Potential Assessment of Oleaginous Fungi for Sustainable Biodiesel Production: Screening, Identification and Lipid Production Optimization


 The present work, aiming to exploit oleaginous fungi for biodiesel production. Ten fungal strains were isolated from two petroleum polluted soil samples and screened for their abilities to accumulate lipid. Lipid rich three species viz, Aspergillus terreus, Aspergillus niger and Aspergillus flavus were found to be the highest lipid producers. Potential isolates were identified at the species level by morphological (macroscopic and microscopic) examination and molecularly confirmed by using 18S rRNA gene sequencing. Improvement of lipid accumulation by optimization of various parameters of culture conditions. The results reported clearly that the most suitable medium conditions for highest lipid production (38.33%) of Aspergillus terreus as the most potent lipid producer composed of 5% sucrose, 0.5 g/L ammonium nitrate with initial pH 6.0, after seven days of incubation in a static condition. The three promising fungal isolates have been taken for fatty acids analysis by gas chromatograph (GC) after transesterification. Fatty acid methyl esters (FAME) profile indicated the presence of higher saturated fatty acid fractions compared to polyunsaturated fatty acids. The total concentration of fatty acids was 107.98, 38.29, and 37.48 mg/100g of lipid accumulated by A. terreus, A. niger and A. flavus, respectively. Gas chromatograph analysis of A. terreus lipid indicated that oleic acid (C18:1, 18.51%) was the most abundant fatty acid, followed by stearic acid (C18:0, 15.91%) and Myristic acid (C14:0, 14.64%), respectively. Therefore, fatty acid profile of A. terreus has confirmed its potentiality as feedstock for producing lipid for biodiesel manufacturing.


Introduction
Massive consumption of fossil fuels has already caused serious concern over global warming caused by greenhouse gases emission and eco-friendly, as biodegradability; a decrease of sulfur and aromatic hydrocarbons content, which reduced their toxic emission of CO, CO 2 during fuel combustion (Demirbas 2008;Knothe 2008;Kotasthan 2017). Biodiesel involves the mixture of fatty acyl methyl/ethyl esters (FAMEs), obtained typically from transesteri cation of vegetable oil that can be used for existing conventional diesel engines regardless of its origin and feedstocks from which it is derived (Alptekin 2017;Patel et al. 2017).
The focus of the present work was to isolate, identify and characterize oleaginous fungal species from oily polluted soil samples, estimate the lipid content of these isolates, study the effects of physical and nutritional parameters to detect and maximize the accumulation of intracellular lipids of the promising fungal strains to enhance the quality of biodiesel. Finally, investigate the fatty acid methyl ester (FAME) composition after transesteri cation of fungal oil. According to the literature, fatty acids from the promising oleaginous isolated fungus are signi cant for biodiesel production.

Materials And Methods
Samples collection and fungi isolation Ten fungal isolates were screened from two different oil-rich soil locations in the front of Moharam-Bek and Amrya gas stations which were polluted by petroleum products e uent in Alexandria, Egypt. Samples were collected in sterile containers and taken to the laboratory for analysis. Serial dilutions of the collected samples were carried out until 10 − 5 folds to isolate oleaginous fungi. One ml of the dilute was pour plated on Potato Dextrose Agar medium (PDA) supplemented with 2 mL of gentamicin and then incubated at 28 ºC for 3-6 days (Kumar et al. 2011). Morphological appearances of the inoculated plates were observed and distinct pure colonies were subcultured on PDA slants and stored at 4 ºC for further study.

Screening of oleaginous fungal isolates for lipid production
Pure fungal isolates were cultured on basal medium (g/L): yeast extract 0.5, MgSO 4 .7H 2 O 0.4, KH 2 PO 4 2.0, CaCl 2 0.5, CuSO 4 5H 2 O 0.05, and 5% glucose (w/v), with initial pH 6.0 to select the highest lipid producers. A portion of mycelium from PDA slant were transferred to a tube containing 10 mL sterilized distillated water and agitated then 0.5 mL of this spore suspension taken by micropipette and added to 250 mL conical asks containing 50 mL of the basal medium and then incubated in 30 °C for seven days under static condition. After incubation the culture fungal growth was harvested by ltration (Whatman no.1), and the mats biomass were collected and washed three times with distilled water to remove the medium residues. The biomass was dried in a hot air oven at 60 °C until constant weight.

