Isolation and Identification of Oleaginous Yeasts from Soils at Bang Phra Reservoir, Chonburi Province
Abstract
Lipid from oleaginous yeasts could be exploited for several applications including the production of biodiesel which is an alternative energy that has been gaining much interest recently. This research aimed to isolate and identify potential oleaginous yeasts from soil samples collected at Bang Phra reservoir in Chonburi province. A total of 39 isolates of yeast were obtained and further screened for intracellular lipid production by Nile red fluorescence assay followed by Sudan Black B staining to observe lipid accumulation in yeast cells. The result showed that there were 8 yeast isolates; I2, J1, J2, J5, L1, L4, M2 and O3 that exhibited high level of intracellular lipid accumulation potential. Identification of yeasts based on nucleotide sequencing of ITS1-5.8S rDNA-ITS2 regions as well as phylogenetic analysis showed that I2, J5 and L1 were identified as Candida tropicalis, J1 and M2 were identified as Cyberlindnera sp., and J2 and O3 were identified as Schwanniomyces vanrijiae var. vanrijiae and L4 was identified as Schwanniomyces sp. These yeast strains belong to the same phylum (Ascomycota) which are divided into 2 families as following; Candida and Schwanniomyces are members of the family Debaryomycetaceae and Cyberlindnera is a member of the family Phaffomycetaceae. This research suggested that soil could be an interesting habitat of potential oleaginous yeasts and this work also provided fundamental and important knowledges for further studies aiming for useful applications. Keywords : microbial lipid ; oleaginous yeasts ; Candida ; Cyberlindnera ; SchwanniomycesReferences
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Aljohani, R., Samarasinghe, H., Ashu, T., & Xu, J. (2018). Diversity and relationships among strains of culturable
yeasts in agricultural soils in Cameroon. Scientific Reports, 8, 15687.
Amornrattanapan, P. & Thongthep, P. (2019). Isolation and screening of oleaginous yeasts capable of using
glycerol as a carbon source. Ramkhamhaeng Research Journal of Sciences and Technology, 22(2),
61-70.
Arous, F., Mechichi, T., Nasri1, M., & Aggelis, G. (2016). Fatty acid biosynthesis during the life cycle of
Debaryomyces etchellsii, Microbiology, 162, 1080-1090.
Ayadi, I., Belghith, H., Gargouri, A., & Guerfali, M. (2018). Screening of new oleaginous yeasts for single cell
oil production, hydrolytic potential exploitation and agro-industrial by-products valorization. Process
Safety and Environmental Protection, 119, 104–114.
Bastos, A. E. R., Moon, D. H., Rossi, A., Trevors, J. T., & Tsai, S. M. (2000). Salt-tolerant phenol degrading
microorganisms isolated from Amazonian soil samples. Archives of Microbiology, 174(5), 346-352.
Boontham, W., Limtong, S., Rosa, C. A., Lopes, M. R., Vital, M. J., & Srisuk, N. (2017). Cyberlindnera tropicalis
fa, sp. nov., a novel yeast isolated from tropical regions. International Journal of Systematic and Evolutionary Microbiology, 67(8), 2569-2573.
Botha, A. (2011). The importance and ecology of yeasts in soil. Soil Biology and Biochemistry, 43, 1-8.
Burdon, K. (1946). Fatty material in bacteria and fungi revealed by staining dried and fixed slide preparations. Journal of Bacteriology, 52(6), 665-678.
Chanchaichaovivat, A. (2015). Yeast and Yeast Technology. (1st ed.). Bangkok: Kao Thai Advertising &
Printing. (in Thai)
Groenewald, M., Lombard, L., de Vries, M., Lopez, A. G., Smith, M., & Crous, P. W. (2018). Diversity of yeast species from Dutch garden soil and the description of six novel Ascomycetes. FEMS Yeast Research, 18(7), 1-14.
Hernández-Chávez, M. J., Clavijo-Giraldo, D. M., Novák, Á., Lozoya-Pérez, N. E., Martínez-Álvarez, J. A.,
Salinas-Marín, R., Hernández, N. V., Martínez-Duncker, I., Gácser, A., & Mora-Montes, H. M.
(2019). Role of protein mannosylation in the Candida tropicalis-Host interaction. Frontiers in
Microbiology, 10, 2743.
Jape, A., Harsulkar, A., & Sapre, V. R. (2014). Modified Sudan Black B staining method for rapid screening of
oleaginous marine yeasts. International Journal of Current Microbiology and Sciences, 3(9), 41-46.
