Abstract
Although gasoline supplies a sizeable portion of the transportation industry’s energy demand, renewable alternatives offer competitive environmental, economical, and innovative benefits. Biomass is a long-standing, renewable energy source that can potentially off-set the demand placed on corn ethanol. Cellulose and corn generate ethanol using similar production schemes; however, ethanol production from corn is much less labor intensive. As a result, cellulosic ethanol production requires a more rigorous four stage process that employs chemicals, enzymes, and microorganisms to convert cellulose into ethanol. Biomass structure limits the ease of cellulose conversion into ethanol. Numerous approaches have been explored to resolve this limitation, and their efficiencies are typically quantified by monitoring improvements in cellulose saccharification with fungal enzymes. While these measurements hold great value, structural characterizations attempt to clarify structural reason(s) for the improved hydrolysis. The information gathered from characterization experiments will help to direct the selection of the most favorable features in biomass for ethanol production.
The three major polymers in biomass are cellulose, hemicellulose, and lignin, and each possesses structural properties that have been linked to recalcitrance through individual or combinatory effects. Accordingly, various instruments and wet chemistry methods are available to measure these properties. Recalcitrance is currently better understood in fungal cellulases than in an alternative, integrated scheme known as consolidated bioprocessing (CBP). Nonetheless, plant cell wall features that influence ethanol production during CBP can also be assessed with the same methodology and instrumentation. Lignin, in particular, has been consistently identified as challenge to cellulose hydrolysis and may pose similar issues in C. thermocellum. This work clarifies biomass features such as lignin content and structure that negatively influence the CBP microorganism Clostridium thermocellum.
To understand the structural properties that are altered by C. thermocellum, the initial investigation compared six Populus trichocarpa before and after CBP. Ethanol yields were recorded paired with characterization data to determine trends in CBP efficiencies. Several properties of cellulose, hemicellulose, and lignin in non-treated and CBP-treated P. trichocarpa were assessed. Gel permeation chromatography (GPC) was used for the molecular weight analysis of cellulose and hemicellulose, while nuclear magnetic resonance spectroscopy (NMR) detailed lignin structure in top, middle, and poor-performing P. trichocarpa. Lastly, total lignin contents were measured with pyrolysis molecular beam mass spectrometry in all natural variants to assess its contributions to recalcitrance. Overall, lignin structure and contents appeared to be related to ethanol yields.
Considering lignin’s role in the initial findings, the second study attempted to detail the impact of lignin S/G ratio on ethanol yields. Two P. trichocarpa with S/G ratio extremes were selected to undergo CBP with C. thermocellum and yielded drastically different hydrolysis activities. Therefore, a series of experiments were conducted to account for the higher solids solubilization in the high S/G ratio P. trichocarpa. Potential inhibitors were identified with GC-MS and screened to measure their concentration differences between the two P. trichocarpa. Lignin S/G ratio was confirmed with NMR spectroscopy, and its molecular weight alongside cellulose molecular weights were also obtained with GPC. Simon’s staining, which requires UV-Visible spectroscopy, was also conducted to determine accessibility differences between samples. Lignin molecular weights and accessibilities pointed towards structural differences in lignin that were the most likely contributors to solubilization differences.
The third study examined the appropriateness of the Simons’ stain in determining accessibility’s role during CBP, as C. thermocellum is believed to carry out hydrolysis at the biomass surface. Simons’ staining partially relies on biomass porosity to measure accessibility and may not be entirely transferable to CBP. Eight P. trichocarpa (four high density and four low density) and were subjected to separate hydrolysis and fermentation (SHF) and CBP. Klason lignin contents were assessed with wet chemistry, while cellulose molecular weights were measured with GPC. Surface roughness measurements were carried out to relate biomass porosity to the biomass surface. Simons’ staining was applied to the control biomass and was paired with the ethanol yields from SHF and CBP data to correlate accessibility to CBP. Klason lignin contents and biomass accessibility appeared to be related to the extent of CBP.
The former three studies confirmed that recalcitrance persists during CBP, and lignin remains a barrier to ethanol production. To clarify, poorer performing biomass generally contained high lignin contents and/or features of lignin that had been previously associated with recalcitrance with fungal cellulases. In the final part of this work, an advanced NMR technique and ATR-FTIR were used to examine the structure of lignin before and after CBP to determine if lignin is altered during CBP. The analyses revealed that lignin structure experiences small, but discernable, changes following CBP, which suggest that C. thermocellum catalyzes changes to lignin.
Each study provided compelling reasoning to strongly consider lignin’s part in limiting CBP efficiencies at the laboratory and eventually the industrial scale. To make cellulosic ethanol production feasible with CBP, lignin structure and content must be manipulated with genetic modifications or carefully selected in natural variants to combat these difficulties.