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
Summary
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.
Abstract
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.
Introduction
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>。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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Wiley mill | Thomas Scientific | 3375E15 (Model 4), or 3383L20 (Mini-mill) | ||
| anti-static bags | Minigrip* | MGST4P02503 | 2.5×3", multiple suppliers available | |
| tin-coated copper wire | McMaster-Carr | 8871K84 | 0.016" diameter, bend-and-stay wire | |
| tea-bags | Herbco | press n' brew teabags | 3.5×5 inches | |
| gluco-amylase | Novozymes | Spirizyme Fuel | ||
| alpha-amylase | Novozymes | Liquozyme SC DS | ||
|
any chemical supplier | reagent grade | ||
| acetic acid | any chemical supplier | reagent grade | ||
| 190 proof (95%) ethanol | any chemical supplier | reagent grade | ||
| hoppers | Freeslate | |||
| 96-well C-276 Hastelloy plates | Aspen Machining (Lafayette, Colorado) | N/A (custom built) | ||
| 1/8” soldering iron tip | Sears | |||
| silicone-adhesive backed Teflon tape | 3M | 5180 | 3" wide (36-yard rolls) | |
| enzyme solution | Novozymes | Cellic CTec2 | ||
| citric acid monohydrate | any chemical supplier | |||
| trisodium citrate dihydrate | any chemical supplier | |||
| disposable, polystyrene 96-well plates | Greiner Bio-One | 655101 | or equivalent; multiple suppliers available | |
| glucose oxidase/peroxidase | Megazyme | K-Gluc | Megazyme D-glucose assay kit | |
| xylose dehydrogenase | Megazyme | K-Xylose | Megazyme D-xylose assay kit | |
| glucose standard solution | Megazyme | K-Gluc | Megazyme D-glucose assay kit | |
| xylose standard solution | Megazyme | K-Xylose | Megazyme D-xylose assay kit | |
| stainless steel sample cups | Frontier Laboratories | PY1-EC80F | ||
| glass fiber sheets | Pall | 66227 | 8×10" sheets–circles punched with standard hole punch | |
| Sugarcane Bagasse Whole Biomass Feedstock | NIST | 8491 | ||
| Eastern Cottonwood (poplar) Whole Biomass Feedstock | NIST | 8492 | ||
| Monterey Pine Whole Biomass Feedstock | NIST | 8493 | ||
| Wheat Straw Whole Biomass Feedstock | NIST | 8494 |
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