Name: ammonia fiber expansion (afex) pretreatment of lignocellulosic biomass
Uploaded: 2020-04-18T13:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass at JoVE.com

This material is based upon work supported in part by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Numbers DE-SC0018409 and DE-FC02-07ER64494. Rebecca Ong acknowledges partial support from Michigan Technological University (startup funding). Shishir Chundawat acknowledges partial support from the US National Science Foundation CBET award (1604421), ORAU Ralph E. Powe Award, and Rutgers School of Engineering (Startup Funding). Bruce Dale acknowledges partial support from Michigan State University AgBioResearch office and also the USDA National Institute of Food and Agriculture. Venkatesh Balan acknowledges partial support from State of Texas and University of Houston (Startup Funding). MBI employees acknowledge partial support from the US Department of Energy and Michigan State University foundation. Lastly, we would like to dedicate this paper to our mentor and co-author Prof. Bruce Dale for inspiring us to collaboratively pursue our dream of making sustainable cellulosic biofuels.

1. Adjusting biomass moisture content
<ol>
	<li>See the Table of Materials outlining all major equipment and materials necessary to perform bench or lab scale AFEX pretreatment using the custom-built tubular AFEX reactor (Figure 2).</li>
	<li>Determine the total moisture content of biomass using a moisture analyzer, or an oven set at 105 &#176;C for 8 h. For the oven method, transfer the samples to a heat-resistant desiccator to cool to prevent water adsorption prior to drying. Perform the process in duplicate or triplicate and calculate the average moisture content.</li>
	<li>For a given dry biomass loading in the reactor (here, it holds 25 g), use the moisture content determined in step 1.2, to calculate how much wet biomass needs to be loaded. 
	<img alt="Equation 1" src="/files/ftp_upload/57488/57488eq1.jpg" />&#160; &#160; &#160; &#160; &#160;[1] 
	Where mwet = total mass of biomass (wet weight basis); mdry = mass of biomass on a dry weight basis; MCTWB = biomass moisture content on a total weight basis</li>
	<li>Weigh out this amount of biomass (mwet) in a plastic container.</li>
	<li>Calculate how much water needs to be mixed with the wet biomass to achieve the desired moisture content. For corn stover, this is typically 0.6 g of H2O per g of dry biomass. 
	<img alt="Equation 2" src="/files/ftp_upload/57488/57488eq2.jpg" />&#160; &#160; &#160; &#160; &#160; [2] 
	Where mwater = mass of water added to the reactor (in addition to the water in the biomass); xwater = AFEX water loading (g:g dry biomass)</li>
	<li>Using a spray bottle, slowly add this amount of water (mwater) to the biomass that had been previously weighed out and mix well by hand.</li>
</ol>
2. Load and assemble the reactor
<ol>
	<li>Assemble the reactor body by placing a cap and Teflon gasket on the bottom of the reactor tube. Bolt a clamp in place, tightening both nuts evenly using a rachet.</li>
	<li>Transfer the wet biomass to the assembled reactor base and place a plug of glass wool at the top of the biomass.</li>
	<li>Place a Teflon gasket on the top of the reactor. Ensure the region is free of biomass and glass wool, which could prevent an effective seal, and place the reactor head on top, maneuvering the thermocouple through the glass wool and biomass.</li>
	<li>Bolt the clamp to the top of the reactor using a ratchet evenly on both sides.</li>
	<li>Weigh the reactor (mreactor) and record the weight.</li>
</ol>
3. Set up the reactor system and fill the ammonia transfer cylinder
<ol>
	<li>Confirm that all equipment is plugged in and operable (temperature controller, temperature monitor, syringe pump, timers).</li>
	<li>Set the timers to desired residence time for each reactor and sample to be run.</li>
	<li>Turn on and, if using a programmable syringe pump, set up the ammonia delivery method on the syringe pump. 
	Step 1: Withdrawal. 
	Step 2: Wait for 15 seconds (to allow time to open and close valves). 
	Step 3: Infuse (to transfer the ammonia into the reactor).
	<ol>
		<li>Save as the AFEX method to allow for easy reuse.</li>
	</ol>
	</li>
	<li>Verify that all valves into and out of the small ammonia cylinder are closed.</li>
	<li>If the cylinder has been used previously and contains residual ammonia/nitrogen, slowly open valve A on the top of the small ammonia cylinder to bleed off any nitrogen and close the valve once liquid ammonia begins to sputter out.</li>
	<li>To fill the small ammonia cylinder, open the large anhydrous ammonia cylinder and all valves on the ammonia line. Slowly open valve (B) near the top of the small ammonia cylinder until the pressure stabilizes. Wait for 5 min before continuing to the next step. Approximately 120 mL of ammonia gets charged from the main cylinder to the transfer cylinder during this time.</li>
	<li>Close all valves between the ammonia tank and the small ammonia cylinder, working from the left to right, beginning from the small cylinder (valve B) and finishing at the main valve on top of the tank.</li>
	<li>Set the nitrogen regulator to 350 psi. Open the valve on the nitrogen cylinder and the valve on the attached regulator. Open valve C on the small ammonia cylinder to slowly add nitrogen, overpressuring the system. Adjust the pressure of the small cylinder to 350 psi, as needed, by adjusting the set point on the regulator. Keep nitrogen lines open while dispensing ammonia.</li>
