Isolation of xylose-utilizing yeasts from oil palm waste for xylitol and ethanol production

Abstract

The energy crisis triggers the use of energy sources that are renewable, such as biomass made from lignocellulosic materials, to produce various chemical compounds for food ingredients and biofuel. The efficient conversion of lignocellulosic biomass into products with added value involves the activity of microorganisms, such as yeasts. For the conversion, microorganisms must be able to use various sugars in lignocellulosic biomass, including pentose sugars, especially xylose. This study aims to isolate xylose-utilizing yeasts and analyze their fermentation activity to produce xylitol and ethanol, as well as their ability to grow in liquid hydrolysate produced from pretreated lignocellulosic biomass. Nineteen yeast isolates could grow on solid and liquid media using solely xylose as a carbon source. All isolates can grow in a xylose medium with incubation at 30 °C, 37 °C, 42 °C, and 45 °C. Six isolates, namely SLI (1), SL3, SL6, SL7, R5, and OPT4B, were chosen based on their considerable growth and high xylose consumption rate in a medium with 50 g/L xylose with incubation at 30 °C for 48 h. Four isolates tested, namely SLI (1), SL6, SL7, and R5, can produce xylitol in media containing xylose carbon sources. The concentration of xylitol produced was determined using high-pressure liquid chromatography (HPLC), and the results ranged from 5.0 to 6.0 g/L. Five isolates tested, namely SLI (1), SL6, SL3, R5, and OPT4B, can produce ethanol. The ethanol content produced was determined using gas chromatography (GC), with concentrations ranging from 0.85 to 1.34 g/L. Three isolates, namely SL1(1), R5, and SL6, were able to produce xylitol and ethanol from xylose as carbon sources and were also able to grow on liquid hydrolyzate from pretreated oil palm trunk waste with the subcritical water method. The three isolates were further analyzed using the 18S rDNA sequence to identify the species and confirm their phylogenetic position. Identification based on DNA sequence analysis revealed that isolates SL1(1) and R5 were Pichia kudriavzevii, while isolate SL6 was Candida xylopsoci. The yeast strains isolated from this study could potentially be used for the bioconversion process of lignocellulosic biomass waste to produce value-added derivative products.

Keywords

Yeast / Xylose / Xylitol / Ethanol / Lignocellulosic-biomass

References

[1] Ali SS, Wu J, Xie R, Zhou F, Sun J, Huang M. Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite. Reticulitermes Chinensis Plos ONE, 2017, 12(7): e0181141.

[2] Almeida JR, Modig T, Petersson A, Hahn-Hägerdal B, Liden G, Gorwa-Grauslund M-F. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol, 2007, 82: 340-349.

[3] Amo K, Arai H, Uebanso T, Fukaya M, Koganei M, Sasaki H, Yamamoto H, Taketani Y, Takeda E. Effects of xylitol on metabolic parameters and visceral fat accumulation. J Clin Biochem Nutr, 2011, 49: 1-7.

[4] Antunes FAF, Milessi TSS, Chandel AK, Moraes V, Rosa CA, Silva SS. Evaluation of a new yeast from Brazilian biodiversity Scheffersomyces shehatae UFMG-HM 522, for pentose sugars conversion into bioethanol. Biochem Biotechnol Reports, 2013, 2(4): 1-7.

[5] Ara´ujo JA, de Abreu-Lima LT, Carreiro SC. Selection and identification of xylose-fermenting yeast strains for ethanol production from lignocellulosic biomass. Boletim Do Centro De Pesquisa De Processamento De Alimentos, 2018, 36(1): 68-79.

[6] Bettiga M, Hahn-Hägerdal B, Gorwa-Grauslund MF (2008) Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains. Biotechnol Biofuels 1:16. https://doi.org/10.1186/1754-6834-1-16

[7] Biswas R, Uellendahl H, Ahring BK. Conversion of C6 and C5 sugars in undetoxified wet exploded bagasse hydrolysates using Scheffersomyces (Pichia) stipitis CBS6054. AMB Express, 2013, 3: 42-47.

