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Optimization and Scale up of CO2-Water Pretreatment of Guayule Biomass Ehsan Moharreri, Tahereh Jafari, Steven L. Suib, Narayanan Srinivasan, Ahmadreza F. Ghobadi, Lu-Kwang Ju, and J.Richard Elliott Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b03318 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 1, 2017

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Industrial & Engineering Chemistry Research

Improved Understanding of CO2-Water Pretreatment of Guayule Biomass by High Solids Ratio Experiments, Rapid Physical Expansion and Examination of Textural Properties

Ehsan Moharreri1,2, Tahereh Jafari1, Steven L. Suib1,3, Narayanan Srinivasan2, Ahmadreza F. Ghobadi2, Lu-Kwang Ju2, J. Richard Elliott2*

1 Institute of Material Science, The University of Connecticut, Storrs, CT, 2 Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, 3 Department of Chemistry, The University of Connecticut, Storrs, CT

*corresponding author: J. Richard Elliott: [email protected]

ACS Paragon Plus Environment


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ABSTRACT In this work, we provide a systematic study of CO2-water pretreatment of guayule biomass to optimize the residual ground bagasse from natural rubber extraction for hydrolysis and fermentation. Guayule biomass is mixed with water then loaded into a 250 mL reactor with exposure to a biphasic environment consisting of a CO2-rich vapor phase and water-rich liquid phase. The pressure is then rapidly released for a “physical expansion” effect. The pretreated biomass is enzymatically hydrolyzed, and the sugar concentration in hydrolysate is measured. Experimental runs are conducted in the temperature range of 145 °C to 210 °C and pressure range of 3.4 MPa to 34 MPa. The solids ratio (dry solids mass/water mass) is between 0.17 and 1.7. The packing density is between 0.03 and 0.2 g biomass per cm3 of the reactor and the


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holding time ranges from 20 to 840 minutes. A four-fold increase in the reactor volume is performed and optimized with a 1-liter vessel. High-pressure carbon dioxide – water pretreatment increases surface area of guayule biomass, introduces ruptured morphological features, and improves enzymatic digestibility. We achieve a total sugar yield of 85% (of theoretical) at two different reactor sizes of 250 mL and 1 L. At the smaller reactor, the optimum operational condition is 180 °C, 26 MPa and 0.5 solid ratio. At the larger reactor, the optimum operational condition is 200 °C, 12 MPa and 0.33 solid ratio.

Keywords: Biomass Pretreatment, CO2-Water, Pretreatment Optimization, Guayule



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INTRODUCTION

Guayule (Parthenium argentatum), is a hardwood desert shrub that synthesizes natural rubber, cis 1,4-polyisoprene, equivalent to that of the Hevea tree (Hevea brasiliensis).1,2 Recent efforts to economically compete with imported Hevea has led to commercial production of guayule in the United States.3,4 Moreover, increased occurrence of life-threatening allergic reactions to products containing Hevea latex has compelled development of guayule latex as a safe alternative.5–8 Worldwide production of natural rubber reached 12 million metric tons in 2013 while the US imported 46000 metric tons mostly from Asia.9 Commercialization of Guayule cuts US dependencies on imported natural rubber and creates a domestic market for the plant residues. Guayule resins have been formulated in preservatives for wood products as well as coatings for its germicidal and fungicidal properties.4,5,10–12 Extraction of resins and latex from guayule leaves up to 80% of the guayule dry mass as a waste crop residue. The result is a ground bagasse and can be used to make plastic composite materials, as a soil amendment, and has been suggested as a feedstock for biorefineries. Producing biofuels from guayule bagasse improves the sustainable commercialization of the crop and contributes to energy supplies.3,4,13 Efficient pretreatment of guayule bagasse as a biorefinery feedstock has been the subject of several studies.2,5,14 Mild pretreatment and a subsequent enzymatic hydrolysis produces more sugars and resulting in requiring less severe detoxification of the hydrolysate.15,16 Dale et al. (2011) utilized ammonia fiber expansion (AFEXTM) pretreatment followed by enzymatic


