Antje C. Spiess

High-throughput screening for ionic liquids dissolving (ligno-)cellulose

The recalcitrance of lignocellulosic biomass poses a major challenge for its sustainable and cost-effective utilization. Therefore, an efficient pretreatment is decisive for processes based on lignocellulose. A green and energy-efficient pretreatment could be the dissolution of lignocellulose in ionic liquids. Several ionic liquids were identified earlier which are capable to dissolve (ligno-)cellulose. However, due to their multitude and high costs, a high-throughput screening on small scale is essential for the determination of the most efficient ionic liquid. In this contribution two high-throughput systems are presented based on extinction or scattered light measurements. Quasi-continuous dissolution profiles allow a direct comparison of up to 96 ionic liquids per experiment in terms of their dissolution kinetics. The screening results indicate that among the ionic liquids tested EMIM Ac is the most efficient for dissolving cellulose. Moreover, it was observed that AMIM Cl is the most effective ionic liquid for dissolving wood chips.

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.

How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

BackgroundIn order to generate biofuels, insoluble cellulosic substrates are pretreated andsubsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safeand environmentally friendly manner is to apply, under mild conditions,non-hydrolyzing proteins such as swollenin - naturally produced in low yields bythe fungus Trichoderma reesei. To yield sufficient swollenin forindustrial applications, the first aim of this study is to present a new way ofproducing recombinant swollenin. The main objective is to show how swolleninquantitatively affects relevant physical properties of cellulosic substrates andhow it affects subsequent hydrolysis.ResultsAfter expression in the yeast Kluyveromyces lactis, the resultingswollenin was purified. The adsorption parameters of the recombinant swolleninonto cellulose were quantified for the first time and were comparable to those ofindividual cellulases from T. reesei. Four different insoluble cellulosicsubstrates were then pretreated with swollenin. At first, it could bequalitatively shown by macroscopic evaluation and microscopy that swollenin causeddeagglomeration of bigger cellulose agglomerates as well as dispersion ofcellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin oncellulose particle size, maximum cellulase adsorption and cellulose crystallinitywere quantified. The pretreatment with swollenin resulted in a significantdecrease in particle size of the cellulosic substrates as well as in theircrystallinity, thereby substantially increasing maximum cellulase adsorption ontothese substrates. Subsequently, the pretreated cellulosic substrates werehydrolyzed with cellulases. Here, pretreatment of cellulosic substrates withswollenin, even in non-saturating concentrations, significantly accelerated thehydrolysis. By correlating particle size and crystallinity of the cellulosicsubstrates with initial hydrolysis rates, it could be shown that theswollenin-induced reduction in particle size and crystallinity resulted in highcellulose hydrolysis rates.ConclusionsRecombinant swollenin can be easily produced with the robust yeast K.lactis. Moreover, swollenin induces deagglomeration of celluloseagglomerates as well as amorphogenesis (decrystallization). For the first time,this study quantifies and elucidates in detail how swollenin affects differentcellulosic substrates and their hydrolysis.

Microgel‐Stabilized Smart Emulsions for Biocatalysis

Emulsions stabilized by stimuli-responsive microgels were used to perform enzyme catalysis. Many substrates are poorly water-soluble, while enzymes naturally require aqueous environments, thus resulting in a two-phase aqueous-organic system. Smart microgels allow an enzyme-catalyzed reaction to be performed in an emulsion that can be broken under controlled conditions to separate the reaction product and to recycle the enzyme (E) and the microgel.

Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

BackgroundIt is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH) and endoglucanases (EG). Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose.ResultsFirst, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir) and kinetics (pseudo-first-order). Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured.It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose.Conclusionsα-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation shows in detail, for the first time, the adsorption of purified cellulases onto α-cellulose, the effect of hydrodynamics on cellulase adsorption and the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of the substrate has to be ensured in order to optimize the cellulase attack. In the future, screenings should be conducted with α-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries.

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 Integrated Catalytic Approach to Fermentable Sugars from Cellulose

