Structural changes of corn stover lignin during acid pretreatment

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70?% at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by (31)P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180?°C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.     

Characterization of extracellular lignocellulolytic enzymes of Coniochaeta sp. during corn stover bioconversion

Biorefinery of renewable lignocellulosic biomass to biochemical and biofuel is a promising technology to mitigate global warming and fuel shortage but hydrolysis of recalcitrant lignocellulose to its constitutive components is the bottleneck of the process. This work isolated and characterized a new lignocellulose degrading filamentous fungus from decomposing wood in mangrove area. The strain was identified as Coniochaeta sp. according to ITS rRNA sequences and its phylogenic analysis. The extracellular lignocellulolytic enzymes of this fungal strain, when grown on corn stover, were profiled by LC–MS/MS and exponentially modified protein abundance index (emPAI) based label-free quantitative proteomics approach. We identified 107 potential lignocellulolytic enzymes and their functional classification revealed unique extracellular enzyme system constituting multienzyme complexes of cellulases (29%), hemicellulases (17%), glycoside hydrolases (10%), proteases and peptidases (24%), lignin degrading enzymes (7%) and hypothetical proteins (13%). The growth behavior, biochemical assay and LC–MS/MS analysis of secretome by isolated fungal strain revealed its lignocellulose degradation potential when cultivated with corn stover as a major carbon source.     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

Life cycle assessment of corn grain and corn stover in the United States

Background, aim, and scope  The goal of this study is to estimate the county-level environmental performance for continuous corn cultivation of corn grain and corn stover grown under the current tillage practices for various corn-growing locations in the US Corn Belt. The environmental performance of corn grain varies with its farming location because of climate, soil properties, cropping management, etc. Corn stover, all of the above ground parts of the corn plant except the grain, would be used as a feedstock for cellulosic ethanol. Materials and methods  Two cropping systems are under investigation: corn produced for grain only without collecting corn stover (referred to as CRN) and corn produced for grain and stover harvest (referred to as CSR). The functional unit in this study is defined as dry biomass, and the reference flow is 1 kg of dry biomass. The system boundary includes processes from cradle to farm gate. The default allocation procedure between corn grain and stover in the CSR system is the system expansion approach. County-level soil organic carbon dynamics, nitrate losses due to leaching, and nitrogen oxide and nitrous oxide emissions are simulated by the DAYCENT model. Life cycle environmental impact categories considered in this study are total fossil energy use, climate change (referred to as greenhouse gas emissions), acidification, and eutrophication. Sensitivities on farming practices and allocation are included. Results  Simulations from the DAYCENT model predict that removing corn stover from soil could decrease nitrogen-related emissions from soil (i.e., N₂O, NO _x , and NO₃ ⁻ leaching). DAYCENT also predicts a reduction in the annual accumulation rates of soil organic carbon (SOC) with corn stover removal. Corn stover has a better environmental performance than corn grain according to all life cycle environmental impacts considered. This is due to lower consumption of agrochemicals and fuel used in the field operations and lower nitrogen-related emissions from the soil. Discussion  The primary source of total fossil energy associated with biomass production is nitrogen fertilizer, accounting for over 30% of the total fossil energy. Nitrogen-related emissions from soil (i.e., N₂O, NO _x , and NO₃ ⁻ leaching) are the primary contributors to all other life cycle environmental impacts considered in this study. Conclusions  The environmental performance of corn grain and corn stover varies with the farming location due to crop management, soil properties, and climate conditions. Several general trends were identified from this study. Corn stover has a lower impact than corn grain in terms of total fossil energy, greenhouse gas emissions, acidification, and eutrophication. Harvesting corn stover reduces nitrogen-related emissions from the soil (i.e., N₂O, NO _x , NO₃ ⁻). The accumulation rate of soil organic carbon is reduced when corn stover is removed, and in some cases, the soil organic carbon level decreases. Harvesting only the cob portion of the stover would reduce the negative impact of stover removal on soil organic carbon sequestration rate while still bringing the benefit of lower nitrogen-related emissions from the soil. No-tillage practices offer higher accumulation rates of soil organic carbon, lower fuel consumption, and lower nitrogen emissions from the soil than the current or conventional tillage practices. Planting winter cover crops could be a way to reduce nitrogen losses from soil and to increase soil organic carbon levels. Recommendations and perspectives  County-level modeling is more accurate in estimating the local environmental burdens associated with biomass production than national- or regional-level modeling. When possible, site-specific experimental information on soil carbon and nitrogen dynamics should be obtained to reflect the system more accurately. The allocation approach between corn grain and stover significantly affects the environmental performance of each. The preferred allocation method is the system expansion approach where incremental fuel usage, additional nutrients in the subsequent growing season, and changes in soil carbon and nitrogen dynamics due to removing corn stover are assigned to only the collected corn stover.     

