Polyunsaturated fatty acids (PUFAs) are used as food and pharmaceutical additives for different biological activities. Omega-3 and -6 PUFAs are used to treat many diseases. Gamma linolenic acid (GLA) has beneficial impacts on health. Dihomogamma linolenic acid (C20:3) and arachidonic acid (C20:4) originate from GLA in n-6 pathway. These PUFAs are converted to eicosanoids, such as leukotrienes and prostaglandins, in humans. Omega-6 PUFAs have nutraceutical and medical applications in reduction of blood lipid levels, inflammation, and atherosclerosis. The lack of omega-6 PUFAs in the diet causes dry hair, hair loss, and poor wound healing (1). The role of docosahexaenoic acid (DHA) and other omega-3 PUFAs in improving cardiovascular diseases has been shown in many in vivo and in vitro experiments (2). Both eicosapentaenoic acid and DHA can improve blood pressure and prevent the development of hypertension (3). Some studies have converted renewable waste sources to edible products, such as enzymes, organic acids, carbohydrates, biodiesel, microbial lipids, and other products (4-10). Vegetable and animal oils can be replaced by microbial oil. Fungi can produce valuable oils containing PUFAs. Recently, microbial lipids have been validated as nutritious for infants (11-12). Several microorganisms, including Cunninghamella echinulata, Mucor rouxii, Mortierella alpina, Mucor circinelloides, Mortierella isabellina, Cryptococcus curvatus, Crypthecodinium cohnii, and Yarrowia lipolytica, produce popular high lipid products (13-17). They use a broad range of common substrates, such as glucose, xylose, lactose, starch, and cellulose, as well as some commercially important industrial and organic wastes. Various oils are used during food preparation in restaurants and factories and particularly in fast food. Oil wastes from oil factories and fried products cause serious environmental problems. For example, oil waste can increase biochemical oxygen demand (BOD) and chemical oxygen demand (COD) in water resources, resulting in severe water pollution. Microbial degradation of oil wastewater has been investigated in earlier studies (5, 18, 19). Papanikolaou et al. (2007) used olive mill wastewater-based media to produce citric acid. Laconi et al. (2007) utilized the same substrate to culture a mixture of fungi to obtain a potentially useful microbial biomass and induce partial bioremediation of a particular waste. In present study, the efficacy of bioconversion various oil wastes to lipids via two oleaginous fungi, namely Cunninghamella echinulata DSM1905 and Rhizopus stolonifer DSM2194, were analyzed on Microscopic observations of the fungi showed the existence of lipid globules. Changes in total lipids, biomass, fatty acids, and pH were examined in a time course. The ability of these fungi to degrade oil wastes were examined by measuring BOD and COD.
Although the capacity of oleaginous fungi to convert lipids and fatty acids has been investigated, the effects of lipid metabolism by fungi on changes in BOD and COD have not been evaluated. In this study, the ability of oleaginous fungi to utilize oil wastes as carbon sources to reduce BOD and COD of the wastes were investigated. Our results may be useful to investigate the abilities of C. echinulata and R. stolonifer to produce value-added lipids.
3. Materials and Methods
All chemicals were obtained from Sigma-Aldrich with analytical grade. The oil wastes from different industries including restaurants and fried food factories were collected in Isfahan, Iran. The samples were abbreviated as F1 and F2 for waste oils from fried food factory of Shilan Kish, F3 for the sample from Naz plant oil factory, and R1 and R2 from two different fast food restaurants.
3.2. Fungal Culture
Cunninghamella echinulat DSM1905 and Rhizopus stolonifer DSM 2194 were
purchased from the German Collection of Microorganisms (DSMZ, Braunschweig, Germany). Preparation of media was carried out according to method described by Papanikolaou et al. (1). The inoculated culture medium contained (perliter
of distilled water) 7.0 g KH2PO4, 2.5 g Na2HPO4, 1.5 g MgSO4, 0.06 g MnSO4, 0.15 g CaCl2, 0.15 g FeCl3, 0.5 g yeast extract, 0.5 g (NH4)2SO4, pH 6.0, and 2% (V/V) oil waste as a carbon source. The media were inoculated by spores (1 × 107) and cultivated at 20ºC for 72 h with shaking at 180 rpm.
Cell dry weight was measured 72 h post inoculation, and mean values from 3 replicates were used for further analysis (14). C. echinulata DSM1905 and R. stolonifer DSM 2194 were passed through Whatman No. 1 filter paper and washed with a large amount of distilled water. To remove unconsumed-oil, an additional washing was carried out using ethanol. The cells were dried down at 105ºC. The dry cell weight was measured gravimetrically.
3.3. Microscopic Observation of Lipid Droplets
Samples were obtained from production media after 72 h and stained by Sudan Black B, according to the method developed by Zhou (18) and visualized under light microscope.
3.4. Extraction and Modification of Lipids
Lipid extraction was performed according to the modified procedure of Bligh and Dyer (1959). Briefly, mycelia were obtained through Whatman No.1 filter paper, washed three times by distilled water and once by ethanol. HCl solution (10 mL of 4M) was added to 1 g mycelia, and incubated at 60ºC for 2 h. The hydrolyzed solution was shaken with 20 mL of chloroform/methanol (1:1) at room temperature for 3 h, and centrifuged at 2000 ×g for 5 min. The upper aqueous phase was separated by a Pasteur pipette and the lower phase containing lipids was evaporated under reduced pressure for 10 min (19).