</ol>
4. Preheat the reactor (for reaction temperatures of &#62;100 &#176;C)
<ol>
	<li>Plug in the temperature monitor to the thermocouple and the heating tape to the temperature controller.</li>
	<li>Manually adjust the temperature controller to bring the reactor up to 60 &#176;C.</li>
</ol>
5. Load the reactor with ammonia
<ol>
	<li>Turn on the syringe pump if not already on.</li>
	<li>Calculate the volume of ammonia required based on the desired ammonia loading (g:g dry biomass) and a previously determined ammonia calibration. 
	<img alt="Equation 3" src="/files/ftp_upload/57488/57488eq3.jpg" />&#160; &#160; &#160; &#160; &#160; [3] 
	NOTE: Because the ammonia pump loads on a volume basis, when first using it, calibrate to convert from the required mass to volume. Follow the same procedure used for AFEX, but end the run (vent the reactor) immediately after loading the ammonia and weighing the reactor. Follow the same procedure for unloading the reactor.</li>
	<li>Set up the method to load the correct amount of ammonia:
	<ol>
		<li>Select the AFEX method from section 3.3.</li>
		<li>Press Step Definition | Step: 1 | Set Target Volume or Time.</li>
		<li>Key in the volume required in mL using the number pad and press the green checkmark.</li>
		<li>If more than 85 mL is required, enter the target volume as half of the amount specified in the spreadsheet and fill the reactor twice using the same syringe volume.</li>
		<li>Repeat steps 5.3.2 through 5.3.4 for &#8220;Step: 3&#8221;.</li>
		<li>Press the back button.</li>
	</ol>
	</li>
	<li>Open valve (D) on the bottom of the small ammonia cylinder towards the exhaust, and then close it once any residual ammonia has exited.</li>
	<li>Open valve (E) on the end of the syringe pump towards the front of the fume hood, and then open valve (F) to release any residual ammonia. Close valves (E) and (F).</li>
	<li>Disconnect the reactor from the temperature monitor and the temperature controller. Attach the reactor to the quick connect.</li>
	<li>Open valve (D) towards the small ammonia cylinder and open valve (E) towards the small ammonia cylinder.</li>
	<li>Press the green arrow on the pump to start the sequence and draw ammonia into the syringe.</li>
	<li>When the syringe stops automatically for the wait period, turn the syringe valve (E) towards the reactor, and the reactor inlet valve so it is pointing towards the quick connect stem. 
	After the delay, the syringe will begin infusing, stopping automatically at the set point.</li>
	<li>If more than 85 mL of ammonia is required, repeat steps 5.7 through 5.9.</li>
	<li>Close the reactor valve and valve (D). Open valve (F) to release residual ammonia from the syringe, and then close valve (F) and close valve (E).</li>
	<li>Open valve (D) towards the exhaust, and then close it once the residual ammonia has left.</li>
	<li>Wearing cryogenic gloves, remove the reactor from the quick connect. Be careful of potential ammonia spray. Use the elephant trunk vent line to vent the released ammonia, if necessary.</li>
	<li>Start the timer for the appropriate reactor.</li>
	<li>Weigh the reactor unit to verify that the appropriate weight of ammonia was added based on the spreadsheet calculations.</li>
</ol>
6. Begin heating and monitor the reaction
<ol>
	<li>Plug in the temperature monitor to the thermocouple and the heating tape to the temperature controller.</li>
	<li>Record the initial temperature and pressure of the reactor following ammonia addition (the start of the residence time).</li>
	<li>Manually adjust the temperature controller to bring the reactor up to the set temperature. The goal is to reach the set point in &#60;5 min.</li>
	<li>Record the pressure and temperature of the reactor every 3 min until the end of the residence time.</li>
	<li>At the end of the residence time, disconnect the reactor from the temperature controller and thermocouple, remove the reactor from the stand, and slowly open the ball release valve inside the fume hood. 
	NOTE: Always wear a face shield during this step.</li>
</ol>
7. Shut down the system
<ol>
	<li>After allowing the reactor to cool for a few minutes, use a ratchet wrench to open the clamps on the reactor.</li>
	<li>Unload the biomass and glass wool from the reactor inside a fume hood. In order to prevent airborne contamination of the biomass as residual ammonia evaporates, it is best to dry inside an enclosed drying box inside a ventilated space.</li>
	<li>Clean the reactor with distilled water until the water runs clear and allow reactors to dry.</li>
	<li>If still open, close all valves on and connecting to the ammonia cylinder.</li>
	<li>Close all valves on nitrogen line.</li>
	<li>Turn off the temperature controller, temperature monitor, balance, syringe pump, and timer. 
	CAUTION: If planning to run more reactions, it is not necessary to vent the small ammonia cylinder. However, if there is no plan to run more experiments, for safety it is best to vent the small cylinder into the hood at the end of the experiment. When doing this, it is important to leave the valves open as the release of ammonia can cause ice formation that may block some lines. As the lines thaw, additional ammonia can be released. Always make sure to have the ventilation functioning while allowing the system to vent. Any ammonia-treated biomass, even if it is not intended to be used, must be dried in the fume hood overnight to allow residual ammonia to evaporate. It cannot be immediately disposed of in the garbage.</li>
</ol>

