Ronny Wahlström

Partial enzymatic hydrolysis of microcrystalline cellulose in ionic liquids by Trichoderma reesei endoglucanases

The enzymatic hydrolysis of cellulose in ionic liquid (IL) containing systems has recently received a lot of interest as a pretreatment in biomass conversion to liquid biofuels and chemicals. In this paper we present a study in which the activity and action of two Trichoderma reesei endoglucanases, Cel7B and Cel5A, were evaluated in aqueous solutions containing 0–90% (v/v) of the ionic liquids 1,3-dimethylimidazolium dimethylphosphate or 1-ethyl-3-methylimidazolium acetate, using microcrystalline cellulose (Avicell) as a model substrate. The degree of hydrolysis was analysed by capillary electrophoresis of the hydrolysates and gel permeation chromatography of the remaining cellulose residues. Both of the employed ionic liquids severely inactivated the T. reesei endoglucanases. Only traces of soluble oligosaccharides were present in hydrolysis mixtures containing 40% (v/v) or more of ionic liquids. The employed ILs were found to have a basic impact on the hydrolysis environment, but it could be concluded that the basicity of the ILs was not the only reason for the cellulase inactivation. The effect of an IL on the cellulose binding module in Cel5A was evaluated by comparing the hydrolysis yields of the intact Cel5A and the Cel5A core lacking the cellulose binding module. In this study the cellulose binding module was found to be the most ionic liquid sensitive part of the enzymes used. Comparative data from the partial hydrolysis of an ionic liquid regenerated cellulose is also reported.

Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions

Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto the cellulosic substrate were studied in the presence of cellulose‐dissolving ILs. Two different ILs, 1,3‐dimethylimidazolium dimethylphosphate ([DMIM]DMP) and 1‐ethyl‐3‐methylimidazolium acetate ([EMIM]AcO), and two monocomponent cellulases, Trichoderma reesei cellobiohydrolase Cel7A and endoglucanase Cel5A, were used in the study. The role and IL sensitivity of the carbohydrate‐binding module (CBM) were studied by performing hydrolysis and binding experiments with both the intact cellulases, and their respective core domains (CDs). Based on hydrolysis yields and substrate binding experiments for the intact enzymes and their CDs in the presence of ILs, the function of the CBM appeared to be very IL sensitive. Binding data suggested that the CBM was more important for the substrate binding of endoglucanase Cel5A than for the binding of cellobiohydrolase Cel7A. The CD of Cel7A was able to bind well to cellulose even without a CBM, whereas Cel5A CD had very low binding affinity. Hydrolysis also occurred with Cel5A CD even if this protein had very low binding affinity in all the studied matrices. Binding and hydrolysis were less affected by the studied ILs for Cel7A than for Cel5A. To our knowledge, this is the first systematic study of IL effects on cellulase substrate binding. Biotechnol. Bioeng. 2014;111: 726–733.

Cellulose hydrolysis with thermo- and alkali-tolerant cellulases in cellulose-dissolving superbase ionic liquids

Pretreatment with ionic liquids (ILs) is known to greatly increase the subsequent biomass hydrolysis with enzymes. However, the presence of even low amounts of ILs has negative effects on cellulase action. Most studies on cellulase inactivation by ILs have focused on imidazolium-based ILs, which until recently were one of the few IL classes known to dissolve cellulose. In this article we describe results of cellulase action in matrices containing ILs belonging to two IL classes recently reported as cellulose solvents. These ILs are based on the organic superbases 1,1,3,3-tetramethylguanidine (TMG) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN). In this study commercial thermo- and alkaline stabile cellulase products were employed, as these were anticipated to also have a higher stability in ILs. For comparison, hydrolysis experiments were also carried out with a well-characterised endoglucanase (Cel5A) from Trichoderma reesei and in matrices containing 1-ethyl-3-methylimidazolium acetate, [EMIM]AcO. Two different substrates were used, microcrystalline cellulose (MCC) and eucalyptus pre-hydrolysis kraft dissolving grade pulp. The hydrolysis yields were on the same level for both of these substrates, but decreases in molecular weight of the cellulose was observed only for the dissolving grade pulp. By using commercial cellulases with good thermo- and alkali-stability some benefits were obtained in terms of IL compatibility. Enzyme thermostability correlated with higher hydrolysis yields in IL-containing matrices, whereas activity at high pH values did not offer benefits in terms of IL tolerance. The new classes of cellulose-dissolving superbase ILs did not differ in terms of cellulase compatibility from the well-studied imidazolium-based ILs. Of the novel superbase ILs tested, [TMGH]AcO was found to inhibit the enzymatic hydrolysis the least.

