Introduction

Yeasts represent valuable source for the production of various industrial products, such as vitamins, enzymes, mono- and polysaccharides, citric acid, alcohols, carotenoids, and lipids (Satyanarayana and Kunze 2009; El-Baz et al. 2011, 2016; Johansen et al. 2019). Polyunsaturated fatty acids (PUFAs) are components of biological membranes; they are essential for membrane structure and function (Stokes et al. 2020). PUFAs serve as precursors for bioactive molecules, like eicosanoids, in mammals, acting as anti-inflammatory agents, such as leukotrienes, prostaglandins, and thromboxane, to mediate inflammations, blood pressure, neurotransmission, and cholesterol metabolism (Funk 2001; Parolini 2020). PUFAs mainly promote many immune functions; thus, they modulate the risk of various diseases (Sorour et al. 2012a, b; Stokes et al. 2020). Moreover, many in vivo studies have revealed the ability of marine ω-3 PUFAs [eicosapentaenoic (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6)] to modulate obesity, Alzheimer’s disease, and Sclerosis progression (Parolini 2020). As a result, the production of PUFAs had attracted great attention because of their health-related benefits (Connor and Connor 2007). In addition, various scientific reports proposed both ω-3 and ω-6 FAs to be included in a balanced diet (Bellou et al. 2016). Therefore, ω-PUFAs are currently sold as supplements, even added to enrich baby’s formulas (Stokes et al. 2020).

On one hand, fish is a source of many bioactive molecules, such as proteins, omega-3 PUFAs, minerals, and vitamins, it is precious, but limited resource facing many problems (Xu et al. 2020). The crucial problem of fish oil is its unsustainability due to the decline of fish stocks, as well as the unpleasant odour, low stability, and the presence of contaminants, such as heavy metals (Bellou et al. 2016). Therefore, the ever-growing population and the limited natural PUFA sources increased research toward finding alternative sustainable resources for PUFA production. In this respect, the marine environment has always been attractive, because of its diversity of microorganisms that can be exploited for various valuable compounds (Gupta et al. 2012; El-Baz et al. 2018).

On the other hand, some microorganisms in the cold saline environment can produce PUFAs, to provide fluidity for their cell membrane, and thus help in their protection and overcome the negative effect of cold (Chintalapati et al. 2004). Therefore, the microbial source plays an important role in supplying PUFAs to meet the market demands being environmentally safe, and more sustainable source (Qiu et al. 2020). Generally, psychrophiles and psychrotrophs can adjust their enzymes and cellular membranes to be metabolically active in the cold environments. The optimum temperature for psychrophilic yeasts usually occurs at 15 °C or lower, and up to 25 °C, but still capable of growing at 0 °C or below (Satyanarayana and Kunze 2009). They can decrease their membrane fluidity through increasing the percentage of monounsaturated FAs and PUFAs (Skerratt et al. 2002; Rossi et al. 2009). In addition, oleaginous microorganisms can grow on many cost-effective substrates, with high growth rates, short life cycle, and easy scale-up (Qiu et al. 2020). However, few microbial oils containing PUFAs are commercially available, such as Arachidonic acid (ARA) by Mortierella alpina fungus (CABIO), gamma-Linolenic acid by Mucor circinelloides fungus (J. and E. Sturge), and EPA by Yarrowia lipolytica yeast (E.I. Du Pont) (Bellou et al. 2016; Galán et al. 2020). Commercial production of microbial oils is mainly restricted to filamentous fungi, yeasts, and microalgae. However, oleaginous fungi belonging to genera Mucor, and Mortierella, can produce some PUFA, such as ɤ-linolenic acid, DHA, EPA, and ARA (Papanikolaou and Aggelis 2019), as well as accumulate high quantities of lipids up to 70% of their weight. However, the major problem lies in the difficulty of their cultivation in submerged cultures where they show rheological problems, and different morphological forms depending on the medium/cultivation conditions, with low lipids accumulations (Troiano et al. 2020). On the other hand, oleaginous yeasts represent fascinating microbial factories, since these heterotrophic microorganisms are able to grow rapidly and accumulate high levels of lipids on variety of raw substrates. Their easy manipulation in fermenters makes them good candidates for bio-refinery practice as compared to other microorganisms. Therefore, studying naturally occurring marine PUFAs-producers can be very promising for new biotechnological applications. In the current study, isolation, screening, and identification of new marine PUFAs-producers were investigated. The effect of incubation temperature, glucose concentration on the biomass, lipids accumulation, and the degree of FAs unsaturation, using the two isolated marine yeast (R. mucilaginosa and L. elongisporus) were studied. The selection of optimum conditions to maximize the biomass and PUFAs production was also determined.

