Name: pretreatment of lignocellulosic biomass with low-cost ionic liquids
Uploaded: 2016-08-10T13:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids at JoVE.com

The authors acknowledge the Grantham Institute for Climate Change and the Environment, Climate-KIC and EPSRC (EP/K038648/1 and EP/K014676/1) for funding and Pierre Bouvier for providing experimental data for pine pretreatments.

Note: The protic ionic liquids used in the process are synthesized in our laboratory, although some might be or become commercially available. The resulting ionic liquids are acidic and corrosive and probably skin/eye irritants (depending on the amine used), and must therefore be handled with care wearing appropriate PPE (lab coat, safety specs, resistant gloves).
1. Preparation
<ol>
	<li>Preparing and storing the lignocellulosic biomass
	<ol>
		<li>Obtain the lignocellulosic biomass prior to the experiment in sufficient quantities, for example 100 g up to 5 kg. 
		Note: Each experiment requires at least 3 g of biomass (1 g each in triplicate).</li>
		<li>Reduce the moisture content shortly after harvest and store the biomass in air-dried form in the laboratory. Air-dry the biomass by spreading the biomass on a table or bench and leaving it for 2 weeks or until it appears dry. Move and turn the biomass during this time to accelerate the process. Perform the fractionation experiments within a year of harvesting. 
		Note: Directly after harvesting, woody biomass can contain up to 50 wt% moisture, while air-dried biomass, which is more stable, contains 5-12 wt% moisture.</li>
		<li>Grind and sieve the biomass to a select particle size range. Store the dried biomass in plastic bags or other suitable containers until use. 
		Note: For the small samples handled in this protocol, a reduced particle size is recommended, for example 180-850 &#181;m.</li>
	</ol>
	</li>
	<li>Determining the moisture content of the biomass (according to the NREL protocol)20
	<ol>
		<li>To determine the moisture content of the biomass, preweigh a piece of aluminum foil (size approximately 5 cm x 5 cm) on an analytical balance and record the weight of the foil (mfoil). Weigh out roughly 100 mg of air dried biomass onto the aluminum foil and record the exact air-dried weight (mADW).</li>
		<li>Fold the aluminum foil to make a packet and place in a fan-assisted oven at 105 &#176;C overnight (at least for 4 hr).</li>
		<li>Take the packet out and place it immediately in a desiccator for 5 minutes, then weigh the packet immediately and record the exact weight of oven-dried weight plus foil (mODW+foil). Calculate the moisture content (in %) of the biomass mcBM according to equation 1: 
		<img alt="Equation 1" src="/files/ftp_upload/54246/54246eq1.jpg" /> &#160; &#160; &#160;&#160; Eq. 1 
		Where mODW+foil is the weight of the oven-dried packet (oven-dried biomass plus foil), mfoil is the weight of the foil and mADW is the air-dried weight of the biomass. All weights should be either in g or in mg.</li>
	</ol>
	</li>
	<li>Synthesis of Ionic Liquid
	<ol>
		<li>In a fume hood or vented enclosure, weigh 1 mole of the amine (triethylamine) into a 1 L round bottom flask with magnetic stir bar. Place the flask in an ice bath on a magnetic stirrer plate. Add a 250 ml addition funnel immediately to minimize evaporation of the amine. 
		Note: Ensuring the correct acid:base ratio is of high importance for achieving reproducibility of the pretreatment experiments.</li>
		<li>Measure out 1 mole of sulfuric acid using a solution of known concentration (in this example 5 mol/L) and a volumetric flask (200 ml). Transfer the sulfuric acid into the addition funnel and rinse any acid adhering to the walls of the volumetric flask into the addition funnel with deionized water.</li>
		<li>Add the sulfuric acid dropwise to the amine while stirring vigorously. Make sure the solution does not heat up, as this would lead to boiling of the amine and an inaccurate ratio of acid to base. Rinse the inside of the addition funnel using deionized water to ensure the acid is transferred quantitatively. 
		Note: Larger batches of ionic liquid solution may be made by increasing the amount of amine and sulfuric acid as well as the flask volume accordingly.</li>
		<li>Evaporate most of the water using a rotary evaporator. The water content should be lower than the water content required for the pretreatment. 
		Note: It is not necessary to completely dry the ionic liquid. It may be beneficial to leave some water in the ionic liquid, as some dried ionic liquids, including triethylammonium hydrogen sulfate, are solids at room temperature. A freeze dryer can also be used to lower the water content.</li>
	</ol>
	</li>
	<li>Confirming and adjusting the Water Content of the IL solution 
	Note: The water content is an important experimental variable. There are three sources that the water in the pretreatment mixture may come from. All of them need to be taken into account: (1) the water contained in the synthesized or bought ionic liquid solution (2) the water contained in the air-dried biomass and (3) any water added with a pipette to achieve the final desired water content.