Sustainability of cellulose dissolution and regeneration in 1,5-diazabicyclo[4.3.0]non-5-enium acetate: a batch simulation of the IONCELL-F process

The recyclability of 1,5-diazabicyclo[4.3.0]non-5-enium acetate ([DBNH][OAc]), as a direct dissolution solvent for cellulose, was evaluated during laboratory scale recycling trials. The main objective was to simulate the conditions of a spinning bath from a Lyocell-type air-gap spinning process, called the IONCELL-F process. The saline solution was then concentrated, recycled and reused as many times as possible before cellulose dissolution was no longer possible. The chemical compositions of the ionic liquid and pulp were recorded throughout the experiments. The results of the experiments showed that [DBNH][OAc] can be recycled from aqueous media with an average recovery rate of 95.6 wt% using basic laboratory equipment, without any further process intensification or optimisation. The recycling of the ionic liquid did not change the chemical composition or degree of polymerisation of the recovered pulp but the colour of the regenerated pulps gradually darkened as the recycling times increased. The ionic liquid was found to hydrolyse 6.0–13.6 mol% per cycle, under these conditions. The build-up of the hydrolysis product, 3-(aminopropyl)-2-pyrrolidonium acetate, killed the dissolution feature at between 30.6–45.6 wt% hydrolysis product. The enzymatic digestibility of the regenerated pulp samples was studied with both a monocomponent endoglucanase and a cellulase mixture. The amount of residual [DBNH][OAc] in the regenerated pulps was determined, by both NMR and capillary electrophoresis. Although hydrolysis of the ionic liquid occurs, this study clearly shows potential for industrial application, with appropriate process equipment and recycling conditions.

Analysis of mono- and oligosaccharides in ionic liquid containing matrices

Ionic liquids (ILs), that is, salts with melting points <100°C, have recently attracted a lot of attention in biomass processing due to their ability to dissolve lignocellulosics. In this work, we studied how two imidazolium-based, hydrophilic, cellulose dissolving ionic liquids 1,3-dimethylimidazolium dimethylphosphate [DMIM]DMP and 1-ethyl-3-methylimidazolium acetate [EMIM]AcO affect the usually employed analytical methods for mono- and oligosaccharides, typical products from hydrolytic treatments of biomass. HPLC methods were severely hampered by the presence of ILs with loss of separation power and severe baseline problems, making their use for saccharide quantification extremely challenging. Problems in DNS photometric assay and chromatography were also encountered at high ionic liquid concentrations and many capillary electrophoresis (CE) methods did not allow an efficient analysis of saccharides in these matrices. In this paper we describe an optimized CE method with pre-column derivatization for the qualitative and quantitative analysis of mono- and oligosaccharides in sample matrices containing moderate (20-40% (v/v)) concentrations of ILs. The IL content and type in the sample matrix was found to affect both peak shape and quantification parameters. Generally, the presence of high IL concentrations (≥20% (v/v)) had a dampening effect on the detection of the analytes. IL in lower concentrations of <20% (v/v) was, however, found to improve peak shape and/or separation in some cases. The optimized CE method has good sensitivity in moderate concentrations of the ionic liquids used, with limits of detection of 5mg/L for cellooligomers up to the size of cellotetraose and 5-20mg/L for cellopentaose and cellohexaose, depending on the matrix. The method was used for analysing the action of a commercial β-glucosidase in ILs and for analysing saccharides in the IL containing hydrolysates from the hydrolysis of microcrystalline cellulose with Trichoderma reesei endoglucanase Cel5A. According to the results, [DMIM]DMP and [EMIM]AcO] showed clear differences in enzyme inactivation.

