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Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars Rutgers University has made this article freely available. Please share how this access benefits you. Your story matters. [https://rucore.libraries.rutgers.edu/rutgers-lib/50917/story/] This work is the AUTHOR'S ORIGINAL (AO) This is the author's original version of a work, which may or may not have been subsequently published. The author accepts full responsibility for the article. Content and layout is as set out by the author. Citation to this Version: Murnen, Hannah K., Balan, Venkatesh, Chundawat, Shishir P. S., Bals, Bryan, Sousa, Leonardo & Dale, Bruce E. (2007). Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars. Biotechnology Progress 23(4), 846-850. Retrieved from doi:10.7282/t34x5bpn. Terms of Use: Copyright for scholarly resources published in RUcore is retained by the copyright holder. By virtue of its appearance in this open access medium, you are free to use this resource, with proper attribution, in educational and other non-commercial settings. Other uses, such as reproduction or republication, may require the permission of the copyright holder. Article begins on next page SOAR is a service of RUcore, the Rutgers University Community Repository RUcore is developed and maintained by Rutgers University Libraries

Optimization of Ammonia Fiber Expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars Hannah K. Murnen, Venkatesh Balan*, Shishir P. S. Chundawat, Bryan Bals and Bruce E. Dale Biomass Conversion Research Laboratory, Department of Chemical Engineering and Material Science, 2527 Engineering Building, East Lansing, MI 48824. * To whom correspondence should be addressed. Tel: 517-432-2665, Fax: 517 432 1105. E-mail: balan@msu.edu Key words: Miscanthus, AFEX pretreatment, enzymatic hydrolysis, biomass, bio-refinery 1

Abstract: Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. Most research conducted so far has focused on the crop yield per hectare, optimal climatic conditions, energy, water and nitrogen input needed for growth. It is clear that Miscanthus has several advantages as a renewable energy crop, the most notable ones being; high yield per hectare, high cellulose content, low water and nitrogen input. However, there is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions such as temperature, moisture, ammonia loading, residence time and enzyme loadings are varied to maximize hydrolysis yields. In addition, further pretreatments such as grinding and soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 ºC, 2:1 ammonia loading, 70 % moisture (dry weight basis), and 5 minute reaction time for water soaked Miscanthus. Approximately 96% glucan and 85% xylan conversions were achieved after 168 hours hydrolysis of 1% glucan loading enzymatic hydrolysis (15 FPU/g glucan of cellulase and 64 p-npgu/g glucan of β-glucosidase along with xylanase and tween-80 supplementation). A complete mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented. 2

Introduction: Miscanthus (Miscanthus x giganteus), also known as Giant Chinese Silver Grass, is a large perennial rhizomatous grass utilizing the C4 biosynthesis pathway (Figure 1A). It can grow over 12 feet tall and has gained considerable interest in Europe as a possible dedicated energy crop, either as fuel for electricity generation or, most notably, conversion to a biofuel such as ethanol. Miscanthus has numerous characteristics that make it advantageous as a dedicated energy crop. It can be grown in poor quality soil with a high yield per acre, and requires little herbicide, nitrogen, and water (1-3). Such characteristics help make the biomass an economically attractive choice to farmers. Numerous agricultural studies throughout Europe have been promising, showing yields ranging from 25 t/acre in Britain and Denmark to 30 t/acre in Spain and Italy (4). Concerns about Miscanthus becoming a destructive invasive species, due to its low resource requirements and fast growth (4), have dampened interest in the crop within the United States. Miscanthus yields tend to be higher than switchgrass (Panicum vergatum), a common energy crop receiving considerable interest in the US. Furthermore, Miscanthus retains high growth efficiency even in cooler climates, thus potentially being preferable in the northern United States (5). Interest in using biomass derived ethanol has grown considerably in the United States, culminating in a national goal to replace 30% of US gasoline with ethanol by 2030 (Energy policy Acts, 2005). Miscanthus may assist in realizing this goal. To date, however, little research has been devoted to the enzymatic hydrolysis of Miscanthus. Using a one-step extrusion/naoh pretreatment process, de Vrije et al. were able to hydrolyze 69% and 38% of the cellulose and hemicellulose respectively into 3

