SEM images were taken for both untreated and pretreated materials using a JEOL Ltd. JSM 6610LV microscope. Prior to SEM analysis samples were prepared by mounting on standard aluminum pin stub mounts with biomass powder adhered via copper tape. Mounted samples were sputter coated with gold prior to analysis using a Hummer 6.2 Sputter System operating at 15 mA under 60 mTorr vacuum for 1 min. SEM images were acquired using a 15 kV accelerating voltage.

XRD was performed using a Rigaku SmartLab diffractometer. Scans were collected at 40 kV and 44 mA with a step size of 0.01°. The degree of cellulose crystallinity can be inferred by the ratio of peak heights for the cellulose I in the 002 plane at 2θ = 22.5 and the amorphous cellulose at 2θ = 16.6 (Segal et al., 1959).

Enzymatic hydrolysis was conducted using a modified version of the procedure described in Selig et al. (2008). Pretreated solids were enzymatically hydrolyzed by adding biomass up to an equivalent of 1.0 g of dry solids to a 50 mL incubation flask with 5 mL of 0.1 M citric acid buffer (pH 4.8), 100 μL of 2 % sodium azide solution, and enough nanopure water to reach a final reaction volume of 10 mL. Enzymes were added at 40 mg g⁻¹ dry biomass for Cellic® CTec2 (Novozymes, Franklin, NC, USA) and 4 mg g⁻¹ biomass for Cellic® HTec2 with enzyme and substrate blanks prepared as controls. To investigate sugar release kinetics, 150 μL aliquots of liquor were removed after 2, 4, 8, 24, 48, and 72 h of incubation at 50°C, filtered through a 0.2 μm filter, and analyzed for monomeric sugars using high performance liquid chromatography (Agilent HPLC Model 1260; Agilent Technologies; Santa Clara, CA). Sugars were analyzed on an Aminex HPX-87P column (BioRad Laboratories; Hercules, CA) with a column temperature of 85 °C using a refractive index detector, a mobile phase of 18 MΩ ultrapure water, and a flow rate of 0.6 mL min⁻¹. The sugars evaluated for this study, defined as the “total sugar yield” included the sum of released glucose, xylose, galactose, arabinose, mannose, and cellobiose divided by those sugars present in the pretreated material multiplied by 100. Duplicate injections were performed for each sample.

The screened ILs, EminAc and BminPF₆, were used to pretreat seven biomass samples including corn stover, miscanthus, pine, sorghum, sugar cane bagasse, switchgrass, and wheat straw. These samples were examined by SEM, XRD, compositional analysis, and enzymatic hydrolysis in order to elucidate the different effect of hydrophilic and hydrophobic IL on various biomass types. The results from the IL pretreatment of the seven biomass types with Emim Ac and BmimPF₆ showed the same general trends as the pine sample. The SEM images for all of the IL treated biomass are shown in Figure 4 where the raw biomass is in the left hand column, biomass treated with Emim Ac is in the middle column, and biomass treated with BmimPF₆ is in the right hand column. Untreated biomass shows a high degree of filamentous structure while Emim Ac treated biomass, for the most part, has a much more amorphous structure. Treatment with BmimPF₆ appears to make the material more crystalline for all biomass types tested in this study.

XRD spectrums for all untreated biomass, biomass pretreated with Emim Ac, and biomass pretreated with BmimPF₆ are presented in Figures 5A–C, respectively. Untreated biomass clearly exhibits a cellulose I peak in the 002 crystalline plane at 2Θ = 22.5°. Treatment with Emim Ac clearly shifts the main peak to 20.7° as cellulose I is transformed to cellulose II.(Sun et al., 2009) Interestingly, reacting higher ash biomass samples (lodgepole pine is low in ash) with BmimPF₆ adds several new peaks to the XRD spectrum as seen in Figure 5C. These peaks are likely the result of the hexafluorophosphate anion complexing with ash species (like sodium and potassium; Huber et al., 1997) in the biomass samples (Wang et al., 2017) which eliminates the recovery of the ionic liquid for recycle and would severely limit the use of these types of IL anions for biomass pretreatment.

Crystallinity index data for the herbaceous biomass can be seen in Table 3. As expected, the cellulose crystallinity for material treated with Emim Ac could not be calculated due to a complete removal of the amorphous cellulose minimum so this data was not added to the table. Interestingly, the crystallinity as measured by the method of Segal et al. generally decreased for the Bmim PF₆ samples. This indicates that an alternative method of measuring cellulose crystallinity would be useful in more carefully evaluating overall cellulose accessibility. Other potential methods include XRD peak deconvolution, and XRD based amorphous peak subtraction, and an NMR C4 peak subtraction method as outlined by Park et al. (2010).

The compositional analysis for the raw biomass, and the biomass after pretreatment with IL can be seen in Table 4. The changes in composition for the various biomass types can be generalized into two different groups. The first group comprises corn stover, miscanthus, sugarcane, switchgrass, and wheat straw. Interestingly, the second group contains pine and sorghum. General changes within these two groups, from the untreated case, is discussed for each type of ionic liquid.

After treatment with BmimPF₆ the first group of biomass showed an average decrease in glucan content of 31% with a standard deviation of 12%. Xylan content showed the same trend as glucan, but to a greater degree, and decreased by an average of 87 ± 12%. Given the large decreases in sugars after treatment with BmimPF₆ it can be expected that the lignin content should increase, and in fact it does increase by an average of 108%. Treatment with Emim Ac produced results that were opposite from the BmimPF₆. In this case the lignin decreased by an average of 43 ± 15% while the glucan and xylan increased by 19.0 ± 15 and 39 ± 16% respectively. These compositional changes are generally in line with what previous literature has seen for treatment with Emim Ac (Brandt et al., 2013).