	<ol>
		<li>Determine the water content of the synthesized or purchased ionic liquid solution by volumetric Karl Fischer titration according to instructions issued by the titrator manufacturer. Add a few drops of the IL into the titrator using a pre-weighed syringe. Enter the weight of liquid added into the titrator and wait until the titrator displays a reading. Record the water content.</li>
		<li>Decide on a water content for the biomass pretreatment. In this experiment, use 20 wt%. Reduce the water content 5 wt% below the desired water content using a rotary evaporator and confirm the new water content by Karl Fischer titration as described in 1.4.1. 
		Note: Good results are obtained with 20 wt% water; however this may not always be the optimum for pretreatment. A higher water content may be chosen in order to reduce solvent cost or to lower viscosity.</li>
	</ol>
	</li>
	<li>Calculations for the experiment.
	<ol>
		<li>Decide on the amount of ionic liquid solution msol,final and a biomass-to-solvent ratio BM/solfinal. Here, use 10 g of ionic liquid solution containing 20 wt% water and a biomass-to-solvent ratio of 1:10 g/g. 
		Note: The tubes used in this protocol can fit up to 18 g of IL solution if the biomass to solvent ratio is 1:10 (wt/wt). A high biomass-to-solvent ratio (up to 1:2 or even 1:1) is favorable from an economic point of view but might compromise the pretreatment efficacy at small scale.</li>
		<li>Determine the amount of (water-free) ionic liquid required for each sample according to the following equation 2: 
		<img alt="Equation 2" src="/files/ftp_upload/54246/54246eq2.jpg" />&#160;&#160;&#160;&#160;&#160; Eq. 2 
		Where mIL is the required amount of ionic liquid in grams, msol,final is the desired amount of ionic liquid solution in grams, and wcfinal is the desired water content (in %) in the ionic liquid solution.</li>
		<li>Next, calculate the amount of synthesized or bought ionic liquid solution to be added into each pressure tube according to the following equation 3: 
		<img alt="Equation 3" src="/files/ftp_upload/54246/54246eq3.jpg" />&#160;&#160;&#160;&#160;&#160; Eq. 3 
		Where msol is the amount of solution to be added into each pressure tube (in grams), mIL is the required amount of ionic liquid (in grams), and wcsol is the water content in the ionic liquid solution (in %) as determined by Karl Fischer titration.</li>
		<li>Determine the amount of biomass (oven-dried weight basis) to be added to the ionic liquid solution using equation 4 below. In this experiment, use 1 g of oven-dried Miscanthus biomass per pressure tube. 
		<img alt="Equation 4" src="/files/ftp_upload/54246/54246eq4.jpg" /> &#160; &#160;&#160; Eq. 4 
		Where ODW is the amount of biomass to be added into each pressure tube (in grams), msol,final is the desired amount of ionic liquid solution (in grams) and BM/solfinal is the desired ratio of biomass-to-ionic liquid solution.</li>
		<li>Determine the weight of air-dried biomass that needs to be added into the tube by using the following equation 5: 
		<img alt="Equation 5" src="/files/ftp_upload/54246/54246eq5.jpg" />&#160;&#160;&#160;&#160;&#160; Eq. 5 
		where ADW is the air-dried weight of the biomass to be added into the tube (in grams), ODW is oven-dried weight of the biomass required for the experiment (determined in 1.5.4 in grams) and mcBM is the value determined in 1.2.3.</li>
		<li>Calculate how much water needs to be added with a pipette to achieve the desired final water content according to the following equation 6: 
		<img alt="Equation 6" src="/files/ftp_upload/54246/54246eq6.jpg" />&#160;&#160;&#160;&#160;&#160; Eq. 6 
		Where mwater is the amount of water to be added, wcfinal is the desired water content in the pretreatment mixture (here 20 wt%), msol.final is the amount of solvent (here 10 g), mcBM is the moisture content of the air-dried biomass, mBM is the amount of air-dried biomass to be added, wcsol is the water content of the synthesized or purchased ionic liquid solution, and msol is the amount of ionic liquid solution.</li>
	</ol>
	</li>
</ol>
2. Pretreatment
Note: The process may be interrupted at any point by leaving the samples at room temperature (for a few days) or in the refrigerator (for longer periods).
<ol>
	<li>Pre-weigh three 15 ml pressure tubes with Teflon caps and silicone O rings. Visually inspect the pressure tubes to ensure they have no cracks or flaws. 
	Note: Larger pressure tubes fitting more ionic liquid and biomass can be used if desired, although pretreatment results will not be directly comparable between samples treated in different vial sizes. We recommend the use of front-sealing caps for better sealing.</li>
	<li>With a 10 ml pipette, add the required amount of ionic liquid solution into the pressure tube standing on the scale. Use cork rings to keep the tube standing up. Record the weight of the ionic liquid solution added. Add the required amount of water to the solution determined in 1.5.6 using a pipette, assuming the density of water to be 1 g/ml.</li>