monomeric sugars (6). This process removes over 75% of the lignin prior to enzymatic hydrolysis, but also removes over 50% of xylan as well (6). An alternative approach that does not require removing lignin is the Ammonia Fiber Expansion (AFEX) pretreatment. This process entails saturating wet biomass with ammonia at high pressures (200-1000 psi) and different temperatures (70-180 o C). After a brief residence time, the pressure is explosively released, effectively disrupting the structure of the biomass. AFEX decrystallizes cellulose, partially hydrolyzes hemicellulose, and depolymerizes lignin (7). With growing demand for ethanol (10-13) has begun the search for suitable energy crops in addition to agricultural residues, that requires less energy, water and nitrogen input, Miscanthus stands out as a potential feed stock. In addition, Miscanthus has higher cellulose content similar to hard wood and hence yields higher sugar per ton of biomass. Our previous work has shown that AFEX followed by enzymatic hydrolysis give near theoretical yields of glucose on different types of agricultural residue (14, 15). Previous work with other herbaceous energy crops, namely switchgrass (8) and dwarf elephant grass (9), has shown high yields of both glucose and xylose after AFEX pretreatment. The goal of this paper is to maximize sugar yields during enzymatic hydrolysis for AFEX pretreated Miscanthus. Pretreatment process conditions such as temperature, moisture, ammonia loading and residence time and enzyme loadings are varied to determine the effect on enzymatic hydrolysis. In addition, further pretreatments such as grinding and water soaking the biomass prior to AFEX as well as washing the material afterwards are also attempted to improve sugar yields. A complete mass balance for the AFEX pretreatment and enzymatic hydrolysis process is also presented. 4

Materials and Methods Lignocellulosic substrate. Miscanthus x giganteus, harvested in spring 2005, was a generous gift from Professor Steve Long, University of Illinois Urbana Champaign (Figure 1A) and stored at room temperature until further use. This material was first milled using a JT Homoloid mill from the Fitzpatrick Company with a 3.175 mm diameter sieve. The milled biomass was further ground down to a desired particle size range using a centrifugal mill (Model ZM 200, Retsch) fitted with various ring sieve attachments. The moisture content was measured using a moisture analyzer (A&D, Model MF-50). Compositional Analysis. The NREL standard protocol for Acid Hydrolysis (LAP- 019) was used to determine the glucan and xylan content. The composition of the untreated Miscanthus was found to be 44% glucan and 19% xylan. The conversions were based on the theoretical glucan and xylan content (44% and 19%, respectively), unless samples were soaked or washed. The glucan composition for soaked or washed biomass was calculated based on the overall mass loss, assuming no glucan was lost during soaking or washing. Soaking. Prior to the AFEX treatment, some of the samples were soaked in water for 24 hours to enhance pretreatment effectiveness. Untreated biomass was presoaked in distilled water with a substrate to water loading of 1:10 (w/w). The wash liquid was removed from the substrate by squeezing the slurry through a filtration cloth (Calbiochem, CA). The moisture content of the soaked Miscanthus was between 60-70% (dwb-dry weight basis) after soaking. In few experiments, the soaked samples were air dried under a hood to achieve the required moisture content before AFEX. 5