Several authors (namely Shishir P S Chundawat, Tim Campbell, Farzaneh Teymouri, Leonardo Sousa, Bruce E Dale, Venkatesh Balan) are inventors/co-inventors on multiple patents filed on ammonia pretreatment and reactor design at MSU/MBI.

The AFEX protocol describes how to process plant materials in the presence of anhydrous ammonia and water at elevated temperatures to increase the digestibility of the pretreatment material by cellulolytic enzymes and/or microbes. AFEX is highly effective on graminoid monocot species (e.g., corn stover, switchgrass, miscanthus, rice straw, wheat straw, and sugarcane bagasse) due to the efficiency of the process to cleave ester linkages that are naturally abundant in these materials31. AFEX is much less effective on biomass derived from dicots and gymnosperms (hardwoods, softwoods, and native forbs)32,33 due to the smaller proportion of lignin-carbohydrate based ester linkages. However, when these linkages are introduced into woody cell walls using plant biotechnology, the AFEX pretreatment process becomes much more effective34.
Cleavage of ester linkages allows certain biomass components to be removed from the material, but redeposited as extractives on the outer cell wall surfaces, resulting in the formation of nanoscale holes that facilitate penetration and action of the cellulolytic enzymes6. AFEX pretreated corn stover showed a roughly 3-fold increase in glucose and xylose release rate following enzymatic hydrolysis under high solids conditions compared to the untreated material. Ammonia pretreatments also produce fewer and far less inhibitory degradation products compared to dilute acid pretreatment35. A previous comparison of AFEX and dilute acid-treated corn stover showed that dilute acid pretreatment produces 316% more acids, 142% more aromatics, and 3,555% more furan aldehydes than AFEX36, all of which can be inhibitory for microorganisms35,37. As AFEX is a dry-to-dry process, there is also no loss of sugars as a dilute liquid stream that cannot economically be utilized during enzymatic hydrolysis. However, this does lead to complications as enzymes with both cellulose-degrading and hemicellulose-degrading capability are required to fully break down the cell wall polysaccharides during enzymatic hydrolysis into mixed fermentable sugars like glucose and xylose. Hemicellulosic oligomers have been reported to inhibit cellulase activity38, which could necessitate a higher enzyme loading to maintain a high final sugar yield. However, optimization of suitable enzyme cocktails can reduce overall enzyme usage during saccharification of AFEX pretreated biomass39,40,41,42,43,44,45. During AFEX pretreatment process the hydrolysis and ammonolysis of ester linkages leads to the formation of acid and amide products in the pretreated biomass (e.g., acetic acid/ acetamide, ferulic acid/ferulamide, coumaric acid/coumarylamide)36. Though formation of amides has been shown to help the fermentation process, their presence at very high concentrations in pretreated feedstock could be a concern if feeding animals pretreated biomas. Pre-hydrolysis of ester linkages with alkali such as NaOH or Ca(OH)2 prior to AFEX pretreatment can be used to address the issue.
There are a number of safety considerations to keep in mind when working with anhydrous ammonia during the AFEX process. Anhydrous ammonia reacts with copper, brass, aluminum, carbon steel, and common fluoroelastomer polymers used in seals (e.g. Viton, etc.). Any tubing or reactor components that may come in contact with ammonia should be made from stainless steel, and gaskets, valve seats, and quick connect seals should be made from Teflon or Kalrez when possible. Ammonia is not considered a toxic chemical, but it is still dangerous due to its hygroscopic and cryogenic properties. It readily targets and can severely damage mucous membranes in the eyes and respiratory system. Ammonia is a cryogenic fluid and ammonia leaks can cause severe frostbite due to direct contact with the gas stream or chilled equipment. Ammonia is immediately dangerous to life and health (IDLH) at concentrations above 300 ppm. Workers should evacuate immediately in the event the concentration exceeds 50 ppm. It is recommended that operators wear a calibrated ammonia monitor to warn of hazardous concentrations in their vicinity. Installing sensors with alarms in the main work area is also advisable. Workers who handle ammonia should be properly trained and wear protective gear such as escape respirators equipped with methylamine cartridges, and cryogenic and heat protective gloves, and be prepared to handle emergency situations. In the event of exposure to anhydrous ammonia, the operator should move to safety and immediately flush the affected area with water for at least 15 min. The ammonia pretreatment process should be conducted inside a fume hood, and the ammonia cylinder should either be stored in a fume hood or ventilated cabinet. Following the experiment, pretreated biomass will have some residual free ammonia and should be either dried in the hood overnight or in a custom ventilated drying box before storage in plastic bags at room temperature for follow-up experiments. Some other key safety considerations include installing an ammonia delivery system with a flow meter that will help to precisely delivery ammonia to the reactor and a reactor designed to handle at least 1.5 times the pressure that the pretreatment process will undergo (e.g., for handling AFEX process at 2 x 106 Pa pressure, the minimum pressure rating of the reactor should be 3 x 106 Pa).
AFEX pretreatment is a promising method to produce highly digestible plant biomass that can be used directly as animal feed or as a feedstock to generate fuels and chemicals. Beyond these two industries, AFEX might find use in other areas such as a bio-renewable feedstock for making biomaterials, or as a feedstock for producing biogas. The laboratory-scale process can be conducted in a laboratory equipped with proper ventilated space and safety precautions, and our current work confirms that this scaled-down AFEX process shows similar results to material generated in a scaled-up and/or pilot AFEX reactor. The lab-scale AFEX process can be used to test feedstocks, processing conditions, and applications in a higher throughput manner, while providing a reasonable expectation of how the process would perform at pilot or industrial scales.

Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that uses volatile ammonia as the main reactant for cellulosic biomass pretreatment. This process was originally invented by Bruce Dale to cost-effectively reduce the recalcitrance of lignocellulosic biomass and enhance biologically-catalyzed pretreated biomass deconstruction into fermentable sugars1,2. Unlike most other aqueous-based thermochemical pretreatments3, AFEX is a dry-to-dry process that causes no significant change in biomass composition and requires no washing step with its associated waste generation and expense. Recovery of excess volatile ammonia has been demonstrated at the pilot scale, resulting in reduced waste generation and processing costs. The pilot-scale packed bed AFEX reactor system developed by MBI (Figure 1) recovers residual ammonia using steam stripping and transfers the hot, concentrated ammonia to a new packed bed4,5. Following AFEX pretreatment, the minor amounts of nitrogen incorporated into the biomass are usable as non-protein nitrogen by ruminant animals and microorganisms. Additionally, by altering the biomass ultrastructure through various physicochemical mechanisms6,7,8, AFEX increases accessibility of the biomass to carbohydrate-active enzymes (CAZymes) and increases the rates of polysaccharides hydrolysis by several-fold8,9, which also increases its digestibility by ruminant animals via their cellulolytic microbiome4,10,11,12. Farmers have long employed a simpler version of this method to increase the digestibility of ruminant forages by incubating the biomass for days or weeks under plastic tarps in the presence of low anhydrous ammonia loadings (&#60;4% w/w basis of dry biomass) and ambient pressures and temperatures10,11.
Anhydrous ammonia was first investigated for its potential to delignify wood in the 1950s and as a pulping chemical in the early 1970s13,14,15,16,17,18. In the early 1980s, pressurized, high-temperature, concentrated ammonia (&#62;30% NH4OH) under sub-critical conditions was first used in the Dale laboratory to enhance the enzymatic digestibility and microbial fermentability of lignocellulosic biomass19. This process underwent several name changes over the years, starting as ammonia freeze explosion, and then ammonia fiber explosion, and finally, ammonia fiber expansion, or simply AFEX. Around this same time (mid-late 1980s), DuPont (now Dow-DuPont) also explored using supercritical and near-critical anhydrous ammonia based pretreatment processes to increase digestibility of biomass20,21,22. In recent decades, there has been increased emphasis on using dilute aqueous ammonia solutions as a pretreatment reagent including ammonia recycle/percolation23 (ARP), soaking in aqueous ammonia (SAA), or the Dow-DuPont process without ammonia recycle24. A few additional methods have looked at use of anhydrous ammonia (low-moisture anhydrous ammonia (LMAA), and low-liquid ammonia pretreatment25 (LAA). &#65279;In the last few years, two new advanced organosolv-type pretreatment technologies utilizing liquid anhydrous ammonia26,27 and ammonia-salt based solutions28 at high liquid to solid loadings were recently developed that enable selective lignin fractionation and high efficiency enzymatic hydrolysis of pretreated cellulosic biomass at ultra-low enzyme loadings. A recent review article has highlighted the similarities and distinct differences between various forms of ammonia-based pretreatments29. However, until recently4, there were no pilot-scale demonstrations of ammonia-based pretreatment processes (like AFEX) that were efficiently coupled with closed-loop chemical recycle of concentrated ammonia used in the process.