	<li>Add the required amount of air-dried lignocellulose by placing a piece of aluminum foil (dimensions 3 cm x 8 cm) on a balance, taring the balance and weighing out the biomass. Tare the balance and add the biomass to the tube. Place the empty foil back on the balance and record the difference. 
	Note: Alternatively, antistatic weighing boats may be used.</li>
	<li>Close the lid with a Teflon cap with silicone O-ring. Check for a good seal without over-tightening. Record the weight of the pressure tubes containing biomass and ionic liquid. Mix the contents of the tube by using a vortex shaker until all of the biomass is in contact with the ionic liquid. 
	Note: Not all O-ring materials withstand being in contact with ionic liquids at elevated temperature. We found that silicone works well.</li>
	<li>Place the pressure tubes in a fan-assisted oven that has been pre-heated to the desired temperature.&#160; For example, leave the tubes for 8 hr at 120 &#176;C or for 1 hr at 150 &#176;C. 
	Note: Time and temperature are important experimental variables. Other time &#8211; temperature combinations can be used.</li>
	<li>Remove the vials from the oven using an oven glove and place them in a fume hood to let them cool to room temperature. Check the weight of the vials after cooling to assure no water escaped during cooking.</li>
</ol>
3. Pulp Wash
<ol>
	<li>Transfer the contents of each tube into a 50 ml centrifuge tube using 40 ml of absolute ethanol. Shake the tube using a vortex shaker for 1 min to mix well and leave the tube at room temperature for at least 1 hr. 
	Note: The separation can also be carried out using filtration, however, less accuracy may be observed for the suggested sample size.</li>
	<li>Shake the tube using a vortex shaker for another 30 sec, then centrifuge the tube for 50 min at 2,000 x g. Separate liquid and solid by careful decanting. Collect the liquid in a clean 250 ml round bottom flask with stir bar.</li>
	<li>Add 40 ml fresh ethanol into the 50 ml tube and repeat step 3.2 three times.
	<ol>
		<li>Remove the ethanol from the ionic liquid by placing the round bottomed flasks on a heating block. Connect each of them to a vacuum pump with a cold trap. Fill the trap with dry ice and set the heating to 40 &#176;C. Switch on stirring and pump. 
		Note: This experiment uses a home-made parallel evaporator set-up based on a parallel synthesis set-up. Parallel evaporators can also be purchased ready-made. Alternatively a rotary evaporator can be used.</li>
	</ol>
	</li>
</ol>
4. Soxhlet Extraction of Pulp
<ol>
	<li>Transfer the wet washed pulp into cellulose thimbles and label each thimble using a pencil.</li>
	<li>Fill 150 ml absolute ethanol into a clean 250 ml round-bottomed flask with stir bar. Insert the sample-containing thimble into a 40 ml Soxhlet extractor, add a condenser and install everything on the parallel extractor work station connected to a recirculating chiller. 
	Note: If ethanol was used to transfer the pulp, add only the difference to 150 ml to the round-bottomed flask.</li>
	<li>When all samples are loaded, begin stirring, set the temperature to 135 &#176;C and turn on the recirculator (temperature setting 18 &#176;C). Extract the pulp samples for 20 hr in total.</li>
	<li>Turn off the heating to allow refluxing to come to a halt. Then switch both stirring and cooling off. Take the thimbles out of the Soxhlet extractor using tweezers and let the wet pulp dry in the thimble overnight in a fume hood.</li>
	<li>Add the liquid from the Soxhlet extraction to the liquid from the biomass wash and continue evaporating the ethanol from the biomass wash with the parallel evaporator or a rotary evaporator at 40 &#176;C.</li>
	<li>Transfer the air-dried pulp from the thimble onto a piece of tared aluminum foil on an analytical balance, record the air-dried weight of the extracted pulp and transfer it into a labelled plastic bag. Try to recover everything while not scraping thimble material off the wall.</li>
	<li>Determine the moisture content of the pulp immediately to calculate the oven-dried yield (as shown previously in step 1.4).</li>
</ol>
5. Lignin Isolation
<ol>
	<li>When all ethanol has evaporated, precipitate the lignin by transferring the ionic liquid from the round-bottom flask into a 50 ml centrifuge tube using 30 ml of water. Mix the suspension and leave for at least 1 hr. Centrifuge for 20 min at 2,000 x g, and separate the solution from the solid by decanting. 
	Note: In this protocol, 3 equivalents of water are used as antisolvent. Less antisolvent can be used, if desired. The washes can be collected, the water removed and the ionic liquid recovered for repeat use.</li>
	<li>Add 30 ml of distilled water to the lignin pellet inside the centrifuge tube.&#160; Repeat the mixing, incubation for 1 hr and centrifugation. Repeat this step for a total of 3 lignin washes.</li>
	<li>Dry the lignin inside the centrifuge tube using a vacuum oven and a pierced lid at 45 &#176;C overnight. To determine the lignin yield, place a piece of aluminum foil on the balance, tare the weight, add lignin from the oven and record the weight immediately. Transfer the lignin into a vial for storage.</li>
</ol>