AFEX Treatment. A bench-top reactor consisted of a 300 ml stainless steel pressure vessel (PARR Instrument Co, IL). The vessel was loaded with Miscanthus adjusted to the appropriate moisture content. Glass beads were used to fill the void volume in the vessel. This step minimized the amount of liquid ammonia vaporized during the process. The vessel was clamped shut and the required amount of ammonia was injected using a preweighed sample cylinder. A 400 W PARR heating mantle was used to heat the reactor and maintain it at the desired temperature (+/- 2 ºC) for the necessary residence time. At the completion of the residence time (5-30 min.), the pressure was explosively released and the vessel cooled. The biomass was removed from the reactor and left in the hood overnight to evaporate the residual ammonia. Figure 1B gives details on the visual appearance of untreated and AFEX treated Miscanthus. Washing. Some of the AFEX treated biomass samples were washed in distilled (deionized) water to substrate loading of 10:1 (w/w). The slurry was mixed for 15 minutes. The wash liquid was removed from the substrate by squeezing the slurry through a filtration cloth (Calbiochem, CA). Enzymatic Hydrolysis. The NREL standard protocol (LAP-009) was followed for enzymatically hydrolyzing the pretreated biomass. All the samples were hydrolyzed in a ph 4.8 citrate buffer at a loading of 1% glucan with the desired cellulase enzyme (Spezyme CP provided by Genencor, CAS 9012-528) at a loading of 15 FPU/g of glucan and β-glucosidase (Novozyme) at a loading of 64 p-npgu/g of glucan. Certain samples were also hydrolyzed using xylanase (Multifect Xylanase, Genencor), pectinase (Multifect Pectinase, Genencor) and Tween-80 surfactant (0.325 g/g glucan). The 6

samples were hydrolyzed at 50 ºC, 90 rpm for a period of 168 hours. Sampling was carried at intervals of 72 and 168 hours to determine glucan and xylan conversions. HPLC Sugar Analysis: A high performance liquid chromatography (HPLC) system was used for sugar analysis. The HPLC system consisted of Waters (Milford, MA) Pump and Waters 410 refractive index detector, an Aminex HPX-87P carbohydrate analysis column (BioRad, Hercules, CA) equipped with a deashing guard cartridge (BioRad). Degassed HPLC grade water was used as the mobile phase at 0.6 ml/min at a column temperature of 85 o C. The injection volume was 10 µl with a run time of 20 min. Mixed sugar standards were used for quantification of cellobiose and other monosaccharides (glucose, xylose, galactose, arabinose and mannose) in the samples. Results and Discussion The AFEX treated material was slightly darker in appearance than the untreated material (Figure 1B), probably due to lignin degradation and re-deposition on the surface of biomass during AFEX. The untreated Miscanthus was hydrolyzed to give less than 5-10% glucan and xylan conversion (data not shown). AFEX pretreatment significantly increased the enzymatic hydrolysis of Miscanthus, giving close to 30-90% conversion, depending on the pretreatment parameters. Therefore, a detailed study of the effect of various pretreatment parameters, such as moisture, ammonia loading and temperature, on the hydrolysis yields was undertaken. Effect of Temperature and Ammonia Loading. Figure 2A shows the effect of increasing pretreatment temperature (100 ºC - 180 ºC) on the enzymatic hydrolysis yields of AFEX-treated Miscanthus, while holding the ammonia to biomass ratio constant either 7

at 1:1 or 2:1 and 60% moisture content (dwb-dry weight basis). The higher ammonia level is clearly beneficial in increasing the maximum glucan conversion at all temperatures. However, the increase in glucan conversion levels off at temperatures above 140 ºC for 2:1 ammonia loading unlike the 1:1 ammonia loading. At higher temperatures (180 ºC) for the 2:1 ammonia loading the glucan conversion drops off substantially. This may be a result of charring or other alkali enhanced degradation of the biomass at high temperatures. The xylan conversion, on the other hand, is fairly constant across 120 ºC 160 ºC in the 2:1 case and only drops off at 180 ºC. This result indicates that the limiting factor for higher glucan-xylan conversions might also be the optimal combination of enzymes along with pretreatment severity. We found that 2:1 ammonia loading was optimal and all further experiments (Figure 2A) were based on that, although further improvements are yet unexplored. Effect of Moisture Content. Figure 2B shows the effect of varying the moisture content of the biomass for a constant ammonia loading (either 1:1 or 2:1) and temperature (140 ºC). For both 2:1 and 1:1 ammonia loading, increasing the moisture content increased the glucan conversion, with the 2:1 ammonia loading giving higher conversions than 1:1 loading. The xylan conversion was more variable, peaking at 80% (dwb) moisture and 2:1 loading. By varying the moisture content for a fixed temperature (140 o C) during the AFEX process, maximum glucan (60%) and xylan (70%) conversion were achieved at 80% moisture (Figure 2B). These results are comparable to the optimum moisture conditions required for AFEX treated switchgrass (8). However, for the 24 hours water soaked sample, the moisture content after removal of the water was 70% 8

