Isolation, Identification, and Physiological Characterization of Indigenous Yeast Species Capable of Efficiently Utilizing Sugarcane Molasses as a Carbon Source

Authors

  • Aamir Sohail State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding, China. Department of Biotechnology, Abdul Wali Khan University Mardan, KP, Pakistan.
  • Kashmala Ihsan Department of Microbiology, Abdul Wali Khan University Mardan, KP, Pakistan.
  • Raham Sher Khan Department of Biotechnology, Abdul Wali Khan University Mardan, KP, Pakistan.
  • Abid Ali Department of Zoology, Abdul Wali Khan University Mardan, KP, Pakistan.
  • Zia-ul Islam State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding, China. Department of Biotechnology, Abdul Wali Khan University Mardan, KP, Pakistan.

DOI:

https://doi.org/10.70749/ijbr.v3i3.860

Keywords:

Sugarcane Molasses, Yeast, Saccharomyces Cerevisiae

Abstract

Molasses, a byproduct of sugar production, contains sugars, ash, and inhibitors, limiting its microbial use. This study screened yeast species for efficient molasses utilization and inhibitor tolerance. Samples from four Khyber Pakhtunkhwa districts yielded 33 yeast strains after scrutiny. Following initial characterization, the strains were identified based on both morphological features and molecular methods involving the amplification of Internal Transcribed Spacer (ITS) regions. By the BLAST analysis, the ITS sequences for Candida tropicalis, Pichia kudriavzevii, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon asahii, and Wickerhamomyces anomalus demonstrated 100% identity, whereas the sequence for Aspergillus fumigatus exhibited a maximum identity of 99.79% with the same species. In the phylogenetic analysis, these sequences were clustered with their respective corresponding species. Since molasses contain sucrose in major quantity, the physiological characterization of these isolated species in synthetic media containing sucrose as a sole carbon source reveals the higher growth efficiency of Torulaspora delbrueckii (OD600nm 5.24, μmax 0.0058 h-1) with second best performance of Trichosporon asahii (OD600nm 4.4, μmax 0.0049 h-1). The lowest grower was Saccharomyces cerevisiae (OD600nm 1.78 μmax 0.00016 h-1) while the remaining species i.e., Aspergillus fumigatus, Candida tropicalis, Pichia kudriavzevii, and Wickerhamomyces anomalus were of intermediate level (OD600nm 3.44, 3.89, 3.81, and 3.77, μmax was 0.0045 h-1, 0.0042 h-1, 0.0042 h-1, 0.0042 h-1 respectively). The isolated yeast species, known for utilizing non-molasses carbon sources, expand our understanding of substrate usage. Their potential as biofactories or genetic resources from natural evolution could aid in engineering industrial yeast strains for biofuel and biochemical production.

Downloads

Download data is not yet available.

References

. Marques, W. L., Raghavendran, V., Stambuk, B. U., & Gombert, A. K. (2015). Sucrose andSaccharomyces cerevisiae: A relationship most sweet. FEMS Yeast Research, 16(1), fov107. https://doi.org/10.1093/femsyr/fov107

Thangavelu, K., Sundararaju, P., Srinivasan, N., & Uthandi, S. (2021). Bioconversion of Sago processing wastewater into biodiesel: Optimization of lipid production by an oleaginous yeast, candida tropicalis ASY2 and its transesterification process using response surface methodology. Microbial Cell Factories, 20(1). https://doi.org/10.1186/s12934-021-01655-7

De La Cruz Pech-Canul, Á., Ortega, D., Garcia-Triana, A., & Lidia Solís-Oviedo, R. (2019). Torulaspora delbrueckii: Towards innovating in the legendary baking and brewing industries. Frontiers and New Trends in the Science of Fermented Food and Beverages. https://doi.org/10.5772/intechopen.83522

Chua, J., Tan, S. J., & Liu, S. (2021). The impact of mixed amino acids supplementation on Torulaspora delbrueckii growth and volatile compound modulation in soy whey alcohol fermentation. Food Research International, 140, 109901. https://doi.org/10.1016/j.foodres.2020.109901