In this paper, we describe in detail the most commonly used AFEX protocol for pretreating cellulosic biomass at the lab scale to produce gram scales of pretreated biomass (e.g., 1 to several 100 g). Typically, biomass is mixed with water (0.1&#8211;2.0 g H2O/g dry biomass) and loaded into a custom-built stainless-steel tubular or Parr type reactors. Anhydrous ammonia is then added (0.3&#8211;2.0 g NH3/g dry biomass) to the reactor and the mixture is heated to the desired reaction temperature (60&#8211;180 &#176;C). Earlier publications on the AFEX process from the 1980s-1990s started the pretreatment residence time (e.g., 5-60 min) immediately after the temperature ramp. However, as the reactions occur as soon as the ammonia is added to the reactor, the current AFEX procedure is to start monitoring the residence time immediately after ammonia addition to the reactor. For temperatures of 90 &#176;C or greater, it is often necessary to preheat the biomass before loading the ammonia in order to keep the initial temperature ramping to a minimum time period (i.e., &#60;5 min). At the completion of the residence time, a valve is opened to rapidly release the pressure, and gas-phase contents into a suitable chemical fume hood. The rapid conversion of ammonia from liquid to gas phase also causes the reactor to cool down. Small reactors (&#60;100 mL reactor volume) can often be unloaded in the fume hood immediately, while larger reactors (&#62;100 mL reactor volume) may need additional time to cool. For user safety, at the larger scale (&#62;100 g ammonia per reactor run), purging with nitrogen is recommended to remove as much residual ammonia as possible from the vessel and assist in cooling the reactor contents before unloading. Typically, no attempt is made at the lab-scale to recycle and/or recover the ammonia. One of the key design challenges for scaling-up the AFEX pretreatment process has been the recycling of ammonia with minimal capital and operating costs. Also, adding liquid ammonia to biomass generally drives partial flashing of the liquid that cools the biomass, requiring heating of the biomass-ammonia mixture before AFEX treatment can begin. Rather than adding ammonia as liquid, adding ammonia vapor to biomass offers two advantages: First, the high porosity of bulk biomass allows ammonia vapor to be transported rapidly, resulting in even ammonia distribution throughout the biomass. Second, ammonia vapor readily and exothermically dissolves into the water entrained in moist biomass, resulting in heat generation that rapidly and evenly heats the biomass. To exploit these advantages, both the MSU Dale lab and MBI have developed AFEX treatment methods using ammonia vapor. The Dale lab has developed the Gaseous Ammonia Pretreatment (GAP) process30, and MBI has developed the packed bed AFEX reactor process (Figure 1)4, which has been demonstrated at the pilot scale. The packed bed AFEX reactor system is capable of semi-batch mode operation with complete recycling of ammonia using a steam stripping method4,5. This novel MBI pilot-scale process exploits the chemical and physical characteristics of ammonia to efficiently pretreat biomass while efficiently recycling the ammonia.
Here, we present a detailed outline for conducting AFEX pretreatment of corn stover at the lab-scale using custom-built 200 mL volume tubular reactors (Figure 2). The AFEX pretreated samples were digested to fermentable sugars using commercially available cellulolytic enzyme cocktails to demonstrate the efficacy of the pretreatment processes. The enzymatic hydrolysis results for the lab-scale AFEX reactor were compared to larger pilot-scale AFEX reactor generated samples. Our goal is to provide a standard operating procedure for the safe and consistent operation of lab-scale pressurized reactors for performing AFEX pretreatment on cellulosic biomass like corn stover. Additional supporting information regarding variations to this lab-scale AFEX pretreatment process (e.g., pilot-scale packed bed AFEX process) are further highlighted in the accompanying supplemental pdf file. A detailed report on the packed bed AFEX process operational steps will be highlighted in a separate publication and is available upon request from MBI-MSU.

