (1), 6-10 (2003).</li><li>Hames, B. R., Thomas, S. R., Sluiter, A. D., Roth, C. J., Templeton, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+biomass+analysis.+New+tools+for+compositional+analysis+of+corn+stover+feedstocks+and+process+intermediates+from+ethanol+production.">Rapid biomass analysis. New tools for compositional analysis of corn stover feedstocks and process intermediates from ethanol production.</a> Appl. Biochem. Biotechnol. 105-108, 5-16 (2003).</li><li>Studer, M. H., 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=Lignin+content+in+natural+Populus+variants+affects+sugar+release.">Lignin content in natural Populus variants affects sugar release.</a> Proc. Natl. Acad. Sci. U. S. A. 108 (15), 6300-6305 (2011).</li><li>Chen, M., Zhao, J., Xia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+different+chemical+pretreatments+of+corn+stover+for+enhancing+enzymatic+digestibility.">Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility.</a> Biomass Bioenergy. 33 (10), 1381-1385 (2009).</li><li>Davison, B. H., Drescher, S. R., Tuskan, G. A., Davis, M. F., Nghiem, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+S/G+ratio+and+lignin+content+in+a+Populus.+family+influences+the+release+of+xylose+by+dilute+acid+hydrolysis.">Variation of S/G ratio and lignin content in a Populus. family influences the release of xylose by dilute acid hydrolysis.</a> Appl. Biochem. Biotechnol. 129-132, 427-435 (2006).</li><li>Li, 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=Lignin+monomer+composition+affects+Arabidopsis+cell-wall+degradability+after+liquid+hot+water+pretreatment.">Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment.</a> Biotechnol. Biofuels. 3, 27-33 (2010).</li><li>Lupoi, J. S., 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=High-throughput+prediction+of+eucalypt+lignin+syringyl/guaiacyl+content+using+multivariate+analysis:+a+comparison+between+mid-infrared,+near-infrared,+and+Raman+spectroscopies+for+model+development.">High-throughput prediction of eucalypt lignin syringyl/guaiacyl content using multivariate analysis: a comparison between mid-infrared, near-infrared, and Raman spectroscopies for model development.</a> Biotechnol. Biofuels. 7, 93 (2014).</li><li>Lupoi, J. S., Smith, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+woody+and+herbaceous+biomasses+lignin+composition+with+1064+nm+dispersive+multichannel+Raman+spectroscopy.">Characterization of woody and herbaceous biomasses lignin composition with 1064 nm dispersive multichannel Raman spectroscopy.</a> Appl. Spectro. 66 (8), 903-910 (2012).</li><li>Sun, L., 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=Rapid+determination+of+syringyl:guaiacyl+ratios+using+FT-Raman+spectroscopy.">Rapid determination of syringyl:guaiacyl ratios using FT-Raman spectroscopy.</a> Biotechnol. Bioeng. 109 (3), 647-656 (2012).</li><li>Lupoi, J. S., 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=High-throughput+prediction+of+Acacia+and+eucalypt+lignin+syringyl/guaiacyl+content+using+FT-Raman+spectroscopy+and+partial+least+squares+modeling.">High-throughput prediction of Acacia and eucalypt lignin syringyl/guaiacyl content using FT-Raman spectroscopy and partial least squares modeling.</a> Bioenerg. Res. in press, (2015).</li><li>Sykes, R., Kodrzycki, B., Tuskan, G., Foutz, K., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within+tree+variability+of+lignin+composition+in+Populus.">Within tree variability of lignin composition in Populus.</a> Wood Sci. Technol. 42 (8), 649-661 (2008).</li><li>Sykes, R., Mielenz, J. 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=Ch.+12.+High-Throughput+Screening+of+Plant+Cell-Wall+Composition+Using+Pyrolysis+Molecular+Beam+Mass+Spectroscopy.">Ch. 12. High-Throughput Screening of Plant Cell-Wall Composition Using Pyrolysis Molecular Beam Mass Spectroscopy.</a> Biofuels: Methods and Protocols. 581, 169-183 (2009).</li><li>Decker, S., Himmel, M. E., 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=Ch.+17.+Reducing+the+effect+of+variable+starch+levels+in+biomass+recalcitrance+screening).">Ch. 17. Reducing the effect of variable starch levels in biomass recalcitrance screening).</a> Biomass Conversion. 908, 181-195 (2012).</li><li>Evans, R. J., Milne, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+pyrolysis+of+biomass.">Molecular characterization of the pyrolysis of biomass.</a> Energy Fuels. 1 (2), 123-137 (1987).</li></ol>

The authors have nothing to disclose.

