Seawater samples, fallen leaves of mangrove trees (Avicennia marina and Rhizophora mucronata) and sediments were collected from coastal waters and mangrove forest of Persian Gulf and Oman Sea in south of Iran. Samples were kept in sterile bags at 4 °C and sent to the laboratory before use.
Yeast strains isolation
Samples were plated on medium containing 10 g/L glucose, 1 g/L peptone, 0.1 g/L yeast extract (YE), natural seawater (50% v/v), 15 g/L agar and supplemented with 300 mg/L streptomycin and penicillin G. The plates were incubated at 30 °C for 3–6 days with regular observation for yeast growth. The obtained single yeast colonies were picked-up and sub-cultured on fresh YEPD medium (20 g/L glucose, 20 g/L peptone, 10 g/L YE, 15 g/L agar and seawater (50% v/v)) and incubated at 30 °C for 48 h to obtain pure cultures .
Light microscopy and morphological characteristics
Yeast strains were grown in YEPD broth medium and incubated at 30°C. Then, morphological characteristics of the isolated yeast strains were determined under light microscope (Zeiss, Germany) during incubation period.
Molecular and phylogenetic analysis
An overnight culture of yeast strain was prepared and used for genomic extraction. Universal primers ITS1 (5´-TCCGTAGGTGAACCTGCG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) were used for amplification of the internal transcribed spacer (ITS). The polymerase chain reaction (PCR) was performed by gradient thermal cycler (Eppendorf, Westbury, NY, USA). The program was set as denaturation for 4 min at 94 °C, 30 cycles of 30 s at 94 °C, 1 min at 55 °C and 90 s at 72 °C. Final extension was 7 min at 72 °C . PCR amplicons were purified and sequenced by Bioneer. Resulting sequences were edited by BioEdit program (BioEdit 7.2) and were searched using Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov). ClustalX program was used for sequence alignment and then generation of phylogenetic tree was done using neighbor-joining method and MEGA4 software . The tree reliability was evaluated by bootstrap analysis of 1000 replicates.
Determination of cell growth and biomass
The growth of yeast cells were determined by measuring optical density (OD) of harvested cells at phosphate buffered saline (PBS) and 600 nm. Cell biomass, expressed as cell dry weight (CDW) was harvested by centrifugation (4000 rpm for 5 min) and pellets were dried at 105 °C for 18 h to constant weight and the weight was determined gravimetrically.
Lipid extractionand TLC analysis
A certain amount of freeze dried cells was suspended in 2 mL distilled water in screw cap test tubes. The cell suspension was ultrasonicated and then 2.5 mL chloroform and 5 mL methanol were added. Cell suspension was sonicated again and homogenized with T10 basic IKA homogenizer. 2.5 mL chloroform and 2.5 mL distilled water were added and vortexed for 30 s. Resulting suspension was centrifuged at 4000 rpm for 15 min to separate two phases. The organic bottom layer was transferred to a pre-weighed tube and the solvent was evaporated. The amount of lipid was determined gravimetrically . Thin layer chromatography (TLC) silica gel plates coated with fluorescent indicator F254 were used for analysis of extracted lipids. Hydrocarbons such as squalene were separated with developing solvent of hexane/chloroform (9:1). Afterward, the TLC plates were exposed to H2SO4 (20%) and then visualized by heating at 70 °C for 60 min .
Fatty acid methyl esters analysis
Total lipid was converted into fatty acid methyl esters (FAMEs) by methanolic sulfuric acid (4% v/v) at 80 °C for 90 min in sealed vials. Then 1 mL of H2O was added and FAMEs were extracted by several hexane extraction (3×2 mL). FAMEs were dried over anhydrous Na2SO4 and solvent was removed by evaporation. The samples were stored at 4 °C prior analysis. Gas chromatography (GC) analysis was performed using Agilent 6890 equipped with a flame-ionization detector (FID) and DB-23 (30 m×0.32 mm, 0.25 µm; Agilent Technologies) capillary column. 0.5 µL of FAMEs sample was injected under splitless injection mode. Nitrogen was used as carrier gas and temperature of injector and detector was set at 300 °C. Column temperature program were 50 °C; 2 min, 10 °C/min to 180 °C; 5 min, 5 °C/min to 240 °C; 7 min. C19:0-FAME (Sigma, USA) was used as an internal standard .
Quantitative determination of squalene by HPLC analysis
High performance liquid chromatography (HPLC) was used for identification and quantification of squalene. Lipids were saponified using 0.5 M potassium hydroxide containing ethanol (0.5 M KOH/EtOH) at 90 °C for 1 h. Then non-saponifiable lipids were extracted with hexane. Afterward, solvent was evaporated and squalene redissolved with 1 mL of acetonitrile/tetrahydrofuran (THF) (9:1, v/v) . Squalene was identified and quantified by HPLC (Agilent, 1100 Series) equipped with a Zorbax, sb-C18 (4.6×250 mm, 5 micron) column. Acetonitrile/THF (80:20, v/v) was considered as mobile phase at a flow rate of 1 mL/min and ran under isocratic conditions. The sample injection volume was 10 μL and the column temperature was set at 30 °C and identification and quantification were done at 210 nm. Squalene (St. Louis, Mo., U.S.A.) was used as external standard for squalene quantification. A standard calibration curve was established by plotting peak area against concentration, by using different concentrations of squalene .
Fourier-transform infrared spectroscopy (FT-IR) analysis
IR spectrum of purified squalene was determined between 4000–400 cm−1 using a Bruker ALPHA FT-IR spectrometer. Three spectral replicates were determined for purified squalene sample.
Strain selection and cultivation
Strain DR37 was isolated based on its characteristic for squalene production. This strain was cultivated in modified YEPD medium (non-optimized medium) (20 g/L glucose, 5 g/L peptone, 5 g/L YE, 15 g/L agar and seawater (50% v/v)) and incubated at 30 °C and 150 rpm.
Optimization of culture conditions
For investigation the effect of various chemical and physical factors on cell growth, lipid and squalene production by Rhodosporidium sp. DR37, five carbon sources (20 g/L) (glucose, sucrose, glycerol, starch and olive oil) and five nitrogen sources (5 g/L) (YE, malt extract (ME), peptone, ammonium chloride and sodium nitrate) were tested. Seawater concentration (50% v/v) was constant in all above experiments. During carbon source experiments, YE and peptone (5 g/L each) were used as nitrogen source. Also, 20 g/L sucrose was used as the carbon source during nitrogen source experiments. Afterward, the effect of different concentrations of seawater (0, 20, 50, 70 and 100% v/v) were assessed along with 20 g/L sucrose and 5 g/L peptone as selected carbon and nitrogen sources, respectively. Also, different concentrations of selected carbon source (sucrose, g/L) (20, 40, 60, 80 and 100) were investigated on cell growth, lipid and squalene production. The effects of various growth parameters, including temperature (25, 30 and 37 °C) and pH (5.0, 7.0 and 9.0) were investigated in medium containing 20 g/L sucrose, 5 g/L peptone and seawater (20% v/v). The experiments were incubated for 3 days at 150 rpm.
Analysis of variance (ANOVA) was used for data analysis with GraphPad Prism 8.0.1 (P<0.05). The results are presented as the mean ± standard deviation of three replicates.