Evaluation of Non-Conventional Yeasts Isolated from Rotten Wood for Hydrolytic Activities and Xylose Fermentation

Abstract

Hydrolysis of lignocellulose to fermentable sugars and their subsequent conversion to ethanol remain great challenges in the biofuel industry. Rotten wood is first colonized by bacteria and molds that possess strong hydrolases. Yeasts are also an important group of microorganisms that may participate in wood hydrolysis. Decaying wood could provide a rich natural reservoir of yeasts possessing promising hydrolytic activities, including xylanases, cellulases, β-glucosidases, or abilities essential for the fermentation of pentose sugars derived from lignocellulose degradation, especially xylose. Therefore, the aim of this work was to screen yeasts isolated from rotten wood samples for the production of hydrolytic enzymes directed at lignocellulose components and the ability to ferment xylose, L-arabinose, and cellobiose. Methods. Yeast strains were isolated from 22 samples of rotten wood and identified by phenotypic characteristics according to Kurtzman et al. Hydrolytic properties and the ability of the isolated strains to ferment xylose, L-arabinose, and cellobiose were determined using conventional methods. Results. 30 strains of yeasts and yeast-like micromycetes were isolated from 22 samples of rotten wood in the Holosiivskyi Forest, Kyiv. Based on phenotypic properties, most of the isolated yeasts belonged to ascomycetous yeasts and were represented by the following genera: Candida (8 strains), Debaryomyces (5 strains), Kluyveromyces (5 strains), Pichia (5 strains), Scheffersomyces (2 strains), Lachancea, Hanseniaspora, Saccharomyces, and Geotrichum/Galactomyces. A strain of yeast-like non-photosynthetic alga Prototheca sp. was also detected. Most of the isolated microfungi (66.6% isolates) exhibited extracellular β-glucosidase activity, two Candida tropicalis strains possessed weak pectinase and xylanase activity. None of the isolates demonstrated extracellular cellulase activity. Two yeast strains preliminarily identified as Scheffersomyces stipitis were able to ferment xylose at a concentration of 20—100 g/L over a wide temperature range up to 37°C. Acetic acid at 0.25—1% (v/v) concentration resulted in the complete inhibition of xylose fermentation. Ethanol production from xylose up to 6 g/L was observed under the microaerobic fermentation conditions for 24 hr at the substrate concentration 40 g/L, but the subsequent fermentation resulted in decreasing ethanol concentration presumably due to ethanol re-assimilation. None of the isolated strains was capable of fermenting cellobiose or L-arabinose under the microaerobic conditions. Conclusions. This work provides the characterization of yeast microbiota of rotten wood that was represented predominantly by ascomycetous yeasts. The dominant extracellular hydrolytic activity of the isolates was β-glucosidase. This is the first report on the isolation of xylose-fermenting yeasts Scheffersomyces stipitis in Ukraine, which comprised 7% of all the microfungi isolated from rotten wood.

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

“…Assessment of the ability of xylose-fermenting yeast for ethanologenesis on media with a mixture of glucose and xylose and initial optimization of the process. Recently isolated xylose-fermenting yeast strain UCM Y-2810 (working code w18 (Ianieva et al, 2022), which belonged to the species Scheffersomyces stipitis according to preliminary phenotypic eva-luations of its physiological and biochemical characteristics, served in this assessment as a model yeast in such experiments. We performed precise molecular genetic identification of this strain to confirm whether it belongs to species S. stipitis.…”

“…According to preliminary biochemical identification, these strains belonged to the species Scheffersomyces stipitis (Pignal) Kurtzman & Suzuki 2010. These strains were isolated in 2021 from decaying wood with working codes w18 and w20.1, and later they were assigned codes of the Ukrainian Collection of Microorganisms UCM Y-2810 and UCM Y-2811, respectively (Ianieva et al, 2022). Of these two strains, the average values of ethanol production on medium with 40 g/L xylose were slightly higher for S. stipitis UCM Y-2810 (6.1 g/L) than for S. stipitis UCM Y-2811 (5.5 g/L).…”

“…Two genetic markers of the ribosomal operon were used for accurate molecular genetic identification of yeast isolate w18 (collection code UCM Y-2810) from rotten wood, which was previously identified by biochemical methods as Scheffersomyces stipitis (formerly known as Pichia stipitis species) (Yamada et al, 1994;Ianieva et al, 2022). There were (1) ITS-fragment containing the 5'-end of the 18S rRNA gene, ITS1, the 5.8S rRNA gene, ITS2, and the 3'-end of the 28S rRNA gene, and (2) D1/D2 locus of the 28S rRNA large subunit gene.…”

“…In the choice of cultivation methods for S. stipitis UCM Y-2810, we relied on the experience of our previous studies of this strain, including data on the duration of its cultivation necessary to obtain ethanol (Ianieva et al, 2022). At this stage of the work, yeast cultivation was carried out on liquid media at 25 °C under following conditions: 1) under static microaerophilic conditions in Dunbar tubes, which are traditionally used for qualitative evaluation of yeast ability for fermentation, with a maximum duration of up to 7 days, as well as 2) under submerged conditions in conical flasks (with total volume 250 mL) on a shaker with rotation rate 120 rpm, with a maximum duration of up to 3 days.…”

Pentose-Fermenting Yeasts in Nature: Ecology, Biodiversity and Applications

“…One of the well-studied xylose-fermenting yeasts Sc. stipitis was found in wood-associated substrates in various geographical locations, including Europe, North, Central America [43], Brazil [13,14], South Africa [11], China [16], and Ukraine [31]. Its close relative Sc.…”