For the second group of biomass types (pine and sorghum) the Emim Ac and Bmim PF₆ showed similar trends. Both of these materials saw an increase (or very little change) in glucan, xylan, and lignin content that corresponds well with the loss of the extractive from the untreated biomass (which is often around 10% for sorghum and 5% for pine) (Inl, 2015). Sorghum has rarely been investigated in the literature and appears to be resistant to dissolution by Bmim Cl (though the authors of that study also saw an increase in lignin content) (Zhang et al., 2011). While somewhat speculative, it is possible that the recalcitrance of sorghum is related to high amounts of p-coumaric and ferulic acids associated with the cell walls creating cross linkages between the lignin and hemicellulose (Billa et al., 1997). The p-coumaric acid would contribute to the formation of the p-hydroxyphenyl (H) lignol and provide recalcitrance in much the same way that the high guaiacyl (G) to syringyl (S) ratio causes recalcitrance in softwoods (Brandt et al., 2013).

While glucan, xylan, and lignin are important components of biomass structure another influential, but lower wt%, component is ash content. It can be seen in Table 4 that the ash content of the biomass increased drastically for the majority of the samples treated with Bmim PF_6. This increase in ash is concomitant with the new peaks appearing in the XRD data in Figure 5. These peaks are likely due to the fact that hydrophobic ILs exhibit a strong metal-complexing ability (Mehdi et al., 2010; Wang et al., 2017) which could lead to the hexafluorophosphate anion binding with the ash present in the biomass, resulting in an increased ash content by making complexes like potassium hexafluorophosphate (Huber et al., 1997). However, while this metal complexation is a detriment to biomass processing the effect could have useful applications in other areas, like the recovery of rare earth elements (Wang et al., 2017).

Overall it can be seen that for less recalcitrant biomass treatment with Emim Ac generally delignifies biomass and increases sugar content while treatment with BmimPF₆ decreases sugars and increases lignin content. It has also been shown that sorghum is recalcitrant in much the same way as pine for treatment with both hydrophilic and hydrophobic ionic liquids. It was also shown that treatment with an IL that contains a metal complexing anion like PF₆ will increase overall ash content by binding with ash species and eliminating the possibility of recovering the IL.

Past research has shown that biomass structure and cellulose crystallinity play an important role in the effectiveness of enzymatic saccharification, where material with a less ordered structure often has a higher sugar release (Li et al., 2010). This is because cellulose crystallinity, lignin content, and hemicellulose-lignin linkages all effect cell wall structure and altering these facets permits enzymatic access for hydrolysis. To investigate the effects of how much the altered structure of the biomass in this study would alter sugar release we performed enzymatic hydrolysis. This enzymatic hydrolysis was also compared to that of untreated biomass and a standard dilute acid pretreatment to assess the overall effectiveness of ionic liquid pretreatment. The enzymatic hydrolysis data for untreated, dilute acid treated, Bmim PF₆ treated, and Emim Ac treated biomass can be seen in Figures 6A–D, respectively. Two points should be noted in this figure: (1) that the legend visible in panel C applies to all of the panels in the figure, and (2) that the theoretical maximum for the sugar yield is the sum of release glucan, xylan, and cellobiose based on the composition data of the pretreated material. If the composition of the untreated material was used in all cases (assuming that the act of pretreating the material reduces the accuracy of the compositional analysis) then the data presented in Figure 6 would change in the following ways: (1) the dilute acid pretreatment data would essentially remain the same, (2) the Bmim PF₆ data would decrease in yield by about 50%, and (3) the Emim Ac data would increase in overall yield by about 20%.

The data in Figure 6A indicates that the ability to release sugars from untreated biomass using enzymatic hydrolysis varies widely by biomass type. However, straight EH was more effective for corn stover and sorghum while being almost completely ineffective for lodgepole pine, miscanthus, and switchgrass. Dilute acid pretreatment on the other hand (panel B) is very consistent for herbaceous materials (including sorghum), yielding about 70% of the available sugars, while being almost completely ineffective for woody feedstocks like pine. Treatment with the hydrophobic ionic liquid Bmim PF₆ (Figure 6C) actually reduces the amount the amount of sugar recovered from the biomass. This is particularly interesting given the fact that the cellulose crystallinity, as determined by the XRD spectrum, showed a reduction in cellulose crystallinity. Traditionally, a decrease in crystallinity is thought to be correlated with an increase in hydrolytic sugar release. However, this result contributes to the evidence that this trend does not always hold true (Park et al., 2010). It is possible that while the cellulose crystallinity has been disrupted the increase in apparent lignin content, as measured by the composition in Table 4, contributes to the resistance to enzymatic hydrolysis. Biomass pretreatment with Emim Ac (Figure 6D) shows significant potential across all biomass types (although the treatment seems to be highly variable compared with dilute acid). Perhaps most favorable aspect of IL pretreatment is the rapid sugar release kinetics. While the biomass treated with dilute acid continues to release sugar over the course of 3 days the Emim Ac treated material has reached the full sugar release in only 1 day (Figure 6). Shorter reaction time could reduce the size of the equipment needed which would improve mixing and heat transfer issues often associated with biomass processing. It is also worth noting that the Emim Ac treated biomass also had more sugars available, based on the compositional analysis, so the fact that it reached such high theoretical yields, in such a short time, is even more impressive. Overall, it appears that using IL as a pretreatment instead of dilute acid provides significant improvements in sugar release kinetics and modest improvements in sugar yield.