是关键的样品制备步骤用于进行高通量筛选实验时获得精确的和可重复的数据如下: 糖释放试验: 在一般情况下，样品在许多范围从几十到几千一次制备。每个主要步骤通常之前向前移动，以最小化的变化样品之间准备进行所有样品。脱上浆最初并不是该协议的一部分，并且可以在某些情况下可以省略。木芯样品，衰老草本材料是一致的低的淀粉和，因此?...

作为非可再生燃料及其相关产品的下降的全球供应，科学家一直面临的挑战是从来自植物的来源1创建类似燃料和化学品。这项工作的一个关键方面是确定哪些植物物种可以适合于生产生物燃料和生物材料2,3。通常情况下，这些原料被评估为木质素，纤维素和半纤维素含量;以及通过具有或不具有随后的酶糖化热，机械和/或化学预处理其易感性解构（顺应）。更详细的分析来确定木质素和半纤维素级分的所需的特定组合物以及最佳酶活性。植物转基因的修改不具有内在的理想性状的生化或热化学转化为所需的商品为​​研究者提供火锅大大扩展源无穷区间原料4。用于量化的植物的化学特征，而对于小样品组相当有用的标准分析方法，是不适合于数百个样品5-7或数千的快速筛选。本文所描述的HTP方法已经发展到快速和有效地评估大量的生物量的变体，以用于热化学和/或酶降解的改变细胞壁不顺应。 重要的是要理解，本文所述的HTP筛选测定没有被设计来最大化转换或收率是非常关键的。的目标是确定在相关的生物量样品的固有不顺应相对差异。其结果是，许多分析步骤不同于"典型"的生物质转化测定中，当目的是获得最大的转化率或程度。例如，低级预处理严重性和较短酶水解时间用于最大化不同样本之间的分配办法。在大多数情况下，相对高的酶负荷，来降低由于实验误差在酶的活性，这可能显著扭曲结果的差异。 用于确定植物细胞壁和单体糖的组合物快速技术解放区以下酶糖化包括机器人，定制的，热化学兼容的96孔板，以及标准实验室方法8-11的修改和器乐的协议，如振动光谱（红外（IR），近红外（NIR），拉曼或）和核磁共振（NMR）12月17日 。这些方法的关键是分离的原料具有高纤维素或低木质素含量，或那些预计产量最高的葡萄糖，木糖，乙醇等 ，这些方法都使能缩小的分析雇用小批量生物质和消耗品，导致减少的实验费用18 </s向上&gt;。这种方法途径的另一特点是，各种实验条件可以迅速，而且在某些情况下同时，进行评价。例如，各种不同的预处理策略或酶鸡尾酒进行检验，从而允许最优化的实验参数，以快速识别和采用。流行的原料，例如玉米秸秆9，杨树8,10，甘蔗渣8，和柳枝8已用这些HTP方法被成功地进行评价。 总木质素和木质素单体组合物通常还定量生物量的性状。在木质素含量的减少已被证明能增加多糖19,20的酶解。该木质素单体的比例（通常报告为紫丁香/愈创木（S / G）的含量）的角色扮演，在植物细胞壁的解构仍在进一步调查中。一些报道已表明，降低的S / G比导致了下面的21水解糖产量增加，而其他的研究揭示出相反的趋势19,22。高通量方法，用于评估木质素及其单体包括振动光谱（IR，NIR，拉曼和23-26）加上多变量分析，和热解分子束质谱（pyMBMS）27,28。 当开发HTP方法筛选生物量，有几个组成因素需要牢记。一个关键方面是该方法的复杂性。什么是该技术所需的技能级别？化学计量学分析中，例如，需要用于构造，评估和维持预测模型的特殊技能。标准的方法表现出不良的准备和数据分析的步骤或使用有毒试剂。模型的发展是一个持续的过程，其中新的数据被合并到模型中随着时间的推移，增加了模型的鲁棒性。另外consid关合作是节省成本和降低的提议的高通量方法实验分析时间。如果该方法是相当快速的，但很昂贵，这可能不是一个可行的技术许多实验室采纳。在这个手稿中所示的方法是标准化的技术，修改，以放大的吞吐能力的变体。这些协议定量测量感兴趣的生物量的性状而不需预测模型的开发。这是这些技术中，由于预测方法的关键属性，同时表现出很强的相关性的标准分析用于开发模型，是不一样精确实际测量为样品利益的量。而使用的基本上是按比例缩小的标准实验室规模的分析方法的版本的方法中，准确度和精度进行交易的速度和吞吐量。大多数情况下，这种结果是由于体积小，移液，重较高的错误;以及增加小号充足异质性样品尺寸减小。虽然大样本组进行筛选和比较，精雕细琢必须作出之间的单独活动和实验室规模的结果进行比较时，应小心。 最耗时的步骤包括生物质的物理操纵。研磨样品可能需要每个样本数分，其中包括清理出样本之间的磨。手动加载，卸载，清理料斗和填充和清空茶叶袋和样品袋也很费力。虽然每个步骤可能需要一分钟或更长，这样做成千上万的样本可能需要几个小时甚至几天的时间。机器人可以加载一个典型的反应器板与生物量在约3至4小时，或6至8天板-1机器人-1。这种情况取决于所使用以及类型和生物质的量要测试的精度参数。填充反应器板，用水，稀酸或酶迅速完成使用液体处理机器人。 P一个板叠（1〜20反应器板）的再处理需要1至3小时时组件，冷却，并拆卸是包括在内。酶水解需要3天，糖分析需要约1小时的准备时间加上每个反应器板10分钟以完成测定和读取结果。的设置预处理和分析天甲每周计划容纳一个合理的工作日程，最小化奇数小时和周末的努力用于测定的人力组成，并允许进行处理，每周〜800〜1000的样品在持续的基础。最大吞吐量取决于几个因素，主要是关于硬件（机器人，反应器板等）和多少"软件"（即，工作人员）可用做手动工作。实际的上限为2500至3000样本/周;但是，输出需要7天一个星期的操作和多个学生实习和技术人员。相比较而言，3000的样品用HPLC将需要大约125天山姆PLE分析加分析手动称量样品进入反应堆和过滤样品前额外的劳动。