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hydrolysis method to achieve up to 50% and 40% yields for glucan to glucose and xylan to xylose respectively.2 Srinivasan and Ju (2010) suggested CO2-water pretreatment combined with enzyme hydrolysis for guayule bagasse. They concluded that supercritical CO2 pretreatment outperforms dilute acid pretreatment and delignification pretreatment with regard to reducing sugars yield. No inhibitory or toxic effect to Trichoderma Reesei Rut C30 was observed when the fungal cells were grown on the hydrolysate.5 In a subsequent study, 14 they conducted a statistical approach to optimize process conditions to obtain yields of glucose and pentose of 56% and 61% respectively. Pretreatment is an important step to increase the structural accessibility and reactivity of lignocellulosic components for subsequent processing while avoiding inhibitory byproducts. Series of technologies have been developed to perform efficient pretreatments such as low-cost ionic liquids17, the well-known AFEX™ process18, autohydrolysis19 and CO2-water pretreatment20,21. CO2-water pretreatment has demonstrated effectiveness for positively impacting digestibility of lignocellulosic biomass through physical swelling, penetration, and disruption as well as chemical hydrolysis.22 Due to the in-situ production of carbonic acid,23 this pretreatment is environmentally friendly since the acid does not remain after depressurization. Besides pretreatment, CO2 integration in a biorefinery has been suggested recently as extraction and as reaction medium.24 Exposure to supercritical CO2 was shown to have swelling effects on some plant material 25

while the rapid release of pressure and CO2 expansion causes disruption of biomass fibers.26

Carbonic acid enhances xylan hydrolysis compared to hot liquid water pretreatment for herbaceous biomass.27,28 Due to the positive effect of water content during pretreatment, there



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will be an increase in yield with a decrease in solids ratio.23,26 Elevated CO2 partial pressure leads to increased levels of carbonic acid, enhancing xylan hydrolysis in herbaceous biomass.28 Sudden physical expansion effect by CO2 release is also strengthened by elevated operating pressure. 26 A positive effect of CO2 pressure on the total sugars yield is expected.29 Pretreatment with high solids ratio is of particular interest from process economic standpoint.30–33 Hydrolysis of polysaccharides during CO2-water pretreatment is expected; therefore, temperature and time – in the right window that avoids production of furan derivatives- should have a positive effect on the downstream enzymatic hydrolysis sugar yield.22 Besides increasing solubility, CO2 pressure exerts a force on the solution increasing its penetration in the biomass structure.22 Pressure, temperature, and time were shown to have a positive impact in enzymatic digestibility of corn stover.29 Increased yield of sugars due to an increase in temperature and holding time in the pretreatment of mixed hardwood, switchgrass, and mixed perennial grasses is followed by a decrease after a certain point due to decomposition of the sugars.32 The change in textural properties during pretreatment can be studied through imaging by scanning electron microscopy (SEM). It has been shown that CO2 pretreatment increases the porosity of corn stover,29 wheat straw,22 empty fruit bunches,34 and rice straw35. High pore volume36,37 has led to higher enzyme accessibility, while effective pretreatment has led to a higher surface area38,39. The objective of the current study is to understand optimization of the process through systematic experimentation and to observe surface transformation during pretreatment. The focus is on maximizing the total sugar yield in hydrolysate produced after enzymatic treatment, while


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exploring a wide range of solid ratios, temperatures, pressures, solid packing density and holding time, on two pretreatment vessel with 250 and 1000 mL.