Materials and methods

Chemicals and media

Yeast extract (Techno Pharmchem, India), dextrose (Alamia company for chemicals, Egypt), peptone, agar, and vanillin (Lobal Chemie, India), Chloroform (Fisher Scientific, UK), Nile-Red (Aldrich Chemicals-Milwaukee, USA), dimethyl sulfoxide (DMSO), methyl alcohol (El-Nasr Pharmaceutical Chemicals Co., Egypt), and phosphoric acid (El-Goumhouria Co., Egypt) were used in this study. All other chemicals and reagents were of analytically reagent grade. Yeast Peptone Dextrose (YPD) was used for yeast isolation, propagation, and maintenance (Yazawa 1996) with the following composition (%): yeast extract 1, peptone 2, glucose 2 (pH 6.0). Basal Broth Medium (BBM) was used for lipids production (Li et al. 2010) with the following composition (%): KH2PO4 0.7, Na2HPO4 0.25, MgSO4.7H2O 0.15, CaCl2 0.015, FeCl3.7H2O 0.015, ZnSO4.7H2O 0.002, (NH4)2SO4 0.05, yeast extract 0.05, and glucose 2.0 (pH 6.0). Basal Defatted Medium (BDM) was used as a minimal medium for lipids production (Gupta et al. 2012) with the following composition (g/L): glucose 30, yeast extract 10, NaCl 30, KCl 0.7, MgCl2 10.8, MgSO4 5.4, and CaCl2 1.0 (pH 5.6). Dalmau plate culture on corn meal agar––containing (g/L) corn meal infusion 2, agar 15, and 7 mL of Tween 80––was used for the identification of yeast (Yarrow 1998).

Sampling and isolation of psychrotrophs

Newly killed fish (Epinephelus areolatus), Epinephelus aeneus, and shrimp samples were collected from the Red and Mediterranean Sea, located at 2° N 38° E/22° N 38° E Coordinates: 22° N 38° E/22° N 38° E, and 35° N  18° E/35° N 18° E Coordinates: 35° N  18° E/35° N 18° E, respectively, Egypt, between the 18th of December and 13th of January, (2014–2015). All Samples were placed in an icebox and used within 24 h (Fig. 1). Psychrophilics were isolated from fresh deep-sea samples, collected in Niskin bottles at a depth of 2 m; the intestine and gills of the samples were aseptically removed, homogenized in 0.9% NaCl, serially diluted, and plated on YPD medium. All plates were incubated in the dark at 5, 15, and 28 ºC for 20–30 days. Pure developed single colonies were obtained by conventional streak plate technique, kept at 4 ºC on slants, and subcultured twice a month. All yeast cultures were routinely stored in glycerol solution (20% v/v) at − 80 °C.

Fig. 1
figure 1

Red Sea (Spotted grouper) fish (A), Mediterranean Sea (White grouper) fish (B), Mediterranean Sea shrimps (C); TTC test for yeast isolates showing different intensity of red color of formazan (D)

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Macroscopic and microscopic identification

Yeast isolates were identified based on their colony size, edge, color, etc., developed on agar media, and their shape under the light microscope. Yeast isolates were predominately identified to the genus level according to their macroscopical and microscopical morphology (Yarrow 1998).

MALDI-TOF/MS Biotyper® identification of selected yeast isolates

Identification was carried out using matrix-assisted laser desorption/ionization time of flight/mass spectrophotometry (MALDI-TOF/MS) Biotyper® (Bruker) (Wieser et al. 2012). 24 h-old yeast culture was mixed with 1 μL of matrix solution, and placed on the steel target plate to co-crystallize the sample. The loaded target plate was placed into the machine, and samples were hit by short laser pulses of MS spectrometer. The microbial sample and the matrix are vaporized by the laser’s energy which ionize the ribosomal proteins of microorganism. The TOF of sample to reach the MS detector is precisely measured, and the ionization degree with the protein’s molecular mass generates their distinct TOF. Based on TOF information, a characteristic spectrum is recorded, which represents a fingerprint for each sample, that is specific for a given species. The computer software displays the identification results after comparison of the generated spectrum with the stored database (Wieser et al. 2012).

Rapid screening of Δ5-desaturase activity using TTC assay

YPD broth media were inoculated with pure single colony of each yeast isolate and were incubated at 15 °C and 28 °C for 7 and 5 days, respectively. Triphenyltetrazolium Chloride (TTC) (0.1% w/v) was added to the culture broth (1:1, v/v), and the mixtures were then incubated for an additional 1 h at 15 °C. The formation of red color of the reduced form (formazan) represents a positive result (Ryan et al. 2010).

Production of lipids

Seed cultures were prepared for the two selected yeast isolates and the two control yeast (Candida lipolytica as positive PUFAs producer, and Saccharomyces boulardii as a negative control). Yeast were inoculated individually into 20 mL of YPD broth and incubated at 15 °C and 28 °C for 72 h. The seed cultures were used to inoculate 180 mL of BBM medium in 500 mL Erlenmeyer flasks, incubated at 15 °C in a rotary shaking incubator (New Brunswick, CA) at 150 rpm for 15 days until the early stage of stationary phase. This medium was used as screening medium and was compared with YPD medium for its ability to stimulate PUFAs production by the two selected yeast isolates. Yeast cells were collected by centrifugation at 5000 × g at 4 °C, washed three times with sterile saline solution, and dried at 80 °C until constant weight. Total lipids were extracted based on Axelsson and Gentili (2014) method, and the lipids content was calculated using the following formula:

$${\text{Lipids content}}\;(\% ) = \frac{{\left[ {{\text{Wt}}{.}\;{\text{of}}\;{\text{tube}}\;{\text{with}}\;{\text{lipids}}\;{\text{after}}\;{\text{extraction}}\;{\text{(g)}} - {\text{Wt}}{.}\;{\text{of}}\;{\text{empty}}\;{\text{tube}}\;{\text{(g)}}} \right]}}{{{\text{Wt}}{.}\;{\text{of}}\;{\text{sample}}\;{\text{(g)}}}}$$

