Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid-liquid partition and potential of enzyme recycling by alkaline washing.

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and ß-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with ß-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and ß-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and ß-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity.

Cellulases without carbohydrate-binding modules in high consistency ethanol production process.

BACKGROUND: Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. RESULTS: High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. CONCLUSIONS: The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

The challenging measurement of protein in complex biomass-derived samples.

Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay-alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant interference from these compounds when using acid hydrolysis, which was not the case when using the alkaline hydrolysis. We evaluated the interference from glucose, cellulose, xylose, xylan, lignin and ethanol on protein determination of BSA, Accellerase(®) 1500 and Cellic(®) CTec2. The experiments demonstrated that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin.

Adsorption of ß-glucosidases in two commercial preparations onto pretreated biomass and lignin.

BACKGROUND: Enzyme recycling is a method to reduce the production costs for advanced bioethanol by lowering the overall use of enzymes. Commercial cellulase preparations consist of many different enzymes that are important for efficient and complete cellulose (and hemicellulose) hydrolysis. This abundance of different activities complicates enzyme recycling since the individual enzymes behave differently in the process. Previously, the general perception was that ß-glucosidases could easily be recycled via the liquid phase, as they have mostly been observed not to adsorb to pretreated biomass or only adsorb to a minor extent. RESULTS: The results from this study with Cellic® CTec2 revealed that the vast majority of the ß-glucosidase activity was lost from the liquid phase and was adsorbed to the residual biomass during hydrolysis and fermentation. Adsorption studies with ß-glucosidases in two commercial preparations (Novozym 188 and Cellic® CTec2) to substrates mimicking the components in pretreated wheat straw revealed that the Aspergillus niger ß-glucosidase in Novozym 188 did not adsorb significantly to any of the components in pretreated wheat straw, whereas the ß-glucosidase in Cellic® CTec2 adsorbed strongly to lignin.The extent of adsorption of ß-glucosidase from Cellic® CTec2 was affected by both type of biomass and pretreatment method. With approximately 65% of the ß-glucosidases from Cellic® CTec2 adsorbed onto lignin from pretreated wheat straw, the activity of the ß-glucosidases in the slurry decreased by only 15%. This demonstrated that some enzyme remained active despite being bound. It was possible to reduce the adsorption of Cellic® CTec2 ß-glucosidase to lignin from pretreated wheat straw by addition of bovine serum albumin or poly(ethylene glycol). CONCLUSIONS: Contrary to the ß-glucosidases in Novozym 188, the ß-glucosidases in Cellic® CTec2 adsorb significantly to lignin. The lignin adsorption observed for Cellic® CTec2 is usually not a problem during hydrolysis and fermentation since most of the catalytic activity is retained. However, adsorption of ß-glucosidases to lignin may prove to be a problem when trying to recycle enzymes in the production of advanced bioethanol.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling.