Certain ionic liquids, molten salts with melting points below 100 °C in which one ion has a delocalized charge and one component is organic, have been shown to be potent biomass solvents, and several biomass conversion processes have been developed using these unique solvents. Depending on the specific ions employed, many solution properties such as pH, ionic strength, and water activity can be altered, and these property changes determine the utility of an ionic liquid for biomass conversion. For example, pretreatment processes based on 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) can completely solubilize whole biomass, producing an easily hydrolyzed polysaccharide stream, while other ionic liquids may dissolve only the xylan or lignin fractions. While the biomass solubility is high and the pH of [C2C1Im][OAc] is in the optimal range of commercial enzyme mixtures, these enzyme mixtures are often inactivated at low levels (∼5-10%) of [C2C1Im][OAc] and thorough washing steps are required to remove the ionic liquid before saccharification, which greatly increases the cost.
More recently, ionic liquids based on widely available and inexpensive choline cations and amino acid-based anions have been shown to effectively pretreat biomass, but many of these approaches result in aqueous solutions too basic to use directly in later enzymatic saccharification steps. Carboxylic acid based anions can produce solutions with pH in the active range of cellulases, but these ionic liquids don't pretreat biomass to any appreciable extent. Research to overcome the problems associated with using ionic liquids in biomass saccharification has focused on the discovery and engineering of enzymes to be more stable and active in solutions of the more efficient ionic liquids such as [C2C1Im][OAc]. Processes using mixtures of enzymes that are active in [C2C1Im][OAc] have been developed, but the number of enzymes characterized as being active in [C2C1Im][OAc] is limited, and optimization of mixtures of recombinant ionic liquid tolerant enzymes that can be produced in industrial scale quantities has not yet been realized.
In addition to their deleterious effects on enzyme activity, many of the ionic liquids that efficiently pretreat biomass severely limit growth of E. coli and fermentation inhibition has been shown for both ionic liquids and the phenolics produced during pretreatment. Other pretreatment conditions also produce inhibitors including acetate, furfural, and HMF. Fermentation of hydrolysate from ionic liquid-based processes then involves either near-complete removal of the ionic liquid (and presumably other inhibitors) under conditions difficult to scale outside the lab or many-fold dilution of the hydrolysate. Current research on fermentation methods are focused on removal or mitigation of inhibitors after pretreatment and one group has successfully engineered an inner membrane transporter discovered in Enterobacter lignolyticus into an E. coli fermentation host to conferred the tolerance needed for it to grow well in the presence of toxic concentrations of [C2C1Im]Cl.
An alternative approach to engineering enzymes and microbes to tolerate IL environments is to develop a solvent system that is compatible with both the enzymes and microorganisms and is very effective at biomass pretreatment. While several amino acid-based ionic liquids have been reported with pH values in the 5 to 6 range, which is in the optimum pH range for typical commercial cellulases, they have been unused due to their poor ability to pretreat biomass. Basic ionic liquids are able to pretreat biomass from biocompatible materials, but often result in solution conditions that are incompatible with enzymes and microbes.
We approached this problem by developing an ionic liquid-based process in which biomass is pretreated under alkaline conditions and the solution conditions are rapidly adjusted to bring the pH to levels at which the enzymes remain stable and active during the hydrolysis step and the fermentation host survives and produces an advanced biofuel, isopentenol. Our aim was to develop a system in which a typical commercial hydrolytic enzyme mixture with a pH optimum in the 5 to 6 range can be used and in which E. coli engineered to produce an advanced biofuel such as isopentenol grows at near its optimal levels. This process uses ionic liquids based on di-carboxylic acids and takes advantage of the two ionization states of di-carboxylic acids to switch back and forth between a basic solution that pretreats biomass effectively to an acidic solution with conditions favorable for cellulases and fermentative organisms. While a few studies have used di-carboxylic acids in the doubly-deprotonated form, they have focused on either the pretreatment step or cellulose dissolution alone and have not focused on developing an adjustable process. This promising technology reversibly adjusts pH from a basic (pH ∼ 11) environment to a slightly acidic environment (pH ∼ 5-6), and generates fermentable sugar yields of 91% and isopentenol titer of 1.2 g L-1 with 34% of maximum theoretical yield.