Lipids estimation in intact yeast cells using Sulfo-Phospho-Vanillin (SPV) assay

Lipids were measured using the modified SPV assay as described by Cheng et al. (2011) and Mishra et al. (2014). Pre-washed yeast cells (10 mg/mL) was transferred to 96-well microplate at 10, 20, 30, and 40 μL aliquots, further diluted to 50 μL with distilled water. 100 μL of sulfuric acid was added, mixed intensively by re-pipetting, and incubated at 90 °C for 20 min. The reaction mixtures were rapidly cooled in ice, and initial pre-vanillin background absorbance was measured spectrophotometry at λ570. SPV reagent (100 μL of 0.2 mg vanillin/mL, 17% phosphoric acid) was added, incubated at 25 °C for 10 min in the dark, and a post-vanillin absorbance was determined. Final SPV response of samples was defined as the difference between final post-vanillin and initial-vanillin absorbance, measured at λ570. Fish oil gelatin capsules (1000 mg, ω-3: 18%EPA, 12%DHA), commercial Flax seed oil (52.9% LNA), and Coconut oil (85.5% saturated FAs) were used as the standard lipids.

Lipids detection using Nile-Red fluorescent dye

Nile-Red solution was freshly prepared by dissolving 0.1 mg of Nile-Red dye in 1 mL of acetone (Kimura et al. 2004) and stored away from light at 4 °C. 1 mL aliquots of the yeast samples were centrifuged for 5 min at 2000 rpm, and cells were re-suspended in 1 mL of 10 mM PBS buffer (pH 7.4). Yeast suspensions (20 μL) were mixed with 400 μL of DMSO (25%), and smears were prepared by spreading 10 μL aliquots of the final cell suspension on a glass slide, then air-dried to fix the yeast cells. 10 μL of Nile-Red solution was added to each smear and kept for 5 min at room temperature. Finally, the excess of Nile-Red dye was washed out using PBS buffer, then examined using a fluorescence light microscope (Leica DMi8 S-platform, Germany), 100 × objective lens at an excitation λ450–500 nm and emission λ528 nm. Lipids were observed as yellow-golden droplets, whose surface area was visually estimated in relation to their cell surface area (Wang et al. 2017).

Extraction of total lipids, FAs methylation, and GLC analysis

Total lipids were extracted as described by Axelsson and Gentili (2014), where 230–300 mg of yeast biomass was extracted by 10 mL solvent (Chloroform:Methanol 2:1, v/v), shaken vigorously for few seconds; then 0.73% solvent:saline solution (Chloroform:Methanol:Saline 2:1:0.8, v/v/v) was added. For FAs methylation, 20 mg of freeze-dried cells were suspended in 2 mL methanolic-HCl (5%) and heated at 70 °C using water bath for 2 h in sealed glass tubes. Tubes were cooled at 25 °C for 30 min; then 1 mL double-distilled water was added and vortexed. To extract the methylated fatty acids (MEFAs), 1 mL hexane was added, vigorously vortexed, and the upper layer was transferred into clean glass vials, dried using nitrogen stream, and stored at − 20 °C. FAs was analyzed at the Regional Center for Food and Feed, Cairo, Egypt, according to Jostensen and Landfald (1997) method. FAs composition was determined using PerkinElmer (Waltham, MA) Clarus-580 GLC equipped with an FID, and an HP88 capillary column (30 m × 0.25 mm i.d, 0.20 μm film thickness). GLC-461 (NuChek Prep, Inc., Elysian, Minnesota, USA) was used as a reference standard to allow the determination of C12–C24 FAs. Standard TAG (C17:0) was included as internal standard. Helium was used as a carrier gas with a flow rate of 15 mL/min. The program conditions were held at 100 °C for 5 min; temperature was raised from 100 to 220 °C at 10 °C/min increment rate, then held at 220 °C for 15 min. The injection volume was 1 μL (10:1 split ratio). The temperature of the injector and the detector were held at 240 °C and 280 °C, respectively. MEFAs peaks were identified as compared to the reference standards injected under the same conditions.

Effect of temperature on lipids production

For preliminary optimization of the lipids content, selected marine yeasts were inoculated into BDM (1:10 v/v) prepared in three groups, and incubated at 7, 15, and 26 °C in a rotary shaking incubator (New Brunswick, CA) at 150 rpm for 20 days. Lipid production was tested every 5 days’ by withdrawing broth samples, washed twice with sterile saline solution after centrifugation at 5000 × g and 4 °C, resuspended in 1 mL saline solution, and stored at − 20 °C for lipids analysis using SPV assay. Yeast cell dry weight CDW (g/L) was measured by drying the cell pellets at 80 °C until constant weight. Lipids were extracted and stored at − 20 °C until FAs analysis by GLC-FID.

