JBEI Researchers Develop Dynamic Visualization Technique to Assess Performance of Ionic Liquid Pretreatments of Biomass

11 July 2009

Fluorescence images of a stem of switchgrass treated with EmimAc ionic liquid showing the section (a) before treatment (b) 20 minutes after treatment (c) 50 minutes after treatment and (d) two hours after treatment when the organized plant cell wall structure has been completely broken down. Source: JBEI. Click to enlarge.

Researchers at the Joint BioEnergy Institute (JBEI), a US Department of Energy Bioenergy Research Center led by the Lawrence Berkeley National Laboratory (Berkeley Lab) have developed a technique, based on the natural auto-fluorescence of plant cell walls, that enables researchers for the first time to visualize dynamically solubilization during an ionic liquid pretreatment of a biomass sample (pristine switchgrass, Panicum virgatum, in the study).

Cellulosic biomass must be pretreated prior to adding enzymes for saccharification of cellulose or hemicelluloses to release sugars for processing into fuel as yields are too low otherwise to be economically competitive. The use of ionic liquids—salts that are liquids rather than crystals at room temperature—to dissolve lignocellulose and later help hydrolyze the resulting liquor into sugars for processing into biofuels, shows promise as a pre-treatment method (e.g., earlier post).

However, the use and understanding of the mechanisms of ionic liquid solvents are still at early stages, and need further exploration before implementation at biorefinery levels. The best ionic liquids in terms of effectiveness are also prohibitively expensive for use on a mass scale. Furthermore, scientists know little beyond the fact that ionic liquids do work. Understanding how ionic liquids are able to dissolve lignocellulosic biomass should pave the way for finding new and better varieties for use in biofuels.

Auto-fluorescence is an intrinsic optical property of biological materials that has been viewed as a nuisance by scientists trying to image specific biological objects. Living cells contain molecules which fluoresce when excited by the right light and this fluorescence can compete with the signals obtained from the fluorophore dyes or markers used to label biological objects of interest. The JBEI researchers turned this “nuisance” into an effective tool.

The auto-fluorescent mapping of cell walls enables researchers to accurately and quickly assess an ionic liquid’s performance without using imaging conventional labor- and time-intensive techniques that require staining, embedding, and processing of biomass. Results of this study were reported in the journal Biotechnology and Bioengineering.

Working with switchgrass and using the ionic liquid known as EmimAc (1-n-ethyl-3-methylimidazolium acetate), which is currently the most effective known to date in terms of pre-treating biomass, we observed a rapid swelling of the secondary plant cell walls within ten minutes of exposure at relatively mild temperatures (120 °C). We attributed the swelling to disruption of inter- and intra-molecular hydrogen bonding between cellulose fibrils and lignin. The swelling was followed by complete dissolution of biomass over three hours. This is the first study to show the process by which biomass solubilization occurs in an ionic liquid pre-treatment using these techniques.

—Blake Simmons, Vice President of JBEI’s Deconstruction Division

Simmons says that once the EmimAc had dissolved the switchgrass biomass into its three components—cellulose and hemicellulose sugars, plus lignin, the woody fiber that gives strength and structure to plant cell walls—the subsequent addition of an anti-solvent, such as water, resulted in the sugars being precipitated out while most of the lignin remained in solution, a requirement for recovering the sugars. This confirmed that the ionic liquid pre-treatment effectively disrupted the recalcitrance of the switchgrass biomass and helped liberate the fermentable sugars.

In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail and provides sugar yields over a relatively short time interval. We are now in the process of evaluating other ionic liquids to discover the optimal combination of cost and performance.

—Blake Simmons

Co-authoring the paper with Simmons were his JBEI colleague Seema Singh, and Kenneth Vogel, of the US Department of Agriculture’s Agricultural Research Service, located in Lincoln, NE. Simmons and Singh also hold appointments with Sandia National Laboratories.

Our approach can be used to evaluate the deconstruction of lignocellulosics in biomass of different chemical compositions, and also to assist in determining the impact of genetically engineered feedstocks. By utilizing this technique, the development and selection of pre-treatment conditions for the selective solubilization and fractionation of either polysaccharides or lignin could be tailored for the development of cost-effective biomass pre-treatments with enhanced yields of sugars.

—Seema Singh

The ultimate goal, Simmons says, is to find an ionic liquid that can efficiently pre-treat biomass, then scale its use up into a cost-effective process for biorefineries. Ideally, he and his colleagues would like to identify a single versatile ionic liquid that is capable of producing enriched polysaccharide and lignin output streams irrespective of feedstock and fuel types.

Right now ionic liquids are a bench-top technique and there are research and engineering obstacles that must be solved before this technology is ready for prime time. But the drivers are clear, and ionic liquids offer processing advantages that no other current commercial pre-treatment technology can provide.

—Blake Simmons

Resources

• Seema Singh, Blake A. Simmons, Kenneth P. Vogel (2009) Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass. Biotechnology and Bioengineering doi: 10.1002/bit.22386

• S. Singh and B. Simmons. “In-situ examination of biomass dissolution and cellulose regeneration enabling cellular level insight of ionic liquid pretreatment process” (poster 3-07, 31^st Symposium on Biotechnology for Fuels and Chemicals, May 2009)