Ensiling corn stover: effect of feedstock preservation on particleboard performance

Ensilage is a truncated solid-state fermentation in which anaerobically produced organic acids accumulate to reduce pH and limit microbial activity. Ensilage can be used to both preserve and pretreat biomass feedstock for further downstream conversion into chemicals, fuels, and/or fiber products. This study examined the ensilage of enzyme-treated corn stover as a feedstock for particleboard manufacturing. Corn stover at three different particle size ranges (<100, <10, and <5 mm) was ensiled with and without a commercial enzyme mixture having a cellulase:hemicellulase ratio of 2.54:1, applied at a hemicellulase rate of 1670 IU/kg dry mass. Triplicate 20 L mini-silos were destructively sampled and analyzed on days 0, 1, 7, 21, 63, and 189. Analysis included produced organic acids and water-soluble carbohydrates, fiber fractions, pH, and microorganisms, including Lactobacillus spp. and clostridia were monitored. On days 0, 21, and 189, the triplicate samples were mixed evenly and assembled into particleboard using 10% ISU 2 resin, a soy-based adhesive. Particleboard panels were subjected to industry standard tests for modulus of rupture (MOR), modulus of elasticity (MOE), internal bonding strength (IB), thickness swell (TS), and water absorption at 2 h boiling and 24 h soaking. Enzyme addition did improve the ensilage process, as indicated by sustained lower pH (P < 0.0001), higher water-soluble carbohydrates (P < 0.05), and increased lactic acid production (P < 0.0001). The middle particle size range (<10 mm) demonstrated the most promising results during the ensilage process. Compared with fresh stover, the ensilage process did increase IB of stover particleboard by 33% (P < 0.05) and decrease water adsorption at 2 h boiling and 24 h soaking significantly (P < 0.05). Particleboard panels produced from substrate ensiled with enzymes showed a significant reduction in water adsorption of 12% at 2 h boiling testing. On the basis of these results, ensilage can be used as a long-term feedstock preservation method for particleboard production from corn stover. Enzyme-amended ensilage not only improved stover preservation but also enhanced the properties of particleboard products.     

Cellulase production from the corn stover fraction based on the organ and tissue

In this study, the biodegradation of fractionated corn stover in solid-state fermentation by Trichoderma reesei YG3 was investigated. Fractions of miscellaneous cells (MC) and fasciculi (FC) tissue from leaf, shell or core were separated using the combined pretreatment method of carding classification after steam explosion. The highest enzyme activities including the filter paper activity, endoglucanase, exoglucanase and β-glucosidase activities, weight loss rate of dry material and biodegradation rate were all observed in the MC tissue fraction from the leaf, which was more nutritious, while lowest activity was observed in the FC tissue fraction from the shell. The maximum filter paper activity and weight loss rate of the dry material were 4.56 and 1.89 times the minimum and the cellulose and hemicellulose biodegradation rates were 51.22 and 39.38% versus 23.85 and 26.51%, respectively. These variances maybe attributed to the heterogeneity of the component in the fractions. A higher weight loss rate corresponded to higher enzymatic activities, whereas cellulose biodegradation was not proportional to cellulase activities. Hemicellulose biodegradation was much slower than cellulose degradation. Here, we demonstrated the importance of fractionation in component biodegradation and utilization of straw.     

Steam activation of chars produced from oat hulls and corn stover

Oat hulls and corn stover were used to produce chars at approximately 500 degrees C. The carbon concentrations of oat hull char and corn stover chars produced were 72.3 and 68.0 wt.%, respectively. Both activation burn-off and Brunauer-Emmett-Teller (BET) surface area appear to exhibit a linear relationship with respect to activation time of oat hulls. As to corn stover activated carbons, there is no linear relationship between activation time and BET surface area. However, activation burn-off of and activation time appear to relate in a linear manner for the activated carbons produced from corn stover chars. Oat hull is better than corn stover as a raw material for the production of activated carbon.     

