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.

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The authors have nothing to disclose.

خطوات تحضير العينة الرئيسية للحصول على بيانات دقيقة وقابلة للتكرار عند إجراء الإنتاجية العالية التجارب العرض كالتالي: السكر الإصدار الفحص: بشكل عام، يتم إعداد العينات في الكثير تتراوح بين ب...

كما المعروض العالمي من الوقود غير المتجددة والتي تراجع المنتجات المرتبطة بها، وقد تحدى العلماء لخلق الوقود مماثلة والمواد الكيميائية من مصادر المشتقة من النباتات 1. أحد الجوانب الرئيسية لهذا العمل هي تحديد أي نوع من النباتات قد تكون مناسبة لإنتاج الوقود الحيوي والمواد الحيوية 2،3. عادة، يتم تقييم هذه المواد الأولية لاللجنين، السليلوز، والمحتوى هيميسيلولوز. فضلا عن قابليتها للالتفكيكية (عناد) من خلال المعالجة الحرارية والميكانيكية، و / أو كيميائية مع أو بدون لاحقة انزيم التسكير. تستخدم تحليلات أكثر تفصيلا لتحديد التشكيل المحدد لاللجنين وهيميسيلولوز الكسور وكذلك الأنشطة الإنزيمية المثلى اللازمة. وقد وفرت التعديلات المعدلة وراثيا من النباتات التي لا تملك في جوهرها الصفات المثالية لتحويل الكيمياء الحيوية أو حراري للسلع المطلوبة للباحثين مصدر توسعت بشكل كبير من وعاءالمواد الأولية ential 4. الطرق التحليلية القياسية لقياس الصفات الكيميائية للنبات، وإن كان مفيدا جدا لمجموعات عينة صغيرة، وغير مناسبة للفحص السريع لمئات أو آلاف من عينات 5-7. وقد تم تطوير أساليب بالمشاركه الموصوفة هنا بسرعة وكفاءة لتقييم أعداد كبيرة من المتغيرات الكتلة الحيوية للتغيرات في جدار الخلية عناد إلى تدهور حراري و / أو الأنزيمية. فمن الأهمية بمكان أن نفهم أن فحوصات الكشف بالمشاركه ووصفت الوثيقة لم يتم تصميمها لتحقيق أقصى قدر من التحويل أو العائد. والهدف من ذلك هو تحديد الاختلافات النسبية في عناد لا يتجزأ من عينات الكتلة الحيوية ذات الصلة. ونتيجة لذلك، فإن العديد من الخطوات التحليل تختلف عن &quot;نموذجية&quot; المقايسات تحويل الكتلة الحيوية، حيث كان الهدف هو الحصول على الحد الأقصى لمعدل التحويل أو حد. على سبيل المثال، يتم استخدام أقل الشدة المعالجة وأقصر الأوقات إنزيم التحلل المائي لتعظيم تختلفبسبب الخلافات بين العينات. في معظم الحالات، يتم استخدام شحنات انزيم عالية نسبيا للحد من الخلافات نظرا لاختلاف التجريبي في نشاط انزيم، والتي يمكن أن تؤثر على النتائج بشكل ملحوظ. تقنيات سريعة لتحديد تكوين خلايا النبات الجدران والسكريات أحادى المحررة يلي التسكير الأنزيمية تشمل الروبوتات، حسب الطلب، متوافقة thermochemically لوحات 96-جيدا، وإدخال تعديلات على أساليب المختبر القياسية 8-11 والبروتوكولات مفيدة، مثل التحليل الطيفي الذبذبات (الأشعة تحت الحمراء (IR)، القريب من الأشعة تحت الحمراء (الجرد)، أو رامان) والرنين المغناطيسي النووي (NMR) 12-17. هذه المنهجيات هي المفتاح لعزل المواد الأولية مع السليلوز عالية أو محتويات اللجنين منخفضة، أو تلك المتوقع أن يحقق أعلى الجلوكوز، الفركتوز، والإيثانول، الخ وقد مكنت هذه الأساليب التحليلات يضيق نطاق التي تستخدم كميات أقل من الكتلة الحيوية والمواد الاستهلاكية، مما يؤدي إلى تخفيضات في التجريبية حساب 18 </sتصل&gt;. ميزة أخرى لهذا النهج المنهجي هي أن ظروف تجريبية مختلفة يمكن أن يكون سريعا، وفي بعض الحالات في وقت واحد، تقييمها. على سبيل المثال، مجموعة متنوعة من مختلف استراتيجيات المعالجة أو الكوكتيلات الانزيم يمكن اختبار، والسماح للمعلمات التجريبية المثلى لتحديدها بسرعة وموظفات. المواد الأولية شعبية، مثل حطب الذرة 9، الحور 8،10، تفل قصب السكر 8، والتبن 8 تم تقييم بنجاح باستخدام هذه الأساليب بالمشاركه. مجموع اللجنين وتكوين أحادى اللجنين كما كميا عادة الصفات الكتلة الحيوية. وقد ثبت تخفيضات في محتوى اللجنين لزيادة هضم الأنزيمية من السكريات 19،20. الدور الذي لنسبة أحادى اللجنين (في كثير من الأحيان كما ذكرت syringyl / guaiacyl (S / G) المحتوى) يلعب في