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The authors have nothing to disclose.

The technique for the fractionation of lignocellulosic biomass presented here produces a cellulose-rich pulp and a lignin. Most of the hemicelluloses are dissolved into the ionic liquid and hydrolyzed, but not recovered. If hemicellulose sugars are desired, a hemicellulose pre-extraction step prior to the Ionosolv delignification may be necessary. It has so far been impossible to fully close the mass balance for the biomass, as it is not possible to identify and quantify all degradation products found in the ionic liquid...

Meeting humanity&#39;s energy demand sustainably is one of the greatest challenges that our civilization faces. Energy use is predicted to double in the next 50 years, putting greater strain on fossil fuel resources. 1 The buildup of greenhouse gases (GHG) in the atmosphere through wide-spread fossil fuel use is particularly problematic, as CO2 generated from combustion of fossil fuels is responsible for 50% of the anthropogenic greenhouse effect. 2 Therefore, large-scale application of renewable and carbon neutral technologies is essential for meeting the increased energy and material needs of future generations. 1, 3
Plant biomass is the most versatile renewable resource, as it can be used to produce heat, electricity as well as carbon-based chemicals, materials and fuels. Primary advantages of lignocellulosic biomass over other biomass types are its abundance, potential for high yields per area of land and often much higher CO2 emission savings, which includes high retention of carbon in the soil. 4, 5 Additional benefits of using biomass include local availability, low capital requirements to convert biomass to energy, and soil erosion prevention. 8
Major producers of lignocellulosic feedstocks are the forestry industry and the agricultural sector as well as municipal waste management. 6 Lignocellulose production has the potential to be expanded, with a mind to limiting deforestation and avoiding the replacement of food crops and release of potential pollutants. 7 For renewable biomass to become a viable widespread source of liquid transportation fuels and chemicals, its processing must become economically competitive with fossil fuel conversion technologies. 9, 10 A key to achieving this is to boost the yield and quality of biomass-derived intermediates while reducing cost.
Lignocellulose contains a high proportion of sugars which can be converted to fuels and chemicals via catalytic and microbial conversions. 11 These sugars are present in lignocellulose in polymeric form as cellulose and hemicellulose. They can be hydrolyzed into glucose and other sugar monomers and then used for producing bioethanol and other bio-derived chemicals and solvents. 12
In order to access the cellulosic sugars, pretreatment of the biomass is necessary through physical, chemical, or combined processes. 4 The pretreatment is arguably the most costly step in the valorization of lignocellulosic biomass. Hence research into improved pretreatment processes is imperative.
Various pretreatment technologies are available. Of particular interest are those that separate the lignin from cellulose (fractionative pretreatment). Lignin, the third major component in lignocellulose, limits access of hydrolyzing agents to cellulose and hemicellulose and reduces the sugar yield per ton of feedstock. 11 The separated lignin can be utilized as an additional biorefinery intermediate if it is isolated in suitable quality. 13 One fractionative process is the Kraft process which is the most common pretreatment for paper/cellulose production. In Kraft pulping, wood chips are placed in a mixture of sodium hydroxide and sodium sulfide and heated at elevated temperatures of around 170 &#176;C under high pressure. 14 The alkaline reactions remove hemicellulose and lignin by breaking the polymers down to short fragments via nucleophilic and base catalysis, and by dissolving the lignin fragments via de-protonation of phenolic hydroxyl/alcohol groups. Another common delignification process is the Organosolv process which also fragments and dissolves the lignin and hemicellulose. Rather than using an alkaline aqueous solution, organic solvents such as ethanol and acetic acid are used at high temperatures ranging between 160-200 &#176;C and pressures from 5-30 bar. Organosolv pretreatment has some advantages over Kraft pulping in that it produces less air and water pollution. 15 Both processes possess some economic challenges, if used for production of chemicals and fuels rather than cellulose. 16 The Ionosolv pretreatment uses ionic liquids, which are salts that have melting points below 100 &#176;C and, as a result of their powerful Coulombic interactions, very low vapor pressures. 17 This eliminates air pollution in the pretreatment process, and enables processing at or near atmospheric pressure.
While most ILs are created in laborious, multi-step syntheses, protic ILs can be synthesized in a one-step process from commodity chemicals, which makes them less expensive; it is estimated that some ILs could be produced at bulk scale for a price of $1.24 per kg which is comparable to common organic solvents such as acetone and toluene. 18 The ability to recycle and reuse these customizable ILs in a process that operates at comparatively lower temperatures and pressures makes this a more benign alternative and an economically attractive candidate for biorefining.
This detailed video protocol demonstrates a lab-scale version of the Ionosolv process for the delignification of lignocellulosic biomass and the eventual enzymatic saccharification of the cellulose-rich pulp as well as the recovery of a high-purity odor-free lignin.19