The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover

Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-β-xylanase III), Xyn 11 (endo-xylanase II), and β-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and β-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity.     

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

Background

Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

Results

In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration.

Conclusion

We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

     

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (～6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.     

Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover

Ethanol can be produced from lignocellulosic biomass using steam pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields, from both hemicellulose and cellulose are critical parameters for an economically-feasible ethanol production process. This study shows that a near-theoretical glucose yield (96-104%) from acid-catalysed steam pretreated corn stover can be obtained if xylanases are used to supplement cellulases during hydrolysis. Xylanases hydrolyse residual hemicellulose, thereby improving the access of enzymes to cellulose. Under these conditions, xylose yields reached 70-74%. When pre-treatment severity was reduced by using autocatalysis instead of acid-catalysed steam pretreatment, xylose yields were increased to 80-86%. Partial delignification of pretreated material was also evaluated as a way to increase the overall sugar yield. The overall glucose yield increased slightly due to delignification but the overall xylose yield decreased due to hemicellulose loss in the delignification step. The data also demonstrate that steam pretreatment is a robust process: corn stover from Europe and North America showed only minor differences in behaviour.     

Chemical and enzymic pretreatment of corn stover to produce soluble fermentation substrates

Corn stover was pretreated with various chemical agents, including sodium hydroxide, sulfuric acid, ethylenediamine, n-butylamine (either alone or in solution with methanol), and acetonitrile or ethanol containing hydrochloric acid. Of these chemicals, n-butylamine was the best reagent for pretreatment of corn stover, considering the degree of loss of total carbohydrate, delignification, cumulative weight loss, cumulative yield of reducing sugars per original total carbohydrate, and the potential ease of recovery and reuse of reagent. In comparison to the other reagents tested, n-butylamine (n-BA) selectively delignified corn stover. The best conditions were as follows: a 12-h presoak of about a 155 g dry wt/L slurry (1 mm average particle size) in 100% n-BA at room temperature, followed by 30 min of refluxing (86.5 degrees C) with 40% (w/w) n-BA-distilled water solution. The cumulative yield of reducing sugars after enzymic hydrolysis was 44.5% of the original total carbohydrate and the cumulative total weight loss (dry basis) was 59%. Degradative loss of total carbohydrate during pretreatment was not detected.     

Effects of biopretreatment of corn stover with white-rot fungus on low-temperature pyrolysis products

The thermal decomposition of biopretreated corn stover during the low temperature has been studied by using the Py-GC/MS analysis and thermogravimetric analysis with the distributed activation energy model (DAEM). Results showed that biopretreatment with white-rot fungus Echinodontium taxodii 2538 can improve the low-temperature pyrolysis of biomass, by increasing the pyrolysis products of cellulose, hemicellulose (furfural and sucrose increased up to 4.68-fold and 2.94-fold respectively) and lignin (biophenyl and 3,7,11,15-tetramethyl-2-hexadecen-1-ol increased 2.45-fold and 4.22-fold, respectively). Calculated by DAEM method, it showed that biopretreatment can decrease the activation energy during the low temperature range, accelerate the reaction rate and start the thermal decomposition with lower temperature. ATR-FTIR results showed that the deconstruction of lignin and the decomposition of the main linkages between hemicellulose and lignin could contribute to the improvement of the pyrolysis at low temperature.     

Effect of anatomical fractionation on the enzymatic hydrolysis of acid and alkaline pretreated corn stover

Due to concerns with biomass collection systems and soil sustainability there are opportunities to investigate the optimal plant fractions to collect for conversion. An ideal feedstock would require a low severity pretreatment to release a maximum amount of sugar during enzymatic hydrolysis. Corn stover fractions were separated manually and analyzed for glucan, xylan, acid soluble lignin, acid insoluble lignin, and ash composition. The stover fractions were also pretreated with either 0%, 0.4%, or 0.8% NaOH for 2 h at room temperature, washed, autoclaved and saccharified. In addition, dilute sulfuric acid pretreated samples underwent simultaneous saccharification and fermentation (SSF) to ethanol. In general, the two pretreatments produced similar trends with cobs, husks, and leaves responding best to the pretreatments, the tops of stalks responding slightly less, and the bottom of the stalks responding the least. For example, corn husks pretreated with 0.8% NaOH released over 90% (standard error of 3.8%) of the available glucan, while only 45% (standard error of 1.1%) of the glucan was produced from identically treated stalk bottoms. Estimates of the theoretical ethanol yield using acid pretreatment followed by SSF were 65% (standard error of 15.9%) for husks and 29% (standard error of 1.8%) for stalk bottoms. This suggests that integration of biomass collection systems to remove sustainable feedstocks could be integrated with the processes within a biorefinery to minimize overall ethanol production costs.     

Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover

Ionic liquid (IL) and ammonia fiber expansion (AFEX) pretreatments were studied to develop the first direct side-by-side comparative assessment on their respective impacts on biomass structure, composition, process mass balance, and enzymatic saccharification efficiency. AFEX pretreatment completely preserves plant carbohydrates, whereas IL pretreatment extracts 76% of hemicellulose. In contrast to AFEX, the native crystal structure of the recovered corn stover from IL pretreatment was significantly disrupted. For both techniques, more than 70% of the theoretical sugar yield was attained after 48 h of hydrolysis using commercial enzyme cocktails. IL pretreatment requires less enzyme loading and a shorter hydrolysis time to reach 90% yields. Hemicellulase addition led to significant improvements in the yields of glucose and xylose for AFEX pretreated corn stover, but not for IL pretreated stover. These results provide new insights into the mechanisms of IL and AFEX pretreatment, as well as the advantages and disadvantages of each.     

Pretreatment of corn stover and hybrid poplar by sodium hydroxide and hydrogen peroxide

Sodium hydroxide and its derivatives are used as pulping reagents, wherein the spent NaOH is recovered in salt form and reused. In this study, use of low concentration NaOH (1–5%) in pretreatment of corn stover and hybrid poplar was investigated. It was done with the understanding that NaOH can be recovered. One of the main objectives in this study is to explore the potential of H₂O₂ with NaOH for pretreatment of high lignin substrate such as hybrid poplar. Pretreatment time has not been optimized in this study but held constant at 24 h. Corn stover, after treatment with NaOH under moderate conditions, attains near quantitative glucan digestibility. On the other hand, hybrid poplar requires treatment at higher temperature and NaOH concentration to attain acceptable level of digestibility. Supplementation of hydrogen peroxide in the pretreatment significantly raises delignification and digestibility of hybrid poplar. It was also helpful in retaining the carbohydrates in the treated solids. Retention of hemicellulose after pretreatment provides a significant economic benefit as it eliminates the need for detoxifying hemicellulose sugars. As the residual xylan remaining after pretreatment is an impediment to enzymatic digestion of glucan, supplementation of xylanase has significantly increased the digestibility of glucan as well as xylan of the treated hybrid poplar. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Flexible biorefinery for producing fermentation sugars, lignin and pulp from corn stover

A new biorefining process is presented that embodies green processing and sustainable development. In the spirit of a true biorefinery, the objective is to convert agricultural residues and other biomass feedstocks into value-added products such as fuel ethanol, dissolving pulp, and lignin for resin production. The continuous biomass fractionation process yields a liquid stream rich in hemicellulosic sugars, a lignin-rich liquid stream, and a solid cellulose stream. This paper generally discusses potential applications of the three streams and specifically provides results on the evaluation of the cellulose stream from corn stover as a source of fermentation sugars and specialty pulp. Enzymatic hydrolysis of this relatively pure cellulose stream requires significantly lower enzyme loadings because of minimal enzyme deactivation from nonspecific binding to lignin. A correlation was shown to exist between lignin removal efficiency and enzymatic digestibility. The cellulose produced was also demonstrated to be a suitable replacement for hardwood pulp, especially in the top ply of a linerboard. Also, the relatively pure nature of the cellulose renders it suitable as raw material for making dissolving pulp. This pulping approach has significantly smaller environmental footprint compared to the industry-standard kraft process because no sulfur- or chlorine-containing compounds are used. Although this option needs some minimal post-processing, it produces a higher value commodity than ethanol and, unlike ethanol, does not need extensive processing such as hydrolysis or fermentation. Potential use of low-molecular weight lignin as a raw material for wood adhesive production is discussed as well as its use as cement and feed binder. As a baseline application the hemicellulosic sugars captured in the hydrolyzate liquor can be used to produce ethanol, but potential utilization of xylose for xylitol fermentation is also feasible. Markets and values of these applications are juxtaposed with market penetration and saturation.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.