تفكيك جدار الخلية النباتية لا يزال قيد التحقيق. وقد أشارت بعض التقارير أن التخفيضات في S / Gأدت نسبة لزيادة الغلة الجلوكوز بعد التحلل 21، في حين أن دراسات أخرى كشف الاتجاه المعاكس 19،22. أساليب إنتاجية عالية لتقييم اللجنين وأحادية لها تشمل التحليل الطيفي الذبذبات (IR نير، ورامان 23-26) إلى جانب التحليل متعدد المتغيرات، والانحلال الحراري شعاع الجزيئي الطيفي (pyMBMS) 27،28. عند وضع أساليب بالمشاركه لفحص الكتلة الحيوية، وتحتاج عدة اعتبارات أساسية ينبغي أن يوضع في الاعتبار. واحد الجوانب الرئيسية هو تعقيد الأسلوب. ما هو مستوى المهارة المطلوب للتقنية؟ تحليلات Chemometric، على سبيل المثال، تتطلب مهارات محددة للبناء، وتقييم، والحفاظ على النماذج التنبؤية. الطرق المعيارية يحمل غير مرغوب فيها خطوات تحليل تحضيرية أو البيانات أو توظف الكواشف السامة. تطوير نماذج عملية مستمرة حيث يتم إدخال بيانات جديدة في النموذج مع مرور الوقت لزيادة متانة النموذج. consid أخرىeration هو وفورات في التكاليف وانخفاض الأوقات تحليل التجريبية من الأساليب الإنتاجية العالية المقترحة. إذا كان الأسلوب هو سريع جدا، ولكن مكلفة جدا، قد لا تكون تقنية مجدية للعديد من المختبرات لاعتماده. الأساليب هو موضح في هذه المخطوطة هي أنواع من تقنيات موحدة أو تعديل لتضخيم القدرات الإنتاجية. هذه البروتوكولات قياس كميا الصفات الكتلة الحيوية التي تهم دون يستلزم تطوير نماذج تنبؤية. هذا هو السمة الرئيسية لهذه التقنيات، منذ أساليب التنبؤ، في حين أنه يظهر الارتباطات القوية مع معيار التحليلات المستخدمة في تطوير النماذج، ليست دقيقة مثل قياس فعلا كمية من الفائدة للعينات. في حين أن الطرق المستخدمة يتم تحجيم أساسا بانخفاض إصدارات الأساليب التحليلية مقاعد البدلاء النطاق القياسية، يتم تداول الدقة والإحكام للسرعة والإنتاجية. في الغالب، هي نتيجة لأخطاء العليا في pipetting لحجم صغير ووزن هذه النتيجة؛ فضلا عن زيادة الصورةوانخفض التجانس وافرة مثل حجم العينة. بينما مجموعات عينة كبيرة يمكن فحص ومقارنة، يجب توخي الحذر الشديد عند إجراء مقارنات بين حملات منفصلة ونتائج مقاعد البدلاء النطاق. وتشمل معظم الخطوات تستغرق وقتا طويلا التلاعب المادي من الكتلة الحيوية. عينات طحن قد يستغرق عدة دقائق لكل عينة، بما في ذلك تنظيف مطحنة بين العينات. تحميل يدويا والتفريغ، والنطاط تنظيف وتعبئة وتفريغ أكياس الشاي وأكياس عينة هي أيضا عمل مكثفة جدا. في حين أن كل خطوة قد يستغرق دقيقة واحدة أو أكثر، والآلاف فعل عينات قد يستغرق عدة ساعات أو حتى أيام. يمكن الروبوتات تحميل لوحة مفاعل نموذجية مع الكتلة الحيوية في حوالي 3-4 ساعة أو 6-8 لوحات اليوم -1 الروبوت -1. هذا الوضع يعتمد على معايير الدقة المستخدمة وكذلك نوع وكمية الكتلة الحيوية لفحصها. ملء وحات مفاعل بالماء، وتمييع حامض، أو يتم الإنزيم بسرعة باستخدام الروبوت معالجة السائل. Pإعادة معالجة من كومة وحة (1-20 وحات مفاعل) ما يستغرق ما بين 1 و 3 ساعة عندما التجمع، ويبرد، وتضمنت التفكيك. التحلل انزيم تستغرق 3 أيام ويتطلب تحليل السكر حوالي 1 ساعة من الوقت الإعدادية بالإضافة إلى 10 دقيقة لكل لوحة مفاعل لاستكمال فحص وقراءة النتائج. جدول الأسبوعي لمجموعة المعالجة والتحليل أيام يستوعب جدول أعمال معقول، والتقليل من الفردية ساعة وعطلة نهاية الأسبوع الجهود لعنصر البشري في الفحص ويسمح للتجهيز ~ 800 إلى 1000 عينة في الأسبوع على أساس مستمر. الحد الأقصى للسرعة تعتمد على عدة عوامل، أهمها كيف هم الكثير من الأجهزة (الروبوتات، المفاعلات لوحات، الخ)، وكيف الكثير من &quot;البرمجيات&quot; (أي الموظفين) المتاحة للقيام بهذا العمل اليدوي. الحد الأعلى العملي هو 2500 إلى 3000 عينات / أسبوع؛ مع ذلك، أن الناتج يتطلب عملية 7 أيام في الأسبوع والطلاب المتدربين متعددة والفنيين. وبالمقارنة، فإن 3000 العينات بواسطة HPLC تتطلب ما يقرب من 125 يوما من سامتحليل التنوير القائل بالإضافة إلى العمل الإضافي وزنها يدويا العينات في المفاعلات وعينات الترشيح قبل التحليل.