Effect of glucose concentration on lipids production

200 mL BDM containing 80 g/L glucose (pH 5.6) was prepared in 500 mL Erlenmeyer flasks capped with rubber plugs. The flasks were inoculated, incubated at 7 °C, and aerated using air pump with a flow rate of 15 mL/min for 20 days. Samples were withdrawn every 5 days’ centrifuged at 5000 × g under cooling at 4 °C and washed twice with sterile saline solution. CDW (g/L) was measured by drying the cell pellets at 80 °C until constant weight. Lipids were extracted and stored at − 20 °C until FAs analysis by GLC-FID.

Statistical analysis

All experiments were conducted in triplicate. Results are expressed as the means ± standard error. The effect of the temperature and time on lipids production were compared by two-way ANOVA analysis using SPSS software (Version 17) at P ≤ 0.05 to determine significant differences.

Results and discussion

TTC assay for rapid isolation and screening of lipids producers

The colorless salt 2,3,5-triphenyl-tetrazolium chloride (TTC) turns red when it is reduced to triphenyl formazan (TF), by Δ5-desaturase enzyme. Some marine microbes use the same enzyme in their metabolic pathway to produce ω-3 PUFAs, converting eicosatetraenoic acid to EPA (Ratledge 2002). Results in Fig. 1 and (Supplementary material file1) show the reaction of different microbial isolates with TTC giving different color intensities at different time intervals. TTC assay is simple and rapid for screening PUFAs producers, and its main advantage is 0% false negative for tested microorganisms (Abd Elrazak et al. 2013). The two yeast isolates with deep red color and fast reaction with TTC test (≤ 30 min) were selected for further optimization of PUFAs production (Supplementary material file 1). TTC assay was used to compare the ability of YPD and BBM screening media to stimulate PUFAs production by the selected yeast isolates. There was no difference in the color intensity for both selected yeast isolates in their reaction with TTC in both media. In eukaryotes, conventional biosynthetic pathway of PUFA is through an aerobic pathway with sequential addition of double bonds to saturated FAs, mainly C18:0 and C16:0, via Δ9 and Δ12 desaturases to produce LA(C18:2), which is further desaturated by Δ15 desaturase to give C18:3 (ALA). A sequence of desaturases inserts double bonds between the Δ9 bond and the carboxyl terminal, while elongases convert ALA to EPA and DHA, and the sequence is Δ6 desaturase → elongase → Δ5 desaturase → elongase → Δ4 desaturase (Guschina and Harwood, 2006; Ryan et al. 2010).

Results (Supplementary material file 1), show that the BBM medium stimulates PUFAs production and increases the enzymes expression necessary for the de-novo pathway of PUFAs production, like Δ5-desaturase enzyme, thus reduced the reaction time of TTC assay (Ratledge 2002). Likewise, Zhu et al. (2004) investigated the relation between the staining intensity of Mortierella alpina using TTC and its arachidonic acid (AA) content, reporting that the staining intensity of mycelia was increased when AA content was increased. The enzyme Δ5-desaturase is part of the FA synthase pathway; it represents the dehydrogenase responsible for the reduction of dihomo-γ-linoleic acid to AA (20-carbon ω-6 PUFA), which is probably responsible for reducing TTC to TF red formazan (Ryan et al. 2010).

Macroscopic and microscopic identification

The white yeast isolate showed moderate growth at 28 °C after 3 days as compared to the orange pigmented yeast isolate which appeared small and faint colonies. The microscopic examination (Fig. 2) of the two selected isolates on Corn-Meal Agar incubated at 15 °C shows as follows: (1) Orange pigmented yeast cells after 30 days of incubation by Dalmau plate culture; pseudomycelium and true hyphae were absent, only blastoconidia appear, reproduction by multilateral budding, single cells, or pairs, short chains or clusters. (2) White yeast cells after 30 days of incubation; pseudomycelium was present, but true hyphae were absent, at × 400 magnification; cells were slender, branched with curved pseudohyphae, short-chain elongated blastospore, with few ascospores (Fig. 2). Based on the morphological properties and mode of reproduction of the two selected yeast isolates, and referring to the standard methods with some modifications (Yarrow 1998; Jones et al. 2009), the two yeast isolates were preliminary identified to the genera level, as Rhodotorula sp. and Saccharomyces sp. belonging to Basidiomycetes and Ascomycetes yeast, respectively.

