Enzymatic hydrolysis ofcellulose in aqueous ionicliquidsPresentation of PhD thesisRonny WahlströmVTT Technical Research Centre of Finland

Place for a photo(no lines around photo)

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Ionic liquids

Definition: Salt with melting temperature < 100 ºCUsually the cation or both the cation or anion are organicThe liquid nature of the salt is due to the ion charge beingspread out on many atoms and the presence of stericallyhindering groups, keeping the ions far from each other nocrystallization is possibleExcellent solvent propertiesNo solvent fume emissions green solvents

Examples of IL ions

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Ionic liquids in biomass processing

Cellulose is difficult to dissolve inconventional solvent systems due toextensive hydrogen bondingSome ionic liquids (ILs) have been foundto dissolve cellulose

• Dry systems neededIn solution, cellulose can be efficientlymodifiedMany chemical modifications have beenreported for cellulose in ILs

• E. g. acylationsFew studies of enzymatic modification ofcarbohydrates in IL solutions

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Enzymatic hydrolysis of cellulose

Enzymatic total hydrolysis of lignocellulosics isa current research hotspot

Challenge: Recalcitrance of substrateMain enzymes: cellulases

EndoglucanasesExoglucanases

Certain classes of ionic liquids (ILs) dissolvebiomass

Pretreatment with ILs greatly increasesbiomass hydrolysability by enzymes

Enzymes tolerate ILs to various degreesDependent on the ILDependent on the actual enzyme

This study: which are the factors limitingenzymatic cellulose hydrolysis in IL solutionsIllustration of the complexity of

lignocellulosic biomass

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Process alternatives for IL-aided enzymatichydrolysis of lignocellosics

Dissolution inIonic liquid

Regeneration +washing

Addition of buffer+ hydrolysis

Hydrolysis

Fermentation

Regeneration pathway

One-pot procedure

Dissolution + hydrolysis + fermentation

Integrated process

Productseparation

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Monocomponent endoglucanases in cellulose-dissolving ILs

T. reesei cellulases in [EMIM]AcO and [DMIM]DMPFollowing MCC dissolution in aqueous ILsMain outcomes:

[DMIM]DMP and [EMIM]AcO highly harmul[DMIM]DMP less inactivating than [EMIM]AcOBasicity of IL may play a significant role in enzyme inactivationEndoglucanases reduced the DP of MCC in high [DMIM]DMP contents

IL basicity in buffer solution

Cellulose dissolution inaqueous ILs

Enzymatic DP reduction in 90 % IL

Wahlström, et al. RSC Adv., 2012, 2 (10), 4472 – 4480

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Development of IL-tolerant saccharide analyticsILs interfere severly with many common analysis methods; therefore an IL-tolerant method had to be developedMethod based on labeling the saccharide reducing ends with UV tagsSeparation by capillary electrophoresisTolerates 20 – 40 % of water-miscible ILs in the sample matrixSeparation of cello- and xylo-oligomers (not manno-oligomers)Appears to be compatible with imidazolium-, TMG- and DBN-based ILs

Effect of IL on saccharide analysis Labeling reaction of saccharides

Wahlström, et al. Carbohydr. Res., 373, 42-51

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Cellulase activity in new cellulose solvents,effect of thermo- and alkalistability

Testing cellulase-compatibility of novel cellulose-dissolving TMG- and DBN-based ILsTwo substrates:

MCCEucalyptus PHK dissolving pulp

Thermo- and alkalistable cellulasesMain outcome:

Thermostablity appears to predict IL-toleranceActivity in high pH does not imply IL-toleranceTMG-ILs inactivating as imidazolium-based ILs, DBN-based ILs are evenmore inactivating

Wahlström et al., RSC Adv., 2013, 3, 20001-20009

Hydrolysis resultsin [EMIM]AcO and[TMGH]AcO

pH activity curves

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IL influence on cellulase substratebinding

Effect of [EMIM]AcO and [DMIM]DMP on cellulase bindingThe role of the carbohydrate-binding module (CBM) in cellulose hydrolysis inIL solutionsOutcome:

CBMs do not promote cellulose hydrolysis in the presence of ILsLarge differences in enzyme function-structure response to ILsBinding more sensitive for endoglucanases than for cellobiohydrolasesBinding not always necessary for hydrolysis to take place

Wahlström et al., Biotech. Bioeng. 2014, 111, 726-733

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Cellulase inactivation kinetics in neat IL

Cellulase inactivation in pulp/[EMIM]AcO or in substrate-free neat [EMIM]AcOor [DMIM]DMP solutions was followedTwo methods:

Reduction of regenerated pulp DP after regeneration caused by cellulaseincuvated 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 slowinactivation in [DMIM]DMP (next slide)

If cellulase retains its activity in some Ils, why is no enzymatic cellulosehydrolysis observed?

Topic for follow-up studies

Ebner and Wahlström et al., J Mol Cat B. 2014, 99, 121-129

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Cellulase inactivation in [EMIM]AcO and[DMIM]DMP

Ebner and Wahlström et al., J Mol Cat B. 2014, 99, 121-129

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Conclusions

Several different factors limiting enzymatic cellulose hydrolysis could beelucidated

pHThermo- and alkalistabilityCellulose 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, soenzyme inactivation is not always the reason to low enzyme performance in ILs

Future studies should concentrate on finding the real inhibitory factor, this could bee.g. IL coating of the cellulose

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PublicationsWahlström, R., Rovio, S., Suurnäkki, A. ”Partial Enzymatic Hydrolysis ofMicrocrystalline Cellulose in Ionic Liquids by Trichoderma reeseiEndoglucanases” RSC Adv, 2012, 2 (10), 4472 – 4480R. Wahlström, S. Rovio, A. Suurnäkki “Analysis of Mono- and Oligo-saccharides in Ionic Liquid Containing Matrices” Carbohydr Res, 373, 42-51R. Wahlström, A. King, A. Parviainen, K. Kruus, A. Suurnäkki ”Cellulosehydrolysis with thermo- and alkali-tolerant cellulases in cellulose-dissolvingsuperbase ionic liquids” RSC Adv, 2013, 3, 20001-20009Wahlström, R., Rahikainen, J., Kruus, K., Suurnäkki, A. "Cellulose hydrolysisand binding with Trichoderma reesei Cel5A and Cel7A and their core domainsin ionic liquid solutions", Biotech Bioeng, 2014, 111, 726-733Ebner, G., Vejdovszky, P., Wahlström, R., Suurnäkki, A., Schrems, M., Kosma,P., Rosenau, .T, Potthast, A. 2013. "The effect of 1-ethyl-3-methylimidazoliumacetate on the enzymatic degradation of cellulose", J Mol Cat B. 2014, 99, 121-129Doctoral thesis Enzymatic hydrolysis of cellulose in aqueous ionic liquids isavailable at http://www.vtt.fi/inf/pdf/science/2014/S52.pdf

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