(dwb) and was used as it is. We could see up to 10% increase in glucan conversion upon soaking Miscanthus in water prior to AFEX (Figure 3). Effect of pre-afex Soaking and post-afex Washing. Ongoing research indicates the critical role of water in AFEX for some materials (unreported results). Figure 3 shows the effect of soaking the sample for 24 hours in water prior to the AFEX treatment. The highest conversion is obtained for 160 ºC, 2:1 ammonia loading, water soaked Miscanthus samples. The water soaking prevented a drop in glucan conversion at higher temperatures (above 140 ºC). Results show that water soaking of Miscanthus prior to AFEX further improves the glucan conversion by 10-15%, as compared to the unsoaked control. Disruption of internal lignocellulosic bonds and forces by solvent swelling will generate internal stresses and improve accessibility to chemicals like ammonia during the pretreatment process (16, 17). Water also helps to solvate the ammonia, releasing hydroxyl ions that catalyze the lignin and hemicellulose depolymerization reactions. There seems to be some sort of equilibrium time required for the water to gain access to the lignocellulosic ultrastructure (16, 17). All samples were water soaked for 24 hours prior to AFEX treatment at conditions of 160 ºC and 2:1 ammonia loading from here onwards. The AFEX corn stover wash stream was found to contain several inhibitory compounds that were identified by HPLC-MS/MS and quantified by HPLC-UV analyses (manuscript submitted). It is likely that washing AFEX treated Miscanthus helps remove some of the degradation products like organic acids, oligosaccharides, ligno-phenolics, etc that may inhibit the enzymatic activity (18). 9

Effect of Residence Time. Figure 4 indicates the effect of residence time on the AFEX process. For the optimization of the moisture content, ammonia loading and temperature, as shown previously, the residence time was kept fixed at 5 minutes. However, it was suspected that a longer residence time might help improve the overall conversions considering the recalcitrant nature of this material. There was an improvement in the glucan yields upon increasing the AFEX residence time from 5 to 30 mins, for a 160 ºC, 2:1 ammonia loading presoaked Miscanthus sample. However, there was no significant improvement in the xylan yield. Increasing the residence time to 30 minutes seemed to have the most beneficial effect beyond which little or no improvement was seen. Similar resident time effect was notices for ground Miscanthus samples (results not shown) because, reduction in particle size increases the surface are and hence reduce the time take for ammonia to penetrate the biomass. Effect of Additives. Figures 5 and 6 show the effect of additives (varying concentrations of xylanases and/or pectinases) on enzymatic hydrolysis using the AFEX treated (160 ºC, 2:1 ammonia loading, presoaked) Miscanthus sample. Xylanase and pectinase are known to hydrolyze xylan and pectin hemicellulose oligomers present surrounding the cellulosic microfibrils (15, 21). By cleaving these components, cellulose microfibril accessibility to cellulolytic enzymes is increased, hence improving overall glucan conversions. These synergistic actions of cellulase, xylanase, pectinase along with β-glucosidase results in higher sugar yields. However, the optimum synergistic concentration tapers off at higher loadings, probably due to non-specific binding of enzymes to substrate (19). The most effective combination of pectinase and xylanase was found to be 1.03 and 0.56 g/100 g of dry biomass, respectively. Tween-80 helps further 10

