Culturable Yeast Diversity Associated with Industrial Cultures of the Microalga Microchloropsis gaditana and Their Ability to Produce Lipids and Biosurfactants

Abstract

The marine oleaginous microalga Microchloropsis gaditana (formerly Nannochloropsis gaditana) exhibits a high capacity to thrive in a broad range of environmental conditions, being predominantly utilized as feed in aquaculture. This article reports the characterization of the culturable yeast population present during the scale-up process of M. gaditana cultivation at Necton S.A. facilities, from 5 L flasks until tubular photobioreactors. The 146 yeast isolates obtained, molecularly identified based on D1/D2 and ITS nucleotide sequences, belong to the species Rhodotorula diobovata, R. mucilaginosa, R. taiwanensis, R. sphaerocarpa, Vishniacozyma carnescens, Moesziomyces aphidis, and Meyerozyma guilliermondii. The yeast abundance was found to increase throughout upscaling stages. The yeast populations isolated from microalgal cultures and water samples share phylogenetically close isolates, indicating a possible common source. The impressive high percentage of red yeasts isolated (90%) is consistent with the recognized role of carotenoid pigments in yeast photoprotection. Sixty yeast isolates were tested for lipid (Nile Red staining) and biosurfactant (oil drop dispersion and emulsification index) production. Results revealed that these capacities are common features. Microbial lipids and biosurfactants have promising biotechnological applications. Moreover, biosurfactants can fulfill various physiological roles and provide advantages in natural environments contributing to the promising use of yeasts as probiotics in microalgae production.

Keywords: Basidiomycota; Rhodotorula; biotechnological applications; microalgae cultivation; yeast isolation; yeasts.

Figure 1

Concentration of culturable yeast cells in the analyzed samples. The concentration, calculated based on colony forming units per milliliter of the original sample (CFU/mL), was obtained for each microalgal culture sample (A) and for each water sample (B). CFUs/mL were calculated by multiplying/dividing the number of yeast colonies obtained from the original sample (direct sample, blue) or from the concentrated sample (concentrated sample, black), by the dilution/concentration factor, whenever necessary. The stars indicate the samples from which yeast colonies were obtained following an enrichment culture step.

Figure 2

Phylogenetic analysis of yeast isolates obtained in this study (one isolate per species per sample). The taxonomic assignment phylogenetic was based on the alignment of sequences of the D1/D2 domain of the 28S rDNA region, inferred by means of the maximum likelihood method and Kimura 2-parameter model. Sequences from the type strains of the different yeast species were included. Isolates obtained from water samples are indicated in blue. The scale bar indicates the number of expected substitutions per site. The numbers provided at the branches are frequencies in percentage with which a given branch appeared in 500 bootstrap replications.

Figure 3

Schematic representation of Microchloropsis gaditana production flow. The samples (Nec_) considered in this study are indicated, as well as the collection date within brackets. Water samples are indicated inside blue squares. The reactor from which the samples were collected is in bold. The small colored squares next to each sample indicate the species isolated from the sample (differentiated by color) and the number of isolates retained from each sample (number within the square). The dates of the passages between the reactors are underlined in dark blue. FLF—Five Liter Flasks, CLM—Column, FP—Flat Panel, PBR—Photobioreactor.

Figure 4

Lipid and biosurfactant production by 60 yeast isolates of the different species retrieved in this study. Lipid production (A) was assessed by Nile Red staining and is indicated in relative fluorescence units (RFU). Biosurfactant production was assessed through the emulsification index (B) and the oil displacement method (C). The chosen isolates were cultivated and the culture tested as described in the Materials and Methods section. For the biosurfactant production assays the positive control (+) was a solution of 1% SDS (w/v) and the negative control (−) was the growth medium. Three reference strains, R. toruloides IST536, R. toruloides IST536 MM15 and R. mucilaginosa IST390 were also used as reference strains for lipid accumulation. The data shown represent the average of three independent experiments, and the error bars indicate standard deviation.