我们要感谢台湾科技部 (104-2628-E-002-008-MY3; 105-2218-155-007; 105-2221-e-002-003-MY3; 105-2221-e-002-227-MY3; 105-2622-e-155-003-CC2) 与国立台湾顶尖大学项目的目标大学 (105R7706) 为资金支持。我们感谢世界银行通过一项高等教育质量提高项目 (HEQEP) 的分项目, #2071 的完整建议, 为这项工作提供部分资金。这项工作也得到了卧龙岗大学 AIIM (金基金) 的部分支持。

在呈现的工作中使用的几种化学物质是有毒和致癌的。在执行 IL 和生物量处理的合成时, 请使用所有适当的安全做法。
1. 酸性 IL 的制备
<ol><li>在50毫升的圆底烧瓶中加入7.625 毫摩尔 13-丙烷 sultone, 然后用橡胶隔膜关闭烧瓶。</li>
	<li>使用注射器 (10 毫升) 在0°c 添加7.625 毫摩尔 1-甲基咪唑到7.625 毫摩尔 13-propanesultone 缓慢 (1 分钟)。</li>
	<li>在完全添加 1-甲基咪唑和 13-propanesultone 后, 加入15毫升的干甲苯和回流的混合物 16 h 在120°c 得到固体两性离子。</li>
	<li>用过滤法将两性离子与甲苯分离, 然后用40毫升甲苯清洗两性离子。为烘干两性离子, 设置烤箱温度到80°c。一旦烤箱温度达到80摄氏度, 保持在烤箱中的样品为4小时, 然后使用干两性离子在下一步。</li>
	<li>用1000µL 微将硫酸加入含有两性离子 (两性离子和硫酸等摩尔) 的圆底烧瓶中。然后将圆底烧瓶连接到回流冷凝器。加热和搅拌的混合物在110°c 12 小时得到理想的 IL。 注: 硫酸与两性离子的反应不含任何溶剂。</li>
	<li>在酸性 IL 合成后, 使用1H 和13C 核磁共振波谱对其进行表征。</li>
</ol>2. 哈米特酸度的测定 (Ho)
<ol><li>在1升容积瓶中添加10毫克的p-硝基苯胺指示器, 然后添加蒸馏水, 使1升溶液。用手摇动溶液2分钟, 离开溶液1小时, 将p-硝基苯胺混合在水中 (空白溶液)。</li>
	<li>添加1.59 毫摩尔的 H + 离子的酸性催化剂 (HCl/H 2, 所以 4/酸性 IL) 到50毫升的 p-硝基苯胺指示剂溶液, 并用手摇动溶液搅拌 (样品溶液). 注意当前工作中使用的所有酸性催化剂 (HCl、H2所以4和酸性 IL) 分别添加在 50 mL 指示器解决方案 (表 1) 中, 用于确定哈米特酸度 (Ho)。</li>
	<li>执行空白溶液的 UV 测量 (p-硝基苯胺溶液) 和样品溶液 (含有p-硝基苯胺溶液的催化剂), 并确定p-硝基苯胺的安迈信。</li>
	<li>最后, 使用p-硝基苯胺的安迈信值和样例解决方案, 计算 unprotonated [I] 和质子 [.+] 指示器解决方案的摩尔浓度。然后使用2以下的公式计算Ho <img alt="Equation 1" src="/files/ftp_upload/57613/57613eq1.jpg">    等式1 其中 pk (I)蒽醌是水中p-硝基苯胺指示器的 pka (pKa = 0.99), [I] 和 [unprotonated+] 分别为摩尔浓度和质子指示器解决方案。</li>
</ol>3. 黄麻生物量分析
<ol><li>
戊聚糖分析 注: 黄麻生物量为烤箱干燥105摄氏度, 在烤箱16小时。<ol>
