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Biofuels Development

Room Temperature Ionic Liquids in Biomass Conversion

The conversion of lignocellulosic biomass from agricultural waste, wood or energy crops into liquid biofuels is necessary to meet the increasing worldwide energy demand1. Highly efficient pretreatment of lignocellulosic biomass for subsequent enzymatic hydrolysis and fermentation is perhaps the most significant limiting step on reduced process cost. Over the past several years, we have studied selective lignin extraction of lignocellulosic biomass using room temperature ionic liquids (RTILs)2. This approach enables efficient extraction of pristine lignin (that may be used as a valuable feedstock for lignochemicals production), while the crystal structure of cellulose and the hydrogen bonding network of lignocellulosic biomass subcomponents is disrupted (Figure BF-1).

We have demonstrated that 1-ethyl-3-methylimidazolium acetate ([Emim] [OAc]) is capable of rendering maple wood flour and corn stover biomass highly susceptible to cellulase action via extraction of a substantial fraction of the native lignin along with substantial loss of native cellulose crystallinity (Figure BF-2A)2. Upon removal of over 60% of the native lignin, >90% of the cellulose was converted enzymatically into reducing sugars suitable for subsequent fermentation. A clear correlation existed between lignin removal and reduced cellulose crystallinity (Figure BF-2B). The residual RTIL could then be recycled multiple times with no loss in effectiveness. Further optimization enabled 50% (w/w) biomass loading to undergo substantial loss in biomass crystallinity with nearly complete cellulose degradation (Figure BF-3)3. Finally, through analysis of the physicochemical properties of the RTIL, we uncovered a quantitative solvent parameter that can be used to fine-tune RTIL composition for optimal pretreatment of lignocellulosic biomass (Figure BF-2)4. Specifically, we determined that the ability to disrupt the crystal structure of cellulose can be predicted by measuring the Kamlet-Taft β parameter of RTILs4. In addition, RTILs can be reused up to 10 cycles of pretreatment without being regenerated.


Figure BF-1. Interaction of ILs with cellulose and lignin. A: The semi- crystalline structure of cellulose is a result of the hydrogen-bonding network among the polysaccharideZ chains. B: Incubation with ILs disrupts cellulose crystalline structure because of the IL's ability to accept hydrogen bonds (blue spheres indicate the cation and green spheres indicate the anion). C: Following pretreatment amorphous cellulose can be depolymerized by cellulases. D: Interaction between lignin and [Emim] cation by π- π interactions.

We are currently focusing on developing a cadre of RTILs with the ability to dissolve lignin, enable lignin recovery, and be efficiently recycled with minimal loss per cycle. Specifically, we are designing RTILs using the Kamlet -Taft parameters of RTIL-water solvents to optimize reduction in cellulose crystallinity and lignin extraction. To date, essentially all research performed has been phenomenological, although properties of the RTILs can be assessed quantitatively. For this reason, we are establishing a new set of fundamental parameters that can be used to fine-tune RTIL pretreatment effectiveness.


Figure BF-2. (A) Influence of pretreatment time on enzymatic hydrolysis of wood flour (from high to low = 70, 42, 32, 19, 14, 8, 5, 0.5, 0 h). (B) Correlation of lignin extraction and decreased cellulose crystallinity.


Figure BF-3. (A) Effect from incubation time on digestibility of pretreated maple wood flour with [EMIM]OAc at 90˚C, and (B) effect of pretreatment temperature on digestibility of pretreated maple wood flour.

Electrobioreactor for Bioconversions

Nearly 20% of known oxidoreductases require cofactors to supply stoichiometric quantities of reducing equivalents. For the majority of these oxidoreductases, NAD(P)H2 is the required cofactor. Since the total pool of intracellular NAD(P) and NAD(P)H2 is relatively small, the cofactor undergoes reduction and oxidation turnovers on the order of 103 to 106 in typical biochemical reactions. People wishing to perform enzyme-catalyzed reductions must supply the reduced cofactor in stoichiometric quantities, which can be quite expensive since the cost of NAD(P)H2 is prohibitive to its extensive use.

We are collaborating with the Prof. Mattheos Koffas (Rensselaer Chemical and Biological Engineering) and Dr. William Armiger (BiochemInsights, Inc.) to develop an eletrobioreactor for enzymatic transformations. Direct electrochemical regeneration of NAD(P)H2 is a relatively simple method for achieving cofactor turnover, and is less expensive than current biological regeneration methods if electrons and protons could be provided directly to the oxidized form of NAD(P) in situ for biocatalytic applications. There are a number of challenges in regenerating NAD(P)H2 electrochemically, including the coproduction of biologically useless and sometimes inhibitory reduced cofactor side products. The amelioration of these dead-end side products is an active area of research in our laboratory, and this could lead to the development of new electrochemical bioreactor technology.

The regeneration of NAD(P)H2 in vivo is another area of interest to our laboratory. Carbon, most commonly in the form of glucose, is biologically reduced by microbial activity to a number of chemical products. However, a portion of the input carbonaceous material must be sacrificially oxidized in order to provide reducing power for downstream reductions. This leads to carbon inefficiency and smaller overall yield of products. By providing cells with extracellular reducing equivalents, we hope to increase carbon efficiency and product yield by minimizing the amount of carbonaceous feedstock that is sacrificially oxidized by the microorganism. A similar approach can be used with enzymes, e.g., dehydrogenases, cytochromes P450, reductases, etc. (Figure BF-4). Combining this approach with metabolic engineering, in an effort called microbial electrosynthesis, could lead to the development of new industrially-relevant strains of microorganisms that are capable of increased product yields and carbon efficiency.


Figure BF-4. Electrochemical reactor for the synthesis of NADPH2 for reduction of oxidized substrates. The figure illustrates the overall electrochemistry and general arrangement of the biocatalytic process. Externally-driven current flow provides reducing power to a redox enzyme via the reduction of the redox cofactor NAD(P) to NAD(P)H2, thereby driving the biocatalytic conversion of a substrate to a reduced product. An optional, generic electron transport mediator can shuttle electrons between the cathode and NAD(P).


  1. M. Mora-Pale, L. Meli, T.V. Dohert, R.J. Linhardt, and J.S. Dordick (2011), "Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass", Biotechnol. Bioeng. 108, 1229-1245.
  2. S.H. Lee, T.V. Doherty, R.J. Linhardt, and J.S. Dordick (2009), "Ionic liquid-mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis", Biotechnol. Bioeng. 102, 1368-1376.
  3. H. Wu, M. Mora-Pale, J. Miao, T.V. Doherty, R.J. Linhardt, and J.S. Dordick (2011), "Facile pretreatment of lignocellulosic biomass at high loadings in room temperature ionic liquids", Biotechnol. Bioeng. 108, 2865-2875.
  4. T.V. Doherty, M. Mora-Pale, S.E. Foley, R.J. Linhardt, and J.S. Dordick (2010), "Ionic liquid solvent properties as predictors of lignocellulose pretreatment efficacy", Green Chem. 12, 1967-1975.