<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.

يتم قياس التأثير المشترك للالمعالجة الحرارية واللاحق انزيم التسكير بوصفها وظيفة من كتلة الجلوكوز والفركتوز صدر في نهاية الفحص. يتم الإبلاغ عن النتائج من حيث ملليغرام من الجلوكوز والفركتوز صدر في كل غرام من الكتلة الحيوية. هذا هو في تناقض صارخ مع البيانات الواردة من ...

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.

الإنتاجية العالية فحص الاختلافات الإستعصاء في Lignocellulosic الكتلة الحيوية: إجمالي اللجنين، اللجنين مونمرات، والأنزيمية السكر الإصدار

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 Views.  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.

Video: 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.

تقرير من خارج الخلية الميكروبية نشاط انزيم في المياه، والتربة، والرواسب باستخدام إنتاجية عالية صفيحة ميكروسكوبية فحوصات

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

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.

الإنتاجية العالية فلوروميتريك قياس التربة المحتملة خارج الخلية أنزيم الأنشطة

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.

شبه عالية الإنتاجية لفحص إمكانية الجفاف التسامح في الخس (<em&gt; خس ساتيفا</em&gt;) مجموعات الأصول الوراثية

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.

المعالجة من Lignocellulosic الكتلة الحيوية مع منخفضة التكلفة أيوني السوائل

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.

الأمونيا الألياف التوسع (AFEX) المعالجة المسبقة للالكتلة الحيوية Lignocellulosic

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).

طريقة جديدة لتحليل بينتوسان الموجودة في الكتلة الأحيائية الجوت وتحويلها إلى أحادية السكر باستخدام السوائل الأيونية الحمضية

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.

الفائق، بروتوكول Microscale لتحليل المعالجة البارامترات والصفات الغذائية في الذرة (ميس زيا ل.)

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-الأبعاد (3D)--طباعة قالب للجنين الزرد الإنتاجية العالية أرايينج

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.

مقايسة الفائق "التنبؤ بالسمية الكيميائية" "الآلي التنميط المظهرية" من ايليجانس كاينورهابديتيس

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.

الفحص سيديروفوري الفائق من العينات البيئية: زراعة الأنسجة، والتربة السائبة، وتربة Rhizosphere

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