Materials and Methods

Materials The guayule biomass was from the waste stream of the natural rubber extraction manufacturing plant at Yulex Corporation (Carlsbad, CA). The company chops the harvested guayule shrubs then extracts the resin and the rubber by hexane and supercritical CO2 at 5000 psi and 100 °C. The extraction was carried out by Supercritical Solutions LLC (Allentown, PA) as described by Cornish et al.40 When the rubber and resin extracted-bagasse went through enzymatic hydrolysis without pretreatment, no more than 15% sugars yield was achieved. The trademarked enzyme Spezyme CP, purchased from Genencor (Hanko, Finland), was used for the enzyme hydrolysis step. The commercial enzyme had the following enzyme activities: cellulase activity of 32.3 FPU/mL, β-glucosidase activity of 26.7 IU/mL and xylanase activity of 12.4 U/mL. 5 This activity is reduced by a factor of about twenty when diluted in the hydrolysis solution. The CO2 was purchased from Praxair in 50 lb gas cylinders.

Pretreatment At the smaller reactor, the CO2-water pretreatment step was carried out in a 250 mL stainless steel vessel with 1.5’’ internal diameter, 8’’ height, and ½’’ wall thickness. The vessel had a ¼” 10V needle valve from Autoclave Engineers (Erie, Pennsylvania) at the inlet and a



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Swagelok (Solon, Ohio) SS-20VM4-F4 ¼’’Integral-Bonnet needle valve at the outlet. The internal pressure of the reactor was measured by a 3” test gauge (Model 1409) from US Gauge (Sellersville, Pennsylvania). The vessel outer wall was covered with a high-temperature heating tape. The vessel was connected to a CN7533, 1/32 Din temperature controller from Omega Engineering (Stamford, Connecticut) and an internally installed 1/16’’ transition joint probe ironconstantan thermocouple from Omega Engineering. The thermocouple was installed in the center of the reactor to measure and control the biomass temperature during the pretreatment process. A 260D syringe pump from ISCO (Lincoln, Nebraska) was used to pump liquid CO2 into the reactor. A 3006D Isotemp bath circulator from Fisher Scientific (Hampton, New Hampshire) was used to liquefy the CO2 content of the syringe pump. The CO2 was directed from the supply cylinder into the pump and was cooled to 7 °C by the bath circulator. In the pretreatment process, the reactor was first heated to the necessary temperature. Then the biomass was soaked in the studied amount of water, put in extraction thimbles, (Whatman®, Piscataway, NJ) and loaded into the reactor. The CO2 was next pumped to the preheated reactor to get the reactor to the pressure and temperature of interest. The reactor was held at that condition for a period before the pressure was quickly released to create the physical expansion effect. The holding time reported in this work starts with pressurization of reaction vessel and ends with the release of pressure. In the larger reactor process, a U46 supercritical CO2 extraction unit from Pressure Products Industries (Warminster, Pennsylvania) was used. The unit contained 4 vessels including two 1 L vessels, one 4 L vessel with a mixer inside, and a 6 L vessel. In the pretreatment process, one of the 1 L vessels was used as the high-pressure pretreatment reactor, while the 4 L vessel


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was used for preheating of the CO2 fluid. Similar to the smaller reactor, CO2 is first cooled to 7 °C and then pumped to desired pressure. (Figure 1)

Characterization

Scanning Electron Microscopy imaging was performed on JEOL 6335F field emission scanning electron microscope (Peabody, MA). Prior to SEM imaging, the samples were coated with AuPt(~60/40%) alloy with a Polaron model E5100 (Quorum Technologies Ltd, Laughton, UK). Nitrogen sorption isotherms were measured with a Quanta Chrome instrument (Autosorb iQ2) instrument (Boynton Beach, FL). Before these surface area and pore volume data measurement, the sample was degassed at a temperature of 150 °C for 6 h. The total pore volume was determined using the adsorption volume at a relative pressure of 0.992. The surface area were calculated using the Brunauer–Emmett–Teller (BET)41 method and the pore sizes and pore volume were obtained based on the Barrett–Joyner–Halenda (BJH)42 method from the desorption branch of the isotherm.

