Single cell oil of oleaginous marine microbes from Saudi Arabian mangroves as a potential feedstock for biodiesel production

2.1 Sampling sites and sample collection

We collected 144 samples from four mangrove sites along the Red Sea and the Arabian Gulf in Saudi Arabia namely: Al-Leith (20° 49′ 10″ N 39° 27′ 26″ E), Jeddah (22° 46′ 32″ N 39° 48′ 25″ E), Syhat (26° 29′ 32″ N 50° 02′ 46″ E) and Yanbu (24°02.55′ N 38°68.89′ E). Collected samples included: 68 decaying leaves of Avicennia marina, 33 decaying thalli of Zostera marina, 14 decaying pneumatophores of A. marina, 9 crab shells, 8 sediment, 7 decaying thalli of Turbinaria ornata and 5 decaying thalli of Cystoseira myrica.

2.2 Isolation of filamentous fungi, yeasts and thraustochytrids

Decaying leaves and pneumatophores of Avicennia marina and decaying thalli of seaweeds were placed in clean plastic bags and brought to the laboratory on the same day. In the laboratory, samples were washed using sterile sea water, cut into small segments (ca 1 cm in length), surface sterilized using 5 % sodium hypochlorite for 1 min, transferred into sterile sea water (1 min), surface dried using sterile filter paper and placed on the surface of GYPTA medium (Abdel-Wahab et al. 2021a). Plates were incubated at 25 °C and examined every 24 h. Microbial growth were transferred to new plates and further purified until axenic cultures were obtained. Pure cultures were maintained on GYP slants (Abdel-Wahab et al. 2021a) and re-subculture every-two months. We also preserved them by cryopreservation at −80 °C and re-subculture every 4 months.

2.3 Sudan Black B test of the isolated microbes

The 84 morphological strains isolated during the present study were tested for their abilities to produce high level of oil using Sudan Black B. Positive strains for oil production were further grown on a larger scale and the percentage of oil were determined using Sulfo-Phospho-Vanlin (SPV) method.

2.4 Quantification of microbial lipids using sulfo-phospho-vanillin (SPV) method

Phosphovanillin (PV) reagent was prepared by dissolving six milligrams of vanillin in 100 mL of hot water and further diluted to 500 mL with phosphoric acid. Oleic and palmitic acids were used as standard and diluted properly with concentrated sulfuric acid to reach 1 mg/mL. Reagents (oleic, palmitic, phosphoric and sulfuric acids and vanillin) were purchased from local company. Twenty-five mg of microbial cells were dissolved in one mL concentrated sulfuric acid, of which 20 µL of the samples were diluted in 180 μL of concentrated sulfuric acid, incubated at 100 °C for 10 min. Samples were cooled to room temperature and 0.5 mL of phosphovanillin reagent was added, incubated at 37 °C for 15 min, cooled to room temperature and stored for 45 min in a dark box for color development. The absorbance was measured at 530 nm in a spectrophotometer (Anschau et al., 2017).

2.5 Lipid extraction and analysis of fatty acids

Biomass and their dry weights were calculated and the lipid was extracted as previously described by Abdel-Wahab et al. (2022). Fatty acids profile of the extracted lipid was determined using methyl esters boron tri-fluoride method (Folch et al., 1957; Abdel-Wahab et al., 2022).

2.6 DNA sequencing and phylogenetic analyses

Pure microbial strains were grown in liquid medium and DNA was extracted using MOBIO microbial DNA extraction kit according to the manufacturer’s instructions. ITS region was amplified and sequenced using ITS1 and ITS4 primers. Partial SSU and LSU rDNA were amplified and sequenced using primers pairs NS1/NS4 and LROR/LR7 respectively (Bunyard et al., 1994). PCR amplification and DNA sequencing were carried out by SolGent Inc., South Korea. The obtained sequences were deposited in GenBank (Figs. 2–3, 5). Sequences were aligned using ClustalX (Thompson et al., 1997). Phylogenetic analyses were carried out using MEGA X (Kumar et al., 2018). Bayesian inference was carried out in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). MrModeltest 2.2 was used to determine the best-fit model for the sequences dataset (Nylander, 2004). The phylogenetic tree in Fig. 2 was visualized using Njplot (Perrière and Gouy, 1996) and edited using Adobe Illustrator CS6.

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Fig. 1

a–f Hortaea werneckii (AL–19) isolated from decaying leaves of Avicennia marina, Al-Leith mangroves, Red Sea. Variously shaped vegetative cells with or without buds and pseudo-hyphae. g–i Rhodotorula mucilaginosa (AL–14) isolated from decaying leaves of Avicennia marina, Al-Leith mangroves, Red Sea. g–i Vegetative cells with or without buds. Bars: a–i = 5 µm.

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Fig. 2

Phylogenetic relationship of Hortaea werneckii strains (AL–19 and AL–20) isolated during the current study. Phylogenetic analyses based on the nucleotide sequences of the LSU rDNA placed the two strains among the other strains of the species. The maximum likelihood (ML) tree (-ln likelihood = 3611.05) was constructed in MEGA X (Kumar et al., 2018). Phylogenetic trees obtained from ML, maximum parsimony (MP) and Bayesian inference posterior probabilities (BYPP) were similar in topology. Bootstrap support on the nodes represents ML and MP ≥ 50 %. Branches with a BYPP of ≥ 95 % are in bold. The two sequences of Hortaea werneckii generated in this study are in green, previous strains of the species from Red Sea are in red.

