Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi
Introduction
Biomass has been recognized as one of the major world renewable energy source and cellulose is its major fraction. Cereal straws, one of the most abundant agricultural wastes, are a relatively slow decomposing substrate because it has high content of lignin and low content of nitrogen. Wheat straw is one of the major crop residues in European countries, with a production of 170 × 106 ton per year, and one of the cheapest and main useful raw materials for lignocellulose biotransformation (Tabka et al., 2006). The ability of several white-rot fungi species to selectively degrade the lignin component of wood has great potential for applications in industries processing lignocellulosic materials to produce cellulose, bio-fuels, or cellulose-enriched forage for ruminants. The main challenge in the biotechnology improvement of biomass is the pre-treatment step (Cardona and Sanchez, 2007).
After holocellulose, lignin is the most abundant biological compound found in nature. However, it is degraded by only a small number of microorganisms, primarily basidiomycetes (Hofrichter et al., 1999). Fungal growth on lignocellulosic substrates has been known for several centuries and has been used traditionally for producing edible mushrooms. Some fungal species have the ability to selectively delignify plant materials leaving a cellulose-enriched residue. White-rot fungi (WRF) produce lignin degrading enzymes that breakdown the lignin “seal” exposing the hemicellulose and cellulose in the wood matrix. Such characteristic could be useful in providing an unprotected carbohydrate for subsequent use, e.g., animal feed or bio-fuel substrate (Rodrigues et al., 2008). A mushroom producing plant coupled with a biorefinery could help generate both food and fuel ultimately benefiting the rural economy and offers the potential for the development of sustainable biomaterials that will lead to a new paradigm (Kalia and Purohit, 2008).
Lignin is chemically difficult to degrade because it is a three-dimensional polymer interconnected through diverse carbon–carbon and other bonds that are not hydrolysable under biological conditions. Plant cell wall degrading enzymes have been classified into enzyme systems such as cellulolytic, ligninolytic and xylanolytic which reflect the nature of the polymers that these enzymes breakdown. The capability to degrade lignin presented by several WRF is due to their extracellular non-specific and non-stereoselective enzyme system (Levin et al., 2008) composed mainly by laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). Lignin peroxidases are strong oxidants that interact directly with non-phenolic lignin structures to cleave them, but cannot penetrate the small pores in sound lignocellulose. Manganese-dependent peroxidases produce small diffusible strong oxidants that can penetrate the substrate. Ferulic acid and p-coumaric acids that are esterified to hemicellulose sugars constitute another limitation to biodegradation of lignocellulosic walls and thus the feruloyl esterase is another key enzyme in the delignification process. Most of the feruloyl esterases have been shown to act synergistically with cellulases, xylanases and pectinases to break down complex plant cell wall carbohydrates (Hermoso et al., 2004).
The main goal of the present work was to investigate the potential of two white-rot fungal species and its enzymatic systems in the delignification of wheat straw. After fungal pre-treatment, the increase in cellulose accessibility towards cellulases was evaluated aiming to determine the susceptibility of the residual carbohydrates to enzymatic hydrolysis.
Section snippets
Fungal strains
Two basidiomycetous strains were used to obtain the enzymatic extracts, being isolate Euc-1, and Irpex lacteus. These WRF were isolated from decaying plant materials collected in North of Portugal and were previously characterized by Dias et al. (2003) and Gomes (2004) respectively. They were maintained on potato dextrose agar (PDA) plates at 4 °C and periodically subcultured.
Enzyme production
The fungi were grown on wheat straw solid media as described earlier (Dinis et al., 2009). Briefly, enzyme extracts were
Ligninolytic potential of fungal isolates
The basidiomycetous fungi Euc-1, and I. lacteus, were pre-selected in previous work for the bio-treatment of wheat straw from a series of four WRF (Fomes fomentarius, Euc-1, I. lacteus, and Phanerochaete chrysosporium) following the criteria of highest efficiency and selectivity of the bio-delignification (Freitas, 2008). During the incubation period (46 days) laccase was only produced by basidiomycete Euc-1 (Fig. 1A). The isolated Euc-1 had a maximum production of laccase (0.10 U ml−1) at the end
Conclusions
The results of this work indicate considerable increase of wheat straw cellulose accessibility (3–4 times) after pre-treatment with basidiomycetes I. lacteus and Euc-1. Both strains revealed low cellulolytic and xylanolytic, but high ligninolytic activities, especially MnP. The high MnP activity seems to be an important pre-requisite for the successful fungal bio-delignification since in the case of I. lacteus, laccase activity was not detected during all pre-treatment period. The initial ratio
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