Influence of the Different Carbon and Nitrogen Sources on the Production of Biodiesel by Oleaginous Fungi Aspergillus terreus, Aspergillus fumigatus

In the present study, the growth and total lipid contents of two oleaginous fungal isolates Aspergillus terreus, Aspergillus fumigatus were compared in different nitrogen and organic carbon sources. Artificially the fungi were cultured on media consisting of various monoor dior polysaccharides and peptone or yeast extract as elementary sources for carbon and nitrogen, respectively. Media containing sucrose /yeast extract or glucose/ yeast extract were the most effective for lipid production from fungal, during two weeks incubation period, the highest biomass of dry weight was (19.6 , 18.8) g / L , (25.8 , 30.5) g /L and lipid yield (1, 0.97 )g/L, (0.65, 0.65) g/ L for two isolates Aspergillus terreus and Aspergillus fumigatus respectively then followed maltose/ yeast extract then starch / yeast extract . Analysis of fatty acid produced by two fungal isolates using Gas Chromatography showed the existence of a wide range of fatty acids, these fatty acids were together saturated and unsaturated. The results revealed that the unevenness in the fatty acids composition mainly depends on the type of carbon and nitrogen sources. The existence of saturated and mono saturated fatty acids in A.fumigatus, A. terreus prove that they are good candidate for biodiesel production.


Introduction:
Many governmental and industrial efforts are looking to find alternative fossil fuels that are renewable and carbon free. One of these alternative is biodiesel derived from microorganisms like algae, bacteria and fungi (1).However, little information has been documented about the lipids utilization from bacteria and fungi for the production of biodiesel (2). Fungi are a good source of lipids used in biodiesel production (3).
Oleaginous microorganisms accumulate lipids Single Cell Oils(S.C.Os) such as yeasts, fungi, bacteria and micro-algae, with more than 20% of their cell mass being consisting of lipids, have appeared as a potential feedstock for biodiesel production (2,4).
There are numerous factors which hearten the use of fungi for production of lipid. For example, the accumulation of lipid in oleaginous fungi has been observed when an essential element in the medium (such as the nitrogen source) comes to be inadequate and the carbon source becomes an addition (5) .
The nitrogen in oleaginous species is favorably directed toward the synthesis of lipid, through the Triacylglycerol (TAG) accumulation that occurs within intracellular lipid bodies (6). The nitrogen depletion in oleaginous species plays a key role in stimulating the accumulation of lipid in Mucor circinelloides and Gliocladium roseum which was done at conditions of stressed like high glucose -to yeast extract ratio and elevation temperature ( 7,8).
Several species of molds and yeasts could accumulate substantial amounts of intra-cellular lipid (9) The production of lipid can be adjusted by adding essential nutrients to culture media and/or by changing culture growth conditions (10). Carbon and nitrogen sources have the effect on the biomass and production of lipid in oleaginous fungi. (11) found glucose in syrup of date and sucrose in liquid medium is most suitable for the biomass growth of A. terreus and lipid production. present study focuses on two oleaginous fungal isolates and optimizes medium conditions in different carbon and nitrogen sources for increasing the activity of oleaginous fungi for the production of biodiesel.

Materials and Methods
Two oleaginous fungal isolates A. terreus, A. fumigatus were obtained from Department of Biology, College of Science., Mustansiriyah university, Baghdad, Iraq which are isolated from different Iraqi environments then cultured on potato dextrose agar at 28º C for 5 days .

Utilizing Different Carbon sources for Lipid Production
Different carbon types were used as a sole source of carbon in basal medium : monosaccharide (glucose, sucrose) disaccharides (maltose) and polysaccharides (starch) the medium prepared by dissolving 20g yeast extract,150g carbon source type in 1000ml distilled water and the medium was sterilized in autoclave for 20 minute under 15 lbs/In 2 pressure at 121℃.

Utilizing Nitrogen source for Lipid Production
Pepton and yeast extract were used as nitrogen source in basal medium both alone. Dissolving 20g type of nitrogen source ,150 g sucrose in 1000ml distilled water and the medium was sterilized in autoclave for 20 minute under 15 lbs/In 2 pressure at 121℃.

Determination of the Biomass for Oleaginous Fungi
The two oleaginous fungal isolates ( A. terreus, A. fumigatus) were cultured in basal medium each500ml of medium was set in one liter Erlenmeyer bottle and supplemented with five discs from fungal inoculum in diameter 5 mm for each fungal isolate at pH 6.0 and incubated for 14 days at 30 ºC, then the fungal growth was noticed on 14 th day. The Biomass was gained using No.2 filter paper (whatman) inside a cabinet for biological safety. The biomass collected was twice washed with water (distilled water) and for 24 h dried at 60 ºC or till the weight became constant then the dry weight was esteemed gravimetrically mg /L (12).

Extraction of Fugal Lipid
The lipids were extracted from dried biomass by using solvent system methanol 2:1(v/v) chloroform: methanol. The isolates samples of fungal dried were sumashed with mortar and pestle by consecutively adding (5ml) of methanol and (10ml) of chloroform then 8ml of this blend was pulled out and added to one gram of biomass then vortexed for 5 minutes. The solution prepared from normal saline containing (NaCl (7.3g) dissolved in 10ml of water) was pulled out (2ml) then added for each tube after being vortexed for 5 min . At 3000 rpm for 15 minutes. The sample tubes were centrifuged and the lower layer of NaCl, water and methanol was excluded by pipette. The remaining of solvent that was dried then the percentage of the cell dry weight to extracted lipids were assessed gravimetrically in mg /L (8).

