(748d) In situ Raman Microscopy to Monitor Changes in Cellulose Crystallinity During Acid Pre-Treatment

Dilute acid pre-treatment is a common technique for decreasing lignocellulosic biomass recalcitrance. During this process hemicellulose is removed, providing structural access for enzymes to hydrolyze cellulose. The effect of acid pre-treatment processes on the cellulose structure is well understood, in particular the change of crystallinity, partly due to uncertainty from sample preparation techniques carried out prior to ex situ analysis. However, cellulose crystallinity is the primary determinant of its recalcitrance to hydrolysis. Raman microscopy has been used extensively to study cellulose structural changes. Furthermore, it is a convenient and versatile tool allowing in situ analysis under hydrothermal environments, as water is mostly Raman transparent. Thus, understanding how cellulose crystallinity changes in situ would allow for improvement of pre-treatment processes by optimizing temperature and acid concentration of treatment.

In this work, we designed and built a custom view-cell reactor capable of operating at conditions of up to 220 °C and 30 bar to investigate cellulose structural changes under pre-treatment conditions. The crystallinity of ball-milled cellulose increased during the first 60 min of dilute acid pretreatment at 150 °C. After 60 min, the increasing crystallinity appeared to cease, coinciding with the appearance of a new band at 1600 cm^-1. To determine whether cellulose decrystallizes under pre-treatment conditions, highly crystalline cellulose was thermally treated in dilute acid. The crystallinity appeared to increase up to a temperature of 160 °C, after which it appeared to decrease. These observations could be attributed to the preferential hydrolysis of amorphous regions in the cellulose structure resulting in an increase in the relative amount of crystalline regions.

To verify the observed increase in crystallinity during dilute acid hydrolysis we carried out control tests in hydrothermal conditions in the absence of acid. At such conditions the hydrolysis rate should be rather low compared to dilute acid. Partially decrystallized cellulose started to fluoresce relatively early, within 20 minutes of reaction time at 150 °C preventing crystallinity analysis. However, similarly to reactions under acidic conditions, a new band at 1584 cm^-1 appeared within 20 minutes of reaction time. Cool-down and washing of the cellulose with water did not completely eliminate the fluorescence and the new band at 1584 cm^-1, suggesting partial water solubility and surface degradation. In the absence of acid, the crystallinity of highly crystalline cellulose appeared to be relatively stable, which is consistent with low hydrolysis rate in the absence of acid.

To understand the nature of the new bands formed during acidic and hydrothermal hydrolysis, we employed IR spectroscopy. We heated cellulose in nitrogen environment and new bands appeared consistent with formation of C=O and C=C at temperatures above 230°C. Further, we compared a Raman spectrum of hydrothermal char, produced by degradation of glucose to the Raman spectra of cellulose under dilute acid and hydrothermal conditions. The G band of the hydrochar appeared at 1582 cm^-1 similar to our observations at 1584 cm^-1 as observed in hydrothermal treatment. These results suggest formation of degradation compounds such as HMF and humins during dilute acid and hydrothermal hydrolysis at temperatures as low as 150 °C.

In situ Ramans studies of the ball-milled and highly crystalline cellulose further understanding of the changes of cellulose structure that occur during dilute acid pre-treatment.