Fig. 2
figure 2

Colony morphology of L. elongisporus on YPD medium (A); cells using simple stain (× 400) (B); branched and curved pseudomycelium under light microscope, simple stained (× 400), arrows refer to ascospores in asci (C). R. mucilaginosa on YPD medium (A’); cells under light microscope (× 400) (B’, C’)

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Identification using MALDI-TOF/MS Biotyper®

MALDI-TOF/MS has been successfully used in research for the determination of molecular mass of peptides and proteins (Marvin et al. 2003). Recently, it has been widely applied and utilized for microorganisms’ identification (Wieser et al. 2012; Ahmed et al. 2021). The selected marine isolates were identified as the Basidiomycetes candidate Rhodotorula mucilaginous and the Ascomycetes candidate Lodderomyces elongisporus (Supplementary material file 2), with score of 2.11 and 2.35, respectively. The high match in the peaks pattern between samples and standards ribosomal proteins verified the species consistency. The software compares the spectra and creates a numerical score-value, based on similarities between the obtained and software database; this score-value provides information for method validity (Wieser et al. 2012). Above 2.0, the score-value is considered valid for identification at the species level. Likewise, Marklein et al. (2009) confirmed that 96% of 250 Candida isolates from 15 different spp. were correctly identified by MALDI-TOF/MS Biotyper®. It is faster than conventional identification methods, and highly accurate for the identification of isolated bacteria and yeast (Wieser et al. 2012; Agustini et al. 2014). MALDI-TOF/MS Biotyper® is a powerful tool for the identification of microbial isolates, it is recently used in clinical microbiology, water, and food applications, it is rapid, cost-effective, and efficient identification technology (Ahmed et al. 2021), as well as it is FDA approved.

Biomass and lipids content determination

Cell dry weight (CDW) of the two selected yeast isolates was significantly increased when grown on BBM screening medium for 15 days. L. elongisporus biomass was increased 4.4-fold from 13.4 to 58.6 mg/mL, while R. mucilaginous biomass was increased 2.5-fold. Also, the biomass was increased 5.6-fold and 2.5-fold for C. lipolytica and S. boulardii, respectively, under the same growth conditions. Results (Fig. 3) showed that the lipids content of C. lipolytica was 57.40%, followed by R. mucilaginous (41.20%), then L. elongisporus (29.0%). These findings are in accordance with Ageitos et al. (2011) who reported that similar amounts of lipids (20–25%) have been produced by different oily yeast genera, such as Yarrowia, Rhodotorula, Candida, Rhodosporidium, Cryptococcus, and Lipomyces using different fermentation conditions. Ratledge (1982) reported that among different spp. of Rhodotorula, the lipid production by R. mucilaginosa reached 28% of its CDW when grown on glucose and sucrose as the carbon source, while R. glutinis was able to accumulate up to 66% lipids when glucose was used as carbon source (Beopoulos et al. 2009). Also, Papanikolaou and Aggelis (2002) reported that Y. lipolytica produced 44% and 40% lipids, after 5 and 10 days, respectively, when grown at 28 °C on medium containing glycerol.

Fig. 3
figure 3

Dry weight and lipids content of C. lipolytica, L. elongisporus, R. mucilaginosa, and S. boulardii after incubation for 15 days at 15 °C using screening Basal Broth Medium

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GLC analysis (Table 1) confirmed the presence of PUFAs, thus verified the positive results of the TTC assay for the two selected marine yeast. Results confirmed the presence of four major FAs, being oleic acid (OA), palmitic acid (PA), stearic acid (SA), and linoleic acid (LA) produced by the four tested yeast. Similarly, Li et al. (2008) reported that the main FAs detected in oleaginous yeast (OY) were myristic acid (MA), PA, SA, OA, LA, and LNA. Results (Table 1) showed that the most prominent FA was OA representing 35.7% of the total FAs. LA was also present in significant amount in all tested yeast strains, R. mucilaginosa produced the highest LA (26.8%) followed by L. elongisporus (20%). Li et al. (2008) reported that C. lipolytica as an OY has a profile of C16:0 (11%), C18:1 (28%), C18:2 (51%), and C18:3 (1%); however, its production of long-chain (LC) PUFAs can only be attained by genetic manipulation (Beopoulos et al. 2009).

Table 1 Fatty acids content (%) of C. lipolytica, L. elongisporus, R. mucilaginosa, and S. boulardii after 15 days incubation on the screening media
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In the present study, LA (C18:2) which is an important precursor in the de-novo pathway of PUFAs production (Ratledge 2002) was detected in relatively high amounts (20% and 26.8%) in the two marine yeast as compared to C. lipolytica (19.8%) (Table 1). The FAs profile (Table 1) showed that R. mucilaginous have 52.5% unsaturated FAs with 26.8% PUFAs, which was close to the FAs profile of the marine R. mucilaginosa AMCQ8A that accumulated 65% lipids with only 6–7% ω-3 content (Gupta et al. 2012). Li et al. (2010) also investigated lipids production by the marine R. mucilaginosa TJY15a, and reported that OA was (54.7–63.5%), while LA was (5.7–11.3%) when grown on various carbon and nitrogen sources. For industrial oil production DuPont (USA) has developed genetically modified Y. lipolytica as an alternative to microalgae-producing EPA (Galán et al. 2020). However, using naturally occurring OY, such as Rhodosporidium toruloides, Rhodotorula glutinis, and Candida curvata, DuPont was able to produce EPA, and the EPA-rich oil has received the GRAS status from FDA (Galán et al. 2020)

The present study is the first one reporting the FAs profile of the local marine L. elongisporus, where only few scientific papers mentioned its applications in industrial biotechnology. In this regard, Wang et al. (2007) isolated the marine yeast, L. elongisporus YF12c, and R. mucilaginosa L10-29 for lipase production. Also, Ma et al. (2015) isolated L. elongisporus SYB-2A that have petroleum-degrading activity. In addition, You et al. (2017) used L. elongisporus to ferment flavor liquor and suggested its usage in the food industry.

