Dilute Acid Hydrolysis Pretreatment

This experiment was conducted using the High Pressure/High Temperature Parr 4571 Reactor, as shown in Figure 3-1. The aspen, balsam, basswood, red maple, and switchgrass were heated in separate experiments from room temperature to 160oC, 175oC, and 190oC in a diluted acid aqueous solution. The detailed procedures followed are included in the JSA “Dilute Acid Hydrolysis Pretreatment”, in Appendix A-2.

Figure 3-1: High Pressure/High Temperature Parr Stirred Reactor Model 4571, 1000 mL capacity

3.6.1 Start-up Procedure

25 grams of each dry biomass sample was mixed with 500 mL of 0.25-1.0% w/w sulfuric acid in a glass insert for the reactor. The glass liner was then placed into the reactor chamber shown in Figure 3-1. The reactor chamber was put into the oven on the moveable cart. The reactors was tightening using the torque-wrench in the pattern of bypass adjacent screw 180 degree from the starting screw and tighten it to approximately 5 ft/lb. The detail sealing instruction is provided by Parr Instruction Company in Manual No. 274M. The instruction is also attached in Appendix B.

After the reactor was clamped tightly, the cooling circuit was connected to the air valve and the agitator cooling circuit was connected to the cooling water. Moreover, the pressure indicator was adjusted to ambient pressure and the initial temperature setpoint was set to 400oC at the controller. After all the controls were set, the reactor was heated up to the desired temperature by turning on the heater switch on the controller. During the heat up period, the temperature and pressure of the oven and the reactor were recorded every 5 minutes.

3.6.2 Run Time Procedure

5 mL of samples were collected at 100oC, 135oC, and thereafter at 3 minutes intervals until the temperature reached the setpoint (160oC, 175oC, and 190oC). Prior to that, approximately 8 mL of samples were removed from the sampling valve and discarded it to eliminate the sample residue from previous sample. The temperature was maintained at the setpoint for 32 minutes by adjusting the airflow to the cooling coil of the reactor. While the temperature was controlled at the setpoint, four 5 mL of samples were collected at 8 minutes intervals. Approximately 8 mL of samples were removed again to eliminate the unreacted solution in the reactor. A graduated cylinder was used to measure the 8 mL samples and a 20-mL vial was used to collect the 5 mL sample afterward. As a result, an approximately 156 mL of solution sampled from the reactor for 12 vials. The vials were labeled and capped tightly. Thus, there was 344 mL of solution left in the reactor at the end of the experiment.

The collected 5-mL samples were placed in an ice bath to stop the reaction and to cool the samples to room temperature. The cooled samples were filtered through 0.2 µm Millipore membrane filter (25 mm dia, “Isopore”) using the 10-mL syringe. The filtered

samples were collected in the 20-mL vial. In the following procedure, the samples were separated into two analysis; monomeric sugars analysis and total sugars analysis. The monomeric sugars content was analyzed without further hydrolysis. However, the total sugars content was analyzed after the further acid hydrolysis using the autoclave.

For monomeric sugars analysis, 1 mL of sample was drawn from the 20-mL vial using the 100-1000 μL Oxford Benchmate II handheld pipette into the HPLC vial (Agilent Screw Cap Vials 100/pk, Part No. 5182-0716). Then, the samples were neutralized using the 6N NaOH. The samples were slowly neutralized to pH 5 – 6 using the pH 1 – 12 Hydrion Papers. The amount of 6N NaOH added to the HPLC vials for neutralization is listed in Table 3-2 below for different acid conditions.

Table 3-2: Neutralization of monomeric sugars samples in various acid conditions

Sample Concentration Neutralization using 6N NaOH 0.25 % Dilute Acid Hydrolysis 7 μL

0.5 % Dilute Acid Hydrolysis 15.5 μL 1.0 % Dilute Acid Hydrolysis 26 μL

As the total sugar analysis was simply extra information to identify the amount of oligomers left in the sample after pretreatment, only the even numbered samples were prepared for the second acid hydrolysis. The pH of each sample was measured and recorded in this step using the pH meter. Then, 1 mL of samples, which were sample 2, 4, 6, 8, 10, and 12, were drawn from the 20-mL vial using the pipette into the amber crimp vial (Hewlett Packard 2 mL vial, Part No. 5181-3376). After that, the samples were acidified to 4% acid concentration using the 96% H2SO4. For each sample, the volume of 96% sulfuric acid required to bring the acid concentration to a 4% final acid concentration was calculated using the equation provided by NREL LAP # 015 and shown in Equation (3-5) below:

% 96 4 2 % 4 % 96 )] 2 / 08 . 98 ] [ ( ) [( C H moles SO H g H V V C V s s + + _× × − × = (3 – 5)

Where as: V96% = volume of 96% acid to be added, mL Vs = initial volume of sample, mL

C4% = concentration of 4% w/w H2SO4, 41.0 g/L C96% = concentration of 96% w/w H2SO4, 1800 g/L

[H+] = the concentration of hydrogen ions in the sample = antilog(-pH) The amount of concentrated sulfuric acid added to the amber crimp vials for acidification is listed in Table 3-3 below for different acid concentration.

Table 3-3: Acidification of total sugar samples for various acid conditions

Sample Concentration Acidification using 96% H2SO4

0.25 % Dilute Acid Hydrolysis 22 μL 0.5 % Dilute Acid Hydrolysis 22 μL 1.0 % Dilute Acid Hydrolysis 20 μL

The acidified samples were crimped tightly and then autoclaved for an hour at 121oC. After completion of the autoclave cycle, allow the hydrolyzates to slowly cool to near room temperature using the cool water before removing the cap. Each sample was subsequently neutralized to pH 5 – 6 using the 6N NaOH. Roughly 133 μL of 6N NaOH was used to neutralize the autoclaved sample.

After neutralizing the monomeric and total sugar samples, the precipitation might be formed in the neutralized sample. The samples were placed into the MARATHON 21K centrifuge (Fisher Scientific) at 8000 rpm for 5 minutes. After completion of centrifuge cycle, the clear liquid was carefully and slowly drawn out from the vials to a

new HPLC vials. The samples were then ready for sugars analysis using the HPLC discussed in the following section.