Elsevier

Bioresource Technology

Volume 102, Issue 6, March 2011, Pages 4552-4558
Bioresource Technology

Effect of endoxylanase and α-l-arabinofuranosidase supplementation on the enzymatic hydrolysis of steam exploded wheat straw

https://doi.org/10.1016/j.biortech.2010.12.112Get rights and content

Abstract

The cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of the bioethanol production process from lignocellulosic biomass. Hemicellulases and other accessory enzymes are becoming crucial to increase enzymatic hydrolysis (EH) yields at low cellulase dosages. The aim of this work was to evaluate the effect of two recombinant hemicellulolytic enzymes on the EH of steam pretreated wheat straw. Pretreatments at two severity conditions were performed and the whole slurry obtained after steam explosion pretreatment was employed as substrate. An endoxylanase (Xln C) from Aspergillus nidulans and an α-l-arabinofuranosidase (AF) from Aspergillus niger, have been applied in combination with cellulase enzymes. A degree of synergism of 29.5% and increases up to 10% in the EH yields were obtained, showing the potential of accessory activities to improve the EH step and make the whole process more effective.

Introduction

Ethanol from lignocellulosic biomass has become a real alternative to be developed as fuel. The process is based on the utilization of carbohydrates contained in plant cell walls in a biological conversion process to produce ethanol. Among lignocellulosic materials, wheat straw is an abundant raw material that can be considered as a waste material; about 60% of its world production could be directed to energetic purposes (Kim and Dale, 2004). Ethanol production using enzymatic hydrolysis (EH) requires a preliminary pretreatment to increase cellulose accessibility to the enzymes (Wyman et al., 2005). Among different pretreatment methods developed to date, steam explosion (SE) is considered an appropriate method for herbaceous biomass (Alvira et al., 2010b). After pretreatment, EH and fermentation are performed separately or simultaneously to obtain ethanol as final product (Alfani et al., 2000).

The cost and hydrolytic efficiency of enzymes employed during the EH are major factors that restrict the commercialization of bioethanol production from lignocellulose. Different enzymatic activities are involved in the complete hydrolysis of carbohydrates contained in lignocellulosic biomass. To convert cellulose into glucose three enzymatic activities are required; endoglucanases hydrolyze internal β-1,4-glucosidic bonds in the cellulose chain, cellobiohydrolases cleave off cellobiose units from the end of the chain and β-glucosidase converts cellobiose into glucose (Himmel et al., 1996). These enzymes work synergistically to hydrolyze cellulose by creating new accessible sites and relieving product inhibition (Jørgensen et al., 2007, Väljamaë, 2002).

Apart from cellulose, the complex structure of lignocellulose contains a significant proportion of hemicellulose and lignin (20–30% and 15–20%, respectively in wheat straw (Prasad et al., 2007)), which have a great effect on the EH process. It has been reported that the presence of hemicellulose in lignocellulosic feedstocks, even in low quantities, can prevent cellulases to degrade the cellulose efficiently (García-Aparicio et al., 2007, Öhgren et al., 2007). Additionally, it is recognized that all sugars (not only glucose) contained in the pretreated substrate should be converted to ethanol by hexose and pentose fermenting yeasts to achieve an economically viable ethanol production process (Hahn-Hägerdal et al., 2007, Tomás-Pejó et al., 2008). In this context, an efficient hydrolysis of the hemicellulosic fraction becomes crucial and supplementation with hemicellulolytic and other accessory enzymes have the potential to increase EH yields and reduce the enzyme dosages and costs.

Utilization of the whole slurry obtained after SE pretreatment entails different advantages and disadvantages: separation and washing process can be avoided diminishing energy and water consumption, and the amount of potential fermentable sugars increases. On the other hand, inhibitors released during SE pretreatment could have a negative effect on the EH (Cantarella et al., 2004, García-Aparicio et al., 2006) and subsequent fermentation (Palmqvist and Hahn-Hägerdal, 2000). In general, more severe conditions imply enhanced accessibility of fibers to enzymatic attack, but also increased degradation and loss of sugars as well as higher formation of compounds potentially toxic for microorganisms (Cantarella et al., 2004, Kabel et al., 2007). Severity reduction makes the pretreatment step more economically viable, but often results in less digestibility of biomass, mostly related to the decrease in hemicellulose removal. Hemicellulases may have an important role to maintain high sugar conversion rates when pretreatment severity conditions are reduced to diminish the costs and losses of sugar associated to the SE pretreatment.