Shariq, M., & Sohail, M. (2019). Application of candida tropicalis MK-160 for the production of xylanase and ethanol. Journal of King Saud University - Science, 31(4), 1189-1194. https://doi.org/10.1016/j.jksus.2018.04.009

Raj, S. B., Ramaswamy, S., & Plapp, B. V. (2014). Yeast alcohol Dehydrogenase structure and catalysis. Biochemistry, 53(36), 5791-5803. https://doi.org/10.1021/bi5006442

Benito, S. (2018). The impact of Torulaspora delbrueckii yeast in winemaking. Applied Microbiology and Biotechnology, 102(7), 3081-3094. https://doi.org/10.1007/s00253-018-8849-0

Raj, K., & Krishnan, C. (2020). Improved Co-production of ethanol and xylitol from low-temperature aqueous ammonia pretreated sugarcane bagasse using two-stage high solids enzymatic hydrolysis and candida tropicalis. Renewable Energy, 153, 392-403. https://doi.org/10.1016/j.renene.2020.02.042

Czarnecka, M., Żarowska, B., Połomska, X., Restuccia, C., & Cirvilleri, G. (2019). Role of biocontrol yeasts Debaryomyces hansenii and Wickerhamomyces anomalus in plants' defence mechanisms against Monilinia fructicola in Apple fruits. Food Microbiology, 83, 1-8. https://doi.org/10.1016/j.fm.2019.04.004

Fan, G., Teng, C., Xu, D., Fu, Z., Minhazul, K. A., Wu, Q., Liu, P., Yang, R., & Li, X. (2019). Enhanced production of Ethyl acetate using Co-culture of Wickerhamomyces anomalus and saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 128(5), 564-570. https://doi.org/10.1016/j.jbiosc.2019.05.002

Oro, L., Feliziani, E., Ciani, M., Romanazzi, G., & Comitini, F. (2018). Volatile organic compounds from Wickerhamomyces anomalus, Metschnikowia pulcherrima and saccharomyces cerevisiae inhibit growth of decay causing fungi and control postharvest diseases of strawberries. International Journal of Food Microbiology, 265, 18-22. https://doi.org/10.1016/j.ijfoodmicro.2017.10.027

Ojha, N., & Das, N. (2018). A statistical approach to optimize the production of Polyhydroxyalkanoates from Wickerhamomyces anomalus VIT-NN01 using response surface methodology. International Journal of Biological Macromolecules, 107, 2157-2170. https://doi.org/10.1016/j.ijbiomac.2017.10.089

Akita, H., Goshima, T., Suzuki, T., Itoiri, Y., Kimura, Z., & Matsushika, A. (2021). Application of Pichia kudriavzevii NBRC1279 and NBRC1664 to simultaneous saccharification and fermentation for Bioethanol production. Fermentation, 7(2), 83. https://doi.org/10.3390/fermentation7020083

Sankh, S., Thiru, M., Saran, S., & Rangaswamy, V. (2013). Biodiesel production from a newly isolated Pichia kudriavzevii strain. Fuel, 106, 690-696. https://doi.org/10.1016/j.fuel.2012.12.014

Dhaliwal, S. S., Oberoi, H. S., Sandhu, S. K., Nanda, D., Kumar, D., & Uppal, S. K. (2011). Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii. Bioresource Technology, 102(10), 5968-5975. https://doi.org/10.1016/j.biortech.2011.02.015

Chan, G. F., Gan, H. M., Ling, H. L., & Rashid, N. A. (2012). Genome sequence of Pichia kudriavzevii M12, a potential producer of Bioethanol and Phytase. Eukaryotic Cell, 11(10), 1300-1301. https://doi.org/10.1128/ec.00229-12

Jones, A. M., Thomas, K. C., & Ingledew, W. M. (1994). Ethanolic fermentation of Blackstrap molasses and sugarcane juice using very high gravity technology. Journal of Agricultural and Food Chemistry, 42(5), 1242-1246. https://doi.org/10.1021/jf00041a037

