Macro-micro fungal cultures synergy for innovative cellulase enzymes production and biomass structural analyses
Graphical abstract
Introduction
Innovation in renewable energy sources that are alternatives to fossil fuels, is a critical worldwide priority for a sustainable development of the global economy [1]. One of the attractive and promising substitutes for fossil fuels is production of bioethanol from biomass [2]. In order to improve the economic feasibility of bioethanol production, extensive studies on use of enzymatic hydrolysis to convert lignocellulosic polymers to fermentable sugars have been undertaken [3]. Bioethanol can be blended at significant concentrations with gasoline or diesel fuels for use in vehicles and is considered to be a sustainable transportation fuel [4], [5]. However, in cellulosic bioethanol production, improving the prospects of crop selection [6] and microorganisms ability to break down the lignocellulosic biomass (LCB) are of paramount importance [7].
In this regard, there are a variety of microorganisms [8], [9], [10] in nature, that are known for producing a combination of enzymes capable of degrading the insoluble cellulose polymer in LCB to soluble sugars, primarily cellobiose and glucose [9]. Enzymes involved in these processes are called cellulases and consist of at least three classes namely, endo-glucanases, cellobiohydrolases and β-glucosidases [10]. The interest in cellulases has particularly increased in recent years with utilization of these enzymes in production of bioethanol from LCB [8], [9], [10]. In this context, we have explored for the first time, the synergistic combination of Asperigillus oryzae (microfungi-MTCC 1212) and Pycnoporus sanguineus (macrofungi) for enhanced and balanced production of cellulase, endo-β-1,4-glucanase and β-glucosidase under solid state fermentation conditions. Solid state fermentation conditions have several advantages which include less infrastructure, ability to utilize cheaper materials for enzyme production, and less skilled manpower leading to a more concentrated product.
Mushrooms or macrofungi have been utilized [11] for enhanced extra-cellular cellulase enzyme production on account of the mechanical pressure created by their elongated hyphae. Sixteen different macrofungi obtained from the culture repository of Indian Institute of Horticulture Research (IIHR), Bangalore, India were evaluated for their ability to produce cellulolytic enzymes [12]. Pycnoporus sanguineus (referred to as PS) also known as Trametes sanquinea in literature [13], was selected based on its maximum radial growth on carboxymethylcellulose (CMC) enriched agar medium. The micro fungus Asperigillus oryzae (referred to as AO) was selected for co-culture studies based on previous experiments conducted at Sri Sathya Sai Institute of Higher learning (SSSIHL) (unpublished work). Moving to the selection of LCB, the use of non-edible feedstock for biofuel production is imperative for growing population [14]. Groundnut shells (GNS) with a chemical composition of 33% cellulose, 24% hemicellulose, 6.5% lignin, 21.2% carbohydrates, 7.3% proteins, was chosen as the substrate in the current study [15]. GNS is one of the most abundantly produced lignocellulosic feed stock; India being the second largest producer of this crop residue in the world. Despite its large availability, cost of production and low product yield of cellulolytic enzymes are limiting factors in its usage for biofuel production [16].
In this context, the SSF technique has emerged as an advantageous economical process over submerged fermentation for cellulase production [17]. The process requires less energy and less water usage as it occurs in absence of free flowing water, using natural polymers (as carbon source) originated from agro-industrial products. The SSF technique was chosen based on an earlier observation of enhanced cellulose production as compared to submerged liquid fermentation. Similar conditions were maintained for mono and co-culture studies on SSF as well. The present study also involved quantification of extracellular enzyme production in mono and co-culture conditions due to fungal colonization of GNS. Structural modifications in the recalcitrant LCB were monitored using spectral and thermal analysis.
Section snippets
Microorganisms and cell suspension
Macrofungal strains were obtained from Mushroom lab, IIHR as sorghum spawn. The mycelial growth on the grains was extracted with distilled water to form a homogenized suspension. Asperigillus oryzae (MTCC 1212) was obtained from National Collection of Industrial Microorganisms, National Chemical laboratory, India. It was cultured on potato dextrose agar (PDA) slants at 30 °C for 7 days, stored at 4 °C and sub-cultured every two weeks. Fungal spore suspension was obtained by vortexing 10 ml of
Strain identification and phylogenic analysis
The Internal Transcribed Region (ITS) region of P. sanguineus was PCR (Polymerase chain reaction) amplified for strain identification with the primers ITS1 and ITS4 [21]. The amplicon was electrophoresed in 1% agarose gel and visualized under UV. Amplicon was purified in Purelink purification column and the concentration was checked in Nanodrop ND 200. Sequencing of the amplicon with forward and reverse primers was performed with ABI 3730xl cycle sequencer. Forward and reverse sequences were
Conclusion
Increase in use of renewable sources of energy contributes towards sustainable development. This can be possible with increased use of biofuels since GNS is an abundantly available LCB and is a preferred solid substrate for the production of cellulase enzyme complex by various fungal strains. In this study, we have chosen a synergistic combination of P. sanguineus (macro fungi with maximal radial growth) with AO (known micro fungi with enhanced cellulase activity), as potent species for lignin
Acknowledgments
SSR acknowledges the funding from DBT-Ramalingaswami fellowship (Sanction Order No. 102/IFD/SAN/1118/2014-15), Govt. of India. PVV is grateful to Kansas State University and Lortscher Endowment for his sabbatical at SSSIHL. Special thanks to Dr. Ron Madl, Research Professor Emeritus, Bioprocessing and Industrial Value Added Program, Kansas State University for proof reading of the manuscript and to Sri Sandeep Patnaik, Dept. of Physics, SSSIHL for helping with measurements on the DSC-TGA
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