<table><tbody><tr><td colspan="4">&lt;strong&gt;IL synthesis&lt;/strong&gt;</td></tr><tr><td>Round bottom flask, with standard ground joint 24/29 NS, 1,000 ml</td><td>Lenz</td><td>3 0024 70</td><td>VWR product code 271-1309</td></tr><tr><td>250 ml Addition Funnel, Graduated, 29/26 Joint Size, 0-4&amp;nbsp;mm PTFE Valve</td><td>GPE</td><td>CG-1714-16</td><td></td></tr><tr><td>Dish-shaped dewar flask, SCH 31 CAL&amp;nbsp;</td><td>KGW-Isotherm</td><td>1197</td><td></td></tr><tr><td>Volumetric flask, 200 ml</td><td>VWR</td><td>612-3745&amp;nbsp;</td><td></td></tr><tr><td>Cork rings, pasteur pipettes and teet, wash bottle with deionised water, large magentic stir bar</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Biomass size reduction&lt;/strong&gt;</td></tr><tr><td>Heavy Duty Cutting Mill SM2000&amp;nbsp;</td><td>Retsch&amp;nbsp;</td><td>Discontinued</td><td>Replaced with Cutting Mill SM 200 (20.728.0001)&amp;nbsp;</td></tr><tr><td>Bottom sieves (10 mesh square holes, for particle size &amp;lt;2 mm)</td><td>Retsch&amp;nbsp;</td><td>03.647.0318</td><td>Part of cutting mill</td></tr><tr><td>Analytical Sieve Shaker AS 200</td><td>Retsch&amp;nbsp;</td><td>30.018.0001</td><td>Part of sieving machine</td></tr><tr><td>Test Sieve 200 mm &amp;Oslash; x 50 mm height ISO 3310/1 (180 &amp;micro;m)</td><td>Retsch&amp;nbsp;</td><td>60.131.000180</td><td>Part of sieving machine</td></tr><tr><td>Test Sieve 200 mm &amp;Oslash; x 50 mm height ISO 3310/1 (850 &amp;micro;m)</td><td>Retsch&amp;nbsp;</td><td>60.131.000850</td><td>Part of sieving machine</td></tr><tr><td>Collecting pan, stainless steel, 200&amp;nbsp;mm &amp;Oslash;, height 50 mm</td><td>Retsch&amp;nbsp;</td><td>69.720.0050</td><td>Part of sieving machine</td></tr><tr><td colspan="4">&lt;strong&gt;Rotary evaporator&lt;/strong&gt;</td></tr><tr><td>Rotary evaporator (Rotavapor R-210)</td><td>Buchi&amp;nbsp;</td><td>Discontinued</td><td>Replaced with Rotavapor R-300</td></tr><tr><td>Water bath (Heating bath B-491)</td><td>Buchi&amp;nbsp;</td><td>48201</td><td>Part of rotary evaporator</td></tr><tr><td>Recirculator&amp;nbsp;</td><td>Julabo</td><td>F25</td><td>Part of rotary evaporator</td></tr><tr><td>Vacuum pump (MPC 101 Z)</td><td>Ilmvac GmbH</td><td>412522</td><td>Part of rotary evaporator</td></tr><tr><td>Vacuum controller (Vacuum Control Box VCB 521)</td><td>Ilmvac GmbH</td><td>600053</td><td>Part of rotary evaporator</td></tr><tr><td colspan="4">&lt;strong&gt;Parallel evaporator&lt;/strong&gt;</td></tr><tr><td>StarFish Base Plate 135&amp;nbsp;mm (for Radleys &amp;amp; IKA)</td><td>Radleys</td><td>RR95010</td><td>Part of parallel evaporator</td></tr><tr><td>Monoblock for 5 x 250&amp;nbsp;ml Flasks</td><td>Radleys</td><td>RR95130&amp;nbsp;</td><td>Part of parallel evaporator</td></tr><tr><td>Telescopic 5-way Clamp with