Bušić, A., Marđetko, N., Kundas, S., Morzak, G., Belskaya, H., Ivančić Šantek, M., Komes, D., Novak, S., & Šantek, B. (2018). Bioethanol production from renewable raw materials and its separation and purification: A review. Food Technology and Biotechnology, 56(3). https://doi.org/10.17113/ftb.56.03.18.5546

Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547-1549. https://doi.org/10.1093/molbev/msy096

Hall, T., Biosciences, I., & Carlsbad, C. J. G. B. B. (2011). BioEdit: an important software for molecular biology. GERF bull biosci, 2(1), 60-61.

Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. https://doi.org/10.1093/nar/22.22.4673

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. https://doi.org/10.1016/s0022-2836(05)80360-2

Raharja, R., Murdiyatmo, U., Sutrisno, A., & Wardani, A. K. (2019). Bioethanol production from sugarcane molasses by instant dry yeast. IOP Conference Series: Earth and Environmental Science, 230, 012076. https://doi.org/10.1088/1755-1315/230/1/012076

Verduyn, C. (1992). Physiology of yeasts in relation to biomass yields. Quantitative Aspects of Growth and Metabolism of Microorganisms, 325-353. https://doi.org/10.1007/978-94-011-2446-1_14

Hřibová, E., Čížková, J., Christelová, P., Taudien, S., De Langhe, E., & Doležel, J. (2011). The ITS1-5.8S-ITS2 sequence region in the musaceae: Structure, diversity and use in molecular phylogeny. PLoS ONE, 6(3), e17863. https://doi.org/10.1371/journal.pone.0017863

Le Borgne, S. (2011). Genetic engineering of industrial strains of saccharomyces cerevisiae. Methods in Molecular Biology, 451-465. https://doi.org/10.1007/978-1-61779-433-9_24

MWESIGYE, P. K., & BARFORD, J. P. (1996). Mechanism of surcrose utilisation by saccharomyces cerevisiae. The Journal of General and Applied Microbiology, 42(4), 297-306. https://doi.org/10.2323/jgam.42.297

MWESIGYE, P. K., & BARFORD, J. P. (1996). Mechanism of surcrose utilisation by saccharomyces cerevisiae. The Journal of General and Applied Microbiology, 42(4), 297-306. https://doi.org/10.2323/jgam.42.297

Rodrigues, C. I., Den Ridder, M., Pabst, M., Gombert, A. K., & Wahl, S. A. (2022). Comparative proteome analysis of differentSaccharomyces cerevisiaestrains during growth on sucrose and glucose. https://doi.org/10.1101/2022.11.03.515096

Solomon, S. (2011). Sugarcane by-products based industries in India. Sugar Tech, 13(4), 408-416. https://doi.org/10.1007/s12355-011-0114-0

Mehmood, A., Sial, M. H., Sharif, S., Hussain, A., Riaz, M., & Shaheen, N. (2020). Forecasting the fisheries production in Pakistan for the year 2017-2026, using Box-Jenkin’s methodology. Pakistan Journal of Agricultural Research, 33(1). https://doi.org/10.17582/journal.pjar/2020/33.1.140.145

Jamir, L., Kumar, V., Kaur, J., Kumar, S., & Singh, H. (2021). Composition, valorization and therapeutical potential of molasses: A critical review. Environmental Technology Reviews, 10(1), 131-142. https://doi.org/10.1080/21622515.2021.1892203

Kumar, V., Naik, B., Choudhary, M., Kumar, A., & Khanduri, N. (2022). Agro-waste as a substrate for the production of pullulanase by penicillium viridicatum under solid-state fermentation. Scientific Reports, 12(1). https://doi.org/10.1038/s41598-022-16854-4

Guaragnella, N., & Bettiga, M. (2021). Acetic acid stress in budding yeast: From molecular mechanisms to applications. Yeast, 38(7), 391-400. https://doi.org/10.1002/yea.3651

