Application of natural fungi in bioconversion of lignocellulosic waste to second-generation ethanol

Abstract

The second generation (2G) or cellulosic ethanol can help with diversification of the use of fossil energy sources. However, as bioconversion of plant waste into 2G bioethanol requires expensive additional steps of pre-treatment/hydrolysis of lignocellulosic materials, and this technology has not yet reached the technological readiness level which would allow it to be scaled-up, this process needs more interdisciplinary and comprehensive studies. This work was aimed at experimental study of a full cycle of successive processes of pre-treatment/saccharification using cellulolytic enzymes of filamentous fungi and fermentation of obtained syrups by xylose-fermenting yeast, using selected natural microorganisms for the fungal-based bioconversion of lignocellulosic agricultural waste to 2G ethanol. Using the Plackett-Burman and Box-Behnken methods of mathematical statistics, the optimal conditions for pre-treatment and enzymatic hydrolysis of wheat straw by a hemi- and cellulolytic multi-enzyme complex of the selected fungal strain Talaromyces funiculosus UCM F-16795 were established: microwave-assisted alkali pre-treatment with sodium hydroxide (NaOH) solutions (concentration range 4.6–4.8%), and saccharification conditions of medium pH 4, temperature 40 °С, hydrolysis duration 18 hours, and dilution of culture liquid with a buffer solution 1:1. The total energy of microwave irradiation 1.2 kJ and the ratio of substrate/enzyme solution 100 mg/1 mL were used. Under optimized conditions, wheat straw hydrolysates contained 5.0–7.5 g/L of reducing sugars, which, according to HPLC assessment, contained 0.7–1.0 g/L of glucose, 2.2–2.9 g/L of xylose and 0.7–0.8 g/L cellobiose. We used the selected strain of xylose-fermenting yeast in fermentation of mixtures of the most important monosaccharides in hydrolysates, xylose and glucose, in the concentration range relevant for syrups obtained by us during the optimized saccharification of lignocellulosic substrates with T. funiculosus enzymes. Based on sequencing and phylogenetic analysis, strain UCM Y-2810 was confirmed as Scheffersomyces stipitis; its nucleotide sequences of ITS region and 28S gene rDNA were deposited in GenBank under the accession numbers OP931914 and OP931915, respectively. The ethanologenic process for S. stipitis UCM Y-2810 was studied according to Box-Behnken design, assessing ethanol concentration by gas chromatography-mass spectrometry. Yeast fermentation under static microaerophilic conditions showed a 1.5 times higher rate of bioethanol production and 1.7 times greater efficiency of ethanologenesis per yeast biomass than for submerged cultivation. Optimization of the process of ethanologenesis resulted in the maximum rate of fermentation mixture of sugars, being 11.30 ± 0.36 g/L of ethanol, with optimal values of factors: 30 g/L of xylose, 5.5 g/L of glucose and cultivation for 5.5 days. It was revealed that the tested glucose concentrations did not significantly affect the process of xylose-fermentation by yeast, and non-competitive inhibition of xylose transport by glucose into yeast cells did not occur. This study demonstrated the potential of a full cycle bioconversion of lignocellulosic waste to 2G ethanol based on use of natural fungal strains and optimization of conditions for all steps.