Effect of temperature on lipids production and growth

R. mucilaginosa and L. elongisporus were incubated at different temperatures (7, 15, and 26 °C) in normal BDM (3% glucose). These cold adapted strains can grow well at 7 °C, but their abundant growth was observed at 15 °C, and up to 26 °C. R. mucilaginosa can grow well in temperature ranging from 7 to 30 °C, while L. elongisporus shows rapid growth up to 35 °C. Therefore, R. mucilaginosa and L. elongisporus can be regarded as facultative psychrophilic strains based on their classification (Margesin 2009). The growth temperature ranges for yeast isolated from cold areas can be surprisingly high, some common spp. can be found in cold and warm habitats, such as Cryptococcus macerans and R. mucilaginosa (De Garcia et al. 2007). Similarly, Butinar et al. (2011) reported that most ascomycetous yeasts isolated from glacier ice can grow well at 25–30 °C in addition to their growth at 4 °C.

A significant increase in the CDW was observed with the increase of temperature at the same day along the whole time course, while no significance was observed at the same temperature throughout the time course (Fig. 4). Although both Egyptian yeast strains had different growth rate over time, an obvious increase in the biomass was achieved through increasing the incubation temperature, where R. mucilaginosa and L. elongisporus reached their maximum biomass of 40 and 78.75 mg/mL, after 10 and 20 days, respectively, when grown at 26 °C (Fig. 4). In contrast, Amaretti et al. (2010) reported that the temperature did not affect the yield of lipids and biomass of Rhodotorula glacialis DBVPG 4785, but only had positive effect on its growth rate; however, R. glacialis is an obligate psychrophilic that cannot grow at 26 °C as compared to the current isolated facultative psychrophilic marine yeast.

Fig. 4
figure 4

Biomass concentration (g/mL) of R. mucilaginosa (A) and L. elongisporus (B) after incubation at 7, 15, and 26 °C, for 20 days on Basal Defatted medium (BDM)

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Effect of glucose concentration on FAs composition

The total lipids content can vary greatly within a species; however, the overall FAs profiles have been quite consistent within a sp. if grown under consistent conditions; thus, it was used to identify yeast before ribosomal sequencing became affordable (Botha and Kock 1993). High-Glucose (8%) HG-BDM induced a positive effect on the total lipids production that reached its maximum of 48% and 54% by R. mucilaginosa and L. elongisporus, respectively, when grown at 15 °C. Relative FAs content (Table 1), showed that the lipid profiles of R. mucilaginosa and L. elongisporus were mainly composed of LC-FAs with 16 and 18 carbon atoms. The distribution of some FAs, namely, C12:0 (lauric acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:3 (LNA), were almost constant during their growth time course. The relative content of OA (C18:1) was decreased from 38% and 32.9% to 34.4% and 25.8% with the increasing incubation temperature from 7 to 26 °C on normal BDM, for L. elongisporus and R. mucilaginosa, respectively. While, further decrease in OA from 38% and 32.9% to 15.5% and 21.8% was observed in HG-BDM, respectively. However, LA content was slightly changed from 15.75 to 10.91% and from 24.5 to 27.63% for L. elongisporus and R. mucilaginosa, respectively, with the increasing glucose concentration to 8%. On the contrary, Gupta et al. (2012) reported the glucose enhancing effect in R. mucilaginosa AMCQ8A when glucose was increased from 2 to 10%, where ALA was increased from 1.7 to 6.21%, while OA and LA were highly increased from 7.96 to 35.71% and from 3.68 to 23.69%, respectively. Overall, a trend of increasing the FAs unsaturation degree was observed for both yeast strains with the decreasing incubation temperature. In contrast, the opposite trend was observed with the increasing glucose concentration from 3 to 8%, at 7 °C (Table 2).

Table 2 Fatty acids content (%) of L. elongisporus and R. mucilaginosa after growth in normal BDM and HG-BDM media
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At low temperature, in order to regulate the membrane fluidity and functionality, R. mucilaginosa and L. elongisporus exploited diverse changes in their lipid’s composition, such as the degree of unsaturation, namely, palmitoleic (C16:1, Δ9), OA (C18:1, Δ9), LA (C18:2, Δ9,12), and α-LNA (C18:3, Δ9,12,15), were increased (Table 2). Likewise, the presence of 16–18 carbons LC-FAs was clearly observed as a common trend at low temperature (0–15 °C). Similarly, Amaretti et al. (2010) reported that both the glucose concentration and the growth temperature influenced the FAs composition of R. mucilaginosa AMCQ8A, where the degree of FAs unsaturation was decreased when the temperature or glucose was increased. In the present study, at low temperature (7 °C), the increase in glucose concentration from 3 to 8%, decreased the degree of unsaturation, namely, OA (C18:1; Δ9) and LA (C18:2, Δ9,12) (Table 2). Also, an increase in the ratio of short-chain unsaturated FAs (C11–C15) was observed for both marine yeast strains in HG-BDM. Likewise, Sitepu et al. (2013) suggested that the variation in the medium composition and the incubation time affect the relative quantities of certain FAs, such as LA (C18:2).