[8] Cagnin L, Gronchi N, Favaro L, Casella S. Selection of superior yeast strains for the fermentation of lignocellulosic steam-exploded residues. Front Microbiol, 2021

[9] Chan GF, Gan HM, Ling HL, Rashid NAA. Genome sequence of Pichia kudriavzevii M12, a potential producer of bioethanol and phytase. Eukaryot Cell, 2012, 11(10): 1300-1301.

[10] Choudhary J, Singh S, Nain L. Review: Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass. Electron J Biotechnol, 2016, 21: 82-92.

[11] Guerrero AB, Ballesteros I, Ballesteros M. The potential of agricultural banana waste for bioethanol production. Fuel, 2018, 213: 176-185.

[12] Guo C, Zhao C, He P, Lu D, Shen A, Jiang N. Screening and characterization of yeasts for xylitol production. J Appl Microbiol, 2006, 101(5): 1096-1104.

[13] Jönsson LJ, Alriksson B, Nilvebrant NO. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels, 2013, 6: 16.

[14] Kalyani DC, Zamanzadeh M, M”uller G, Horn SJ. Biofuel production from Birch wood by combining high solid loading simultaneous saccharification and fermentation and anaerobic digestion. Appl Energy, 2017, 193: 210-219.

[15] Kim JH, Block DE, Mills DA. Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl Microbiol Biotechnol, 2010, 88(5): 1077-1085.

[16] Kishore P, Kehlenbrink S, Hu M, Zhang K, Gutierrez-Juarez R, Koppaka S, El-Maghrabi MR, Hawkins M. Xylitol prevents NEFA-induced insulin resistance in rats. Diabetologia, 2012, 55: 1808-1812.

[17] Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol, 2004, 66(1): 10-26.

[18] Koutinas M, Patsalou M, Stavrinou S, Vyrides I. High temperature alcoholic fermentation of orange peel by the newly isolated thermotolerant Pichia kudriavzevii KVMP10. Lett Appl Microbiol, 2015, 62: 75-83.

[19] Kumar A, Singh LK, Ghosh S. Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicelulose acid hydrolysate to ethanol by Pichia stipitis. Biores Technol, 2009, 100: 3293-3297.

[20] Kusumawati N, Wardani AK, Zubaidah E, Sumarlan SH. Isolation, screening and identification of potential cellulolytic and xylanolytic mold from oil palm waste. IOP Conf Ser Earth Environ Sci, 2020, 475: 012083.

[21] Laluce C, Schenberg ACG, Gallardo JCM, Coradello LFC, Pombeiro-Sponchiado SR. Advances and developments in strategies to improve strains of Saccharomyces cerevisiae and processes to obtain the lignocellulosic ethanol—a review. Appl Biochem Biotechnol, 2012, 166: 1908-1926.

[22] Liang M, He QP, Jeffries TW, Wang J. Elucidating xylose metabolism of Scheffersomyces stipitis by integrating principal component analysis with flux balance analysis. Proc Am Control Conf, 2013

[23] Martini C, Tauk-Tornisielo SM, Codato CB, Bastos RG, Ceccato-Antonini SR. A strain of Meyerozyma guilliermondii isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media. World J Microbiol Biotechnol, 2016, 32(5): 80-89.

[24] Martins GM, Bocchini-Martins DA, Bezzerra-Bussoli C, Pagnocca FC, Boscolo M, Monteiro DA, da Silva R, Gomes E. The isolation of pentose-assimilating yeasts and their xylose fermentation potential. Braz J Microbiol, 2018, 49: 162-168.

[25] Mattam AJ, Kuila A, Suralikerimath N, Choudary N, Rao PVC, Velankar HR. Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain. Biotechnol Biofuels, 2016, 9: 157.

[26] Moremi ME, Van-Rensburg ELJ, La-Grange DC. The improvement of bioethanol production by pentose-fermenting yeasts isolated from herbal preparations, the gut of dung beetles, and Marula Wine. Int J Microbiol, 2020

[27] Murata Y, Danjarean H, Fujimoto K, Kosugi A, Arai T, Ibrahim WA. Ethanol fermentation by the thermotolerant yeast, Kluyveromyces marxianus TISTR5925, of extracted sap from old oil palm trunk. AIMS Energy, 2015, 3: 201-213.

[28] Navnit LM, Unnati P. Isolation and screening of wild yeasts for maximum xylitol production. Int J Life Sci Special Issue, 2015, A5: 11-18.

