Development of renewable liquid fuels, like bioethanol and advanced biofuels will reduce reliance on fossil fuels...Cellulosic biomass provides a more sustainable source of fermentable sugar and it is estimated that a billion tons are available annually in the US. Roughly half of that biomass is composed of cellulose that, after hydrolysis to glucose, can be fermented into cellulosic biofuels...Biomass recalcitrance is a difficult barrier to commercial deployment of cellulosic biofuels, yet there are a few promising pretreatments currently available or under development.

...for example, pretreatment with ionic liquids (ILs), such as 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) dramatically reduces biomass recalcitrance and enhances the enzymatic hydrolysis of fermentable sugars.

—Park et al.

However, problems associated with ILs include:

1. IL-pretreatment is frequently conducted using 100% IL at high temperatures (120–160 °C);

2. ILs are currently expensive so any viable bioprocessing scheme must include efficient IL recycling;

3. this pretreatment configuration requires extensive washing of the biomass post-pretreatment to completely remove ILs, which can inhibit downstream saccharification and fermentation; and

4. Washing has a negative impact in this scheme, increasing costs through energy-intensive evaporation or reverse osmosis recycling of ILs.

Pretreatment with...lower IL concentrations presents the possibility to explore alternate, potentially more inexpensive, bioprocessing configurations in which the washing step is removed.

In this study, we used thermophilic enzymes to develop an IL-tolerant cellulase cocktail, called “JTherm”, which is compatible with [a] single-pot bioprocessing configuration...We took a hybrid approach where we combined native enzymes produced by a thermophilic bacterial community with recombinant thermostable enzymes. In addition, to validate the IL-pretreatment bioprocessing scheme...we assessed the impacts of hydrolyzates of IL-pretreated biomass produced by both JTherm and a commercial cocktail on fuel production in an E. coli strain engineered to produce FAEEs, a fuel equivalent to biodiesel.

—Park et al.

The JBEI team created their JTherm cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. JTherm liberates sugars from biomass in the presence of up to 20% ionic liquid.

This cellulase cocktail stands alone in its ability to function so efficiently in the presence of [C2mim][OAc], one of the most potent ionic liquids used for biomass pretreatment. Several other studies have investigated IL-tolerant cellulase cocktails, but those studies either use ILs that pretreat biomass less effectively than [C2mim][OAc] or excessive mounts of cellulase enzymes, both of which are unlikely to lead to commercially viable technologies.

In addition, to explore the possibility of whether microbial inhibitors could be generated during enzymatic hydrolysis of IL-pretreated biomass, hydrolysates were generated by JTherm and CTec2 and fed to an E. coli strain engineered to produce FAEE biodiesel. To our knowledge, this is the first report of the production of an advanced biofuel from IL-pretreated biomass using a metabolically engineered organism. Like S. cerevisiae, E.coli is inhibited by ILs, and by unidentified, possibly biomass-derived, inhibitors. This study both confirms the feasibility of using IL-pretreatment to produce biofuels and shows that thermotolerant enzymes can be used to develop IL-tolerant enzymes cocktails that can potentially lead to the development of inexpensive IL-based biomass-to-biofuels technologies.

—Park et al.

Resources

• Park JI, Steen EJ, Burd H, Evans SS, Redding-Johnson AM, et al. (2012) A Thermophilic Ionic Liquid-Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels. PLoS ONE 7(5): e37010. doi: 10.1371/journal.pone.0037010