<li>在1升圆底烧瓶中加入3克的烘箱干黄麻生物量, 然后在其中加入100毫升的 3.85 N HCl 溶液。</li>
		<li>将烧瓶连接到蒸馏装置, 开始搅拌和加热, 使溶液开始沸腾。</li>
		<li>在含有黄麻生物量和 HCl 溶液的圆底烧瓶中加入250毫升的 3.85 N HCl 滴状。</li>
		<li>在蒸馏过程中, 通过添加 3.85 N HCl 溶液滴状, 维持恒定体积 (100 毫升) 在圆底烧瓶中。</li>
		<li>当收集220毫升馏出物时, 停止实验。然后用蒸馏水将所收集的馏分稀释到500毫升。</li>
		<li>用紫外可见光光谱仪分析样品, 并记录 280 nm 的吸光度。</li>
		<li>使用吸光度和稀释值根据以下公式确定戊聚糖%: <img alt="Equation 2" src="/files/ftp_upload/57613/57613eq2.jpg">    等式2 注: 此方法称为纸浆和造纸工业 (TAPPI) 方法的技术协会, 用于戊聚糖分析9, 19.重复实验两到三次, 取戊聚糖的平均值。如有必要, 稀释所收集的馏出物, 使吸光度达到最佳极限。</li>
	</ol>
</li>
	<li>
木质素分析 注: 去除黄麻生物量中的水分, 然后用它进行木质素分析。将黄麻的生物量保持在105摄氏度的烤箱中, 以去除湿气。<ol>
<li>将1克的黄麻生物量添加到50毫升瓶中, 然后在含有黄麻生物量的小瓶中添加15毫升 72小波% H2,4 。搅拌的混合物使用热板与搅拌设施在30°c 2 小时。</li>
		<li>在1升圆底烧瓶中加入150毫升蒸馏水, 将所消化的生物质样品 (存在于瓶子中) 转移到烧瓶中。</li>
		<li>用195毫升水冲洗瓶子, 将洗涤过的液体转移到含有消化生物量的1升圆底烧瓶中。</li>
		<li>将溶液回流4小时, 然后将圆底烧瓶冷却至室温。等待12小时的不溶木质素和灰分定居下来。</li>
		<li>用 G2 坩埚过滤溶液, 得到不溶木素与灰分。然后用150毫升的热水清洗不溶解的固体, 使其无酸性。</li>
		<li>干燥固体 (木质素 + 灰) 在60°c 为16小时在烤箱和进一步烘干它105°c 为 1 h 在烤箱。</li>
		<li>将样品保存在干燥中, 并在样品冷却时取重量。在这个阶段获得的木质素含有灰分, 因此被称为未被修正的木质素。</li>
		<li>在空气存在的情况下, 将获得的样品加热650摄氏度, 在5小时内进行灰分校正。使用下面的公式确定灰分校正: <img alt="Equation 3" src="/files/ftp_upload/57613/57613eq3.jpg">    等式3
</li>
	</ol>
</li>
</ol>4. 将戊聚糖从黄麻生物量转化为糖
<ol><li>将2克的黄麻生物量添加到高压和高温间歇反应器 (160 毫升帕尔反应器)。加60毫升水和0.24 克酸性 IL, 将温度升高到160摄氏度。</li>
	<li>将搅拌速度设置为 200 rpm, 而反应器加热高达160摄氏度。一旦达到160摄氏度温度, 增加搅拌速度到 600 rpm。</li>
	<li>继续反应为 1 h。然后, 将搅拌速度降低到 200 rpm 并停止加热。</li>
	<li>让反应堆冷却到室温。停止搅拌, 打开反应器, 从反应混合物中分离出固体。采用高效液相色谱法对反应混合物进行分析。</li>
</ol>