Comparison of three deep eutectic solvents and 1-ethyl-3-methylimidazolium acetate in the pretreatment of lignocellulose: effect on enzyme stability, lignocellulose digestibility and one-pot hydrolysis

Certain ionic liquids (ILs) are well-known pretreatment chemicals for lignocellulosic substrates prior to enzymatic total hydrolysis. Deep eutectic solvents (DESs) are closely related to ILs in many properties, but are easier and on occasion cheaper to synthesize and have been claimed to be less inactivating to enzymes used in the hydrolysis, and less toxic for the environment and to micro-organisms used in fermentation. The use of DESs as lignocellulose pretreatment chemicals has not been studied to a similar extent as the use of ILs. In this study, the stability of three Trichoderma reesei cellulases (the endoglucanases Cel5A and Cel7B and the cellobiohydrolase Cel7A) and one T. reesei xylanase (Xyn11) was compared in concentrated solutions (85% w/w) of three DESs (choline chloride : boric acid in molar ratio 5 : 2, choline chloride : glycerol 1 : 1 and betaine : glycerol 1 : 1) and 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO), a powerful lignocellulose-dissolving IL. The pretreatment efficiency of these chemicals was further compared in a mild pretreatment (90% w/w DES or [EMIM]AcO, 80 °C, 24 h, 5% (w/w) lignocellulose consistency) of four different substrates; microcrystalline cellulose, eucalyptus dissolving pulp, shredded wheat straw and spruce saw dust. After pretreatment, the enzymatic digestibility of the pretreated substrates was evaluated in the enzymatic total hydrolysis in three different setups, including hydrolysis of the washed pretreated substrates in buffer, and of the pretreated substrates in solutions containing 30% (w/w) and 80% (w/w) of DES or [EMIM]AcO. The stability analysis identified glycerol-containing DESs to be highly stabilizing for the cellulases, but their pretreatment efficiency was limited. [EMIM]AcO had a high pretreatment efficiency, but was highly inactivating for the used cellulases. The presence of DES or [EMIM]AcO led in all cases to decreased enzymatic hydrolysis yields. Thus, good enzymatic stability in a certain DES does not directly implicate good performance in the hydrolysis of solid lignocellulosic substrates in that DES.

Clustered Single Cellulosic Fiber Dissolution Kinetics and Mechanisms through Optical Microscopy under Limited Dissolving Conditions

Herein, we describe a new method of assessing the kinetics of dissolution of single fibers by dissolution under limited dissolving conditions. The dissolution is followed by optical microscopy under limited dissolving conditions. Videos of the dissolution were processed in ImageJ to yield kinetics for dissolution, based on the disappearance of pixels associated with intact fibers. Data processing was performed using the Python language, utilizing available scientific libraries. The methods of processing the data include clustering of the single fiber data, identifying clusters associated with different fiber types, producing average dissolution traces and also extraction of practical parameters, such as, time taken to dissolve 25, 50, 75, 95, and 99.5% of the clustered fibers. In addition to these simple parameters, exponential fitting was also performed yielding rate constants for fiber dissolution. Fits for sample and cluster averages were variable, although demonstrating first-order kinetics for dissolution overall. To illustrate this process, two reference pulps (a bleached softwood kraft pulp and a bleached hardwood pre-hydrolysis kraft pulp) and their cellulase-treated versions were analyzed. As expected, differences in the kinetics and dissolution mechanisms between these samples were observed. Our initial interpretations are presented, based on the combined mechanistic observations and single fiber dissolution kinetics for these different samples. While the dissolution mechanisms observed were similar to those published previously, the more direct link of mechanistic information with the kinetics improve our understanding of cell wall structure and pre-treatments, toward improved processability.