Place for a photo (no lines around photo) Enzymatic hydrolysis of cellulose in aqueous ionic liquids Presentation of PhD thesis Ronny Wahlström VTT Technical Research Centre of Finland
Ionic liquids Definition: Salt with melting temperature < 100 ºC Usually the cation or both the cation or anion are organic The liquid nature of the salt is due to the ion charge being spread out on many atoms and the presence of sterically hindering groups, keeping the ions far from each other no crystallization is possible Excellent solvent properties No solvent fume emissions green solvents Examples of IL ions 23/05/2014 2
Ionic liquids in biomass processing Cellulose is difficult to dissolve in conventional solvent systems due to extensive hydrogen bonding Some ionic liquids (ILs) have been found to dissolve cellulose Dry systems needed In solution, cellulose can be efficiently modified Many chemical modifications have been reported for cellulose in ILs E. g. acylations Few studies of enzymatic modification of carbohydrates in IL solutions 23/05/2014 3
Enzymatic hydrolysis of cellulose Illustration of the complexity of lignocellulosic biomass Enzymatic total hydrolysis of lignocellulosics is a current research hotspot Challenge: Recalcitrance of substrate Main enzymes: cellulases Endoglucanases Exoglucanases Certain classes of ionic liquids (ILs) dissolve biomass Pretreatment with ILs greatly increases biomass hydrolysability by enzymes Enzymes tolerate ILs to various degrees Dependent on the IL Dependent on the actual enzyme This study: which are the factors limiting enzymatic cellulose hydrolysis in IL solutions 23/05/2014 4
Process alternatives for IL-aided enzymatic hydrolysis of lignocellosics Regeneration pathway Dissolution in Ionic liquid Regeneration + washing Addition of buffer + hydrolysis Hydrolysis Product separation Fermentation One-pot procedure Dissolution + hydrolysis + fermentation Integrated process 23/05/2014 5
Monocomponent endoglucanases in cellulosedissolving ILs T. reesei cellulases in [EMIM]AcO and [DMIM]DMP Following MCC dissolution in aqueous ILs Main outcomes: [DMIM]DMP and [EMIM]AcO highly harmul [DMIM]DMP less inactivating than [EMIM]AcO Basicity of IL may play a significant role in enzyme inactivation Cellulose dissolution in aqueous ILs Endoglucanases reduced the DP of MCC in high [DMIM]DMP contents IL basicity in buffer solution Wahlström, et al. RSC Adv., 2012, 2 (10), 4472 4480 Enzymatic DP reduction in 90 % IL 23/05/2014 6
Development of IL-tolerant saccharide analytics ILs interfere severly with many common analysis methods; therefore an ILtolerant method had to be developed Method based on labeling the saccharide reducing ends with UV tags Separation by capillary electrophoresis Tolerates 20 40 % of water-miscible ILs in the sample matrix Separation of cello- and xylo-oligomers (not manno-oligomers) Appears to be compatible with imidazolium-, TMG- and DBN-based ILs Wahlström, et al. Carbohydr. Res., 373, 42-51 Effect of IL on saccharide analysis Labeling reaction of saccharides 23/05/2014 7
Cellulase activity in new cellulose solvents, effect of thermo- and alkalistability Testing cellulase-compatibility of novel cellulose-dissolving TMG- and DBNbased ILs Two substrates: MCC Eucalyptus PHK dissolving pulp Thermo- and alkalistable cellulases Main outcome: Thermostablity appears to predict IL-tolerance Activity in high ph does not imply IL-tolerance TMG-ILs inactivating as imidazolium-based ILs, DBN-based ILs are even more inactivating ph activity curves Wahlström et al., RSC Adv., 2013, 3, 20001-20009 Hydrolysis results in [EMIM]AcO and [TMGH]AcO 23/05/2014 8
IL influence on cellulase substrate binding Effect of [EMIM]AcO and [DMIM]DMP on cellulase binding The role of the carbohydrate-binding module (CBM) in cellulose hydrolysis in IL solutions Outcome: CBMs do not promote cellulose hydrolysis in the presence of ILs Large differences in enzyme function-structure response to ILs Binding more sensitive for endoglucanases than for cellobiohydrolases Binding not always necessary for hydrolysis to take place Wahlström et al., Biotech. Bioeng. 2014, 111, 726-733 23/05/2014 9
Cellulase inactivation kinetics in neat IL Cellulase inactivation in pulp/[emim]aco or in substrate-free neat [EMIM]AcO or [DMIM]DMP solutions was followed Two methods: Reduction of regenerated pulp DP after regeneration caused by cellulase incuvated in IL together with the pulp (BOKU) Reduction of cellulase activity on CMC after incubation in [EMIM]AcO and [DMIM]DMP Main outcome: Fast and irreversible inactivation of cellulase in [EMIM]AcO, very slow inactivation in [DMIM]DMP (next slide) If cellulase retains its activity in some Ils, why is no enzymatic cellulose hydrolysis observed? Topic for follow-up studies Ebner and Wahlström et al., J Mol Cat B. 2014, 99, 121-129 23/05/2014 10
Cellulase inactivation in [EMIM]AcO and [DMIM]DMP 23/05/2014 11 Ebner and Wahlström et al., J Mol Cat B. 2014, 99, 121-129
Conclusions Several different factors limiting enzymatic cellulose hydrolysis could be elucidated ph Thermo- and alkalistability Cellulose substrate binding in IL solutions, role of the CBM Analytical methods were developed for saccharide analysis in IL solutions Novel IL classes evaluated for enzyme-compatibility Cellulase was shown to retain steady long-term activity in [DMIM]DMP, so enzyme inactivation is not always the reason to low enzyme performance in ILs Future studies should concentrate on finding the real inhibitory factor, this could be e.g. IL coating of the cellulose 23/05/2014 12
Publications Wahlström, R., Rovio, S., Suurnäkki, A. Partial Enzymatic Hydrolysis of Microcrystalline Cellulose in Ionic Liquids by Trichoderma reesei Endoglucanases RSC Adv, 2012, 2 (10), 4472 4480 R. Wahlström, S. Rovio, A. Suurnäkki Analysis of Mono- and Oligosaccharides in Ionic Liquid Containing Matrices Carbohydr Res, 373, 42-51 R. Wahlström, A. King, A. Parviainen, K. Kruus, A. Suurnäkki Cellulose hydrolysis with thermo- and alkali-tolerant cellulases in cellulose-dissolving superbase ionic liquids RSC Adv, 2013, 3, 20001-20009 Wahlström, R., Rahikainen, J., Kruus, K., Suurnäkki, A. "Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions", Biotech Bioeng, 2014, 111, 726-733 Ebner, G., Vejdovszky, P., Wahlström, R., Suurnäkki, A., Schrems, M., Kosma, P., Rosenau,.T, Potthast, A. 2013. "The effect of 1-ethyl-3-methylimidazolium acetate on the enzymatic degradation of cellulose", J Mol Cat B. 2014, 99, 121-129 Doctoral thesis Enzymatic hydrolysis of cellulose in aqueous ionic liquids is available at http://www.vtt.fi/inf/pdf/science/2014/s52.pdf 23/05/2014 13
TECHNOLOGY FOR BUSINESS