Kaewwichian, R., & Khamthaiklang, S. (2017). Yeast in mangrove forest soil from the central Thailand and its
ability in the degradation of starch, carboxymethylcellulose and xylan. Burapha Science Journal, 22(Special issue; The 9th National Science Reseach Conference), 411-422. (in Thai)
Kurtzman, C. P., Fell, J. W., & Boekhout, T. (2011). Gene sequence analyses and other DNA-based methods
for yeast species recognition. In C. P. Kurtzman, J. W. Fell, & T. Boekhout. (Eds), The Yeasts; A taxonomic study. (pp. 137-144). Elsevier.
Kurtzman, C. P., Robnett, C. J. and 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, 939-954.
Leesing, R., & Nontaso, Ng. (2011). Isolation and cultivation of oleaginous yeast for microbial oil production.
KKU Research Journal, 16(2), 112-126. (in Thai)
Li, Q., Du, W., & Liu, D. (2008). Perspectives of microbial oils for biodiesel production. Applied Microbiology
and Biotechnology, 80, 749-756.
Limtong, S. (2006). Yeast: Diversity and Biotechnology. (1st ed.). Bangkok: Kasetsart University Press. (in Thai)
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(2016). Yeast communities of Formica aquilonia colonies. Microbiology, 85(1), 124-129.
Miranda, C., Bettencourt, S., Pozdniakova, T., Pereira, J., Sampaio, P., Franco-Duarte, R., & Pais, C. (2020). Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts
using acetic acid as carbon source. BMC Microbiology, 20(1), 60.
Ochsenreither, K., Glück C., Stressler T., Fischer L., & Syldatk C. (2016). Production strategies and applications of
microbial single cell oils. Frontiers in Microbiology, 7, 1539.
Pan, L. X., Yang, D. F., Shao, L., Li, W., Chen, G. G., & Liang, Z. Q. (2009). Isolation of the oleaginous yeasts
from the soil and studies of their lipid producing capacities. Food Technology and Biotechnology, 47, 215-220.
Pawar, P. P., Odaneth, A. A., Vadgama, R. N., & Lali, A. M. (2019). Simultaneous lipid biosynthesis and recovery
for oleaginous yeast Yarrowia lipolytica. Biotechnology for Biofuels, 12, 237.
Pongcharoen, P., Chawneua, J., & Tawong, W. (2018). High temperature alcoholic fermentation by new
thermotolerant yeast strains Pichia kudriavzevii isolated from sugarcane field soil. Agriculture and Natural Resources, 52, 511-518.
Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production.
Biochimie, 86(11), 807-815.
Ratledge, C., & Cohen, Z. (2008). Microbial and algal oils: Do they have a future for biodiesel or as commodity
oils? Lipid Technology, 20, 155-160.
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. (3rd ed.). New York:
Cold Spring Harbor Laboratory Press.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A. & Chen, W. (2012).
Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. PNAS, 109(16), 6241- 6246.
Sitepu, I. R., Garay, L. A., Sestric, R., Levin, D., Block, D. E., German, J. B., & Boundy-Mills, K. L. (2014).
Oleaginous yeasts for biodiesel: Current and future trends in biology and production. Biotechnology Advances, 32, 1336-1360.
Sitepu, I. R., Ignatia, L., Franz, A. K., Wong, D. M., Faulina, S. A., Tsui, M., Kanti, A., & Mills, K. B. (2012). An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. Journal of Microbiological Methods, 91, 321-328.
Suzuki, M., & Kurtzman, C. P. (2011). Schwanniomyces Klöcker emend. M. Suzuki & Kurtzman (2010). In C. P.
Kurtzman, J. W. Fell, & T. Boekhout. (Eds.), The Yeasts; A Taxonomic Study. (pp. 785-794). London: Elsevier.
White, T. J., Bruns, T., Lee, S. J. W. T., & Taylor, J. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In M. A., Innis, D. H., Gelfand, J. J., Sninsky, & T. J., White. (Eds.), PCR protocols: A Guide to Methods and Applications. (pp. 315 - 322). New York: Academic Press.
Yaguchi, A., Franaszek, N., O’Neill, K., Lee, S., Sitepu, I., Boundy‑Mills, K. & Blenner, M. (2020). Identification
of oleaginous yeasts that metabolize aromatic compounds. Journal of Industrial Microbiology and Biotechnology, https://doi.org/10.1007/s10295-020-02269-5.