<table><tbody><tr><td>Biomek FX Automated Workstation</td><td>Beckman Coulter</td><td>Biomek FX</td><td>Automated Liquid Handler</td></tr><tr><td>Wiley mill</td><td>Thomas Scientific</td><td>3375E15 (Model 4), or 3383L20 (Mini-mill)</td><td></td></tr><tr><td>anti-static bags</td><td>Minigrip*</td><td>MGST4P02503</td><td>2.5x3&quot;, multiple suppliers available</td></tr><tr><td>tin-coated copper wire</td><td>McMaster-Carr</td><td>8871K84</td><td>0.016&quot; diameter, bend-and-stay wire</td></tr><tr><td>tea-bags</td><td>Herbco</td><td>press n&#039; brew teabags</td><td>3.5x5 inches</td></tr><tr><td>gluco-amylase</td><td>Novozymes</td><td>Spirizyme Fuel&amp;nbsp;</td><td></td></tr><tr><td>alpha-amylase</td><td>Novozymes</td><td>Liquozyme SC DS</td><td></td></tr><tr><td>sodium acetate trihydrate</td><td>any chemical supplier</td><td></td><td>reagent grade</td></tr><tr><td>acetic acid</td><td>any chemical supplier</td><td></td><td>reagent grade</td></tr><tr><td>190 proof (95%) ethanol</td><td>any chemical supplier</td><td></td><td>reagent grade</td></tr><tr><td>hoppers</td><td>Freeslate</td><td></td><td></td></tr><tr><td>96-well C-276 Hastelloy plates</td><td>Aspen Machining (Lafayette, Colorado)</td><td>N/A (custom built)</td><td></td></tr><tr><td>1/8&amp;rdquo; soldering iron tip</td><td>Sears</td><td></td><td></td></tr><tr><td>silicone-adhesive backed Teflon tape</td><td>3M</td><td>5180</td><td>3&quot; wide (36-yard rolls)</td></tr><tr><td>enzyme solution</td><td>Novozymes</td><td></td><td>Cellic CTec2</td></tr><tr><td>citric acid monohydrate</td><td>any chemical supplier</td><td></td><td></td></tr><tr><td>trisodium citrate dihydrate</td><td>any chemical supplier</td><td></td><td></td></tr><tr><td>disposable, polystyrene 96-well plates</td><td>Greiner Bio-One</td><td>655101</td><td>or equivalent; multiple suppliers available</td></tr><tr><td>glucose oxidase/peroxidase&amp;nbsp;</td><td>Megazyme</td><td>K-Gluc</td><td>Megazyme D-glucose assay kit</td></tr><tr><td>xylose dehydrogenase</td><td>Megazyme</td><td>K-Xylose</td><td>Megazyme D-xylose assay kit</td></tr><tr><td>glucose standard solution</td><td>Megazyme</td><td>K-Gluc</td><td>Megazyme D-glucose assay kit</td></tr><tr><td>xylose standard solution</td><td>Megazyme</td><td>K-Xylose</td><td>Megazyme D-xylose assay kit</td></tr><tr><td>stainless steel sample cups</td><td>Frontier Laboratories</td><td>PY1-EC80F</td><td></td></tr><tr><td>glass fiber sheets</td><td>Pall</td><td>66227</td><td>8x10&quot; sheets; circles punched with standard hole punch</td></tr><tr><td>Sugarcane Bagasse Whole Biomass Feedstock</td><td>NIST</td><td>8491</td><td></td></tr><tr><td>Eastern Cottonwood (poplar) Whole Biomass Feedstock</td><td>NIST</td><td>8492</td><td></td></tr><tr><td>Monterey Pine Whole Biomass Feedstock</td><td>NIST</td><td>8493</td><td></td></tr><tr><td>Wheat Straw Whole Biomass Feedstock</td><td>NIST</td><td>8494</td><td></td></tr></tbody></table>