Considering the high variability of hemicellulose, the structural modifications of insoluble residual fragments and the wide range of oligomers produced during the pretreatment, the role of hemicellulases in the EH of pretreated lignocellulosic biomass becomes a challenge. Hemicellulases involved in herbaceous biomass hydrolysis include endoxylanases, xylosidases, esterases and arabinosidases. These enzymes work in concert and their synergistic effect on hemicellulose degradation is being widely studied. Xylanase and β-xylosidase supplementation has been reported to improve EH of corn stover pretreated by different technologies (Li et al., 2010, Öhgren et al., 2007, Kumar and Wyman, 2009). Xylanase positive effect was also observed on the EH of steam pretreated substrates as barley straw (García-Aparicio et al., 2007) or spruce (Várnai et al., 2010) even when xylan content was low. Tabka et al. (2006) showed a synergistic action of cellulase, xylanase and feruloyl esterase on the enzymatic saccharification of wheat straw. A synergistic action of α-l-arabinofuranosidase with hemicellulolytic enzymes has been also shown in the degradation of agricultural residues (Raweesri et al., 2008).

In wheat straw hemicellulose on average 11 out of 100 xylose residues are linked to an arabinose (Kabel et al., 2007). Hydrothermal pretreatments such as SE usually remove arabinose from the xylan backbone, however certain amount of arabinose can remain linked to xylan or to xylooligomers in the pretreated material. These arabinose residues may hinder the action of endoxylanase catalyzing internal breakdown of the xylan chain. α-l-arabinofuranosidases remove arabinose residues, favouring xylan debranching and degradation but also helps to disrupt lignin–carbohydrate binding since arabinose residues are believed to take part in lignin–hemicellulose ether bonds (Sun et al., 2005). Efficient hydrolysis and removal of hemicellulosic fraction could have a synergistic effect by increasing accessibility of cellulases to the cellulose fibres and improving the EH process, making possible a reduction of the enzyme dosages.

In this work, two recombinant hemicellulolytic enzymes; an endoxylanase (Xln C) and an α-l-arabinofuranosidase (AF) have been studied in combination with low dosages of commercial cellulase enzymes to evaluate their effect and potential application on the EH of the whole slurry obtained from SE pretreatment of wheat straw.

Section snippets

Enzymes

Two recombinant hemicellulolytic enzymes were studied: an endoxylanase (XlnC) from Aspergillus nidulans (Fernández-Espinar et al., 1992, Pérez-González et al., 1996) and an α-l-arabinofuranosidase (AF) from Aspergillus niger (Sánchez-Torres et al., 1996), which were kindly supplied by Biopolis S.L. (Spain). Both enzymes were expressed heterologously and produced in Saccharomyces cerevisiae strains under the control of a constitutive yeast promoter, resulting in the construction of recombinant

Enzyme characterization

The enzyme preparations were characterized by measuring different enzymatic activities and protein concentration. Specific enzyme activities and protein content are shown in Table 1. It was observed that XlnC and AF did not have side activities apart from their respective endoxylanase and α-l-arabinofuranosidase activity; XlnC presented 46.7 IU xylanase/mg protein while AF presented 3.4 IU α-l-arabinofuranosidase/mg protein.

Cellulase preparations were also characterized. Higher cellulase (FPU)

Conclusions

A positive effect of XlnC and AF supplementation on the EH of pretreated wheat straw was shown. Cellulase preparations including recently developed Cellic CTEC and Accellerase 1500 presented a suboptimal endoxylanase activity and insignificant AF activity. Both activities work synergistically and their supplementation resulted in higher sugar release. XlnC and AF were also tested using wheat straw pretreated at milder conditions, showing the possibility to reduce pretreatment severity not

Acknowledgements

This work was supported by “Abengoa Bioenergy New Technologies” within the CENIT I+DEA Project financed by CDTI (Spanish Ministry of Science and Innovation). The cellulase preparations used in this work were provided by Novozymes (Denmark) and Genencor (USA). Recombinant enzymes (AF and XlnC) were kindly provided by Biopolis S.L. (Spain). Authors also thank O. De Juan and I. Higueras for their help in HPLC samples analysis.

References (34)

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