Helene Wulfhorst

Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of native and modified cellulose

New strategies are needed to efficiently convert non-food biomass to glucose as a platform chemical. One promising approach is to use ionic liquids to first dissolve lignocellulose. Yet, in the presence of such solvents, the enzymes that catalyze cellulose hydrolysis become compromised in their activity. However, this decreased cellulase activity has not been examined in detail. Thus, the aim of this study was to investigate how the ionic liquid precisely affects cellulase activity and stability with regard to different cellulose substrates. Hereby, four ionic liquids were screened to identify which one best minimized the loss of enzyme activity. Then, this best ionic liquid was tested on one insoluble and two soluble cellulose substrates. Subsequently, the relevant parameters of solution viscosity and ionic strength were evaluated with respect to enzyme activity and stability. Finally the residual ionic liquid concentration from the precipitation of α-cellulose was varied. The best ionic liquid was found to be 1,3-dimethylimidazolium dimethylphosphate with the highest retained activity of 30% on the α-cellulose substrate in the presence of 10% (v/v) ionic liquid. Most importantly, an increase in viscosity and ionic strength contributed to the decrease in enzyme activity which nonetheless retained their stability. The hydrolysis of precipitated α-cellulose from ionic liquid showed significant higher reaction rates but reduced sugar yields when residual ionic liquid was present. None the less, it should be possible to effectively produce glucose from precipitated cellulose without needing to wash off all residual ionic liquid when optimized cellulase mixtures are used.

Membrane-based recovery of glucose from enzymatic hydrolysis of ionic liquid pretreated cellulose

In this work, a membrane-based downstream process for the recovery of glucose from cellulose hydrolysis is described and evaluated. The cellulose is pretreated with the ionic liquid 1,3-dimethyl-imidazolium dimethylphosphate to reduce its crystallinity. After enzymatic conversion of cellulose to glucose the hydrolysate is filtered with an ultrafiltration membrane to remove residual particulates and enzymes. Nanofiltration is applied to purify the glucose from molecular intermediates, such as cellobiose originating from the hydrolysis reaction. Finally, the ionic liquid is removed from the hydrolysate via electrodialysis. Technically, these process steps are feasible. An economic analysis of the process reveals that the selling price of glucose from this production process is about 2.75 €/kg which is too high as compared to the current market price.

An efficient process for the saccharification of wood chips by combined ionic liquid pretreatment and enzymatic hydrolysis

A process concept combining pretreatment of wood in ionic liquids and subsequent enzymatic hydrolysis to sugars is herein investigated to identify operating conditions which allow for (i) the processing of larger wood chips of 10 mm length, (ii) low temperature, (iii) high sugar yield, and (iv) short processing time. A careful quantitative study of the interaction of pretreatment and hydrolysis reveals that hydrolysis is most effective if beech chips are first disintegrated in [EMIM][Ac] at 115 °C for 1.5 h. The cellulose conversion varies between 70.5 wt% and 90.2wt% for hydrolysis times between 5 h and 72 h. A complete recovery of cellulose and xylan resulting in a total saccharification of 65 wt% of the wood chips could be demonstrated. It is shown that short pretreatment times are required to enable high sugar yield as well as to limit product degradation.

Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection.

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.

Screening for Enzyme Activity In Turbid Suspensions with Scattered Light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (Vmax,app and Km,app) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.

Population balance modelling of homogeneous and heterogeneous cellulose hydrolysis

Abstract Chemical or enzymatic cellulose hydrolysis makes earths most abundant natural resource, lignocellulose, available for further processing to chemicals, materials, or fuels. In this contribution, population balance models are evaluated with respect to their ability to describe the kinetics of depolymerisation in a mechanistically sound way for both chemical and enzymatic cellulose hydrolysis. This integrative approach should support the rational development and optimisation of these processes in future.

Enzymatic degradation of lignocellulose for synthesis of biofuels and other value-added products

Wood is a renewable source for biofuels and chemicals. An efficient pretreatment is required to destroy the highly ordered and complex structure of wood fibres and to improve their enzymatic degradability. To understand the effectiveness of pretreatment on enzymatic degradability, high-throughput analysis of cellulose kinetics using insoluble cellulosic substrate is required. The BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of cellulose samples in microtiter plates. It is used to monitor the hydrolysis of three different cellulosic substrates catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast). Moreover, the reduction of crystallinity and particle size is a key determining factor for an efficient hydrolysis of cellulose particles in heterogeneous system. To increase the sugar release, crystallinity and particle size were decreased by the dissolution of spruce wood in the ionic liquid EMIM Ac resulting in high conversion and reaction rates. Additionally, the enzymatic action on lignin model substrates is characterised using an activity assay and cyclic voltammetry.