<table><tbody><tr><td>&lt;strong&gt;Safety Equipment/PPE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonia Monitor</td><td>CanarySense</td><td>BW GAXT-A-DL</td><td>Single gas detector, Ammonia (NH3), 0 to 100 ppm</td></tr><tr><td>Cryogenic gloves</td><td>Amazon</td><td>B01L8WA238/B01L8WA1H0/B01L8WA1O8</td><td>Keep hands protected when handling liquid ammonia</td></tr><tr><td>Ear muffs</td><td>3M</td><td>H7A</td><td>Ear muffs to protect hearing when releasing ammonia at end of pretreatment</td></tr><tr><td>Face shield</td><td>-</td><td>-</td><td>Wear while handling ammonia</td></tr><tr><td>Heat protective gloves</td><td>Grainger</td><td>2EWX1/2EWX2/2EWX3</td><td>Showa heat resistant gloves, max temperature 500&amp;deg;F</td></tr><tr><td>Nitrile gloves</td><td>-</td><td>-</td><td>Wear while mixing biomass to prevent contamination</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anhydrous Ammonia Compressed Gas Cylinder</td><td>-</td><td>-</td><td>An anhydrous ammonia compressed gas cylinder with a dip tube is required for this process. The dip tube is essential in order to withdraw liquid ammonia from the cylinder.</td></tr><tr><td>Distilled water</td><td>-</td><td>-</td><td>Used to add water to the biomass to achieve the desired water loading</td></tr><tr><td>Milled or Chopped Corn Stover</td><td>-</td><td>-</td><td>Corn stover is not readily commercially available. Contact local farmers or agricultural extension if you wish to locate some.</td></tr><tr><td>Nitrogen Compressed Gas Cylinder</td><td>-</td><td>-</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonia Cylinder Adapter</td><td>-</td><td>-</td><td>CGA fitting that depends on the gas cylinder. Matheson is a good source. Some require teflon gaskets. This connects the cylinder to the ammonia delivery system. A regulator is not necessary as the system uses liquid ammonia.</td></tr><tr><td>Ammonia Delivery System (Figure 4)</td><td>Swagelok</td><td>Misc.</td><td>Stainless steel pressure cylinder and components, valves, check valves, and gauges were used for all lines potentially in contact with ammonia.</td></tr><tr><td>Analytical Balance</td><td>Sartorius</td><td>CPA4202S</td><td>Balance used for preparing biomass and weighing the reactors. Toploading balance, 4200g x 0.01g</td></tr><tr><td>Chemraz O-rings</td><td>Harvard Apparatus</td><td>5013091</td><td>Ammonia-resistant o-rings for the SS syringe</td></tr><tr><td>Custom Tubular Reactors (Figure 3)</td><td>Parts were purchased from McMaster-Carr, Swagelok, Omega, and Motion Industries (Dixon Fittings)</td><td>Misc.</td><td>To be compatible with ammonia, the custom reactor was constructed from stainless steel components (sanitary tube and fittings, compression fittings, quick connect, pressure gauge, thermocouple), and teflon gaskets. The maximum pressure rating of the vessel is 1500 psig, which is the maximum pressure rating of the bolted sanitary clamps.