Velcro</td><td>Radleys</td><td>RR95400</td><td>Part of parallel evaporator</td></tr><tr><td>Gas/Vacuum Manifold with connectors</td><td>Radleys</td><td>RR95510&amp;nbsp;</td><td>Part of parallel evaporator</td></tr><tr><td>650&amp;nbsp;mm Rod</td><td>Radleys</td><td>RR95665&amp;nbsp;</td><td>Part of parallel evaporator</td></tr><tr><td>Quick Release Male, R/A Barbed 6.4&amp;nbsp;mm + Shut-off (3.2&amp;nbsp;mm ID)</td><td>Radleys</td><td>RR95520</td><td>Part of parallel evaporator</td></tr><tr><td>Stirrer/hot plate</td><td>Radleys</td><td>RR98072</td><td>Part of soxhlet extractor</td></tr><tr><td>Temperature controller</td><td>Radleys</td><td>RR98073</td><td>Part of soxhlet extractor</td></tr><tr><td>Elliptical Stirring Bar 15&amp;nbsp;mm Rare Earth</td><td>Radleys</td><td>RR98097&amp;nbsp;</td><td>Part of parallel evaporator</td></tr><tr><td>Vacuum cold trap, plastic coated, PTFE stopcock</td><td>Chemglass</td><td>CG-4519-01</td><td>Part of parallel evaporator</td></tr><tr><td>Vacuum pump (MPC 101 Z)</td><td>Ilmvac GmbH</td><td>412522</td><td>Part of parallel evaporator</td></tr><tr><td>Tygon tubing E-3603, 6.40&amp;nbsp;mm (internal) 12.80&amp;nbsp;mm (external)</td><td>Saint-Gobain/VWR</td><td>228-1292&amp;nbsp;</td><td>Part of parallel evaporator</td></tr><tr><td colspan="4">&lt;strong&gt;Parallel Soxhlet extractor&lt;/strong&gt;</td></tr><tr><td>StarFish Base Plate 135&amp;nbsp;mm (for Radleys &amp;amp; IKA)</td><td>Radleys</td><td>RR95010&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Monoblock for 5 x 250&amp;nbsp;ml Flasks</td><td>Radleys</td><td>RR95130&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Telescopic 5-way Clamp with Velcro</td><td>Radleys</td><td>RR95400&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Telescopic 5-way Clamp with Silicone Strap and Long Handle</td><td>Radleys</td><td>RR95410&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Water Manifold with connectors</td><td>Radleys</td><td>RR95500&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>650&amp;nbsp;mm Rod</td><td>Radleys</td><td>RR95665&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Quick Release Male, R/A Barbed 6.4&amp;nbsp;mm + Shut-off (3.2&amp;nbsp;mm ID)</td><td>Radleys</td><td>RR95520&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Coil condensers with standard ground joints 29/32 NS</td><td>Lenz</td><td>5.2503.04&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Extractor Soxhlet 40&amp;nbsp;ml borosilicate glass 29/32 socket 24/29 cone</td><td>Quickfit</td><td>EX5/43&amp;nbsp;</td><td>Part of soxhlet extractor</td></tr><tr><td>Stirrer/hot plate</td><td>Radleys</td><td>RR98072</td><td>Part of soxhlet extractor</td></tr><tr><td>Temperature controller</td><td>Radleys</td><td>RR98073</td><td>Part of soxhlet extractor</td></tr><tr><td>Recirculator</td><td>Grant</td><td>LTC1</td><td>Part of