Enzymatic Hydrolysis The pretreated samples were subject to enzymatic hydrolysis; each system consisted of 5 g of pretreated dry based biomass and 100 mL of a citrate buffer (pH=4.8, prepared by 0.05 M sodium citrate and 0.05 M citrate acid). To prevent bacterial growth 5 mg of sodium azide was added. Enzymatic hydrolysis of the pretreated biomass was performed as described by Srinivasan et al.,5 except for the difference in the enzyme activity, which within the hydrolysis



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solution was 1.84 FPU/mL, 1.52 β-glucosidase IU/mL and 0.7 xylanase U/mL and the enzymatic hydrolysis was conducted for 48 hours.

Analytical Methods The guayule bagasse used in this study had approximately 34% (weight) cellulose and 18% hemicellulose, 43% acid insoluble materials, 1% Acid soluble lignin, 4% Other. 43 The application of CO2-water high pressure pretreatment to materials so high in acid-insoluble content is unusual. Nonetheless, the availability of the bagasse in ground and ready form is what makes the economics of the process appealing. With 50 g/L pretreated bagasse as the starting material for enzymatic hydrolysis, the yields of the total reducing sugars (from the combined pretreatment and enzymatic hydrolysis steps) can be evaluated by the following equation:

= ×(. .) × 100

Where Ys is the total reducing sugars yield percent, Cs is the measured concentration (g/L) from the enzymatic hydrolysate, and denotes total reducing sugars. The Dinitrosalicylic Acid (DNS) method was used to measure the total reducing sugar concentration, as described by Srinivasan and Ju.5 The samples taken periodically during enzymatic hydrolysis of the pretreated biomass were centrifuged with 12,000g for 10 minutes with a Centrifuge 5415D from Eppendorf (Hamburg, Germany). The supernatant was collected diluted, filtered, and analyzed according to the method established by Ghose 44. Four standard solutions of 2, 1, 0.5 and 0.25 g/mL glucose were used for calibration of the DNS tests.


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RESULTS

Operating Conditions Solids ratio is the weight ratio of dry biomass to water added to the pretreatment reactor. Packing density (or loading) is defined as the weight of biomass per unit volume of the reactor. Results presented in this section were mainly from the smaller reactor experiments made with a holding time of 35 minutes and packing density at 0.06 g/mL –which are close to the optimal conditions, unless otherwise indicated. At a temperature of 170°C, it takes about 35 minutes to reach maximum yield. Due to a low density of the biomass, experiments are limited to packing density of 0.2 g/mL. Packing density of 0.06 g/mL gives the highest yield at 180 °C, 26.2 MPa and 0.5 solids ratio. Figure 2 illustrates the effect of temperature at two levels of solids ratio of 0.5 and 1.0 (dry solids mass/water mass) at 26.2 MPa. The total reducing sugar yield increased with temperature in a range of 145 °C to 190 °C but decreased at above 190 °C, giving a maximum yield of roughly 60% at solids ratio of 1.0. The decline in yield could be due to sugar degradation. A similar effect by temperature on reducing sugar yield was seen for lower solids ratio of 0.5. However, the lower solids ratio gave a higher maximum reducing sugar yield of 85% at a lower optimal temperature of 180°C. At 200°C, the yield was highest at the elevated pressure of 34.5 MPa, when compared to the two lower pressures of 26.2 MPa and 6.9 MPa. On the other hand, at the lower temperature of 145°C, the change in pressure did not have a significant effect on the yield.



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The effect of solids ratio is shown at high and low temperatures in Figure 3 at the smaller reactor. The total reducing sugar yield exhibits a maximum at a solids ratio of 0.5 at lower temperatures. For the solids ratio in the range of 0.1-0.5 and higher temperatures, yields are near 85% for 24.6 MPa, a 0.06 packing density, and holding time of 35 minutes. The error bars were obtained from standard deviation of three pretreatment processes at the same operating condition. The error from other experimental steps (enzymatic hydrolysis and DNS test) was proved to be minor compared to the experimental error in the pretreatment step. The highest yield was obtained at a packing density value near 0.06 for the pretreatment made at 26.2 MPa, 180°C and solids ratio of 0.5. The highest yield at the packing density of 0.06 could be the result of heat transfer effects. Excessively dense packing could hinder uniform heat transfer while if it is too low it could lead to drying of the feed. Based on these studies, packing density values of 0.06-0.10 g/mL is recommended.