Yurkov, A. M. (2018). Yeasts of the soil-obscure but precious. Yeast, 35(5), 369-378.
Yurkov, A. M., Kemler, M., & Begerow, D. (2012). Assessment of yeast diversity in soils under different
management regimes. Fungal Ecology, 5(1), 24-35.
Zhu, L. Y., Zong M. H., & Wu, H. (2008). Efficient lipid production with Trichosporon fermentans and its use for
biodiesel preparation. Bioresource Technology, 99, 7881-7885.
Zuza-Alves, D. L., Silva-Rocha, W. P., & Chaves, G. M. (2017). An update on Candida tropicalis based
on basic and clinical approaches. Frontiers in Microbiology, 8, 1927.
Applied Microbiology and Biotechnology, 90, 1219-1227.
Aljohani, R., Samarasinghe, H., Ashu, T., & Xu, J. (2018). Diversity and relationships among strains of culturable
yeasts in agricultural soils in Cameroon. Scientific Reports, 8, 15687.
Amornrattanapan, P. & Thongthep, P. (2019). Isolation and screening of oleaginous yeasts capable of using
glycerol as a carbon source. Ramkhamhaeng Research Journal of Sciences and Technology, 22(2),
61-70.
Arous, F., Mechichi, T., Nasri1, M., & Aggelis, G. (2016). Fatty acid biosynthesis during the life cycle of
Debaryomyces etchellsii, Microbiology, 162, 1080-1090.
Ayadi, I., Belghith, H., Gargouri, A., & Guerfali, M. (2018). Screening of new oleaginous yeasts for single cell
oil production, hydrolytic potential exploitation and agro-industrial by-products valorization. Process
Safety and Environmental Protection, 119, 104–114.
Bastos, A. E. R., Moon, D. H., Rossi, A., Trevors, J. T., & Tsai, S. M. (2000). Salt-tolerant phenol degrading
microorganisms isolated from Amazonian soil samples. Archives of Microbiology, 174(5), 346-352.
Boontham, W., Limtong, S., Rosa, C. A., Lopes, M. R., Vital, M. J., & Srisuk, N. (2017). Cyberlindnera tropicalis
fa, sp. nov., a novel yeast isolated from tropical regions. International Journal of Systematic and Evolutionary Microbiology, 67(8), 2569-2573.
Botha, A. (2011). The importance and ecology of yeasts in soil. Soil Biology and Biochemistry, 43, 1-8.
Burdon, K. (1946). Fatty material in bacteria and fungi revealed by staining dried and fixed slide preparations. Journal of Bacteriology, 52(6), 665-678.
Chanchaichaovivat, A. (2015). Yeast and Yeast Technology. (1st ed.). Bangkok: Kao Thai Advertising &
Printing. (in Thai)
Groenewald, M., Lombard, L., de Vries, M., Lopez, A. G., Smith, M., & Crous, P. W. (2018). Diversity of yeast species from Dutch garden soil and the description of six novel Ascomycetes. FEMS Yeast Research, 18(7), 1-14.
Hernández-Chávez, M. J., Clavijo-Giraldo, D. M., Novák, Á., Lozoya-Pérez, N. E., Martínez-Álvarez, J. A.,
Salinas-Marín, R., Hernández, N. V., Martínez-Duncker, I., Gácser, A., & Mora-Montes, H. M.
(2019). Role of protein mannosylation in the Candida tropicalis-Host interaction. Frontiers in
Microbiology, 10, 2743.
Jape, A., Harsulkar, A., & Sapre, V. R. (2014). Modified Sudan Black B staining method for rapid screening of
oleaginous marine yeasts. International Journal of Current Microbiology and Sciences, 3(9), 41-46.
Kaewwichian, R., & Khamthaiklang, S. (2017). Yeast in mangrove forest soil from the central Thailand and its
ability in the degradation of starch, carboxymethylcellulose and xylan. Burapha Science Journal, 22(Special issue; The 9th National Science Reseach Conference), 411-422. (in Thai)
Kurtzman, C. P., Fell, J. W., & Boekhout, T. (2011). Gene sequence analyses and other DNA-based methods
for yeast species recognition. In C. P. Kurtzman, J. W. Fell, & T. Boekhout. (Eds), The Yeasts; A taxonomic study. (pp. 137-144). Elsevier.
Kurtzman, C. P., Robnett, C. J. and 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, 939-954.
Leesing, R., & Nontaso, Ng. (2011). Isolation and cultivation of oleaginous yeast for microbial oil production.