<ol>
	<li>Demirba&#351;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomass+resource+facilities+and+biomass+conversion+processing+for+fuels+and+chemicals.">Biomass resource facilities and biomass conversion processing for fuels and chemicals.</a> Energy Convers. Manage. 42 (11), 1357-1378 (2001).</li><li>Matsagar, B. M., Dhepe, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Br&#246;nsted+acidic+ionic+liquid-catalyzed+conversion+of+hemicellulose+into+sugars.">Br&#246;nsted acidic ionic liquid-catalyzed conversion of hemicellulose into sugars.</a> Catal. Sci. Technol. 5 (1), 531-539 (2015).</li><li>Matsagar, B. M., Dhepe, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cations,+anions+and+H++concentration+of+acidic+ionic+liquids+on+the+valorization+of+polysaccharides+into+furfural.">Effects of cations, anions and H+ concentration of acidic ionic liquids on the valorization of polysaccharides into furfural.</a> New J Chem. 41 (14), 6137-6144 (2017).</li><li>Costa Lopes, A. M., Morais, A. R. C., &#321;ukasik, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+Catalytic+Strategies+for+C5-Sugars+and+Biomass+Hemicellulose+Conversion+Towards+Furfural+Production.">Sustainable Catalytic Strategies for C5-Sugars and Biomass Hemicellulose Conversion Towards Furfural Production.</a> Production of Platform Chemicals from Sustainable Resources. , 45-80 (2017).</li><li>Matsagar, B. M., Munshi, M. K., Kelkar, A. A., Dhepe, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+concentrated+sugar+solutions+into+5-hydroxymethyl+furfural+and+furfural+using+Bronsted+acidic+ionic+liquids.">Conversion of concentrated sugar solutions into 5-hydroxymethyl furfural and furfural using Bronsted acidic ionic liquids.</a> Catal. Sci. Technol. 5 (12), 5086-5090 (2015).</li><li>G&#252;rb&#252;z, E. I., 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=Conversion+of+Hemicellulose+into+Furfural+Using+Solid+Acid+Catalysts+in+&#947;-Valerolactone.">Conversion of Hemicellulose into Furfural Using Solid Acid Catalysts in &#947;-Valerolactone.</a> Angew Chem Int Ed. 52 (4), 1270-1274 (2013).</li><li>Filiciotto, L., Balu, A. M., Van der Waal, J. C., Luque, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+insights+into+the+production+of+biomass-derived+side+products+methyl+levulinate,+furfural+and+humins.">Catalytic insights into the production of biomass-derived side products methyl levulinate, furfural and humins.</a> Catal Today. 302, 2-15 (2017).</li><li>Matsagar, B. M., 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=Direct+Production+of+Furfural+in+One-pot+Fashion+from+Raw+Biomass+Using+Br&#248;nsted+Acidic+Ionic+Liquids.">Direct Production of Furfural in One-pot Fashion from Raw Biomass Using Br&#248;nsted Acidic Ionic Liquids.</a> Sci. Rep. 7 (1), 13508 (2017).</li><li>Gschwend, F. J. V., 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=Pretreatment+of+Lignocellulosic+Biomass+with+Low-cost+Ionic+Liquids.">Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids.</a> J Vis Exp. (114), e54246 (2016).</li><li>Xu, F., 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=Transforming+biomass+conversion+with+ionic+liquids:+process+intensification+and+the+development+of+a+high-gravity,+one-pot+process+for+the+production+of+cellulosic+ethanol.">Transforming biomass conversion with ionic liquids: process intensification and the development of a high-gravity, one-pot process for the production of cellulosic ethanol.</a> Energy Environ. Sci. 9 (3), 1042-1049 (2016).</li><li>Sun, 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=One-pot+integrated+biofuel+production+using+low-cost+biocompatible+protic+ionic+liquids.">One-pot integrated biofuel production using low-cost biocompatible protic ionic liquids.</a> Green Chem. 19 (13), 3152-3163 (2017).</li><li>Nguyen, C. V., 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=Combined+treatments+for+producing+5-hydroxymethylfurfural+(HMF)+from+lignocellulosic+biomass.">Combined treatments for producing 5-hydroxymethylfurfural (HMF) from lignocellulosic biomass.</a> Catal Today. 278 (Part 2), 344-349 (2016).</li><li>Yan, N., Yuan, Y., Dykeman, R., Kou, Y., Dyson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodeoxygenation+of+Lignin-Derived+Phenols+into+Alkanes+by+Using+Nanoparticle+Catalysts+Combined+with+Br&#248;nsted+Acidic+Ionic+Liquids.">Hydrodeoxygenation of Lignin-Derived Phenols into Alkanes by Using Nanoparticle Catalysts Combined with Br&#248;nsted Acidic Ionic Liquids.</a> Angew Chem Int Ed. 49 (32), 5549-5553 (2010).</li><li>Weerachanchai, P., Lee, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recyclability+of+an+ionic+liquid+for+biomass+pretreatment.">Recyclability of an ionic liquid for biomass pretreatment.</a> Bioresour. Technol. 169 (Supplement C), 336-343 (2014).</li><li>Shill, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+liquid+pretreatment+of+cellulosic+biomass:+Enzymatic+hydrolysis+and+ionic+liquid+recycle.">Ionic liquid pretreatment of cellulosic biomass: Enzymatic hydrolysis and ionic liquid recycle.</a> Biotechnol Bioeng. 108 (3), 511-520 (2011).</li><li>Tadesse, H., Luque, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+on+biomass+pretreatment+using+ionic+liquids:+An+overview.">Advances on biomass pretreatment using ionic liquids: An overview.</a> Energy Environ. Sci. 4 (10), 3913-3929 (2011).</li><li>Agirrezabal-Telleria, I., Gandarias, I., Arias, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+furfural+from+pentosan-rich+biomass:+Analysis+of+process+parameters+during+simultaneous+furfural+stripping.">Production of furfural from pentosan-rich biomass: Analysis of process parameters during simultaneous furfural stripping.</a> Bioresour. Technol. 143 (Supplement C), 258-264 (2013).</li><li>Yingying, 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=An+Improved+Method+for+Determination+of+Pentosans+in+Pulps+using+Dual-Wavelength+Spectroscopy.">An Improved Method for Determination of Pentosans in Pulps using Dual-Wavelength Spectroscopy.</a> BioResources. 11 (3), 6801-6807 (2016).</li><li>Kumar, A. K., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+updates+on+different+methods+of+pretreatment+of+lignocellulosic+feedstocks:+a+review.">Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review.</a> Bioresour. Bioprocess. 4 (1), 7 (2017).</li><li>Kumar, P., Barrett, D. M., Delwiche, M. J., Stroeve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Pretreatment+of+Lignocellulosic+Biomass+for+Efficient+Hydrolysis+and+Biofuel+Production.">Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production.</a> Ind. Eng. Chem. Res. 48 (8), 3713-3729 (2009).</li></ol>