Physical Expansion The rapid release of pressure can provide up to 15% increase in the yield at 180 °C and 0.5 solids ratio and 26.2 MPa. Results of a study on the physical expansion effect are illustrated in Figure 4, which shows the contribution of physical expansion to higher yields. The experimental variability is quite significant when considering the physical expansion effect. When dealing with morphological and textural properties, nitrogen adsorption studies and SEM imaging indicated a significant impact of CO2 treatment, but was inconclusive with respect to the impact of rapid physical expansion.


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Phase Equilibrium during Pretreatment To make reasonable assumptions for future modeling purposes, there needs to be a clear understanding of the phase equilibrium during pretreatment. The experimental measurement of the amount of CO2 in the smaller reactor and the calculated amount of CO2 for the larger reactor, makes it possible to determine whether the reaction environment is biphasic. The biphasic environment consists of water rich liquid phase and CO2 rich vapor phase in equilibrium. Each experiment was determined to be in the biphasic region during their holding times except those conducted with the initial solids ratio of 2.5 and temperatures of 190 °C and higher. The enthalpy of the mixture during the holding time was also calculated using Peng-Robinson (1) equation of state with a binary interactive coefficient of kij = -0.02 for CO2 and water. By considering the pressure to be atmospheric post-expansion, the temperature was estimated assuming isenthalpic expansion. As a result, calculations indicate that most experiments were carried out at biphasic environment. Detailed calculations for determining the amount of CO2 for the larger reactor experiments is presented in the supporting information.

Reactor Size Due to mechanical sealing limitations, the pressure could not exceed 12.4 MPa in the apparatus and hence in the larger reactor experiments. Therefore, all the experiments at the larger reactor were conducted at 12.4 MPa. Figure 5 shows the effect of temperature and solids ratio on total reducing sugar yield at two reactor sizes. For both, temperature is controlled by thermocouples placed internally at the center of pretreatment vessels and heating is conducted through the walls. Therefore, temperature gradient over the radius of the vessel must increase



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with the vessel size. For pretreatment experiments were temperature plays a crucial role, this could lead to different values of yield at different sizes. In each reactor size, the difference between yields for the two temperatures of 170 °C and 200 °C diminishes as the solids ratio increases. At higher solids ratio, larger reactor pretreatments leads to higher yields compared to the smaller reactor for both temperatures. However, to reach to highest yields at the larger reactor, temperature should be set to 200 °C and solids ratio should be lowered to (0.33-0.5) range.

Discussion The highest total sugar yields were achieved with the physical expansion experiments at the following conditions: 26.2 MPa, 180 °C and 200 °C, 0.5 and 0.25 solids ratio, 0.06 packing density, and holding times of 35 and 50 min. These conditions led to 85% and 87% total reducing sugar yields. The volume increase from 250 mL to a 1000 mL reactor confirms similar optimal conditions but the conditions in the larger reactor are favored by slightly higher solids ratios. Decreasing solids ratio to 0.3-0.5 has a positive effect on yield at higher temperatures (i.e. 180 to 200) while the effect flattens afterward. Going to lower temperatures (i.e. 170 and 160), solids ratios below 0.5 cause a decrease in yield. This is in agreement with earlier works that have shown solids ratio is not favorable below a certain level.26 The role of high-pressure CO2 and moisture is complementary in this pretreatment process. Liquid hot water pretreatment without CO2 at 180°C, 1 MPa, 35 min and 0.06 g/mL with the same biomass was performed and resulted in 77% yield in total reducing sugar yield which is 8% lower than when high-pressure CO2 and 0.5 solids ratio were utilized.