The future of biofuels relies on the safe, low-cost, and efficient breakdown of cellulose into fermentable sugars. Potentially, being the major component of biomass, cellulose is a practically inexhaustible source of glucose, opening up new horizons for the production of bioethanol, tailor-made biofuels, and platform chemicals; however, the recalcitrance of cellulose is one of the most serious challenges for the development of biorefineries. 4] Dissolving the biopolymer in ionic liquids (ILs) helps to enhance its reactivity. We have shown earlier that cellulose depolymerizes controllably under acidic conditions in ILs. Herein, we use this approach as one part of an effective, integrated strategy to produce fermentable sugars from cellulose in almost quantitative yield. The approach consists of three essential stages: First, cellulose is partially depolymerized to cello-oligomers in an IL using an acid catalyst. Next, the oligomers are precipitated from the IL. Finally, the cello-oligomers are hydrolyzed by enzymes in water. Most strikingly, this integration of chemoand enzymatic catalysis allows almost complete conversion of cellulose to glucose and cellobiose within only a few hours. Performing the acid-catalyzed depolymerization of cellulose using a solid catalyst (e.g. , Amberlyst 15DRY) allows to control the reaction progress effectively. As a result, cello-oligomers with a tunable degree of polymerization can be conveniently produced. In practice, stopping the process at the cello-oligomer stage is highly advantageous, because the cello-oligomers are easily separable from the IL solution simply by the addition of water. Recycling of the catalyst and the IL is possible. In contrast, the separation of fermentable sugars (e.g. , glucose and cellobiose) from ILs is difficult and very expensive because of their high solubility in ILs. Figure 1 shows the appearance of cello-oligomers isolated from 1-butyl-3-methylimidazolium chloride (BMIMCl). In the regenerated cello-oligomers, the water content is around 95 wt %. The high degree of swelling suggests that the cellulosic chains are highly accessible. The residual ionic liquid content found in the samples was below 0.1 %. Cellulases selectively hydrolyze cellulose to fermentable sugars. Yet, enzymatic hydrolysis suffers from two major drawbacks: the slow reaction rate and the incomplete conversion of cellulose. The accessibility of the cellulosic chains to the cellulases has been identified as one of the major limiting factors for efficient enzymatic reaction. Recent reports have shown that the use of cellulose regenerated from ILs accelerates the enzymatic hydrolysis and improves the yield of fermentable sugars; however, a high conversion of cellulose is only reached after 20 to 50 h. To investigate whether the acid pretreatment in an IL has positive effects on the enzymatic hydrolysis, enzymatic hydrolysis experiments using cello-oligomers with different degrees of polymerization (DP) were performed. Figure 2 shows the performance of a commercial cellulose preparation (Celluclast, T. reseei) in the enzymatic hydrolysis of Figure 1. Making cellulose accessible. Both flasks contain the equivalent of 1 g of dry cellulose.

Directed laccase evolution for improved ionic liquid resistance

In nature, the biodegradation of lignin is a challenging process since lignin is highly cross-linked and poorly water-soluble. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) play a key role in the enzymatic degradation of lignin and ionic liquids (ILs) have been used successfully to dissolve lignin. One limitation in lignin degradation using laccases is their low activity/resistance in the presence of ILs. In order to improve the resistance of laccase in IL, a directed evolution protocol based on the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))-screening assay in 96-well microtiter plate format was developed. 1-Ethyl-3-methylimidazolium ethylsulfate ([EMIM] [EtSO4]) can dissolve lignin efficiently and its anion does not inhibit laccase. The stability of the ABTS radical cation was not affected in the presence of [EMIM] [EtSO4]. Therefore, ([EMIM] [EtSO4]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2 and CVLG1) from T. versicolor (Trametes versicolor) were expressed in Saccharomyces cerevisiae and finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO4]. After two rounds of directed evolution, the lcc2 variant M3 (Phe265Ser/Ala318Val) displayed about 4.5-fold higher activity than the lcc2 wild type (WT) in the presence of 15% (v/v) [EMIM] [EtSO4] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC50 value of [EMIM] [EtSO4] towards M3 increases from 392 mM (lcc2 WT) to 497 mM.

Laccases for biorefinery applications: a critical review on challenges and perspectives

Modern biorefinery concepts focus on lignocellulosic biomass as a feedstock for the production of next generation biofuels and platform chemicals. Lignocellulose is a recalcitrant composite consisting of several tightly packed components which are stuck together by the phenolic polymer lignin hampering the access to the carbohydrate compounds of biomass. Certain saprophytic organisms are able to degrade lignin by the use of an enzymatic cocktail. Laccases have been found to play a major role during lignin degradation and have therefore been intensively researched with regard to potential applications for biomass processing. Within this review, we go along the process chain of a third generation biorefinery and highlight the process steps which could benefit from laccase applications. Laccases can assist the pretreatment of biomass and promote the subsequent enzymatic hydrolysis of cellulose by the oxidative modification of residual lignin on the biomass surface. In combination with mediator molecules laccases are often reported being able to catalyze the depolymerization of lignin. Studies with lignin model compounds confirm the chemical possibility of a laccase-catalyzed cleavage of lignin bonds, but the strong polymerization activity of laccase counters the decomposition of lignin by repolymerizing the degradation products. Therefore, it is a key challenge to shift the catalytic performance of laccase towards lignin cleavage by optimizing the process conditions. Another field of application for laccases is the detoxification of biomass hydrolyzates by the oxidative elimination of lignin-derived phenolics which inhibit hydrolytic enzymes and are toxic for fermentation organisms. This review critically discusses the potential applications for laccases in biorefinery processes and emphasizes the challenges and perspectives which go along with the use of this enzyme for the technical utilization of lignocellulose.

Study on Mesophilic and Thermophilic Alcohol Dehydrogenases in Gas-Phase Reaction

The initial reaction rate and the thermostability of the mesophilic alcohol dehydrogenase (ADH) from Lactobacillus brevis (LBADH), and the thermophilic ADH from Thermoanaerobacter sp. (ADH T) in gas‐phase reaction were compared. The effects of water activity, cofactor‐to‐protein molar ratio, and reaction temperature on the reduction of acetophenone to 1‐phenylethanol were studied. An optimal water activity of 0.55 in terms of productivity was found for both ADHs. The cofactor‐to‐protein molar ratio was chosen slightly higher than equimolar to increase both activity and thermostability. An excellent optimal productivity of 1000 g·L−1·d−1 for LBADH and 600 g·L−1·d−1for ADH T was found at 60 °C, while the highest total turnover numbers with respect to the enzyme were achieved at 30 °C and amounted to 4.2 million for LBADH and 1.7 million for ADH T, respectively. Interestingly, the ADH from the mesophilic L. brevis showed the higher thermostability in the nonconventional medium gas phase.