Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

The overall goal of this experimental method is to use low-cost ionic liquids for isolating lignin and cellulose from lignocellulosic biomass and understand factors affecting the separation and isolation of these biopolymers. This method can help answer key questions in the biorefinery field, such as how to isolate cellulose and a novel lignin using low-cost ionic liquids. Our technique utilizes unique solvent properties of ionic liquids, but our advantage is that these ionic liquids can be easily synthesized using low-cost starting materials.

Visual demonstration of this method is critical as there are numerous steps that are a lot easier to learn and carry out once you've seen it, rather than reading a page-long description. Demonstrating this procedure will be Florence, Clem, Wei-Chien, and Lisa. All of whom are graduate students in my laboratory.

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 ionic liquid 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. Reduce the water content to five weight percent below the desired water content using a rotary evaporator, and confirm the new water content by Karl Fischer titration as before. Use 10 grams of ionic liquid solution containing 20 weight percent water and a biomass to solvent ratio of one to 10, and perform the necessary experimental calculations as described in the text protocol.

Pre-weigh three 15 milliliter pressure tubes with Teflon caps and silicone O-rings. Visually inspect the pressure tubes to ensure they have no cracks or flaws. With a 10 milliliter 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. Then add the calculated amount of water to the solution using a pipette, assuming the density of water to be one gram per milliliter.

Add the required amount of air-dried liquid lignocellulose by placing a piece of aluminum foil on a balance, tearing the balance, and weighing out the biomass. Tear the balance again and add the biomass to the tube. Place the empty foil back on the balance and record difference.

Close the lid using a Teflon cap with the silicone O-ring. Check for a good seal without over-tightening. Record the weight of the pressure tubes containing the biomass and ionic liquid.

Then mix the contents of the tube by using a vortex shaker until all of the biomass is in contact with the ionic liquid. Place the pressure tubes in a fan-assisted oven that has been preheated to the desired temperature. For example, leave the tubes for eight hours at 120 degrees Celsius or for one hour at 150 degrees Celsius.

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. Transfer the contents of each tube into a 50 milliliter centrifuge tube using 40 milliliters of absolute ethanol.

Shake the tube using a vortex shaker for one minute to mix well, and leave the tube at room temperature for at least one hour. After vortexing for another 30 seconds, centrifuge the tube for 50 minutes at 2, 000 times G.Separate the liquid and solid by careful decanting. Collect the liquid in a clean 250 milliliter round bottom flask with a stir bar.

Add 40 milliliters of fresh ethanol into the 15 milliliter falcon tube and repeat the mixing and centrifugation step three times. Remove the ethanol from the ionic liquid by placing the round bottom flasks on a heating block. Connect each flask to a vacuum pump with a cold trap.

Fill the trap with dry ice and set the heating to 40 degrees Celsius. Then switch on the stirring and the pump. Transfer the wet-washed pulp into cellulose thimbles and label each thimble using a pencil.

Fill 150 milliliters of absolute ethanol into a clean 250 milliliter round bottom flask with a stir bar. Insert the sample-containing thimble into a 40 milliliter soxhlet extractor and add a condenser. Install everything on the parallel extractor work station connected to a recirculating tiller.

When all samples are loaded, begin stirring, set the temperature to 135 degrees Celsius and turn on the recirculator. After 20 hours of extraction, 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 using tweazers, and let the wet pulp dry in the thimble overnight in a fume hood. 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 degrees Celsius. After transferring the air-dried pulp from the thimble onto a piece of teared aluminum foil on an analytical balance, record the air-dried weight of the extracted pulp and transfer it into a labeled plastic bag.

Try to recover everything while not scraping thimble material off the wall. Determine the moisture content of the pulp immediately to calculate the oven-dried yield as described in the text protocol. When all of the ethanol has evaporated, precipitate the lignin by transferring the ionic liquid from the round bottom flask into a 50 milliliter centrifuge tube using 30 milliliters of water.

Mix the suspension and leave it for at least one hour. Then centrifuge the suspension for 20 minutes at 2, 000 times G, and separate the solution from the solid by decanting. Add 30 milliliters of distilled water to the lignin pellet inside the centrifuge tube.

Repeat the mixing incubation for one hour and centrifugation. Repeat this step for a total of three lignin washes. Dry the lignin inside the centrifuge tube using a vacuum oven and a pierced lid at 45 degrees Celsius overnight.

To determine the lignin yield, place a piece of aluminum foil on the balance, tear the weight, add lignin from the oven, and record the weight immediately. Finally, transfer the lignin into a vial for storage. The amount of pulp and lignin isolated after pretreatment depends on the reaction conditions, the feedstock, and the ionic liquid.

A typical time-course is presented here, where pulp yields initially decrease as lignin and hemicelluloses are extracted, while lignin yields increase over time. The lignin is isolated as a brown solid that had a faint but pleasant smell. The properties of the recovered cellulose pulp strongly depend on the conditions chosen.

Running the pretreatment for too long or at too high temperatures will result in the degradation of the cellulose and formation of pseudo-lignin. More differences between the pulp properties become evident when subjecting it to further analysis, such as compositional analysis or enzymatic saccharification. Shown here is the compositional analysis of pine pulp and miscanthus pulp pretreated using same tenmerature and time.

As can be seen, the pulp composition is very different as a result of the different types of biomass reacting differently to the same conditions. After watching this video, you should have a good understanding of how to perform pretreatment using low-cost ionic liquids and how to study different experimented parameters. Once mastered, this technique can be completed in around five days.

This includes a lot of downtime and waiting, so other tasks can be performed in parallel. Following this procedure, other methods, such as compositional analysis of the pulp, saccharification of the pulp, or lignin NMR, and molecular weight analysis can be carried out. These will further characterize the isolated materials.

I had the idea for this method when I discovered that certain ionic liquids move lignin from lignocellulosic biomass after absorbing moisture from air. By varying the water content before and after the pretreatment, I found that I could isolate the lignin easily by adding water after the extraction. Don't forget that working with acids and elevated temperatures can be hazardous, and personal protective equipment, such as lab specs, lab coats, and appropriate gloves, should always be worn while performing this procedure.