我们没有什么可透露的。

将黄麻生物量转化为 C5 糖单体的戊聚糖表现为使用各种均质 Brønsted 酸性催化剂, 如H2,因此4、HCl 和酸性 IL。并将酸性 il 的催化效果与无酸度的 il (1-丁基 3-咪唑氯化氯) 进行了比较。所有反应都是在160摄氏度的巴里高压釜中进行的。酸性 IL 的使用表明, 与这项工作中使用的均质酸 (矿物酸 H2, 所以4和 HCl) 相比, 戊聚糖转化率最高。结果表明, 酸性 IL 具有较高的 C5 糖产量 (76%), 而矿物酸表现出较低的产量 (HCl 49% 和 H2, 所以4 57% 的 C5 糖产量) 戊聚糖转化为糖。矿物酸催化剂和酸性 IL 用于类似的酸量 (1.59 毫摩尔的 H +), 以避免不同催化剂酸度的影响.使用非酸性 IL 和无催化剂进行的反应表明, C5 糖产量极低。这意味着酸性 IL 是更好的催化剂戊聚糖转化成糖单体相比, 矿物酸。此外, il 的酸度对于这种反应是必不可少的, 因为类似类型的非酸性 il 在这个反应中不活跃。
酸性 IL 也可用于纤维素生物量中戊聚糖的分析, 因为它产生的 C5 糖单体 (76%) 和糠醛 (12%) 的产量非常高。与使用 3.85 HCl 和较长反应时间 (ca 24 h) 的3.1 节中描述的方法相比, 此方法更优越。使用酸性 IL 获得的糖可以进一步转化为呋喃 (糠醛和各种呋喃衍生物) 或氢化成木糖醇或 arabitol。更重要的是, 使用这种方法可以恢复 C5 糖作为戊聚糖水解产物。但是, 从3.1 节中描述的方法无法恢复戊聚糖, 因为戊聚糖在集中 HCl 19 中降解为呋喃。该 ILs 有较低的蒸汽压力, 因此, 在过程中, 有减少的 IL 蒸发的可能性, 这使得这个过程环境更安全。此外, hcl 的腐蚀性和回收是 hcl 预处理20、21的主要问题。另一方面, 利用催化量的酸性 IL 在戊聚糖转化过程中可以回收。
哈米特酸度 (Ho) 结果表明, 酸性 IL 具有较高的酸强度 (ho = 1.57), 与 H2, 所以4 (Ho = 1.62);因此, 它的性能优于 H2, 因此4催化剂。然而, 酸性 IL 的酸强度比 HCl 低。然而, 它的性能优于 HCl 催化剂, 因为它有利于更好的离子偶极子相互作用的多糖, 目前在纤维素生物量2。此外, 目前工作中使用的酸性 IL 温度稳定在300摄氏度以下 (使用热重分析), 而水热法稳定低于180°c 温度 (0.6 克酸性 il 在60毫升水中加热180摄氏度) 3.
此外, 利用 Klason 法对黄麻生物量中木质素的分离进行了研究 (3.2 节)。目前工作中使用的黄麻生物量包含14个重量% 木质素。从黄麻生物量分离的木质素是纯净的, 含有较少的灰分 (< 1%), 进一步可以转化为芳香单体。
戊聚糖和木质素浓度的分析是使用矿物酸 (HCl 和H2所以4) 完成的。此外, 用于黄麻生物量戊聚糖的酸性 il 在 C5 糖 (76%) 和糠醛 (12%) 和5-10% 寡聚物的转化率上表现优异, 在水中使用少量酸性 il 进行反应, 没有任何外部压力和预处理。此外, 酸性 IL 具有超过90% 的戊聚糖转换 (戊聚糖的转换是用 C5 糖、糠醛和寡聚物的产量来计算的)。
本文提出了黄麻生物量中戊聚糖的转化为 C5 糖的方法, 但该方法也可用于黄麻生物量中戊聚糖浓度的测定。另外, 利用本方法可以确定其他各种纤维素生物量中的戊聚糖浓度。

