A new yeast strain for valorisation of vinasse, a rum distillery waste product

Abstract

Background: Waste valorisation refers to processes of reusing or recycling waste materials to create valuable products. In the Rum distillery industry, the primary waste byproducts include bagasse, a solid waste made up of sugar cane residue and vinasse, a thick and acidic liquid. Although vinasse has been repurposed in agricultural fields, it has also contributed to both soil and ocean pollution. Despite several potential solutions having been suggested, an effective and environmentally safe use for vinasse has yet to be found.

Results: The valorisation of vinasse for biofuel production was explored by assessing its potential as a growth medium for lipid production by non-conventional yeasts. The oleaginous yeast strain Yarrowia lipolytica, known for its lipid production capabilities, was initially tested on vinasse but required further adaptation and optimization. To circumvent this, we isolated a novel yeast strain from old vinasse waste, named V1, which demonstrated strong growth potential. The growth conditions of V1, including temperature and acidity, were characterized, and its potential for bioengineering was evaluated. This strain exhibited resistance to highly acidic pH levels and higher temperatures when cultivated on YPV, an artificial laboratory medium designed to mimic the acidity and glycerol content of vinasse. Whole genome sequencing (WGS) identified V1 as Pichia kudriavzevii. We demonstrated that V1 could be transformed with Yarrowia lipolytica vectors using the classical yeast heat shock protocol, thus enabling potential genetic engineering. Finally, lipid content in V1 was analysed in different conditions, confirming the strain's potential for biofuel production.

Conclusions: Pichia kudriavzevii is not a traditional yeast, but its ability to adapt and grow under extreme pH and higher temperature conditions makes it a promising candidate for rum industry waste management applications. This strain could potentially be utilised to convert vinasse and other food waste products into valuable biofuels. Although further research is required to engineer and optimize this novel strain for vinasse cultivation, our findings highlight its great potential as a micro-factory in rum-producing regions and high locations, where agricultural waste is in need of valorisation solutions.

Keywords: Pichia kudriavzevii; Yarrowia lipolytica; Biofuel; Rum distillery; Vinasse.

Fig. 1

Liquid growth curve of yeast strains on YPV at either 28 ℃ or 37 ℃. V1 is Pichia kudriavzevii isolated from vinasse, and PK is commercial Pichia kudriavzevii

Fig. 2

Temperature resistance of yeast strains. All yeast strains were grown at 28 ℃ in YPD media then serial diluted and spotted on either YPD or YPV media and incubated for 24 h and 48 h at various temperatures. V1 strain is the only strain growing at temperature above 40 ℃. V1: Pichia kudriavzevii from this study PK: commercial Pichia kudriavzevii, YL: Yarrowia lipolytica, Sc: Saccharomyces cerevisiae

Fig. 3

V1 is resistant to acidic pH. All yeast strains were grown at 28 ℃ in YPD then serial diluted and spotted on either YPD or YPV media and incubated for 24 h and 48 h at various pH. V1: Pichia kudriavzevii from this study PK: commercial Pichia kudriavzevii, YL: Yarrowia lipolytica, Sc: Saccharomyces cerevisiae

Fig. 4

V1 is sensitive to hygromycin and Nourseothricine. Various dilution of V1 and PK were spotted on YPD containing various antibiotics (300ug/ml) and growth was observed after 24 h or 48 h at 28℃. V1: Pichia kudriavzevii from this study PK: commercial Pichia kudriavzevii. Dilution of yeast culture before plating is mentioned

Fig. 5

Yeast transformation results with Y. lipolytica plasmid. A Wild-type Y. lipolytica was transformed with a plasmid containing a cassette of genes for the production of β-carotene [28] and selected on YPD with hygromycin. Colonies were then streaked onto YPD without antibiotic next to an untransformed control to show the colour change. B Growth of V1 (P. kudriavzevii isolated from vinasse) untransformed (V1) or transformed with the same Y. lipolytica plasmid (V1T) as in (A), and selected on two concentrations of hygromycin, showing V1T slow growth at lower hygromycin concentration but without colour change

Fig. 6

GO enrichment in genes involved in V1 lipid metabolism. The figure and table showing the enzymes from V1 containing high levels of SNPs involved in the free fatty acids (FFA) synthesis pathway. SNPs are enriched in genes in green. The lipid metabolism of P. kudriavzevii is not known; therefore, the left panel is adapted from the lipid biosynthesis pathway in Y. lipolytica (adapted from Bredeweg et al. [56]). WGS can be found in supplemental Table S2

Fig. 7

Principal component analysis and distribution of normalised abundances of features in V1 samples compared to Y. lipolytica samples, both grown at 28 °C. A, B Progenesis analysis from negative ion electrospray data, while (C) and (D) show the analysis from positive ion electrospray data. A, C PCA plots showing separation of the data sets; the triplicates for V1 at 28 °C are blue circles and triplicates for YL at 28 °C are purple circles. All the features are indicated by the grey cloud and features with power > 0.95 in red. B, D Distribution of normalized abundance between the 3 replicates in each of the two data sets (triplicates for V1 28 °C in the blue box and triplicates for YL 28 °C in the purple box)

Fig. 8

Principal component analysis and distribution of normalised abundances of features in V1 samples grown at 28 °C compared to 37 °C. A, B Progenesis analysis from negative ion electrospray data, while (C) and (D) show the analysis from positive ion electrospray data. A, C PCA plots showing separation of the data sets; the triplicates for V1 at 28 °C are blue circles and triplicates for V1 at 37 °C are purple circles. All the features are indicated by the grey cloud and features with power > 0.95 in red. B, D Distribution of normalized abundance between the 3 replicates in each of the two data sets (triplicates for V1 28 °C in the blue box and triplicates for V1 37 °C in the purple box)

Fig. 9

Relative quantification of free fatty acid (FFA) content in YL, V1 28 ℃, V1 37 ℃. A Each free fatty acid calculated as a percentage of all the lipid classes (FFAs, Lysolipids, phospholipids and triacylglycerols) containing that specific fatty acyl chain, determined from the MS² extracted ion chromatogram in negative ion mode. B Each free fatty acid calculated as a percentage of all the FFAs analysed in each sample, determined from the data used to plot (A). For statistical analysis p < 0.01 are represented by two stars. p < 0.05 are represented by one star