Kiselev, E. G., Demidenko, A. V., Zhila, N. O., Shishatskaya, E. I., & Volova, T. G. (2022). Sugar beet molasses as a potential C-substrate for PHA production by Cupriavidus necator. Bioengineering, 9(4), 154. https://doi.org/10.3390/bioengineering9040154

Santiago, C., Rito, T., Vieira, D., Fernandes, T., Pais, C., Sousa, M. J., Soares, P., & Franco-Duarte, R. (2021). Improvement of Torulaspora delbrueckii genome annotation: Towards the exploitation of Genomic features of a Biotechnologically relevant yeast. Journal of Fungi, 7(4), 287. https://doi.org/10.3390/jof7040287

Kurtzman, C. P., Robnett, C. J., & Basehoar-Powers, E. (2008). Phylogenetic relationships among species of pichia, issatchenkia and williopsis determined from multigene sequence analysis, and the proposal of barnettozyma gen. nov., lindnera gen. nov. and wickerhamomyces gen. nov. FEMS Yeast Research, 8(6), 939-954. https://doi.org/10.1111/j.1567-1364.2008.00419.x

Kurtzman, C. P. (2010). Phylogeny of the ascomycetous yeasts and the renaming of Pichia anomala to Wickerhamomyces anomalus. Antonie van Leeuwenhoek, 99(1), 13-23. https://doi.org/10.1007/s10482-010-9505-6

Choudhry, R., Hodgins, M. B., Van der Kwast, T. H., Brinkmann, A. O., & Boersma, W. J. (1992). Localization of androgen receptors in human skin by immunohistochemistry: Implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands. Journal of Endocrinology, 133(3), 467-NP. https://doi.org/10.1677/joe.0.1330467

Jahanbani, J., Sandvik, L., Lyberg, T., & Ahlfors, E. (2009). Evaluation of oral mucosal lesions in 598 referred Iranian patients. The Open Dentistry Journal, 3(1), 42-47. https://doi.org/10.2174/1874210600903010042

Dos Santos, P. J., Bessa, C. F., De Aguiar, M. C., & Do Carmo, M. A. (2003). Cross‐sectional study of oral mucosal conditions among a central Amazonian Indian community, Brazil. Journal of Oral Pathology & Medicine, 33(1), 7-12. https://doi.org/10.1111/j.1600-0714.2004.00003.x

GARDES, M., & BRUNS, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts. Molecular Ecology, 2(2), 113-118. https://doi.org/10.1111/j.1365-294x.1993.tb00005.x

Martin, K. J., & Rygiewicz, P. T. (2005). Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology, 5(1). https://doi.org/10.1186/1471-2180-5-28

Lazcano, O., Speights Jr, V. O., Strickler, J. G., Bilbao, J. E., Becker, J., & Diaz, J. (1993). Combined histochemical stains in the differential diagnosis of Cryptococcus neoformans. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc, 6(1), 80-84. https://europepmc.org/article/med/7678937

Pylvänäinen, I. (2005). A parametric approach to yeast growth curve estimation and standardization. Chalmers Tekniska Hogskola (Sweden).

Khosravi-Darani, K., Mokhtari, Z., Amai, T., & Tanaka, K. (2013). Microbial production of poly(hydroxybutyrate) from C1 carbon sources. Applied Microbiology and Biotechnology, 97(4), 1407-1424. https://doi.org/10.1007/s00253-012-4649-0

Lazcano, O., Speights Jr, V. O., Strickler, J. G., Bilbao, J. E., Becker, J., & Diaz, J. (1993). Combined histochemical stains in the differential diagnosis of Cryptococcus neoformans. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc, 6(1), 80-84. https://europepmc.org/article/med/7678937

Downloads

Published

2025-03-18

How to Cite

Sohail, A., Ihsan, K., Khan, R. S., Ali, A., & Zia-ul Islam. (2025). Isolation, Identification, and Physiological Characterization of Indigenous Yeast Species Capable of Efficiently Utilizing Sugarcane Molasses as a Carbon Source. Indus Journal of Bioscience Research, 3(3), 265-274. https://doi.org/10.70749/ijbr.v3i3.860