Ammonia fiber expansion pretreatment, also known as AFEX, is a chemical processing method that can be used to generate renewable fuels, chemicals, or animal feed from plant cell walls, also known as lignocellulosic biomass. While AFEX has been used on a variety of lignocellulosic materials, it is most effective on grasses and grass-based agricultural residues. During AFEX, highly concentrated ammonium hydroxide reacts with a biomass to break ester linkages in the plant cell wall and introduce nano to microscale pores.
This facilitates enzymatic deconstruction of the cell wall polysaccharides. The end result is a plant material that is more easily biochemically converted to fuels and chemicals or that can be used as a highly digestible animal feed. AFEX has a number of key advantages compared to other pretreatment processes.
First, AFEX has low water usage and is fairly unique in that it generates a mostly dry material that can be densified and easily shipped. The AFEX process also produces minimal amounts of microbial inhibitors such as furans which makes it highly compatible with microbial fermentation processes. It is also possible to recover the majority of the pretreatment chemical ammonia which improves the process economics.
Pilot scale AFEX pretreatment and ammonia recovery has been demonstrated at one ton per day capacity in the MBI pilot plant located in Lansing, Michigan. The following protocol will describe the steps involved in laboratory-scale operation of the AFEX process. Demonstrating this technique will be Jacob Aguado, one of the undergraduate students working in our research group.
To begin, first determine the moisture content of the untreated biomass using a moisture analyzer. Weigh out 25 grams of biomass and using a spray bottle, adjust the moisture content of the plant biomass using distilled water to reach your target moisture content. For corn stover, this is typically 0.6 grams of water per gram of dry biomass.
Mix the sample by hand. Assemble the reactor body by placing a cap and Teflon gasket on the bottom of the reactor tube. Transfer the wet biomass to the assembled reactor base and place a plug of glass wool at the top of the biomass.
Place a Teflon gasket on the top of the reactor. Ensure that the region is free of biomass and glass wool which could prevent an effective seal and place the reactor head on top maneuvering thermocouple through the glass wool and biomass. Bolt the clamp to the top of the reactor using a ratchet evenly on both sides.
Weigh the reactor and record the weight. Confirm that all equipment is plugged in and operable, set the timer to the desired residence time for each reactor and the sample to be run. Turn on the programmable syringe pump and set up the ammonia delivery method and save for convenience.
Verify that all valves into and out of the small ammonia cylinder are closed. If the cylinder has been used previously and contains residual ammonia or 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. To fill the small ammonia cylinder, open the large anhydrous ammonia cylinder and all valves in the ammonia line.
Slowly open valve B near the top of the small ammonia cylinder until the pressure stabilizes. Wait for five minutes before continuing to the next step. Close all valves between the ammonia tank and the small ammonia cylinder working from the left to the right beginning from the small cylinder valve B and finishing at the main valve on top of the ammonia tank.
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 over pressuring 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. For reaction temperatures that are greater than 100 degrees Celsius, you need to preheat the biomass and reactor before adding the ammonia.
Plug in the temperature monitor to the thermocouple and the heating tape to the temperature controller. Manually adjust the temperature controller to bring the reactor up to 60 degrees Celsius. Calculate the volume of ammonia required based on the desired ammonia loading and a previously determined ammonia calibration.
Set up the method in the syringe pump to load the correct amount of ammonia, then to residual ammonia from the lines between the small ammonia cylinder and the syringe pump by opening and closing the valves on the bottom of the ammonia cylinder toward the exhaust and the valves on the syringe pump line. Disconnect the reactor from the temperature monitor and the temperature controller. Attach the reactor to the quick connect.
Open valve D towards the small ammonia cylinder and valve E towards the small ammonia cylinder. Press the green arrow on the pump to start the sequence and draw ammonia into the syringe. When the syringe stops automatically for the wait period, turn the syringe valve 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. Close the reactor valve and valve D.Open valve F to release residual ammonia from the syringe, then close valve F and close valve E.Open valve D towards the exhaust, then close it once the residual ammonia has left. While wearing cryogenic gloves, remove the reactor from the quick connect.
Be careful of potential ammonia spray. Start the timer for the appropriate reactor. Weigh the reactor unit to verify that the appropriate weight of ammonia was added based on spreadsheet calculations.
Plug in the temperature monitor to the thermocouple and the heating tape to the temperature controller. Record the initial temperature and pressure of the reactor following ammonia addition. Manually adjust the temperature controller to bring the reactor up to the set temperature.
The goal is to reach the set point in less than five minutes. Record the pressure and temperature of the reactor every three minutes until the end of the residence time. 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.
Always wear a face shield during this step. After allowing the reactor to cool for a few minutes, use a ratchet wrench to open the clamps in the reactor. Unload the biomass and glass wool from the reactor while inside a fume hood.
In order to prevent airborne contamination of the biomass as residual ammonia evaporates, it is best to dry the biomass inside an enclosed drying box inside a ventilated space. If still open, close all valves on and connecting to the ammonia cylinder and all valves on the nitrogen line. Following AFEX pretreatment, the biomass will be darker in color, but otherwise visually unchanged.
AFEX generates a highly digestible material at a variety of scales besides the one outlined in this protocol. We pretreated the same corn stover sample using our 200 milliliter packed bed bench scale system, a five gallon stirred power reactor and MBI's pilot reactor that is able to process one ton of biomass per day. The conditions used for the two smaller reactors were one gram of ammonia per gram of dry biomass, 0.6 grams of water per gram of dry biomass for 30 minutes at 100 degrees Celsius.
Pilot scale AFEX was carried out on the same material at the same conditions with the exception that the ammonia loading was 0.6 grams of ammonia per gram of dry biomass. Samples were enzymatically hydrolyzed for 72 hours at 6%glucan loading, pH of five, 50 degrees Celsius, and 250 rpm in a shaking incubator. A commercial cocktail of enzymes was loaded that consisted of 60%cellulase and 40%hemi-cellulase at 15 milligrams of enzyme protein per gram of glucan.
Our results demonstrate that AFEX pretreatment significantly increases the yield of fermentable sugars compared to untreated biomass. Furthermore, the 72-hour hydrolysis yields for biomass pretreated using the three processes are comparable to each other. Following the AFEX process, the pretreated materials can be stored indefinitely as long as they remain dry and used for various conversion experiments such as high-solids enzymatic hydrolysis and fermentation, consolidated bioprocessing or animal feed testing.
A key consideration during the implementation of AFEX centers on the safe use of anhydrous ammonia. AFEX experiments should always be conducted inside a ventilated space and researchers should take appropriate safety precautions to prevent ammonia exposure and be familiar with how to respond in the event of an emergency. In conclusion, ammonia fiber expansion pretreatment is a highly promising method to reduce biomass recalcitrants and more efficiently produce fuels, chemicals, and animal feed from renewable plant-based raw materials.
