Hot Water (HW) Pretreatment with Tubes

Hot Water (HW) pretreatment with tubes

Step I: Making the tubes ready

1. Take a tube and look for the cap with the same number as the tube.

2. Close one end of the tube using holder (in pretreatment lab) and wrench.

3. Check moisture content (MC1; solid fraction= 1-MC) of biomass using moisture analyzer or oven (105oC; overnight) following the NREL protocol.

4. Calculate the amount of biomass required to get the appropriate amount of dry biomass, which would vary based on pretreatment mixture’s final total weight and solids loading.

a. For an example, for X wt% solids loading with Y g total weight, the amount of as received (or untreated) biomass required would be (Z) = [ Y*X/(100*(1-MC))] g

5. Weigh appropriate amount of biomass on weighing paper on sensitive balance and record the weight.

6. Carefully add biomass to the tube. To add biomass comfortably, make a funnel out of a weighing paper and tape the edges.

7. Add appropriate amount of water to obtain the required solids loading.

a. For instance, for an X wt% solid loading and total mass of Y gram, the amount of water would be (g) = Y- Z

8. After closing the caps with hands, screw both the ends with holder and wrench very tightly.

9. Shake the tubes to disperse water evenly with biomass and now tubes are ready for treatment, however, in general, the biomass is allowed long enough, at least for 4 hours, to come into equilibrium with water for better pretreatment.

10. It is preferred to store them upright in bench-top centrifuge cups holders for overnight soaking.

Step II: Fluidized sand bed treatment

1. Switch the sand bath on at least an hour before the experiment and increase the fluidization by increasing the air supply. Make sure you wear glasses, shoes, and have high temperature gloves on to protect yourself from the hot sand.

1 The average value of duplicates should be used for % solid content.  For determining the solids content using moisture analyzer, refer to the SOP for that.  For determining the solids content using oven, refer to the SOP for that. 2. Set the goal temperature on thermostat to about a couple of degrees (2 – 3°C) higher than the desired temperature.

3. Initially, the thermometer rod and temperature logger need to be tested. For this, put the thermometer rod in the porous iron basket, which should be attached to an iron rod, longer than the fluidizer’s diameter, to prevent basket from sinking into the bath, and lower the basket into the fluidized bed. Use tongs for placing or removing anything from the sand bath. The temperature logger should show temperature similar to that set in the thermostat. Then the equipment is ready to start experiments. The basket with thermometer is removed from the sand bath and placed in water bath to be cooled for further experiments.

4. The tubes with biomass and water in it are placed in the basket in an order for easy identification as they are removed with time through the course of experiment. The basket is immersed in the fluidized bed. The basket can accommodate about 10 tubes of 10 ml volume which can be used for five different time points taking duplicate at each time point

5. A stop watch is started immediately after the basket is placed inside the fluidized bed. Time is noted until temperature in the sand bath reaches 1°C less than the target temperature.

6. The stop watch is stopped and the temperature logger is started simultaneously. The time given on temperature logger can be used as a measurement for reaction time.

7. After the designated time period, e.g., 5, 10, 20 mins etc., remove tubes from the basket using a tong and lower the basket back inside the bed. The tubes are immediately placed in the adjacent water bath for sudden cooling. For the final time, the whole basket can be taken out of the sand bath and immersed in water.

8. Note average, minimum, and maximum temperature for each time.

9. Unplug the thermostat, and reduce air to the system.

10. To turn the sand bath off, turn the heaters off, cover the bed with a lid, and let air flow at a reduced rate for at least 2 hours. Otherwise, there are chances that the heat will damage the heating coils.

Step III: Filtration process

1. Find regular filter paper, small plastic porous cups to hold the filter paper, funnel fitting the bottom of the cup, a rubber ring, and a beaker that can be connected to the vacuum via a tube.

2. Weigh the filter cup together with filter paper and then connect the filter assembly to the vacuum manifold. 3. Cup + filter should be weighed prior to use as given below

4. Group the duplicate tubes removed at each time point and carefully unscrew them using wrench and holder on the table.

5. Turn the vacuum pump on and then empty the slurry onto the filter paper. Use a spatula to get as much liquid from the tubes as possible. The solids remain on the filter paper while the liquid vacuumed into the flask.

6. The filtrate should be collected with proper labeling in 15 ml centrifuge tubes for further analysis and stored in refrigerator. Check the filtrate pH and note it down

7. The residual solids from tubes are removed by washing with DI water and poured onto the filter paper. The walls of the tubes are scrapped to ensure the removal of all the remaining solid. The solids are further washed to remove any residual sugars and/ or inhibitors. The filtrate is discarded.

8. Solids are pressed to remove water and weighed along with the cups. The bottom of the cups should be wiped before weighing.

9. Solids are scrapped of the filter paper into Ziploc bags and stored in the fridge until further analysis. Care should be taken not to scrap off the filter paper while removing the wet solids cake from it.

Step IV: Beyond the pretreatment

Liquids:

1. The pH of the pretreatment liquor should be measured. Then following NREL’s protocol, about 10 ml of it is transferred in 125 ml pressure bottles. Also, as per NREL’s protocol, a range of sugar recovery standards should be prepared.

2. The amount of 72wt% H2SO4 is added to the bottles to bring the final concentration of acid to 4wt %. The pretreatment liquid and sugar recovery standards bottles are closed with rubber septa and

aluminum stoppers and then autoclaved at 121°C for 1 hour. Amount of H2SO4 added is calculated based on the pH (equation given as follows). 3. After the bottles cool down to room temperature, the samples are filtered/ neutralized and run on HPLC for the determination of total and monomeric sugars and other compounds.

Pretreated Solids:

1. Moisture/ solids content should be determined following NREL’s protocol either using an oven set at 105 or moisture analyzer.

2. To determine the solids composition, approximately 4 to 5 g of wet biomass, assuming that vacuum filtered solids generally contain about 20-25% solids, should be dried at 45-50C for couple of days.