Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source

Abstract

Background Over the last years oleaginous yeasts have been studied for several energetic, oleochemical, medical and pharmaceutical purposes. However, only a small number of yeasts are known and have been deeply exploited. The search for new isolates with high oleaginous capacity becomes imperative, as well as the use of alternative and ecological carbon sources for yeast growth. Results In the present study a high-throughput screening comprising 366 distinct yeast isolates was performed by applying an optimised protocol based on two approaches: (I) yeast cultivation on solid medium using acetic acid as carbon source, (II) neutral lipid estimation by fluorimetry using the lipophilic dye Nile red. Conclusions Results showed that, with the proposed methodology, the oleaginous potential of yeasts with broad taxonomic diversity and variety of growth characteristics was discriminated. Furthermore, this work clearly demonstrated the association of the oleaginous yeast character to the strain level, contrarily to the species-level linkage, as usually stated.

Clavispora santaluciae f.a., sp. nov., a novel ascomycetous yeast species isolated from grapes

“…This capacity is due to the presence of amylolytic enzymes that are required for starch utilization. This fact highlights the commercial potential of these strains of Clavispora santaluciae to use alternative carbon sources as energy, with applications for example in the production of biomass or single cell oils with reduced costs [22].…”

Single Cell Oil Production by Oleaginous Yeasts Grown in Synthetic and Waste-Derived Volatile Fatty Acids

“…Moreover, oleaginous yeasts able to grow on a broad array of carbon sources increase their economic interest. For the present work, four yeast species were chosen, based on lipid production and biomass results, previously obtained in glucose and acetic acid media [ 8 ]: Apiotrichum brassicae V134, Candida tropicalis V139, Metschnikowia pulcherrima V213, and Pichia kudriavzevii V194. A. brassicae is a synonym of the already described oleaginous yeast Trichosporon brassicae [ 28 ]; however, exploitation of its oleaginous character is still scarce, despite the oil-bearing character of the Apiotrichum genus described in previous studies [ 29 , 30 ].…”

“…Yeast biomass was measured as OD 640 and by dry cell weight (DCW). Intracellular lipid accumulation was estimated by a fluorescence assay using the lipophilic dye Nile red (>98.0% Sigma Aldrich, Steinheim, Germany) and quantified as percentage of lipids by DCW (% w / w ), according to Miranda et al [ 8 ].…”

“…In 2014, 70 oleaginous yeast species were described as oil-bearing microorganisms [ 5 ]. In our previous work [ 8 ], we developed a screening method to rapidly detect oleaginous species, which allowed the species Millerozyma farinosa , Trigonopsis cantarellii , and Geotrichum candidum , for the first time, to be defined as oleaginous, and to confirm the oleaginous character of many others. Notwithstanding, it is worth mentioning that lipid content and profile may not be species-dependent, varying amongst strains of the same species, as reported in recent works [ 8 , 9 ].…”

Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

“…There are few high throughput methodologies for screening oleaginous microorganisms, mainly yeasts. The most used protocols are Kimura et al (2004) , modified by Sitepu et al (2019) , followed by recent publications with modifications proposed by Rostron and Lawrence (2017) ; Zhao et al (2019) , and Miranda et al (2020) . Our main objective was to evaluate the stability of Nile red fluorescence in different solvents using a protocol based on Sitepu et al (2019) and a microplate reader equipment.…”