Pretreatment of cotton stalks by synergistic interaction of Daedalea flavida and Phlebia radiata in co-culture for improvement in delignification and saccharification
Introduction
Non-food energy crops and agro-forest residues has potential for the use in production of low cost biofuels, an alternative fuel to the fossil fuels, but a polyphenyl propanoid structure i.e. lignin shielding of the plant cell wall limits the enzymatic hydrolysis of cellulose and hemicellulose (fermentable carbohydrates) present in lignocellulosic biomass (Fermor, 1993). Pretreatment of lignocellulosic biomass is necessary step to overcome the hindrance of lignin (Zhang et al., 2007). An effective pretreatment process is required to remove lignin from lignocellulosic biomass and to reduce cellulose crystallinity to enhance the accessibility of fermentable carbohydrates for enzymatic hydrolysis (Taniguchi et al., 2005). Biological methods of pretreatment are most environment-friendly and cost-effective method compared to chemical and physical methods. In biological pretreatment, white-rot fungi (WRF) are the most potential candidate for selective lignin degradation. This unique property of WRF is due to their ability to produce various lignolytic enzymes; mainly laccase (phenol oxidase), lignin peroxidase (LiP) and manganese peroxidase (MnP). Lignolytic enzymes produced by WRF are needed to breakdown the lignin polymer into soluble compounds that are further mineralize (completely) into CO2 & H2O and have been explored widely on different types of lignocellulosic feed stocks (Boyle et al., 1992).
The mechanism of action of individual lignolytic enzyme in degradation of lignin model compounds has been reported, but the sequential role of lignolytic enzymes in lignin degradation during pretreatment of lignocellulosic biomass is poorly understood (Wong, 2009). WRF are the best lignin degraders, but not all of them produce all lignolytic enzymes, resulting slow lignin degradation. High utilization of fermentable carbohydrates due to non-selective lignin degradation is also major problem of fungal pretreatment (Sharma and Arora, 2014). The simultaneous expression of the lignolytic enzymes is essential for the effective lignin degradation. Low enzyme expression and mismatching of lignolytic enzyme profiles during pretreatment leads to low lignin degradation of lignocellulosic biomass and require long pretreatment time as well (Wan and Li, 2010).
Microbial interaction during co-culture may enhance or inhibit the production of various biochemicals. In synergistic interactions, species act in coordination to degrade the same substrate (Boddy, 2000, Chi et al., 2007). The basidiomycetes behave antagonistically if they are grown in the same medium. A series of secondary fungal metabolites are produced mainly in dual cultures of the basidiomycetes, which exert growth-inhibiting activities against other fungi (Sonnenbichler et al., 1994). The induction of novel isozymes as well as elevated expressions of various enzymes had been reported during co-culture of microorganisms (Kuhar et al., 2015). WRF in co-culture had expressed high lignolytic enzyme activities due to synergistic effect and expression of isozymes (Qi-He et al., 2011, Score et al., 1997). The combinations of different metabolic pathways of microorganisms present in nature are responsible for bioconversion and biotransformation of various substrates (Dong et al., 2012). It may be possible that co-cultivation of interacting fungi over expresses lignolytic enzymes during pretreatment process giving synergistic and combinatorial effect for efficient lignin degradation. To the best of our knowledge, improvement of delignification process through co-culture of fungi has not been studied till date. The objective of the study was to design a fungal co-culture strategy to improve the delignification of cotton stalks in less pretreatment time for higher enzymatic saccharification.
Section snippets
Materials
Fungal culture media (Yeast extract glucose agar, malt extract agar and potato dextrose agar), tannic acid, pyrogallol, birch wood xylan, carboxymethyl cellulose (CMC), p-nitrophenyl- β-D glucopyranoside, standard sugars (HPLC-grade) and N-acetyl glucosamine (NAG) were purchased from HiMedia laboratories Ltd, Mumbai, India. Veratryl alcohol was purchased from Sigma Aldrich Co, USA. 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) was purchased from MP Biomedicals, USA. All other
Screening of selective lignin degrading fungal strains
Four best selective lignin degrading fungal strains out of eight were screened on the basis of their lignocellulolytic abilities in petri plates and SV during pretreatment by SSF.
The lignolytic ability, laccase activity, peroxidase activity and cellulolytic activity of fungal strains were analyzed from the diameter of characteristic colored zones produced on the full grown plates of fungal strains during tannic acid, guaiacol, pyrogallol and Congo red dye tests. After observing the diameter of
Conclusion
Selective lignin degrading fungi Daedalea flavida MTCC 145 (DF-2) and Phlebia radiata MTCC 2791 (PR) in co-culture showed synergistic interaction resulting over expression and combinatorial effect of lignolytic enzymes during pretreatment of lignocellulosic cotton stalks. Increased lignolytic activity resulted in higher delignification of cotton stalks by co-culture in shorter pretreatment time (half) compared to the monocultures. The glucose released by enzymatic saccharification of cotton
Acknowledgements
Mr. Harmanpreet Meehnian gratefully acknowledges Ministry of Human Resource Development, Govt. of India for providing the fellowship during the study. All authors are highly thankful to National Institute of Technology (NIT), Jalandhar for providing grants and administrative supports for the study.
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