Analysis by Gas Chromatography and Biodiesel Production
The total lipids from dried biomass in the previous item were extracted.The compositions of fatty acid of the produced lipid by isolates of fungi that were extracted from the previous items were determined by depending upon the method of (13) (analysis of fatty acid methyl esters (FAMEs)) with some change. By trans-esterification reaction, the FAMEs were produced. 2.5% V/V H 2 SO 4 /CH 3 OH and 2 ml of methanol was added as a catalyst to 100 mg of the crude lipid.The reaction was proceeded at 90 C for 45 min (water bath). Then, 2 mL n-hexane and 1 mL H 2 O were added. The Fatty Acid Methyl Esters were liquefied into the n-hexane. The solution at 2000 rpm was submitted to centrifugation for 15 min to get FAMEs within the compact the water from the hexane phase , then transmitted into ampoules made glass by using pipettes of Pasteur. The fatty acid methyl esters in n-hexane were examined after additing 0.1ml of solution (methanol 1% W/ V, 1ml of heptane and KOH,) to the Fatty Acid Methyl Esters by using a gas chromatograph. The weight proportions of the fatty acids were analyzed, a gas chromatography was used to (GC) analyzer (made in Kyoto, Japan GC-17A model by Shimadzu Inc.,) with the Chromatography Data Management system (Advantech Inc., Taipei, Taiwan), (6890N, Agilent, Japan) equipped with an supelco wax tm 10 capillary column (Agilent, Japan, length 30 m 0.32 mm I.D, 0.25 Mm flim thikness, substance fused silica) and a flame ionized detector (FID).The functional settings were as follows : temperature of oven 180 C , and helium air were used as carrier gas. The detector temperatures 260 C and the injector temperatures 250 C .

Results and Discussion
Biomass and total lipid yields The yield of lipid and the highest biomass of A. terreus isolate were determined , after 14 days of growth in liquid medium under the different carbon (glucose, maltose, sucrose, starch) and rich, nitrogen source (yeast extract). The results indicated a quantitative biomass. The highest value of the dry weight of biomass was 19.6 g/l, 18.8 g/l to carbon sources sucrose and glucose respectively and the lowest value was 15.2 g /l at starch carbon source, while for the medium culture which consisted of carbon source, sucrose and pepton as nitrogen source the total biomass recorded 8.0 g/L (Fig. 1).

Figure 1. Dry Weight of fungal biomass g /l of A. terreus in basal medium at different Carbon and Nitrogen sources, pH adjacent to7 and incubated at 120 rpm at 30 ̊ C for 14 days.
The lipids extracted from biomass was estimated .The maximum harvest of lipids was 1 g /L by using sucrose as source of carbon and yeast extract as source of nitrogen 0.9 g/ L (Fig. 2).

Figure 2. Total Lipid (g / L) of A. terreus in basal medium
at different Carbon and Nitrogen sources, pH adjacent to7 and incubated at 120 rpm at 30 ̊ C for 14 days.
While The best biomass Productivity,A. fumigatus showed the optimium conditions in liquid medium enriched with maltose as carbon source. The dry weight of biomass recorded the highest value at 30.5 g /L and 26.7 g /L with glucose and yeast extract respectively as nitrogen source as seen in Fig. 3.

Figure 3. Dry Weight of fungal biomass g /l of A. fumigatus in basal medium at different Carbon and Nitrogen sources , pH adjacent to7 and incubated at 120 rpm at 30 ̊ C for 14 days.
However the highest value of lipid extracted from liquid medium was enriched with carbon source sucrose 1 g/L and yeast extract 0.9 g/ as nitrogen source as shown in Fig. 4. Among the various carbon sources tested, the disaccharides sucrose (consist from two glucose molecules ) and monosacchride glucose were the best organic carbon source for A. fumigatus and A. terreus and yeast extract the better nitrogen source for biomass and total lipid yield in comparison to other organic carbon and nitrogen sources. The most commonly carbon source employed for growing fungal oleaginous and lipid production is the glucose Moreover, the fatty acids composition of the lipids was affected by the concentration of initial glucose .These results agree with other studies around getting lipids for the production of biodiesel from a number oleaginous fungi isolates specially Aspergillus spp. (14,15,16,11).

The production of Biodiesel and Gas Chromatography -GC analysis
The extraction of lipids for two fungal isolates to fatty acid methyl esters were detected by GC -Gas Chromatography .As exposed in Table1 and 2, the profiles of fatty acid by Gas Chromatography with RT (Retention Time) revealed the presence of Oleic acid and Palmitic acid mostly in two isolates selected in different carbon sources: glucose, sucrose, maltose and starch which provided the maximum concentrations between the others kinds of Stearic acid , fatty acids, Linolienic. Palmitic acid, or hexadecanoic acid, is the most common fatty acid (saturated) and it is a main constituent of the biodiesel (17).
Mainly, fungal oils contained higher fraction of saturated fatty acids like palmatic, stearic and myristic acid than unsaturated fatty acids oleic and linoleic acid in the fatty acid methyl esters analysis. The biodiesel quality is dependent on the fatty acid profile of the oil used as feedstock for its production. Therefore, the production of biodiesel, the lipids obtained should be analyzed and compared with standard specifications for renewable diesel (18,19).

Conclusion:
The results of present study reveal that the Yeast Extract Sucrose (YES) medium was the best medium for growth and lipid production of oleaginous fungal isolates Aspergillus terreus, Aspergillus fumigatus. The hydrocarbon profile of mycodiesel extracted from oleaginous fungi have number of compounds normally associated with diesel fuel which containing saturated more than unsaturated fatty acids