生物量作为可再生能源和化学来源具有很大的潜力, 因为它是可持续的、廉价的、同样分布不像化石资源, 这使得它成为替代化石原料的最有希望的候选国之一。纤维素生物量的估计产量为每年1460亿公吨1。纤维素生物量主要由木质素、纤维素和半纤维素组成, 为其三主要成分。木质素是由 phenylpropanoid 单位制成的芳香聚合物;另一方面, 纤维素和半纤维素是纤维素生物量的多糖部分。纤维素由β (1→4) 糖苷链连接的葡萄糖单元组成, 而半纤维素由β (1→4)、β (1→3) 和β (1→6) 糖苷键2、3组成, 由 C5 糖、C6 糖和糖酸共同构成。与各种纤维素生物量 (蔗渣, 稻壳, 麦草,等), 黄麻木质纤维素的生物量也产生了很大数量 (ca. 98% 2014年) 在亚洲与世界黄麻总产量相比。印度生产 1.96 x 106公吨黄麻生物量, 而孟加拉国生产 1.34 x 106公吨的黄麻生物量, 与世界黄麻生物量总产量 (3.39 x 106公吨) 相比, 在 2014年4。这种非食用生物量的利用不会与粮食需求发生冲突。因此, 将其用作合成各种增值化学品 (木糖、阿拉伯糖、糠醛、5-hydroxymethylfurfural (HMF)、等) 的原料是有益的. 根据美国能源部的数据, 糠醛和 HMF 被认为是来自生物量5的前30块建筑化学物质中的一些。糠醛是从木糖或直接从半纤维素得到的, 可以转化为许多重要的化学物质。糠醇、甲基呋喃和四氢呋喃是从糠醛6中获得的重要化学物质。因此, 将纤维素生物量 (如黄麻生物量) 转化为 C5 糖和其他重要化学物质是一个重要的课题。
关于将纤维素生物量转化为增值化学品的各种催化方法, 可提供广泛的报告。矿物酸 (HCl 和 H2因此4) 和异质催化剂 (Amberlyst、HMOR、HUSY、SAPO-44、等) 被大量用于将半纤维素和纤维素生物量转化为糖 (戊和己糖依赖糖)和呋喃 (糠醛和 HMF)7,8。矿物酸的复用性和腐蚀是一个重要问题。然而, 随着固体酸催化剂的出现, 由于反应发生在催化剂表面, 因此需要较高的温度和压力。为了克服这些问题, 最近报告了测算的生物量作为催化剂或溶剂9,10,11,12,13,14。使用 IL 作为溶剂不是一个更好的方法, 因为它的成本较高, 并降低蒸汽压力的 ILs, 造成了困难的产品分离。因此, 必须在水溶剂系统中使用可回收 IL 作为催化剂 (少量), 以便将生物质转化为增值化学品。
在这里, 我们提出了使用 1-甲基 3-(3-磺丙基) 鎓硫酸氢酸性 IL 作为催化剂, 以直接转化为戊聚糖在黄麻生物量成糖单体, 没有任何预处理。通常, 纤维素生物量10、15、16、17的预处理报告了 ils, 而大量的 ils 用于生物量预处理。因此, 使用 IL 作为催化剂并将纤维素生物量转化为化学物质, 而无需任何额外的处理, 总是有利的。此外, 在目前的工作中, 用 Klason 方法计算了黄麻生物量中的木质素浓度, 可转化为各种芳香单体18。