</td></tr><tr><td>Drying Box</td><td>-</td><td>-</td><td>Optional: an enclosed system for drying is necessary if planning to do microbial experiments to avoid contamination. Avoid drying at elevated temperatures.</td></tr><tr><td>High Pressure Syringe Pump</td><td>Harvard Apparatus</td><td>70-3311</td><td>Infuse/Withrdraw PHD ULTRA HPSI Programmable Syringe Pump for transferring liquid ammonia</td></tr><tr><td>Moisture Analyzer</td><td>Sartorius</td><td>MA35</td><td>Moisture analyzer for determining moisture content of biomass prior to pretreatment.</td></tr><tr><td>Nitrogen Delivery</td><td>Misc.</td><td>Misc.</td><td>Nitrogen compressed gas cylinder, inert gas regulator (at least 1000 psig max pressure rating), lines, and valves.</td></tr><tr><td>Ratchet wrench and 7/8&quot; socket</td><td>-</td><td>-</td><td>Ratchet and socket to quickly tighten and open bolts on the sanitary clamp. Can be purchased anywhere.</td></tr><tr><td>Retractable Thermocouple Cables</td><td>Omega</td><td>RSC-K-3-4-5</td><td>Retractable thermocouple cable. You need one for each reactor.</td></tr><tr><td>Stainless Steel Syringe</td><td>Harvard Apparatus</td><td>702261</td><td>Stainless steel syringe for tranferring ammonia to the reactors.</td></tr><tr><td>Temperature Monitor</td><td>Omega</td><td>HH12B</td><td>Dual input temperature monitor. You need one for every two reactors.</td></tr><tr><td>Voltage Controller</td><td>McMaster-Carr</td><td>6994K11</td><td>Variable-Voltage Transformer for controlling heating to the reactors. You need one for each reactor.</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metal Scoops, Spoons and/or Spatulas</td><td>-</td><td>-</td><td>For transferring biomass for weighing, mixing, transferring into the reactor and removing from the reactor at the end of the run</td></tr><tr><td>Plastic Bowls or Tubs</td><td>-</td><td>-</td><td>Used for mixing the biomass with the water. Any bowl or tub could be used.</td></tr><tr><td>Spray Bottle</td><td>-</td><td>-</td><td>Used to add water to the biomass to achieve the desired water loading</td></tr><tr><td>Wide-Mouth Funnel</td><td>-</td><td>-</td><td>Any funnel that has a bottom opening 0.5-1.0 inches diameter.</td></tr><tr><td>Wooden Dowel</td><td>-</td><td>-</td><td>1-1.5&quot; diameter wooden dowel to assist with loading/unloading the reactor</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass Wool</td><td>Sigma-Aldrich</td><td>CLS3950-454G</td><td>For packing the top of the reactor to prevent biomass escape and clogging the tubing</td></tr><tr><td>Plastic Press-to-Close Bags</td><td>McMaster-Carr</td><td>1959T24</td><td>Bags for storing processed samples and for transferring to drying box</td></tr><tr><td>Plastic Tote</td><td>-</td><td>-</td><td>Used to transfer pretreated biomass to an alternate location for drying</td></tr><tr><td>Plastic Weighboats or Metal Trays</td><td>-</td><td>-</td><td>Used to catch the biomass when removing from the reactors, and for storing the samples while drying</td></tr></tbody></table>