[29] Ndubuisi IA, Nweze JE, Onoyima NJ, Yoshinori M, Ogbonna JC. Ethanol production from cassava pulp by a newly isolated thermotolerant Pichia kudriavzevii LC375240. Energy Power Eng, 2018, 10: 457-474.

[30] Nguyen QA, Yang J, Bae HJ. Bioethanol production from individual and mixed agricultural biomass residues. Ind Crops Prod, 2017, 95: 718-725.

[31] Phong HX, Klanrit P, Dung NTP, Yamada M, Thanonkeo P. Isolation and characterization of thermotolerant yeasts for the production of second-generation bioethanol. Annal Microbiol, 2019, 69: 765-776.

[32] Pilap W, Thanonkeo S, Klanrit P, Thanonkeo P. The potential of multistress tolerant yeast, Saccharomycodes ludwigii, for second-generation bioethanol production. Sci Reports, 2022, 12: 22062.

[33] Pongcharoen P, Chawneua J, Tawong W. High temperature alcoholic fermentation by new thermotolerant yeast strains Pichia kudriavzevii isolated from sugarcane field soil. Agric Nat Resour, 2018, 52: 511-518.

[34] Rahman KHA, Ismail KSK, Najimudin N. Growth of thermotolerant Pichia kudriavzevii UniMAP 3–1 strain for ethanol production using xylose and glucose at different fermentation temperatures. IOP Conf Ser Earth Environ Sci, 2021, 765(1): 01210.

[35] Roehr M. The biotechnology of ethanol, 2001, Weinheim: WileyVCH Verlag GmbH & Co. KGaA.

[36] Ruchala J, Sibirny A. Pentose metabolism and conversion to biofuels and high- value chemicals in yeasts. FEMS Microbiol Rev, 2021, 45: 1-44.

[37] Senatham S, Chamduang T, Kaewchingduang Y, Thammasittirong A, Srisodsuk M, Elliston A, Roberts IN, Waldron KW, Thammasittirong SN. Enhanced xylose fermentation and hydrolysate inhibitor tolerance of Scheffersomyces shehatae for efficient ethanol production from non-detoxified lignocellulosic hydrolysate. Springerplus, 2016, 5: 1040.

[38] Singh LK, Chaudhary G, Majumder CB, Ghosh S. Utilization of hemicellulosic fraction of lignocellulosic material for bioethanol production. Adv Appl Sci Res, 2011, 2(5): 508-521.

[39] Talukder AA, Easmin F, Mahmud SA, Yamada M. Thermotolerant yeasts capable of producing bioethanol: isolation from natural fermented sources, identification and characterization. Biotechnol Biotechnol Equip, 2016, 30(6): 1106-1114.

[40] Talukder AA, Adnan N, Siddiqa A, Miah R, Tuli JF, Khan ST, Dey SK, Lertwattanasakul N, Yamada M. Fuel ethanol production using xylose assimilating and high ethanol producing thermosensitive Saccharomyces cerevisiae isolated from date palm juice in Bangladesh. Biocatal Agric Biotechnol, 2019, 18: 1-7.

[41] Tanimura A, Nakamura T, Watanabe I, Ogawa J, Shima J. Isolation of a novel strain of Candida shehatae for ethanol production at elevated temperature. Springerplus, 2012, 1: 27.

[42] Tye YY, Lee KT, Abdullah WNW, Leh CP. The world availability of non-wood lignocellulosic biomass for the production of cellulosic ethanol and potential pretreatments for the enhancement of enzymatic saccharification. Renew Sustain Energy Rev, 2016, 60: 155-172.

[43] Ulya D, Astuti R, Meryandini A. The ethanol production activity of indigenous thermotolerant yeast Pichia kudriavzevii 1P4. Microbiol Indonesia, 2018, 14(4): 121-128.

[44] Wu J, Hu J, Zhao S, He M, Hu G, Ge G, Peng N. Single-cell protein and xylitol production by a novel yeast Strain Candida intermedia FL023 from lignocellulosic hydrolysates and xylose. Appl Biochem Biotechnol, 2018, 185: 163-178.

Funding

Ministry of Research, Technology and Higher Education, the Republic of Indonesia (115G/WM01.5/N/2019)