<table>
	<tbody>
		<tr>
			<td>1-Methylimidazole</td>
			<td>Sigma Aldrich</td>
			<td>M50834</td>
			<td></td>
		</tr>
		<tr>
			<td>1,3-Propanesultone</td>
			<td>Sigma Aldrich</td>
			<td>P50706</td>
			<td>Moisture sensitive</td>
		</tr>
		<tr>
			<td>p-nitroaniline</td>
			<td>Sigma Aldrich</td>
			<td>185310</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>J. T. Baker</td>
			<td>9460-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Honeywell-Fluka</td>
			<td>30743</td>
			<td>Highly corrosive</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Honeywell-Fluka</td>
			<td>30719</td>
			<td>Highly corrosive</td>
		</tr>
		<tr>
			<td>1-butyl-3-methylimidazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>900856</td>
			<td>Highly hygroscopic</td>
		</tr>
		<tr>
			<td>D(+)-Xylose</td>
			<td>Acros Organics</td>
			<td>141001000</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Arabinose</td>
			<td>Acros Organics</td>
			<td>104981000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Spectrometer</td>
			<td>JASCO</td>
			<td>V-670</td>
			<td></td>
		</tr>
		<tr>
			<td>Parr reactor</td>
			<td>Parr USA</td>
			<td>Seriese 4560</td>
			<td></td>
		</tr>
		<tr>
			<td>Parr reactor controller</td>
			<td>Parr USA</td>
			<td>Seriese 4848</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure liquid chromatography (HPLC)</td>
			<td>JASCO</td>
			<td>Seriese LC-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital hot plate stirrer</td>
			<td>Thermo Scientific</td>
			<td>SP142020-33Q Cimarec</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven furnace</td>
			<td>Thermal Scientific</td>
			<td>FB1400 Thermolyne blast oven furnace</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel method for the pentosan analysis present in jute biomass and its conversion into sugar monomers using acidic ionic liquid

近年来, 离子液体 (ILs) 被用于生物质测算, 因为它们具有热稳定性、较低的蒸气压、不可燃性、较高的热容量、可调谐的溶解度和酸度等显著特性。通过利用 Brønsted 酸性 1-甲基-3-(3-磺丙基)-鎓硫酸氢的催化量, 我们演示了在一锅工艺中从黄麻生物量的戊聚糖中合成 C5 糖 (木糖和阿拉伯糖) 的方法。酸性 IL 是在实验室合成的, 其特点是使用核磁共振光谱技术来了解其纯度。测定了不同的保水性能, 如酸强度、热、热液稳定性等, 表明催化剂在较高温度下稳定 (250 摄氏度), 具有很高的耐酸强度 (Ho 1.57)。酸性 IL 将超过90% 的戊聚糖转化为糖和糠醛。因此, 本研究提出的方法也可用于其他纤维素生物量戊聚糖浓度的评价。