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.

Following AFEX pretreatment, the biomass is darker in color, but otherwise visually unchanged (Figure 3). AFEX process generates a highly digestible material at a variety of scales besides the one outlined in this protocol. Here, we pretreated the same corn stover sample in our small 200 mL, packed-bed, bench-scale system; a larger 5 gallon, stirred Parr reactor; and MBI&#8217;s pilot reactor. The conditions used for the two smaller reactors (i.e., 200 mL and 5 gallon scale) were 1.0 g NH3:g dry biomass, 0.6 g H2O:g dry biomass, for 30 min at 100 &#177; 5 &#176;C. Pilot-scale AFEX4 was carried out on the same material at 0.6 g NH3:g dry biomass, 0.6 g H2O:g dry biomass, for 30 min at 100 &#177; 5 &#176;C. Details regarding the protocols used for conducting AFEX pretreatment at larger scales are provided in the supporting information (see Supplemental File 1). The following &#8216;Quality Control Criteria&#8217; have been established based on target temperature for AFEX pretreatment. If after reaching the set point, the reactor temperature goes outside &#177; 10 &#176;C from the set point, the experiment must be aborted. If the target temperature (within 5 &#176;C) is not reached within 5 min after ammonia pumping, abort the experiment. In addition, pretreatment efficacy for the AFEX process can be tested using cellulolytic enzyme cocktails to hydrolyze the accessible polysaccharides into fermentable sugars. Samples were enzymatically hydrolyzed for 72 hours at 6% glucan loading, pH 5.0, 50 &#176;C, and 250 rpm in a shaking incubator. A commercial cocktail of enzymes consisting of 60% cellulase (CTec3):40% hemicellulase (HTec3 or NS22246) on a fixed total protein loading basis loaded at 15 mg enzyme/g glucan was employed for all saccharification assays. The results (Figure 4) demonstrate that AFEX pretreatment significantly increases the yield of fermentable sugars in all cases. Furthermore, the cellulose/xylan hydrolysis yields for biomass pretreated using the lab-scale AFEX process is comparable to the larger 5-gallon Parr reactor and MBI&#8217;s pilot-scale packed bed AFEX process.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57488/57488fig1.jpg" /> 
Figure 1. Schematic outline of steps involved in the pilot scale operation of MBI&#8217;s AFEX reactor for pretreating lignocellulosic biomass fully-integrated with efficient ammonia recycle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/57488fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57488/57488fig2.jpg" /> 
Figure 2. Schematics of lab-scale of A) ammonia delivery system and B) small 200 mL AFEX pretreatment reactor utilized to perform AFEX process outlined in the video protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/57488fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57488/57488fig3.jpg" /> 
Figure 3. AFEX pretreated biomass has a very similar gross morphology compared to untreated biomass, apart from being slightly darker in color. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/57488fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57488/57488fig4.jpg" /> 
Figure 4. Glucose and xylose yields obtained after 72 h enzymatic hydrolysis of 6% glucan loading AFEX treated corn stover is shown here. All saccharification assays were carried out in duplicate with mean values (m) reported here. Standard deviations (1s) are reported here as error bars. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/57488fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Additional protocols <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/Supplementary_Information.pdf" target="_blank">Please click here to download this file.</a>
Supplemental Table 1: Ammonia delivery system and strut frame <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57488/Supplemental_Table_1.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass at JoVE.com

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.

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 ...

ammonia-fiber-expansion-afex-pretreatment-of-lignocellulosic-biomass

Nanyang Technological University

Research

JoVE Journal

Environment

13.4K Views.  Rutgers-State University of New Jersey. Ammoniak vezel expansie pretreatment, ook bekend als AFEX, is een chemische verwerkingsmethode die kan worden gebruikt om hernieuwbare brandstoffen, chemicaliën, of diervoeder te genereren uit plantencelwanden, ook bekend als lignocellulosic biomassa. Hoewel AFEX is gebruikt op een verscheidenheid van lignocellulose materialen, het is het meest effectief op grassen en gras gebaseerde landbouwresten. Tijdens AFEX reageert sterk geconcentreerd ammoniumhydroxide met een biomassa om esterkoppeli...

Video: Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass

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 ...

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.

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

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

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Biology

Twin-Screw Extrusion Process to Produce Renewable Fiberboards

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as source of mechanical reinforcement in fully bio-based fiberboards was developed. Various lignocellulosic crop by-products have already been successfully pre-treated through this process, e.g., cereal straws (especially rice), coriander straw, shives from oleaginous flax straw, and bark of both amaranth and sunflower stems.
The extrusion process results in a marked increase in the average fiber aspect ratio, leading to improved mechanical properties of fiberboards. The twin-screw extruder can also be fitted with a filtration module at the end of the barrel. The continuous extraction of various chemicals (e.g., free sugars, hemicelluloses, volatiles from essential oil fractions, etc.) from the lignocellulosic substrate, and the fiber refining can, therefore, be performed simultaneously.
The extruder can also be used for its mixing ability: a natural binder (e.g., Organosolv lignins, protein-based oilcakes, starch, etc.) can be added to the refined fibers at the end of the screw profile. The obtained premix is ready to be molded through hot pressing, with the natural binder contributing to fiberboard cohesion. Such a combined process in a single extruder pass improves the production time, production cost, and may lead to reduction in plant production size. Because all the operations are performed in a single step, fiber morphology is better preserved, thanks to a reduced residence time of the material inside the extruder, resulting in enhanced material performances. Such one-step extrusion operation may be at the origin of a valuable industrial process intensification.
Compared to commercial wood-based materials, these fully bio-based fiberboards do not emit any formaldehyde, and they could find various applications, e.g., intermediate containers, furniture, domestic flooring, shelving, general construction, etc.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pretreatment on lignocellulosic biomass was developed, which leads to an increased average fiber aspect ratio. A natural binder can also be added continuously after fiber refining, leading to bio-based fiberboards with improved mechanical properties after hot pressing of the obtained extruded material.

A versatile twin-screw extrusion process to provide an efficient thermo-mechano-chemical pre-treatment on lignocellulosic biomass before using it as ...

Engineering

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 ...

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

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