Direct ethanol production from cellulosic materials by consolidated biological processing using the wood rot fungus Schizophyllum commune
Introduction
Second-generation bioethanol is a renewable biofuel made from lignocellulosic biomass that does not compete with food and forage use (Wang et al., 2009). Lignocellulosic biomass includes crop residues and un-utilised forest resources and is mainly composed of cellulose, hemicellulose and lignin. Bioethanol production from cellulosic biomass requires pretreatment such as delignification and saccharification. Physicochemical pretreatment such as sulphite pretreatment (Zhu et al., 2009, Luo et al., 2010, Wang et al., 2009), diluted acid pretreatment (Mosier et al., 2005, Yan et al., 2009), and steam explosion (Horn and Eijsink, 2010, Sassner et al., 2005) can solubilise lignin and produce fermentable sugars from polysaccharides but can also form furfural, which is a strong fermentation inhibitor (Nilsson et al., 2005, Ewanick et al., 2007, Sassner et al., 2005). Enzymatic pretreatment is preferable for fermentation because inhibitors are not generated. There have been several reports on microbial pretreatment using lignin-degrading fungi (Bak et al., 2010, Holmgren and Sellstedt, 2008). Delignification enables access to cellulose and hemicellulose by saccharification enzymes (Ryu et al., 2013).
Similar to the case for saccharification, simultaneous saccharification and fermentation (SSF) processes, in which a two-step process is conducted in a single vessel, are being actively studied (Narra et al., 2015, Olofsson et al., 2008, Kádár et al., 2004). SSF is considered to be effective in reducing costs but a lower efficiency in raw material utilisation will increase costs during bioethanol production from lignocellulose. Saccharomyces cerevisiae is one of the most commonly used microbes in the brewing industry and for ethanol production but it cannot ferment xylose, which is the most abundant pentose in hemicellulose (Ho et al., 1998, Sedlak and Ho, 2004). Many genetically modified S. cerevisiae strains and ethanol fermenting bacteria have been produced to ferment xylose (Ho et al., 1998, Bertilsson et al., 2009). Moreover, the capability to produce fermentable sugars has been added to these fermenting microbes (Lynd et al., 2005, Wingren et al., 2003). Such multifunctional microbes can play multiple roles, such as saccharification of polysaccharides and fermentation of both glucose and xylose, so they are used in consolidated bio-processing (CBP) (de Almeida et al., 2013, Anasontzis and Christakopoulos, 2014, Huang et al., 2014).
Several wood rot basidiomycete, which include lignin degrading groups, have been reported to produce ethanol directly (Okamoto et al., 2010, Okamoto et al., 2014, Mizuno et al., 2009). In a previous report, we selected wood rot fungi that produced ethanol with high efficiency (Horisawa and Nishida, 2014). Schizophyllum commune NBRC4928 gave the highest yields amongst the strains we tested. White rot basidiomycetes, which can degrade lignin and polysaccharides, may possess multiple functionalities, such as delignification, saccharification, and ethanol fermentation. S. commune is a very common white-rot fungus that is distributed all over the world. In the present study, ethanol conversion efficiencies from various sugars during fermentation using S. commune NBRC 4928 were estimated. Direct fermentation tests on S. commune NBRC4928 were conducted using cellulose, hemicellulose and wood chips as raw materials.
Section snippets
Fungal strains and preparation of inocula
S. commune NBRC 4928 was subcultured on potato dextrose agar (PDA, Nissui, Tokyo, Japan) plates at 25 °C. Mycelial discs, 12 mm in diameter, were prepared as inocula. Thanatephorus cucumeris IWA5b was employed as lignin-degradable fungus for co-culturing with S. commune NBRC 4928. T. cucumeris IWA5b was collected as a spore in environmental air by catching on an agar plate. The spore was incubated to make a colony and then identified as a basidiomycete by observing clamp connection of hyphae.
Ethanol production from various sugars
Ethanol production by S. commune NBRC 4928 was observed in fermentation tests using glucose, mannose and galactose, xylose and cellobiose, respectively, as raw materials. Table 1 shows the maximum concentration of ethanol from each sugar, the actual yield per 1 g of sugar and the percentage of theoretical yield obtained by comparing the actual and theoretical yields. The actual yield per sugar was calculated by comparing the maximum concentration found in the fermentation test (Fig. 1) with the
Conclusions
The wood-rot fungus S. commune NBRC 4928 converted various monosaccharides including xylose to ethanol under semi-anaerobic condition. This strain also produced ethanol directly from cellulose and hemicellulose. The results suggest a possibility of applying for SSF or CBP system. Cocultivating a lignin-degrading fungus T. cucumeris with S. commune increased ethanol production from cedar wood chip without a removal process of lignin. Further investigation of fermentation condition in static
Acknowledgements
We express our appreciation to Mr. Tatsuyoshi Nishida and Mr. Masashi Mizobuchi of Kochi University of Technology, for their technical support.
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