improve the glucan and xylan yields. Tween-80 is a non-ionic surfactant that prevents irreversible binding of lignin to hydrolytic enzymes (20). With the combination of pectinase or xylanase, along with Tween-80, a glucan and xylan conversion of 90-95% and 80-85%, respectively, was achieved. The combination of both xylanase and pectinase did not seem to have much impact on the conversions (Figure 6). This could be due to competition between the enzymes or due to non specific binding of enzyme on the substrate (19). Effect of Size Reduction. The pre-milled Miscanthus was further ground using a 80 micron sieve to study the effect of size reduction on AFEX. Reducing biomass particle size was found to have a beneficial effect on AFEX pretreatment (120 o C, 2:1, 60% moisture and 5 min. residence time) and enzymatic hydrolysis of Miscanthus. There is a substantial improvement in glucan (15-20%) and xylan (10-15%) yields for the ground samples compared to un-ground sample (Figure 7). Similar improvements in glucan yields were seen for ground AFEX treated corn stover (18). Two factors probably contribute most to the enhancement in hydrolytic yield upon size reduction; namely improved enzyme and pretreatment chemical (i.e. ammonia) accessibility to lignocellulosic ultra structure. The role of particle size on glucan conversion has been reported before (18). The primary purpose for grinding the biomass prior to AFEX was to study its suitability in conducting microplate-based high throughput hydrolytic experiments (work under progress). Mass Balance. In order to fully understand the process, a detailed mass balance was done for each step (Figure 8). The samples were soaked prior to AFEX treatment and the optimal AFEX conditions used were 160 ºC, 2:1 ammonia loading, and 5 min. reaction 11

time. Subsequent to AFEX, the washing step resulted in a 10-11% mass loss. While, the soaking process prior to AFEX treatment resulted in a 1% mass loss. The AFEX process was assumed to have made no contribution to the mass balance, considering previous work (15). An enzyme loading of 15 FPU/g glucan of cellulase (Spezyme CP), 64 p- NPGU/g glucan of β-glucosidase (Novozyme 188) with additives such as xylanase (0.53 g/100 g of Miscanthus), pectinase (1.06 g/100 g of Miscanthus) and tween 80 (0.35 g/g glucan) were used to hydrolyze the AFEX treated and untreated biomass. The samples were hydrolyzed at 50 ºC, 200 rpm for a period of 168 hours. Close to 88% glucan and 80% xylan conversion was achieved at the end of 168 hours for 1% glucan loading based enzymatic hydrolysis. A 20 fold increase in the glucose and xylose concentration is seen for the AFEX treated hydrolysate compared to the untreated hydrolysate (Figure 8). The overall glucan conversions using mixed enzyme combinations of xylanase and pectinase are lower compared to individual enzyme action (Figures 5 and 6). Thus the slightly lower glucan yield compared to when xylanase or pectinase are used alone. Conclusion Several AFEX pretreatment parameters were varied while screening the best AFEX conditions for Miscanthus. Two different ammonia to biomass loadings (i.e., 1:1 and 2:1) were screened, along with varying temperatures (100-180 ºC) and moisture contents (20-80% based on dwb). Grinding the pre-milled Miscanthus to 80 microns prior to AFEX also helped improve glucan and xylan conversion by 10-20%. It was found that water soaking the biomass prior to AFEX (pre-afex soaking) followed by washing the treated material (post-afex washing) and hydrolyzing with cellulolytic enzymes supplemented 12

by additives (i.e. xylanase and/or pectinase and tween-80) results in close to theoretical glucan and xylan conversions. A complete mass balance for this process was also carried out. Glucan and xylan conversions close to 90-95% and 80-85%, respectively, were possible upon hydrolysis of AFEX treated Miscanthus using cellulases (15 FPU/ g of glucan) and other additives, such as xylanase (0.53 g/100 g of Miscanthus) or pectinase (1.03 g/100 g of Miscanthus) and Tween-80 (0.35 g/g of glucan). Further improvements in the AFEX process and varying enzyme mixtures will help further improve the glucan and xylan conversions at lower pretreatment severity. Currently under development is a microplate-based enzyme assay to help screen suitable enzyme mixtures. High solid loading based hydrolysis and fermentation of the AFEX treated Miscanthus hydrolysate is currently under progress. Acknowledgement This research was conducted at the Biomass Conversion Research Lab (BCRL), Michigan State University, supported by funds from SPG Proposal (MSU Research Foundation). The authors would like to thank Leonardo D sousa for helping draft the figures. Special thanks to Dr. Steven P. Long for authorizing use of the Miscanthus photograph and for providing the material. We would also like to acknowledge MBI for helping grind the biomass. 13