KKU Research Journal, 16(2), 112-126. (in Thai)
Li, Q., Du, W., & Liu, D. (2008). Perspectives of microbial oils for biodiesel production. Applied Microbiology
and Biotechnology, 80, 749-756.
Limtong, S. (2006). Yeast: Diversity and Biotechnology. (1st ed.). Bangkok: Kasetsart University Press. (in Thai)
Maksimova, I. A., Glushakova, A. M., Kachalkin, A. V., Chernov, I. Y., Panteleeva, S. N., & Reznikova, Z. I.
(2016). Yeast communities of Formica aquilonia colonies. Microbiology, 85(1), 124-129.
Miranda, C., Bettencourt, S., Pozdniakova, T., Pereira, J., Sampaio, P., Franco-Duarte, R., & Pais, C. (2020). Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts
using acetic acid as carbon source. BMC Microbiology, 20(1), 60.
Ochsenreither, K., Glück C., Stressler T., Fischer L., & Syldatk C. (2016). Production strategies and applications of
microbial single cell oils. Frontiers in Microbiology, 7, 1539.
Pan, L. X., Yang, D. F., Shao, L., Li, W., Chen, G. G., & Liang, Z. Q. (2009). Isolation of the oleaginous yeasts
from the soil and studies of their lipid producing capacities. Food Technology and Biotechnology, 47, 215-220.
Pawar, P. P., Odaneth, A. A., Vadgama, R. N., & Lali, A. M. (2019). Simultaneous lipid biosynthesis and recovery
for oleaginous yeast Yarrowia lipolytica. Biotechnology for Biofuels, 12, 237.
Pongcharoen, P., Chawneua, J., & Tawong, W. (2018). High temperature alcoholic fermentation by new
thermotolerant yeast strains Pichia kudriavzevii isolated from sugarcane field soil. Agriculture and Natural Resources, 52, 511-518.
Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production.
Biochimie, 86(11), 807-815.
Ratledge, C., & Cohen, Z. (2008). Microbial and algal oils: Do they have a future for biodiesel or as commodity
oils? Lipid Technology, 20, 155-160.
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. (3rd ed.). New York:
Cold Spring Harbor Laboratory Press.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A. & Chen, W. (2012).
Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. PNAS, 109(16), 6241- 6246.
Sitepu, I. R., Garay, L. A., Sestric, R., Levin, D., Block, D. E., German, J. B., & Boundy-Mills, K. L. (2014).
Oleaginous yeasts for biodiesel: Current and future trends in biology and production. Biotechnology Advances, 32, 1336-1360.
Sitepu, I. R., Ignatia, L., Franz, A. K., Wong, D. M., Faulina, S. A., Tsui, M., Kanti, A., & Mills, K. B. (2012). An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. Journal of Microbiological Methods, 91, 321-328.
Suzuki, M., & Kurtzman, C. P. (2011). Schwanniomyces Klöcker emend. M. Suzuki & Kurtzman (2010). In C. P.
Kurtzman, J. W. Fell, & T. Boekhout. (Eds.), The Yeasts; A Taxonomic Study. (pp. 785-794). London: Elsevier.
White, T. J., Bruns, T., Lee, S. J. W. T., & Taylor, J. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In M. A., Innis, D. H., Gelfand, J. J., Sninsky, & T. J., White. (Eds.), PCR protocols: A Guide to Methods and Applications. (pp. 315 - 322). New York: Academic Press.
Yaguchi, A., Franaszek, N., O’Neill, K., Lee, S., Sitepu, I., Boundy‑Mills, K. & Blenner, M. (2020). Identification
of oleaginous yeasts that metabolize aromatic compounds. Journal of Industrial Microbiology and Biotechnology, https://doi.org/10.1007/s10295-020-02269-5.
Yurkov, A. M. (2018). Yeasts of the soil-obscure but precious. Yeast, 35(5), 369-378.
Yurkov, A. M., Kemler, M., & Begerow, D. (2012). Assessment of yeast diversity in soils under different
management regimes. Fungal Ecology, 5(1), 24-35.
Zhu, L. Y., Zong M. H., & Wu, H. (2008). Efficient lipid production with Trichosporon fermentans and its use for
biodiesel preparation. Bioresource Technology, 99, 7881-7885.
Zuza-Alves, D. L., Silva-Rocha, W. P., & Chaves, G. M. (2017). An update on Candida tropicalis based
on basic and clinical approaches. Frontiers in Microbiology, 8, 1927.
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2021-05-05
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