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Fig. 3

Phylogenetic relationship of Rhodotorula mucilaginosa strains (AL–14 and AL–15) isolated during the current study. Phylogenetic analyses based on the nucleotide sequences of the LSU rDNA placed the two strains among the other strains of the species. The maximum likelihood (ML) tree (-ln likelihood = 2318.03) was constructed in MEGA X (Kumar et al., 2018). The maximum parsimonious data set consisted of 29 taxa include: 18 sequences of Rhodotorula mucilaginosa strains, 4 sequences of other Rhodotorula, 5 sequences of other taxa of Sporidiobolaceae and two taxa belong to Chrysozymaceae (outgroup). Phylogenetic trees obtained from ML, maximum parsimony (MP) and Bayesian inference posterior probabilities (BYPP) were similar in topology. Bootstrap support on the nodes represents ML and MP ≥ 50 %. Branches with a BYPP of ≥ 95 % are in bold. The two sequences of Rhodotorula mucilaginosa generated in this study are in red.

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Fig. 4

Aurantiochytrium spp. isolated from decaying leaves of Avicennia marina from Syhat mangroves. a–c Aurantiochytrium sp. (SY–78) Variously shaped sporangia. d–g Aurantiochytrium sp. (SY–85). d–e Variously shaped sporangia. f–g Zoospores. Bars: a–g = 5 µm.

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Fig. 5

Phylogenetic relationship of oleaginous thraustochytrid strains (AL–22, AL–23, SY–78, SY–85 and YB–39) isolated during the current study from Al-Leith, Syhat and Yanbu mangroves. Phylogenetic analyses based on the nucleotide sequences of the ITS region placed the five strains in a well-supported node basal to other strains of the genus Aurantiochytrium. The maximum likelihood (ML) tree (-ln likelihood = 1185.76) was constructed in MEGA X (Kumar et al., 2018). Phylogenetic trees obtained from ML, maximum parsimony (MP) and Bayesian inference posterior probabilities (BYPP) were similar in topology. Bootstrap support on the nodes represents ML and MP ≥ 50 %. Branches with a BYPP of ≥ 95 % are in bold. The newly generated sequences in this study are in red.

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Bayesian inference.

3.1 Diversity of oleaginous filamentous fungi and yeasts from Saudi mangroves

A total of 961 isolates (include 759 filamentous fungi and 202 yeasts) were isolated from 144 submerged marine samples that include: 68 decaying leaves of Avicennia marina, 33 decaying thalli of Zostera marina, 14 decaying pneumatophores of Avicennia marina, 9 crab shells, 8 sediment, 7 decaying thalli of Turbinaria ornata and 5 decaying thalli of Cystoseira myrica. Samples were collected from four mangrove sites: Al-Leith, Jeddah and Yanbu (Red Sea) and Syhat (Arabian Gulf). Isolated fungi were grouped into 62 morphological types that include: 21 yeasts and 41 filamentous fungi. Aspergillus and Penicillium species were the most common fungi and represented by 337 and 330 isolates respectively. Other recorded genera were: Cladosporium, Lasiodiplodia, Mortierella and Rhizopus. Forty-three isolates did not fruit in culture and were grouped into ten morphological types (Table 1).

The 202 isolates of yeasts were cultured from 48 samples of decaying leaves of Avicennia marina and seaweeds. The yeast isolates were grouped into 21 morphological types, of which four morphological types were identified using molecular phylogenetics of ribosomal genes.

3.2 Diversity of oleaginous thraustochytrids from the Saudi mangroves

Fifty-four isolates of thraustochytrids were cultured from the four mangrove sites namely: Al-Leith, Jeddah and Yanbu (Red Sea) and Syhat (Arabian Gulf). Isolated thraustochytrids were grouped into 22 strains. Isolated thraustochytrids belonged to two genera: Aurantiochytrium and Thraustochytrium (Table 2). Thraustochytrids that produced high biomass and high levels of lipid were chosen for further study. Selected strains were grown on a larger volume of culture media and their lipid were extracted and the fatty acids and their derivatives were determined using GC/MS.

3.3 Molecular phylogenetics of yeasts and thraustochytrids

Four oleaginous yeast strains were selected and their taxonomical placements were determined based on LSU rDNA and ITS and were placed in Rhodotorula mucilaginosa and Hortaea werneckii (Table 1, Figs. 1–3). DNA was extracted from the most promising thraustochytrid isolates and their taxonomical placements were determined based on ITS sequences. Thraustochytrids from the Arabian Gulf and the Red Sea formed a separate node basal to other known Aurantiochytrium species and might represent undescribed taxa (Figs. 4-5).

3.4 Lipid profile of yeasts and thraustochytrid strains

Two oleaginous yeasts: Hortaea werneckii (Horta) Nishim. & Miyaji (AL–19) and Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison (AL–14) and four Aurantiochytrium strains: AL–22, AL–23, SY–78 and SY–85 produced high dry weight ranged between 32 and 49.3 g/L of which 35.2–62 % lipid. These promising results can be improved by changing the growth conditions using different combinations of different C/N ratio. Fatty acid profiles of the six strains on GC/MS were determined. Palmitic acid was the major fatty acid in the lipid of the four thraustochytrid isolates and ranged between 5.71 and 82 % of the total fatty acids, while it was not recorded from the lipid of the two yeast isolates, 9-Octadecenamide, (Z)- was the major fatty acid amide in the lipid of the two isolates and two thraustochytid isolates and ranged between 26.94 and 56.63 %, followed by 13-Docosenamide, (Z)- (20.44–34.99 %) from the same four isolates. Other major lipid compounds were: Hexadecanamide (4.35–7.19 %), Cholestrol (7.24–15.07 %), Butylated Hydroxytoluene (3.46–15.76 %), Octadecanamide (3.95–7.9 %), Phenol, 2-(1,1-dimethylethyl)-5-methyl- (1.78–10.33 %) and Pentadecanoic acid (7.47 %) (Tables 3).