References and Notes (1) Clifton-Brown, J. C.; Lewandowski, I.; Andersson, B. Performance of 15 Miscanthus genotypes at five sites in Europe. Agronomy J. 2001, 93, 1013 1019. (2) Heaton, E.; Clifton-Brown, J.; Voigt, T.; Jones, M.; Long, S. Mitigation and Adaptation Strategies for Global Change 2004, 9, 433-451. (3) Heaton E.; Voigt, T.; Long, S. P. A quantitative review comparing the yields of two candidate C-4 perennial biomass crops in relation to nitrogen, temperature and water. Biomass and Bioenergy, 2004, 27, 21 30. (4) Scurlock, J. Miscanthus: A Review of European Experience with a Novel Energy Crop. Oak Ridge National Laboratory, 1999. (5) Beale, C.V.; Bint, D.A.; Long, S.P. Journal of Experimental Botany 1996, 47, 267-273. (6) de Vrije, T.; de Haas, G.; Tan, G.; Keijsers, E.; Claassen, P. International Journal of Hydrogen Economy 2002, 27, 1381-1390. (7) Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Lee, Y.; Holtzapple, M.; Ladisch, M. Bioresource Technology 2005, 96, 673-686. (8) Alizadeh, H.; Teymouri, F.; Gilbert, T.; Dale, B. Applied Biochemistry and Biotechnology 2005, 121-124, 1133-1141. (9) Ferrer, A.; Byers, F.M.; Sulbarán De Ferrer, B.; Dale, B.E.; Aiello, C. Applied Biochemistry and Biotechnology, 2000, 84-86, 163-179. 14

(10) Knauf, M.; Moniruzzaman, M. Lignocellulosic biomass processing: A perspective, Int. Sugar J. 2004, 106, 147-150. (11) Gray, K. A.; Zhao, L.; Emptage, M. Bioethanol, Current Opinion in Chemical Biology 2006, 10, 141 146. (12) Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresource Technol. 2002, 83, 1 11. (13) From Niche to Nation: Ethanol Industry Outlook 2006; Renewable Fuels Association Washington D.C.: 2006, p4. (14) Sulbaran-de-Ferrer, B.; Aristiguieta, M.; Dale, B.; Ferrer, A.; Ojeda-de-Rodriguez, G. Enzymatic hydrolysis of ammonia-treated rice straw. Appl. Biochem. Biotechnol. 2003, 105-108, 155-164. (15) Teymouri, F.; Laureano-Perez, L.; Alizadeh, H.; Dale, B. E. Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Bioresource Technol. 2005, 96, 2014 2018 (16) Ye, D.; Farriol, X. Improving accessibility and reactivity of celluloses of annual plants for the synthesis of methylcellulose, Cellulose, 2005, 12, 507-515. (17) Boluk, Y. Acid-base interactions and swelling of cellulose fibers in organic liquids, Cellulose, 2005, 12, 577-593. (18) Chundawat S. P. S., Venkatesh, B.; Dale, B. E. Effect of Particle Size Based Separation of Milled Corn Stover on AFEX Pretreatment and Enzymatic Digestibility. Biotech Bioeng. 2006 (In Press) 15

(19) Jeoh, T.; Wilson, D.B.; Walker, L.P. Cooperative and competitive binding in synergistic mixtures of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A. Biotechnol Prog. 2002, 18, 760-769. (20) Kim, S.B.; Kim, H.J.; Kim, C.J. Enhancement of the enzymatic digestibility of waste newspaper using Tween. Appl. Biochem. Biotchnol. 2006, 129-132, 486-495. (21) Foster, B. L.; Dale, B. E.; Doran-Peterson, J. B. Enzymatic hydrolysis of ammoniatreated sugar beet pulp. Appl Biochem Biotechnol. 2001, 91-93, 269-82. 16