high-throughput screening of recalcitrance variations in lignocellulosic biomass: total lignin, lignin monomers, and enzymatic sugar release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

热化学预处理和随后的酶糖化的组合效果测量为葡萄糖和木糖的在测定结束释放的质量的函数。结果列在葡萄糖和木糖的每克生物量释放毫克方面。与此形成鲜明对比的从工作台规模的测定法，基于所述原料的组成分析其通常报告为％的理论产率报告的数据。因为它是尚未实用进行组成分析数千个每周样品，数据最好是报告为以质量计转化水平。只要这允许合理样品评价为比较是生物量密切相关?...

Watch this Scientific Journal Video about High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release at JoVE.com

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

high-throughput-screening-recalcitrance-variations-lignocellulosic

Research

JoVE Journal

Environment

9.9K 次观看.  National Renewable Energy Laboratory. Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

视频: High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

生物工程

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

轻度乙醇萃取高β-o-4 的木质素及其对解聚产率的影响

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

化学

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.

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.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

生物学

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of raw materials and energy. For these novel and sustainable biorefinery concepts, it is important to use catalysts and solvents that are aligned with the principles of Green Chemistry. Therefore, the implementation of biogenic alternatives can be a promising solution. The lignocellulose pretreatment and fractionation process presented herein-OrganoCat-is an integrated fractionation of lignocellulose into its main components using biogenic acids such as 2,5-furandicarboxylic acid as catalyst. Hemicelluloses and other non-cellulosic polysaccharides are selectively depolymerized by the diluted acid and dissolved, while the crystalline cellulose remains in the solid pulp. In the presence of a second organic phase consisting of biogenic 2-methyltetrahydrofuran, disentangled lignin is extracted in situ. The process allows for the efficient fractionation of the three main components-lignin, cellulose, and non-cellulosic sugars. This helps to focus on the quality of the lignin, the improvement of enzymatic hydrolysis of the cellulose-enriched pulp, and the mild non-cellulosic sugar extraction with low degradation.

OrganoCat is a method for the pretreatment and fractionation of lignocellulose under mild conditions into lignin, fermentable sugars, and cellulose pulp. In a biogenic, biphasic solvent system of water and 2-methyltetrahydrofuran with 2,5-furancarboxylic acid as catalyst, the OrganoCat products are separated in situ for straightforward product recovery.

使用有机催化工艺分馏木质纤维素生物质

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of ...

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

超高速利格宁从不寻常的地中海木质细胞残留物中提取

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

无标签原位成像在植物细胞壁的木质化

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

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