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Surface Transformations under Pretreatment Table 1 indicates the obtained surface area for pretreated and unpretreated samples. The CO2 pretreated sample has greater surface area than unpretreated sample by a factor of 10. Figure 6a displays the adsorption-desorption isotherm of biomass samples which confirms that the pretreated sample has higher surface area than biomass feed. Pore size distribution indicates that large pores of 150 nm are developed in the biomass after pretreatment with CO2 expansion. (Figure 6b). Hydrothermally treated biomass was analyzed for surface area (Table 1) and morphology (S-3) as control experiments to highlight the effect of CO2 pretreatment. Among various factors limiting the hydrolysis of biomass, the accessible surface area and pore size are important, affecting the efficiency of enzymatic digestibility.46 The effects of pretreatment on the textural properties of biomass materials have rarely been studied.38,39 Hydrothermal and ionic liquid pretreatment effectively changed the surface area and activity of biomass materials. In the pretreatment stage, cracks developed and fragmentation results in increasing the surface area of biomass.38 Based on BET data, surface area and pore size distribution changes after pretreatment, which confirms the effect of CO2 pretreatment on the biomass feed (Table 1). SEM images also indicated porous structure development in the biomass after the pretreatment stage (Figure 7), verifying again the effectiveness of the pretreatment approach. The microstructure of unpretreated biomass was screened extensively under Scanning Electron Microscope. Among various features, arrays of dome-like morphology (which are likely from the stalk tissue) are chosen in this study to exemplify surface transformation. These reverse



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domes with a radius of about 1 µm are intact in the unpretreated bagasse (Figure 7 (a,c), S-2 1 and S-2). In the pretreated bagasse, arrays of ruptured domes emerged which were not seen in unpretreated feed (Figure 7 (b,d), S-4). Comparing the pretreated samples with and without physical expansion implies that there hydrolyzing/dissolving effect is the main contributing factor for the morphological disruptions. However, physical expansion led to sever morphological disruptions that were not detected in any other types of samples. (Figure S-5 and Table S-5). Optimizing yields

To optimize yields, practical constraints were considered 200°C, 34 MPa for the smaller reactor and 12.4 MPa for the larger reactor, a packing density of 0.1 g/mL, and no less than 0.33 for solids ratio. The limits on temperature and pressure are set by the equipment’s operating conditions. The packing density cannot exceed 0.1 g/mL otherwise, the vessel will overfill or be too tightly compressed inside. Factorial designs were used to optimize the yield by adjusting operational variables. As suggested by Figures 3 and 5, higher temperatures and lower solids ratios improve the sugars yield. Similarly, higher pressures and lower packing densities favored sugar yield. The maximum yield under these constraints was 85% on both reactor sizes.


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Conclusions

CO2-water pretreatment leads to significant textural transformation of the guayule biomass, such as opening up of dome-like surface features observed by SEM imaging. The pretreatment also led to more than a 10-fold increased surface area exhibited by nitrogen adsorption measurements. At optimal conditions in combination with enzymatic hydrolysis, the pretreatment results in yielding 85% of total reducing sugars. Solids ratio and temperature have the highest impact on the sugar yield, particularly near the optimal condition. A four-fold reactor volume increase of the pretreatment process moved the optimal point towards higher temperatures and lower solids ratio. The positive contribution of rapid CO2 physical expansion to the sugars yield during the pretreatment was up 15%. Phase equilibrium calculations showed that a biphasic environment preserves after isenthalpic expansion for solids ratio below 2.5 and temperature below 190 °C. Variations of yield with solids ratio showed that at temperatures of 170 °C and lower, sugar yield first increased and then decreased beyond solids ratio of 0.5. At higher temperatures the sugar yield monotonically decreased with solids ratio. At the larger reactor, and high solids ratio, differences between high and mid-range temperature became less significant on the yield. Larger reactor experiments showed that moving from solids ratio of 0.3 to 1.7 decreases the yield only about 20% while concentrating the product about 5 times. Considering the economic appeal of concentrated pretreatment product for less energy-intensive downstream processing, we recommend high solids ratio and mid-range temperature.

