الفهرس 
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Abstract
This study deals with isolation of soil origin oleaginous filamentous fungi with valued unsaturated fatty acids and investigates the effect of various parameters on biomass and lipids production of the isolated fungi. Also study the feasibility of using different wastes as an alternative carbon sources for oil production. So, about 95 fungi were isolated from different Egyptian localities and from these isolates two fungal isolates exhibited the highest lipid content, so they were selected for further studies and were identified as Alternaria citri and Pyrenophora nobleae.
Accordingly, to maximize lipids production, various nutritional, environmental and cultivation parameters were optimized using one variable at a time (OVAT) strategy followed by employing a Plackett-Burman statistical design (PBD) and Response surface methodology (RSM). Results of OVAT indicated that the best carbon source, nitrogen source, aeration conditions and temperature for maximum lipid accumulation by each fungus were sucrose, yeast extract, static conditions and 30 °C, respectively. On the other hand, the best carbon/nitrogen ratios were 30:1 and 30:2 for Alternaria citri and Pyrenophora nobleae, respectively.
Results of PBD exposed that sucrose, yeast extract and incubation days were found to be critical factors affecting lipid accumulation of Alternaria citri. While, incubation time, FeSO4.7H2O, pH and yeast extract were the most significant factors influencing lipid accumulation of Pyrenophora nobleae. Therefore, central composite design (CCD) was employed to optimize the optimum levels of these significant factors.
Under optimized conditions, Alternaria citri give a total biomass of 11.8 g/L with a lipid content of 50.3 % (corresponding to a lipid yield 5.94 g/L), after 160 h of incubation at 30 °C, pH 6.5, sucrose 35 g/L, yeast extract 2.33 g/L, KH2PO4 2 g/L, MgSO4.7H2O 0.25 g/L, KCl 0.25 g/L and FeSO4.7H2O 0.02 g/L.
On the other hand, Pyrenophora nobleae give a total biomass of 12 g/L with a lipid content of 40.75 % (corresponding to a lipid yield of 4.89 g/L), after 144 h of incubation at 30 °C, pH 8.5, sucrose 45 g/L, yeast extract 2 g/L, KH2PO4 2 g/L, MgSO4.7H2O 1 g/L, KCl 1 g/L and FeSO4.7H2O 0.015 g/L. Under optimized conditions the fatty acid methyl ester profile (FAME) for Alternaria citri revealed a percentage of 59.68 of unsaturated fatty acids (USFAs), with linolenic acid as the most dominant one (32.62 %). Whereas, a percentage of 41.99 USFAs for Pyrenophora nobleae with palmitic acid as the dominant one (35.06 %).
The Possibility of using different wastes as substrates for growth for lipid production for each fungus was studied. Results proved that orange peel and molasses represent potentially valuable sources for single-cell oil with desirable quantities of valuable unsaturated fatty acids. For Alternaria citri the fungus give oil yield of 4.5 g/L with orange peel and 5.6 g/L with molasses. Moreover, Pyrenophora nobleae give oil yield of 3.65 g/L with orange peel and 4.14 g/L with molasses.
Large scale production studies were performed by cultivation of each fungus on sucrose and molasses in a 5L bioreactor, and results revealed that cultivation of Alternaria citri on sucrose and molasses in bioreactor favored biomass formation while lipid yield was lowered compared with shaked flask. Similarly, bioreactor trial of Pyrenophora nobleae indicated an increase in biomass when sucrose was utilized as a carbon source. In contrast, when the fungus was cultivated on molasses a decrease in biomass and increase in lipid yield was recorded. Interestingly, it was observed that bioreactor trials of Alternaria citri cultivated on molasses and Pyrenophora nobleae cultivated on sucrose resulted in increment in percentage of USFAs to nearly 62 %.
Molecular detection of specific genes responsible for lipid biosynthesis showed that malic enzyme and acetyl coenzyme-A carboxylase were amplified at 2000 bp and 1000 bp, respectively for each fungus. While, primers of ATP citrate lyase gene and elongase doesn’t give any ampilcons.