soxhlet extractor</td></tr><tr><td>Cellulose extraction thimble</td><td>Whatman</td><td>2280-228</td><td></td></tr><tr><td>Tweezers</td><td>Excelta</td><td>20A-S-SE</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Vacuum drying oven&lt;/strong&gt;</td></tr><tr><td>Vacuum drying oven</td><td>Binder</td><td>VD 23</td><td>Part of vacuum oven</td></tr><tr><td>Dewar vessel 2&amp;nbsp;L 100&amp;nbsp;x&amp;nbsp;290&amp;nbsp;mm with handle</td><td>KGW-Isotherm</td><td>10613</td><td>Part of vacuum oven</td></tr><tr><td>Vacuum Trap</td><td>GPE</td><td>CG-4532-01&amp;nbsp;</td><td>Part of vacuum oven</td></tr><tr><td colspan="4">&lt;strong&gt;Other equipment&lt;/strong&gt;</td></tr><tr><td>Analytical balance</td><td>A&amp;amp;D</td><td>GH-252</td><td>accuracy to &amp;plusmn; 0.1 mg</td></tr><tr><td>Volumetric Karl Fischer titrator</td><td>Mettler Toledo</td><td>V20</td><td></td></tr><tr><td>10 ml disposable pipette</td><td>Corning Inc</td><td>Costar 4101 10 mL Stripette</td><td></td></tr><tr><td>Eppendorf Research plus pipette, variable volume, volume 100-1,000&amp;nbsp;&amp;mu;l</td><td>Eppendorf</td><td>3120000062</td><td></td></tr><tr><td>Desiccator</td><td>Jencons</td><td>JENC250-028BOM</td><td></td></tr><tr><td>Ace pressure tube bushing type, Front seal, volume 15&amp;nbsp;ml</td><td>Ace Glass</td><td>8648-04&amp;nbsp;</td><td></td></tr><tr><td>Ace O-rings, silicone, 2.6&amp;nbsp;mm, I.D. 9.2&amp;nbsp;mm&amp;nbsp;</td><td>Ace Glass</td><td>7855216</td><td>O-ring for pressure tube</td></tr><tr><td>Vortex shaker</td><td>VWR International</td><td>444-1378 (UK)</td><td></td></tr><tr><td>Fan-assisted convection oven</td><td>ThermoScientific</td><td>HeraTherm OMH60</td><td></td></tr><tr><td>Oven glove (Crusader Flex)</td><td>Ansel Edmont</td><td>42-325</td><td></td></tr><tr><td>250 ml Round bottom flask single neck ground joint 24/29 (Pyrex)</td><td>Quickfit&amp;nbsp;</td><td>FR250/3S</td><td></td></tr><tr><td>Rotaflo stopcock adapter with cone 24/29</td><td>Rotaflo England</td><td>MF11/2/SC</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>Heraeus/Kendro</td><td>HERA 76002844</td><td></td></tr><tr><td>Centrifuge (Mega Star 3.0)</td><td>VWR&amp;nbsp;</td><td>521-1751</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20820.464</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>T0886</td><td></td></tr><tr><td>Sulfuric acid 5 mol/L (10&amp;nbsp;N) AVS TITRINORM volumetric solution Safe-break bottle 2.5&amp;nbsp;L</td><td>VWR</td><td>191665V</td><td></td></tr><tr><td>Purified water (15 M&amp;Omega; ressitance)</td><td>Elga</td><td>CENTRA R200</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Lignocellulosic biomass&lt;/strong&gt;</td></tr><tr><td>Miscanthus X gigantheus</td><td></td><td></td><td></td></tr><tr><td>Pinus sylvestris</td><td></td><td></td><td></td></tr></tbody></table>