从生物量中回收的戊聚糖和木质素的确切数量取决于纤维素生物量的类型。从不同的地方收集的纤维素生物量的相似类型可以有不同浓度的戊聚糖和木质素。本研究中使用的黄麻生物量包含20个戊聚糖% 和 14 个小波% 木质素。
图 1显示了矿物酸 (H2、4和 HCl) 和酸性 IL 对黄麻生物量转化为 C5 糖的催化活性的比较。反应是在水中进行的160°c (1 小时) 使用相同的酸量的酸性催化剂 (即, 1.59 毫摩尔的 h +).非酸性 il 和酸性 il 用于类似的摩尔浓度 (0.79 毫摩尔)。催化活性与无任何 Brønsted 酸度的 IL (1 丁基 3-咪唑氯化氯) 进一步比较。
图 2阐释了本研究中使用的酸性 IL 的1H 和13C 核磁共振特征。酸性 il 的核磁共振 (1H 和13C) 的光谱显示除酸性 il 外没有额外的峰值;这证实了合成的酸性 IL 是纯的。图 3显示了在木质素分离之前黄麻生物量的 xrd 和从黄麻生物量中分离的木质素的 xrd。
表 1提供了哈米特酸度函数 (Ho) 对所有催化剂的分析。分析是使用p-硝基苯胺指示器提供的酸强度信息进行的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57613/57613fig1.jpg"> 图 1: 黄麻生物量中戊聚糖的转化为 C5 糖和糠醛。反应条件: 黄麻生物量2克, 催化剂1.59 毫摩尔 H + (il 和酸性 il 使用同摩尔即, 0.79 毫摩尔), 60 毫升水, 160 °c, 1 H <a href="/files/ftp_upload/57613/57613fig1large.jpg" target="_blank">. 请单击此处查看此图的较大</a>版本。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57613/57613fig2.jpg"> 图 2: 1H 和13C 核磁共振的酸性 IL (1-甲基-3-(3-磺丙基)-鎓硫酸氢)。<a href="/files/ftp_upload/57613/57613fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57613/57613fig3.jpg"> 图 3: x 射线衍射.(a) 黄麻生物量的 xrd 和 (b) 从黄麻生物量中提取木质素的 xrd。<a href="/files/ftp_upload/57613/57613fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>催化剂</td>
			<td>max
</td>
			<td>[I]%</td>
			<td>[+]%</td>
			<td>H0</td>
		</tr>
<tr>
<td>空白</td>
			<td>0.991</td>
			<td>100</td>
			<td>0</td>
			<td>--</td>
		</tr>
<tr>
<td>盐酸</td>
			<td>0.753</td>
			<td>76</td>
			<td>24</td>
			<td>1。5</td>
		</tr>
<tr>
<td>H2, 因此4
</td>
			<td>0。8</td>
			<td>80.72</td>
			<td>19.28</td>
			<td>1.62</td>
		</tr>
<tr>
<td>酸性 IL</td>
			<td>0.787</td>
			<td>79。4</td>
			<td>20。6</td>
			<td>1.57</td>
		</tr>
<tr>
<td>非酸性 IL</td>
			<td>0.991</td>
			<td>100</td>
			<td>--</td>
			<td>--</td>
		</tr>
</tbody></table>表 1:各种催化剂的哈米特酸度函数 (Ho) 的测定。在所有测量中, 催化剂 (1.59 毫摩尔 H +) 与50毫升的 p-硝基苯胺溶液混合在水中 (10 毫克的 p-硝基苯胺在水的1升中加入, p-硝基苯胺 = 0.99) 的 pKa.

Watch this Scientific Journal Video about A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid at JoVE.com

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

我们提出了一个协议, 以合成 C5 糖 (木糖和阿拉伯糖) 从可再生的不可食用的纤维素生物量 (即, 黄麻) 与存在 Brønsted 酸性离子液体 (捞) 作为催化剂在水中.该催化剂比传统的矿物酸催化剂具有更好的催化性能 (H2, 因此4和 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, ...

a-novel-method-for-pentosan-analysis-present-jute-biomass-its

Nanyang Technological University

Research

JoVE Journal

Environment

7.2K 次观看.  National Taiwan University. 我们提出了一个协议, 以合成 C5 糖 (木糖和阿拉伯糖) 从可再生的不可食用的纤维素生物量 (即, 黄麻) 与存在 Brønsted 酸性离子液体 (捞) 作为催化剂在水中.该催化剂比传统的矿物酸催化剂具有更好的催化性能 (H2, 因此4和 HCl)。

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

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

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.

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 ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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 ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

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 Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.