Supporting Information



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Calculation of CO2 amount during pretreatment, tabulated description of experimental parameters and yields for factorial designs, SEM images of non-pretreated, hydrothermally treated and CO2-water pretreated biomass, variations of sugars yield with holding time and packing density

ACKNOWLEDGMENT This work was partially supported by US Department of Agriculture under the Biomass Research and Development Initiative (Grant No. 68-3A75-7-610). Dr. Katrina Cornish (Senior VP, R&D, Yulex Corporation) provided us the guayule bagasse. The authors are sincerely grateful for their help.

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Figures Figure 1. Schematics of the larger reactor system Figure 2. Variation in total reducing sugars yield with temperature at 1.0 and 0.5 solids ratios, 26.2 MPa, biomass packing density of 0.06 g/mL and 35 minutes holding time. Figure 3. The effect of solids ratio on total reducing sugars yield at (a) 170 and 180 °C temperatures and (b) 160 and 200 °C temperatures at 26.2 MPa, biomass packing density of 0.06 g/mL and 35 minutes holding time. Figure 4. The effect of physical expansion on total sugar yield at packing density of 0.06 and pressure of 26.2 MPa and holding time of 35 minutes. All the above experiments were run at 0.5 solids ratio except the higher temperature one which was run at 0.67. Figure 5. Comparison between larger reactor and smaller reactor when total reducing sugars yield varies with solids ratio both at pressure of 12.4 MPa. Larger reactor experiment was carried out at packing density of 0.1 g/mL while smaller reactor was carried out at 0.06 g/mL. Figure 6. Nitrogen adsorption-desorption isotherms (a) and pore size distribution of biomass samples (b). Unpretreated biomass (Raw) vs pretreated (EXP) with type II isotherm Figure 7. SEM images of unpretreated biomass at 1000x and 5000x magnification (a) and (c) show the array of dome-like morphology ruptured to hole arrays after pretreatment (b) and (d) Tables Table 1. Textural properties of biomass samples before and after pretreatment.


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Exhaust Pump

1L Pretreatment vessel

4 L heating vessel with mixer

Chiller Control Panel

T, P

Figure 1.


Carbon Dioxide

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



90

Solid Ratio = 1 Solid Ratio = 0.5

80

Total Reducing Sugars Yield [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

70

60

50

40

30

20 130

150

170

190

Temperature [°C]

Figure 2.


210

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100

90

90

T=170 °C

80

80

180 °C

70

Total Reducing Sugars Yield (%)

Total Reducing Sugars Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70 60 50 40 30

T = 160 °C

200 °C

60 50 40 30 20

20 10 10 0

0 0

0.5

1

1.5

2

2.5

0

3

0.5

1

1.5

Solids Ratio (g biomass/ g water)

Solids Ratio (g biomass/g water)

(a)

(b) Figure 3.


2


100.00 With Sudden Expansion Without Sudden Expansion

90.00 80.00 Total Reducing Sugars Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 T= 200 °C t=35 T= 180 °C t=35 T= 170 °C t=35 min P=26.2 min P=26.2 min P=26.2 Mpa Solids Mpa Solids Mpa Solids Ratio=0.67 Ratio=0.5 Ratio=0.5

Figure 4.


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100 90

Total Reducing Sugars Yield [%]

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80 70 60 50 40 30

small 170 C large 170 C small 200 C large 200 C

20 10 0 0

1 Solid Ratio

Figure 5.


2


15

Volume@STP (cc/g)

Raw EXP 10

5

0 0.0

0.2

0.4

0.6

0.8

1.0

P/P0 (a)

0.025

Raw EXP

0.020

dV(logd)

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0.015

0.010

0.005

0.000

0

100

200

300

400

Diameter (nm)

(b) Figure 6.


500

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(a)

(b)

(c)

(d)

Figure 7.

Table 1. Materials Surface area (m2.g-1) feed (non-pretreated) 0.27 a Hydrothermally pretreated 0.60 CO2-water pretreatedb 3.03 a. without CO2, 180 °C, 0.5 solids ratio, 35 minutes, b. 180 °C, 0.5 solids ratio, 35 minutes, 26.2 MPa, with sudden pressure release.


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