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 exact amount of lignin removal and lignin precipitation, recovered pulp and glucose yield depend on the type of biomass used, the temperature at which the treatment is run and the duration of the treatment. Short pretreatment times and low temperatures lead to incomplete pretreatment while at higher temperatures the cellulose becomes unstable in the ionic liquid, leading to hydrolysis and degradation. The selected ionic liquid also plays an important role in the outcome of the fractio...

Watch this Scientific Journal Video about Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids at JoVE.com

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

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.

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

pretreatment-of-lignocellulosic-biomass-with-low-cost-ionic-liquids

Universidad San Sebastian

Research

JoVE Journal

Environment

17.9K Views.  Imperial College London. 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.

Video: Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Chemistry

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.

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

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).

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

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

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

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

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

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

Bioengineering

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

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

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

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

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

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

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

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

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

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

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

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

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Biology

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol.&#160;The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast.&#160;Ethanol production is monitored using an ethanol probe.&#160;The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc.&#160;An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol.&#160;As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available.
With an increased emphasis on &#8216;inquiry-based learning&#8221;, scientific probes are becoming more popular.&#160;Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions.&#160;Another advantage is that these are small and lightweight and can be taken into the field for measurements.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

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Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

High-throughput Saccharification Assay for Lignocellulosic Materials

Biofuels: Producing Ethanol from Cellulosic Material