Extraction of fugal lipid and determination of total lipid content
The lipids were extracted from the screened fungal isolates dried biomass by the method of Bligh and Dyer method (1959) with slight modi cation using chloroform: methanol 2:1 (v/v). One hundred milligrams of the dried biomass was crushed with a mortar and pestle. Mixture of 10 mL of chloroform, and 5 mL of methanol were mixed, 8 mL from this mixture withdrew and added to a known amount of crushed dried mycelium then vortexed for 5 minutes. Saline solution (7.3 g of NaCl, 10 mL of distillated water) was prepared, withdrew 2 ml of the prepared saline solution then added to each sample tube (containing dried mycelium) then vertexing for 5 minutes. Samples centrifuged at 3000 rpm for 15 minutes and the lower layer of methanol, water and NaCl was removed by Pasteur pipette, residual of solvent was dried then estimated gravimetrically mg/L and determine the ratio of extracted lipids in compare to the cell dry weight (Magdum et al. 2015).
Percentage of lipid content (%) = weight of lipid (g) / weight of dried biomass (g) x 100 Morphological and molecular identi cation of the most potent fungal isolates The promising lipid producers were kindly identi ed morphologically at the Mycological Center, Assiut University, Assiut, Egypt.
Identi cation was performed considering some speci c morphologic characteristics, such as colony diameter, color, texture appearance, and microscopic examination was done by lactophenol cotton blue staining including conidiophore, vesicle, metulae, phialides and conidia (Larone 2002).
The promising selected oleaginous fungi were molecularly identi ed based on the 18S rRNA sequences. Fungal strains were cultivated on PDA medium at 28 °C for 5 days. A small amount of the fresh culture was scraped and suspended in 100 µL autoclaved distilled water in a sterile Eppendorf vial (2 ml capacity) and boiled in a water bath at 100 °C for 15 minutes. The non-living fungal strain was sent to the Molecular Biology Research Unit, Assiut University, Egypt for DNA extraction using patho gene-spin DNA/RNA extraction kit provided by Intron Biotechnology Company, Korea. The (ITS) region of the rRNA gene was ampli ed by polymerase chain reaction (PCR) at SolGent Company, Daejeon South Korea using two universal fungal primers ITS1 (forward) and ITS4 (reverse). Primers have the following composition: ITS1 (5' -TCC GTA GGT GAA CCT GCG G − 3'), and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC -3'). The puri ed PCR product (amplicon) was recon rmed using a size nucleotide marker (100 base pairs) by electrophoreses on 1% agarose gel. Then these bands were eluted and sequenced with the incorporation of dideoxynucleotides (dd NTPs) in the reaction mixture. Each sample was sequenced in the sense and antisense directions using ITS1 and ITS4 primers (White et al. 1990). Sequences were further analyzed using Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website. The phylogenetic tree was constructed by the neighbour-joining program in MEGA 5.05 software.
Optimization of culture conditions to maximize lipid production To enhance biodiesel production from the tested fungal strains, the in uence of the studied nutritional and environmental parameters on the fungal growth and lipid production are discussed under static condition for the promising fungal isolates (A. terreus, A. niger and A. avus).

Effect of carbon and nitrogen sources on lipid production
The effect of different carbon sources on the lipid production was studied using the basal culture medium supplemented with (5%) glucose as a control. An equimolar amount of eight different carbon sources were tested as fructose, lactose, sucrose, maltose, dextrose, starch, wheat bran, and corn. Also for nitrogen source, yeast extract was replaced on equal nitrogen bases (0.5 g/L) by six different nitrogen sources as ammonium nitrate, sodium nitrate, peptone, ammonium acetate, casein, and soybean one at a time.