LA is an essential FA that cannot be synthesized by humans or animals, but must be ingested for good health; therefore, yeasts capable to produce significant amounts of ω-6 FAs can be considered as good candidates for its commercial production. Although some microorganisms produce PUFAs naturally, native microorganisms produce low yields, which are usually far below the level for commercial production. Therefore, more research efforts are needed to explore new candidates and modify them through metabolic engineering to accumulate higher amounts of lipids enriched in PUFAs (Galán et al. 2020). In addition, Xue et al. (2013) reported that wild oleaginous yeast strains are not able to produce very LC-PUFAs. In same regard, PUFAs, such as ɤ-LNA (C18:3) and stearidonic acid (C18:4), were detected in low amounts (< 0.4%), or α- LNA (C18:3) as 6.2% in some marine yeast (Wang et al. 2007; Gupta et al. 2012). Interestingly, results in Table 2 showed the presence of LC-FAs, such as 15-Docosenoic acid (C22:1, ω7) and Tricosanoic acid (C23:0) in the FAs profile of marine L. elongisporus with significant amount of 12.12% and 21.49%, respectively, on normal BDM. These FAs were present in the marine environment, as well as in the marine standard oil used for FAs identification in marine samples (Restek-Corporation 2018; Dikma-Technologies 2021). However, few scientific papers reported the isolation of L. elongisporus from marine habitat (Wang et al. 2007; Ma et al. 2015). In the present study, L. elongisporus represents an oleaginous Egyptian marine yeast, and this is the first study to report its FAs profile and its capability for lipids accumulation under different cultural conditions.

Lipids quantification using SPV assay

To validate a simple and sensitive micro-scale assay, three different oils were used to design standard calibration curves, including Fish, Flax seed, and Coconut oils. Selection of the standard oils depends mainly on understanding the major lipids content in the marine yeasts taking into consideration the saturated/unsaturated FAs distributions (Rossi et al. 2009). To detect the production of PUFAs in the designed experiment, Fish oil was used as standard oil because of its EPA (18%) and DHA (12%) content, while Flax seed oil was used because of its LNA content (52.9%) and Coconut oil for its saturated FAs content (85.5%). Calibration curves were generated by measuring the absorbance at λ570 and were plotted against the lipid’s quantity. The three calibration curves showed a very strong linear relationship with R2 values of 0.9903, 0.9882, and 0.9717 using Flax seed, Fish, and Coconut oils, respectively (Fig. 5). In general, the increase in standard oil concentration was accompanied with the gradual increase in the pink color intensity. Furthermore, responses of the three standard oils were obviously varied, which indicates that they were significantly affected by their lipid’s composition. Results (Supplementary material file 3) showed that total lipids content of both marine yeasts had a good coefficient of variation typically ≤ 7% with respect to the SPV results with an exception of L. elongisporus at 7 °C, which is probably due to its high amount of MUFAs (47%), as compared to PUFAs (16%) (Table 2). Similarly, other studies reported that the SPV assay was used in the quantification of total intracellular lipids of microalgal, bacterial, and yeast cells (Wang et al. 2009; Cheng et al. 2011; Mishra et al. 2014; Dien et al. 2016).

Fig. 5
figure 5

Absorbance linearity of three different standard oils, Fish (A), Flax (B), and Coconut (C) using SPV assay. Absorbance was measured at λ570, data points represent the mean of two replicates. Representative color intensity in SPV assay with the three standard lipids (5–200 µg)

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In the current study, R. mucilaginosa and L. elongisporus were grown at three incubation temperatures (7, 15, 26 °C). All samples showed an increase in the color intensity with biomass increase (Fig. 6). The lipids content was calculated using the standard oils curves. Assay linearity was tested for each of the yeast strain by plotting amounts of measured lipids versus amounts of yeast biomass (Supplementary material file 3). The highest lipids production occurred after 15 days of incubation in BDM at 7 °C by R. mucilaginosa and L. elongisporus using SPV assay.

Fig. 6
figure 6

SPV assay reaction and color development with set of experiments conducted at 15 °C (plate I) and 26 °C (plate II). R. mucilaginosa (A, a), L. elongisporus (B, b) were cultured in three media (M1, M2, M3) for 20 days. (A, B: background colors), and (a, b: SPV assay colors). M1; Basal medium, M2; Basal Defatted medium, M3; High-glucose medium, D; day

