(599e) Towards An Improved Understanding of the Effects of Ethanol Organosolv Pretreatment On Buddleja Davidii

The earth's finite fossil fuel reserves and the increase in demand for energy have raised many concerns which have led researchers to focus on the development of new technologies for the production of sustainable and renewable energy. Lignocellulosic bioethanol is one of the promising alternative sources of energy, as it is produced from renewable materials that do not directly compete with food and can be grown on non-agricultural lands. The process of producing bioethanol requires three main steps: pretreatment, enzymatic hydrolysis, and fermentation. Pretreatment is considered to be the most important step because it dictates the susceptibility of the biomass to the downstream processes, hydrolysis and fermentation.

The purpose of pretreatment is to alter the structure of the biomass so that cellulose, which is entrapped in the lignin and hemicellulose matrix, can become more amenable to enzymatic deconstruction. Some of the desired characteristics of a pretreatment are: high recovery of carbohydrates, modification of lignin, hydrolysis of hemicelluloses, minimum sugar degradation, and allowing high digestibility of cellulose by cellulases. Ethanol organosolv pretreatment has been shown to be effective on softwoods and hardwoods because it addresses these requirements.

In this pretreatment, the material is treated in an aqueous-organic solvent mixture with the addition of an inorganic acid catalyst, H₂SO₄, and heated at high temperatures for 40 ? 60 min. After pretreatment, three fractions are recovered: Ethanol Organosolv Lignin (EOL) fraction, solid fraction, and water-soluble fraction. The lignin that is dissolved in the organophilic phase is referred to as EOL; the solid fraction contains mainly cellulose and some residual lignin and hemicelluloses; and the water-soluble fraction has the soluble lignin, monomeric and oligomeric hemicelluloses.

The focus of this research is to better understand the organosolv pretreatment chemistry by exploring the fundamental characteristics of pretreated biomass in the context of developing an efficient bioconversion of cellulose to glucose. Pretreatment of Buddleja davidii was performed at three conditions (A, B, and C), which produced substrates with different compositions and/or enzymatic hydrolysis profiles. The most sever condition, condition C caused more delignification and carbohydrate hydrolysis/degradation than conditions A and B. Results showed higher %EOL, higher % furfurals, and lower Klason lignin (KL) content in the solid fraction for condition C, when compared to the other two conditions. Glucose recovery was ~85% of the original glucose in the wood for the three conditions.

However, although the wood in condition C was delignified more than in condition B, ~19% KL and ~9% KL for substrate B and C respectively, their enzymatic hydrolysis profiles were similar. Both of these substrates were readily digestible by cellulases. This was interesting because it is believed that lignin has a large impact on the efficiency of enzymatic hydrolysis. A set of experiments were conducted in order to explain this behavior. The idea was to study the structure of cellulose, crystallinity of cellulose, and its degree of polymerization (DP), which are another three important factors that affect enzymatic hydrolysis. Gel-permeation chromatography (GPC) was used to determine the DP; the ultrastructure and crystallinity index (CrI) of cellulose were investigated using solid-state CP/MAS ¹³C NMR, in terms of the relative amounts of cellulose I_α, cellulose I_β, para-crystalline cellulose, and celluloses at accessible and inaccessible surfaces.

Results showed that efficient organosolv pretreatment was capable of reducing DP and CrI of cellulose, and convert the stable crystalline cellulose dimorphs (I_α/I_β) to the more reactive para-crystalline and amorphous celluloses. These outcomes made the cellulose more accessible to enzymatic attacks due to two reasons. First, lower DP of cellulose means higher number of reducing chain ends of cellulose, which is an enhancement for efficient enzymatic deconstruction, because cellobiohydrolase enzyme acts mainly on the reducing chain ends of cellulose. It is one the main enzymes used to convert cellulose to glucose besides endoglucanase and cellobiase. Second, shorter chains of cellulose and less crystalline cellulose cause the formation of weaker networks that lack strong intermolecular hydrogen bonding, allowing for easier digestibility by cellulases.

In this study, a plant called Buddleja davidii was used as a resource for bioethanol production because it has unique properties to become an agro-energy crop. B. davidii exhibits a very wide range of growth habitat and is well adapted to growing in poor soil conditions. The plant is perennial, has moderate growth dimensions, and is susceptible to very few pests or diseases.

We have assessed this new bioresource by determining its composition and by elucidating the chemical structures of both lignin and cellulose. Results from compositional analysis indicate that B. davidii has low ash and extractives content, low cellulose (35%), high hemicellulose (34%), and relatively high lignin content (30%). The predominant crystalline form of cellulose is para-crystalline cellulose (33%) and the crystallinity index is determined to be 0.55. Structural characterization of lignin has been performed using quantitative ¹³C NMR and ³¹P NMR spectroscopy. ¹³C NMR reveals that the lignin in B. davidii is of the hardwood type with a syringyl to guaiacyl ratio of 19:81.

The characteristics of B. davidii can be categorized into attractive and undesired features. The positive ones include: the wide range of growth habitat, the favorable growth dimensions, and the low degree of polymerization of cellulose (1000 DP). The negatives ones include: the low cellulose content, the high hemicellulose and lignin contents, and the high crystallinity index. The high and low descriptions were based on comparing B. davidii to other softwoods and hardwoods. For instance, spruce has 27% lignin, 41% cellulose, 31% hemicellulose, 0.45 crystallinity index, and 3300 DP. Aspen has 21% lignin, 48% cellulose, 27% hemicellulose, 0.47 crystallinity index, and 2500 DP.

In summary, the results of this study provide fundamental investigation of the biomass chemistry, the organosolv pretreatment technology, and its ability to prepare solid cellulosic biomass for conversion by enzymatic hydrolysis. This presentation also summarizes the attractive features and challenges associated with using B. davidii for bioethanol production.