Effect of initial pH and incubation period on lipid production
The initial pH of the culture medium was adjusted with 1N HCl or 1N NaOH before autoclaving at different values ranging from 4 to 9 under static condition for 7 days. The effect of incubation period was detected at different time intervals 3, 5, 7 and 9 days.

Effect of incubation temperature on lipid production
The effect of different incubation temperatures was evaluated as following 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C for 7 days.

Methylation of lipid and transesteri cation
The transesteri cation reaction of the obtained tested lipids was performed according to Radwan (1978) with slight modi cation. The lipid sample (5 mg) is dissolved in benzene (2 ml) in a test tube tted with a condenser, and 1% sulfuric acid in methanol (2.0 ml) is added, close the tube well and place in water bath at 90 °C for an hour and half. Cool, add 8 mL water and 5 mL petroleum ether shake and separate out the ethereal layer in a dry tube.

Gas chromatography (GC)
Gas chromatographic analysis of FAMEs for promising fungal isolates were performed using Hewlett Packard (HP) 6890 GC system at Central Laboratories of General Health Institute, Alexandria, Egypt.

Results And Discussion
Screening of different oleaginous fungal strains for their lipid production Ten locally fungal isolates were obtained, ve from Moharam-Bek and ve from Amrya oily polluted soil gas stations, Alexandria, Egypt.
The selected fungal isolates were tested for their growth and lipid accumulation under static condition ( Table 1). The highest lipid value   Phylogenetic tree was constructed after alignment with the related strains with a percentage of similarity (Fig. 2).
Optimization of culture conditions for the highest lipid production Optimization of the culture conditions was a signi cant strategy to enhance the lipid accumulation with economical cost e ciency (Jiru et al. 2017).

Effect of different carbon sources on lipid production
The present results (

Effect of different nitrogen sources on lipid production
The results as shown in Table 3   The external pH of the medium was an important environmental factor affecting plasma membrane permeability, metabolic activity, and  (2014) who recorded that pH values ranging between 5 and 6 were found to be the suitable pH range for most oleaginous fungal growth and lipid production. In uence of incubation period on lipid production Incubation period has an observable effect on biomass lipid production. Lipid content of each strain differs depending upon its speci c growth rate. In the current study, the lipid accumulation and dry biomass increased gradually during the rst seven days of incubation and reached its maximum values (38.33%, 0.24 g/50 mL) and (16.84%, 0.19 g/50 mL) at the 7th day of incubation for A. terreus and A. avus, respectively (Table 5)  In uence of incubation temperature on lipid production In the case of studying the effect of incubation temperature in the present survey, the highest fungal biomass and lipid productivity were  Fatty acid composition of the potent fungal lipid producer The fatty acid composition of Aspergillus terreus as the most potent fungal isolate was cited in (Fig. 4, Table 6).  In the current study, a higher number of saturated fatty acids than polyunsaturated fatty acids was observed that indicates the fungal oil obtained has properties similar to those of biodiesel, which con rms its superior biodiesel quality (

Conclusion
Experimental data revealed the goals of the work, promising fungal isolates Aspergillus terreus, Aspergillus niger and Aspergillus avus exhibited satisfactory lipid accumulation and constructed for biodiesel production. Optimization of cultural conditions for maximum lipid production (38.33%) was achieved on the seventh day of growth at 30 °C incubation temperature in a static condition, using 5% sucrose, 0.5 g/L ammonium nitrate with initial pH 6.0 for Aspergillus terreus as a highest lipid producer. FAME pro le indicated the presence of higher saturated fatty acid fraction compared to unsaturated fatty acids of the tested fungal species. The total concentration of fatty acids was 107.98, 38.29, and 37.48 mg/100 g of lipid accumulated by A. terreus, A. niger and A. avus, respectively. Gas chromatograph analysis of A. terreus lipid as the potent fungal strain revealed that oleic acid (C18:1, 18.51%) was the most abundant fatty acid, followed by stearic acid (C18:0, 15.91%) and Myristic acid (C14:0, 14.64%). Therefore, fatty acid pro le of A. terreus has con rmed its potentiality as a new commercial biodiesel feedstock.

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Competing interests:
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