Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform.

Cutaneotrichosporon curvatum and Yarrowia lipolytica can accumulate microbial oils using short-chain fatty acids (SCFA) as carbon sources. SCFAs-rich media often contain significant amounts of nitrogen that prevent high carbon:nitrogen (C:N) ratios necessary to boost lipid production. This work assessed the intrinsic ability of C. curvatum and Y. lipolytica to produce high amounts of microbial oils from these unusual carbon sources. Results demonstrated that minor differences in SCFA concentration (only 2 g/L) had a significant effect on yeast growth and lipid production. A C:N of 80 promoted yeast growth at all SCFA concentrations and favored SCFA consumption at 19 g/L SCFAs. The different SCFA uptake preferences in C. curvatum and Y. lipolytica highlighted the importance of considering the SCFA profile to select a suitable yeast strain for microbial oils production. At the most challenging SCFA concentration (19 g/L), 57.2% ±1.6% (w/w) and 78.4 ± 0.6% (w/w) lipid content were obtained in C. curvatum and Y. lipolytica, respectively. These values are among the highest reported for wild-type strains. To circumvent the challenges associated with media with high nitrogen content, this report also proved struvite precipitation as an effective method for increasing lipid production (from 17.9 ± 3.9% (w/w) to 41.9 ± 2.6% (w/w)) after nitrogen removal in food waste-derived media.

Slight variations in SCFA concentrations have a relevant effect on yeast lipid productionHigh nitrogen availability is crucial to promote cell growth at very high SCFA concentrationsC:N effect on cell growth and lipid production is specie-specific and may depend on yeast robustnessYeast strains have diverse SCFA preferences and differently metabolize these acidsStruvite precipitation effectively removes nitrogen from real digestates increasing C:N.

Phosphate limitation as crucial factor to enhance yeast lipid production from short-chain fatty acids.

Microbial lipids for chemical synthesis are commonly obtained from sugar-based substrates which in most cases is not economically viable. As a low-cost carbon source, short-chain fatty acids (SCFAs) that can be obtained from food wastes offer an interesting alternative for achieving an affordable lipid production process. In this study, SCFAs were employed to accumulate lipids using Yarrowia lipolytica ACA DC 50109. For this purpose, different amounts of SCFAs, sulfate, phosphate and carbon: phosphate ratios were used in both synthetic and real SCFAs-rich media. Although sulfate limitation did not increase lipid accumulation, phosphate limitation was proved to be an optimal strategy for increasing lipid content and lipid yields in both synthetic and real media, reaching a lipid productivity up to 8.95 g/L h. Remarkably, the highest lipid yield (0.30 g/g) was achieved under phosphate absence condition (0 g/L). This fact demonstrated the suitability of using low-phosphate concentrations to boost lipid production from SCFAs.

Key role of fluorescence quantum yield in Nile Red staining method for determining intracellular lipids in yeast strains.

BACKGROUND: Microbial lipids are found to be an interesting green alternative to expand available oil sources for the chemical industry. Yeasts are considered a promising platform for sustainable lipid production. Remarkably, some oleaginous yeasts have even shown the ability to grow and accumulate lipids using unusual carbon sources derived from organic wastes, such as volatile fatty acids. Recent research efforts have been focused on developing rapid and accurate fluorometric methods for the quantification of intracellular yeast lipids. Nevertheless, the current methods are often tedious and/or exhibit low reproducibility. RESULTS: This work evaluated the reliability of different fluorescence measurements (fluorescence intensity, total area and fluorescence quantum yield) using Nile Red as lipid dye in two yeast strains (Yarrowia lipolytica ACA-DC 50109 and Cutaneotrichosporon curvatum NRRL-Y-1511). Different standard curves were obtained for each yeast specie. Fermentation tests were carried with 6-month difference to evaluate the effect of the fluorometer lamp lifetime on lipid quantification. CONCLUSIONS: Fluorescence quantum yield presented the most consistent measurements along time and the closer estimations when compared with lipids obtained by conventional methods (extraction and gravimetrical determination). The need of using fluorescence quantum yield to estimate intracellular lipids, which is not the common trend in studies focused on microbial lipid production, was stressed. The information here provided will surely enable more accurate results comparison.