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>

<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> U.S. Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. , (2011).</li><li>Henry, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Plant Resources for Food, Fuel and Conservation. , 53-80 (2009).</li><li>Henry, R. J. <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+plant+biomass+resources+available+for+replacement+of+fossil+oil.">Evaluation of plant biomass resources available for replacement of fossil oil.</a> Plant Biotechnol. J. 8 (3), 288-293 (2010).</li><li>Furtado, 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=Modifying+plants+for+biofuel+and+biomaterial+production.">Modifying plants for biofuel and biomaterial production.</a> Plant Biotechnol. J. 12 (9), 1246-1258 (2014).</li><li>Sluiter, J. B., Ruiz, R. O., Scarlata, C. J., Sluiter, A. D., 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=Compositional+analysis+of+lignocellulosic+feedstocks.+1.+Review+and+description+of+methods.">Compositional analysis of lignocellulosic feedstocks. 1. Review and description of methods.</a> J. Agric. Food Chem. 58 (16), 9043-9053 (2010).</li><li>Lupoi, J. S., Singh, S., Simmons, B. A., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Lignocellulosic+Biomass+Using+Analytical+Spectroscopy:+an+Evolution+to+High-Throughput+Techniques.">Assessment of Lignocellulosic Biomass Using Analytical Spectroscopy: an Evolution to High-Throughput Techniques.</a> Bioenerg. Res. 7 (1), 1-23 (2014).</li><li>Lapierre, C., Monties, B., Rolando, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioacidolysis+of+lignin:+comparison+with+acidolysis.">Thioacidolysis of lignin: comparison with acidolysis.</a> J. Wood Chem. Technol. 5 (2), 277-292 (1985).</li><li>DeMartini, J. D., Studer, M. H., Wyman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+and+automatable+high-throughput+compositional+analysis+of+biomass.">Small-scale and automatable high-throughput compositional analysis of biomass.</a> Biotechnol. Bioeng. 108 (2), 306-312 (2010).</li><li>Selig, M. 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=High+throughput+determination+of+glucan+and+xylan+fractions+in+lignocelluloses.">High throughput determination of glucan and xylan fractions in lignocelluloses.</a> Biotechnol. Lett. 33 (5), 961-967 (2011).</li><li>Selig, M. 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=Lignocellulose+recalcitrance+screening+by+integrated+high-throughput+hydrothermal+pretreatment+and+enzymatic+saccharification.">Lignocellulose recalcitrance screening by integrated high-throughput hydrothermal pretreatment and enzymatic saccharification.</a> Ind. Biotechnol. 6 (2), 104-111 (2010).</li><li>Studer, M. H., De Martini, J. D., Brethauer, S., McKenzie, H. L., Wyman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+high-throughput+screening+system+to+identify+cellulosic+biomass,+pretreatments,+and+enzyme+formulations+that+enhance+sugar+release.">Engineering of a high-throughput screening system to identify cellulosic biomass, pretreatments, and enzyme formulations that enhance sugar release.</a> Biotechnol. Bioeng. 105 (2), 231-238 (2009).</li><li>Gjersing, E., Happs, R. M., Sykes, R. W., Doeppke, C., Davis, M. F. <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+sugar+content+in+biomass+hydrolysates+using+nuclear+magnetic+resonance+spectroscopy.">Rapid determination of sugar content in biomass hydrolysates using nuclear magnetic resonance spectroscopy.</a> Biotechnol. Bioeng. 110 (3), 721-728 (2013).</li><li>Templeton, D. W., Sluiter, A. D., Hayward, T. K., Hames, B. R., Thomas, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+corn+stover+composition+and+sources+of+variability+via+NIRS.">Assessing corn stover composition and sources of variability via NIRS.</a> Cellulose (Dordrecht, Netherlands). 16 (4), 621-639 (2009).</li><li>Tucker, M. P., 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=Fourier+transform+infrared+quantification+of+sugars+in+pretreated+biomass+liquors.">Fourier transform infrared quantification of sugars in pretreated biomass liquors.</a> Appl. Biochem. Biotechnol. 84-86, 39-50 (2000).</li><li>Wolfrum, E. J., Sluiter, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+multivariate+calibration+models+for+corn+stover+feedstock+and+dilute-acid+pretreated+corn+stover.">Improved multivariate calibration models for corn stover feedstock and dilute-acid pretreated corn stover.</a> Cellulose (Dordrecht, Netherlands). 16 (4), 567-576 (2009).</li><li>Ona, T., 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=Non-destructive+determination+of+wood+constituents+by+Fourier-transform+Raman+spectroscopy.">Non-destructive determination of wood constituents by Fourier-transform Raman spectroscopy.</a> J. Wood Chem. 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...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<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&#34;, multiple suppliers available</td>
		</tr>
		<tr>
			<td>tin-coated copper wire</td>
			<td>McMaster-Carr</td>
			<td>8871K84</td>
			<td>0.016&#34; diameter, bend-and-stay wire</td>
		</tr>
		<tr>
			<td>tea-bags</td>
			<td>Herbco</td>
			<td>press n&#39; brew teabags</td>
			<td>3.5x5 inches</td>
		</tr>
		<tr>
			<td>gluco-amylase</td>
			<td>Novozymes</td>
			<td>Spirizyme Fuel&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha-amylase</td>
			<td>Novozymes</td>
			<td>Liquozyme SC DS</td>
			<td></td>
		</tr>
		<tr>
			<td align="left">
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<td>sodium acetate trihydrate</td>
					</tr>
				</tbody>
			</table>
			</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&#8221; 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&#34; 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&#160;</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&#34; 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

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

科研

环境科学

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

潜在土壤胞外酶活性的高通量荧光测量

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

半高通量筛选潜在的抗干旱的生菜（<em&gt;莴苣</em&gt;）种质资源

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

木质纤维素生物质预处理低成本离子液体

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass

Ammonia fiber expansion (AFEX) is a thermochemical pretreatment technology that can convert lignocellulosic biomass (e.g., corn stover, rice straw, and sugarcane bagasse) into a highly digestible feedstock for both biofuels and animal feed applications. Here, we describe a laboratory-scale method for conducting AFEX pretreatment on lignocellulosic biomass.

氨纤维膨胀（AFEX）预处理利诺纤维素生物量

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

玉米加工参数和营养品质分析的高通量、微尺度协议 (玉蜀黍)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

从环境样品中筛选高通量的安德森菌: 植物组织、散装土壤和根际土壤

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...