Figure Captions Figure 1. Miscanthus, untreated and pretreated samples for biomass conversion. (A) Giant Miscanthus (Miscanthus x giganteus), a hybrid grass that can grow over 12 feet high, may be a valuable renewable fuel source for the future. (B) Milled Miscanthus samples; untreated (UT), un-soaked and AFEX treated (US-AFEX) and water soaked and AFEX treated (WS-AFEX). Figure 2. Effect of varying AFEX conditions on glucan and xylan yields upon enzymatic hydrolysis. Here, the effect of varying temperature for fixed 60% moisture content (A) and varying moisture content (dry weight basis) for a fixed pretreatment temperature (140 o C) (B) is shown, for two different ammonia to biomass (BM) loadings during the AFEX process (5 minutes residence time). The corresponding glucan (white shade) and xylan (grey shade) conversion after 72 (un-hatched) and 168 hours (hatched) of enzymatic hydrolysis are shown with duplicate error bars. Figure 3. Glucan and xylan yields after enzymatic hydrolysis for (a) un-soaked and, (b) 24 hour water soaked Miscanthus prior to AFEX (2:1 ammonia to biomass, 70% moisture on dry weight basis, 5 minutes residence time) at varying temperatures. Here, the corresponding glucan (white shade) and xylan (grey shade) conversion after 72 (unhatched) and 168 hours (hatched) of enzymatic hydrolysis are shown with duplicate error bars. 17

Figure 4. Glucan and xylan yields after enzymatic hydrolysis for different residence times during the AFEX Process (160 o C, 2:1 ammonia to biomass and 70% moisture on dry weight basis) for 24 h water soaked Miscanthus sample. Here, 5, 15 and 30 represents waiting period once the desired temperature is reached during the AFEX process and their corresponding glucan (white shade) and xylan (grey shade) conversions after 72 (un-hatched) and 168 hours (hatched) of enzymatic hydrolysis. Figure 5. Effect of varying supplementation of xylanase (A) and pectinase (B), along with cellulase and β-glucosidase during enzymatic hydrolysis. Miscanthus was soaked in water for 24 h prior to AFEX (160 o C, 2:1, 60% moisture on dry weight basis, 5 minutes residence time) and washed prior to hydrolysis. The corresponding glucan (white shade) and xylan (grey shade) conversion after 72 (un-hatched) and 168 hours (hatched) of enzymatic hydrolysis are given. Here, T, represents Tween-80 supplementation. Figure 6. Effect of combination of xylanase and pectinase, along with cellulase and β- glucosidase during enzymatic hydrolysis for AFEX process (conditions same as in figure 5) on Miscanthus sample. The corresponding glucan (white shade) and xylan (grey shade) conversion after 72 (un-hatched) and 168 hours (hatched) of enzymatic hydrolysis are given. Here, (a) no Tween-80 and (b) with Tween-80 supplementation respectively. 18

Figure 7. Effect of further grinding (under 80 microns) and enzymatic hydrolysis (using 15 FPU/g glucan of cellulase and 64 pnpgu/g glucan of β-glucosidase) for AFEX treated Miscanthus (120 o C, 60% moisture on dry weight basis and 2:1 ammonia to biomass loading, 5 min residence time). Figure 8. Flow chart showing mass balance during pretreatment and hydrolysis process for Miscanthus. (A) AFEX treatment at 70% moisture content (dwb), 2:1 ammonia loading and 160 o C and (B) untreated sample. Enzymes (C, Cellulase; X, Xylanase; P, Pectinase) and additives (T, Tween 80) used are listed below; 15 FPU/g glucan of cellulase (C), 64 p-npgu/g glucan of β-glucosidase, 0.56 g/100 g dry Miscanthus of xylanase (X), 1.03 g/100 g dry Miscanthus of pectinase (P) and Tween-80 (0.35 g/glucan). 19

Figure 1. A B UT US-AFEX WS-AFEX 20

Figure 2 21

Figure 3 22

Figure 4 23

Figure 5 24

Figure 6 25

Figure 7 26

Milled iscanthus 100g dry basis) Water Enzymes (C, X, P) Ammonia And Additives (T) Water Treated Water A Miscanthus 88.16 g Soak AFEX Wash Hydrolysis System 98.98 g 98.98 g Residual 10.82 g 9.49 g Figure 8 Hydrolyzate liquid Sugars 47.04 g glucose 17.48 g xylose Enzymes (C, X, P) And Additives (T) B Untreated Miscanthus 98.98 g Residual Hydrolysis Hydrolyzate liquid Sugars 2.56 g glucose 0.83 g xylose 93.81 g 27