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Results in Fig. 7 showed the relationship between SPV results of both marine yeast, and their PUFAs and MUFA contents, when incubated at different temperatures. In agreement with the chemical reaction, an obvious relationship between FAs composition and SPV results was found, when LA and OA were the only PUFAs and MUFA; the SPV response was closely associated to the LA amount in R. mucilaginosa (Table 2 and Fig. 7). Depending on the chemical nature of SPV reaction, only a single carbonium ion is formed per molecule, where multiple ones would not be stable and steric hindrance can occur in case of multiple unsaturated compounds. Therefore, OA shows highly reaction response, and more intense color than LA and LNA (Knight et al. 1972). In case of multiple unsaturated FAs, the steric hindrance was observed in the presence of PUFA (LA and ALA) with OA alone or OA with Palmitoleic acid (MUFAs), which resulted in reaction response of 24–26% as compared to (30–32%) in the presence of PUFAs and MUFAs for both yeast strains grown at 15 °C (Table 2 and Fig. 7). In addition, high presence of OA with other MUFAs (Palmitoleic acid) and 15-Docosenoic acid (C22:1) showed a higher reaction response of SPV (20–26%) as compared to LA alone (0–16%) in L. elongisporus. Overall, when an appropriate reference oil is used and compared with tested biological samples, the SPV reaction can provide meaningful estimates of the overall lipids content of the tested samples (Dien et al. 2016).

Fig. 7
figure 7

Lipids content and fatty acids composition of R. mucilaginosa (A), L. elongisporus (B) grown on normal BDM medium at 7, 15, 26 °C, and their corresponding SPV results. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; and PUFAs, polyunsaturated fatty acids

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Visualization of lipid droplets using Nile-Red fluorescent dye

Nile-Red fluorescent dye is widely used to determine lipids accumulation in yeast (Wang et al. 2017). Nile-Red dye can be applied to cells, where it dissolves preferentially in lipids, and its fluorescence can be observed only in the stained substances (Greenspan et al. 1985). Staining of R. mucilaginosa and L. elongisporus cells showed large spherical, fluorescent cytoplasmic structures (Fig. 8). Although these structures varied in size, fluorescence intensity, and distribution, their presence gave a good indicator for high lipid production. In order to test the effect of low temperature on the accumulation of lipids, both R. mucilaginosa, L. elongisporus, and the two control (C. lipolytica and S. boulardii) were stained by Nile-Red dye, after incubation for 15 days on normal BDM at 7 °C. Results in Fig. 8 showed yellow-golden fluorescent lipids that can be viewed in the stained cells. The Nile-Red stained cells of S. boulardii exhibit numerous small discrete bodies distributed throughout its cytoplasm, although large individual lipid bodies were observed alone. However, stained cells of C. lipolytica did not exhibit clear yellow-golden fluorescence, in spite of apparent diffused fluorescence inside and outside its cells (Fig. 8). Similarly, Diniz Rufino et al. (2014) reported that the low intensity of fluorescent lipids and the diffusion of Nile-Red dye outside Y. lipolytica cells might be due to its pathogenicity nature that accumulates cell biomass in its thick mucoid secretion of hydrophobic FAs. Thus, the interaction of Nile-Red and the yeast mucoid secretion appears as low intensity fluorescence around it (Fig. 8). Nile-Red staining confirmed the effect of BDM on increasing the lipids accumulation at low temperature in R. mucilaginosa and L. elongisporus cells by the presence of yellow-golden fluorescent lipid droplets (Fig. 8). Based on the yellow-golden fluorescence (Poli et al. 2013), the four examined yeast strains were classified according to the area covered by lipid droplets (yellow-gold) inside the cell; R. mucilaginosa had lipid droplets filling ≥ 50% of its cell area, L. elongisporus had lipid droplets filling 30–50%, and S. boulardii had lipid droplets filling up to 30% (Fig. 8), as previously described by Poli et al. (2013). Therefore, Nile-Red can be recommended for staining intercellular lipids.

Fig. 8
figure 8

The four examined yeast strains were classified according to the area covered by lipid droplets (yellow-gold) inside the cell visualized with oil immersion objective lenses (100x). L. elongisporus (A), R. mucilaginosa (B), S. boulardii (C), and C. lipolytica (D); all photos are shown in fluorescent view (right) and grey background (left)

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Conclusion

This study explored the production of lipids using two newly isolated marine yeast, Rhodotorula mucilaginosa and Lodderomyces elongisporus, in terms of growth temperature and C:N ratio. Both strains can accumulate high amounts of lipids (48–54%) when cultured in BDM with high C:N (8:1) at 15 °C. The unsaturation degree of FAs was decreased by raising the temperature from 7 to 26 °C, especially total PUFAs. An obvious increase in the biomass (40–78.75 mg/mL) was achieved through increasing the incubation temperature to 26 °C. Their FAs profile and lipids content suggest their usage as alternative source for high-value edible oil, and could be considered as a promising source for the production of single cell oils, since they can grow and produce lipids over a wide range of temperatures. Significant amounts of LC-15-Docosenoic acid (C22:1, ω7) and Tricosanoic acid (C23:0) were detected as 12.12% and 21.49%, respectively, in the FAs profile of L. elongisporus, when incubated at 26 °C on normal BDM. Normal BDM (3% glucose) and 15 °C were the best conditions to increase the production of PUFAs by both strains. This study is considered promising application for the newly isolated marine oleaginous L. elongisporus that can accumulate high amounts of lipids, with considerable amounts of LC-FAs through appropriate fermentation conditions. The outcomes of this study are promising and can serve as basis for the development of new biotechnological applications using the new marine Egyptian yeast.