The extracted fatty acids were modified to fatty acid methyl esters (FAMEs) according to the method of Christie (20). Lipid (50 mg) was dissolved in 1 mL toluene in a test tube fitted with a condenser. Sulfuric acid (2 mL) in methanol (1% v/v) was added to the lipid mixture and maintained at 50ºC for 24 h. The methylated fatty acids were separated from the aqueous layer by adding 5 mL of NaCl (5%) and dissolved in n-hexane. The separated n-hexane layer washed by 4 mL of water containing potassium bicarbonate (2%) and dried over anhydrous sodium sulfate. Solution is filtered and the solvent removed under reduced pressure in a rotary film evaporator.
3.5. FAMEs analysis by Gas Chromatography (GC)
GC was carried out using Agilent 19091J-413 Series Gas Chromatograph equipped with a FID and the capillary column DB-23 (USA). The temperatures of injector and detector were maintained at 260 and 300ºC, respectively. The oven was programmed as 100ºC: 2 min; 160°C: 3 min; 215ºC: 2 min; 217ºC: 2 min; 218ºC: 2 min;, and 260ºC for 2 min. Nitrogen was used as the carrier gas at a flow rate of 1.5 mL/min. The injection volume was 1 mL, with a split ratio of 50:1. The standard FAMEs (Sigma-Aldrich) including C18:2 (Linoleic acid), C18:3 (GLA), C18:3 (alpha- linolenic acid), C20:4 (ARA), C20:5 (EPA), C22:6 (DHA) were assayed by GC and the standard elution profile was obtained. The Production of FAMEs from the extractions of C. echinulata DSM1905 and R. stolonifer DSM 2194 were assayed by comparing retention time from extracts to those of standard FAMEs.
3.6. BOD and COD Determinations
BOD was determined using the modified iodometric method and COD was determined by the standard closed reflux method according to the procedures described by Clesceri et al. (1998). The BOD and COD degradation efficiency was defined as the reducing amount of BOD and COD versus the amount of initial values (21).
3.7. Statistical Analysis
All the experiments were performed in triplicate, and data presented as means ± SE. A one-way ANOVA was used toevaluatewhethertherewasastatistically significant difference between data. Statistical analysis was performed using SPSS 15.0 (SPSS Inc., USA), and tests were considered significant at p < 0.05.
4.1. Cell Culture and Lipid Assay
The fungal strains of C. echinulata DSM 1905 and R. stolonifer DSM2194 were cultured under nitrogen-limited conditions in flasks containing five different oil wastes (2%), and the dry cell weights were measured gravimetrically. Oil particles were observed in both strains and appeared as black droplets after Sudan Black B staining (Figure 1).
All oil wastes were subjected to assays for total lipids, biomass, and pH at the indicated time intervals (Figure 2). The maximum yields of all oil wastes were achieved 72 h after inoculation.
4.2. Fatty Acid Determination and Gas Chromatography (GC)
A fatty acid analysis of cellular lipids was performed in both strains. The fungal cells were disrupted, and the lipids were extracted to assay the types and quantities of lipids. The lipid-containing fractions were separated and modified to obtain fatty acid methyl esters (FAMEs). The FAMEs were subjected to GC analysis. The GC results were analyzed based on the standard peaks obtained from FAME standards (Table 1).
Both R. stolonifer and C. echinulata produced lipids. The yield of C. echinulata lipid production was about 65% in F3 oil waste medium, but in F1, F2, and R1 wastes was less than that of R. stolonifer. Linoleate and GLA were the typical omega-6 fatty acids produced by the two strains in all oil waste media. C. echinulata cultured in F3 oil waste medium demonstrated maximum linoleate content (46.7%), but minimum GLA content (0.63%) of total FAMEs. Linolenic acid was produced by both strains in F3 and R1, and DHA appeared in F1 for the two strains. ARA concentration varied in relation to media and strain. Initial pH was 6.1, but decreased to 3.9 after 72 h and remained at this value until 192 h (Figure 2).
4.3. BOD and COD Assay
BOD and COD in all oil wastes decreased significantly by both strains. The changes in BOD over 5 days are shown in (Figure 3).
Figure 4 shows the reduction of BOD5 in all oil wastes, and the final BODs were 67%-75% and 37%-55% of the initial values for C. echinulata and R. stolonifer, respectively. About 98% reduction in COD was achieved by C. echinulata in F5 and other decreases in COD were remarkable. In contrast, R. stolonifer decreased COD in F2, F3, and R2 (Figure 5).
Oil wastes from factories and restaurants may provide suitable carbon sources for microorganisms to produce lipids. Sewage sludge has been used to produce oil by Papanikolaou and Aggelis (2002). Fungi and yeasts can grow on waste products, such as glycerol, which is a biodiesel byproduct (22). Hydrophilic materials and molasses have been applied to single Cunninghamella echinulata cells to make oil (23). Tomato hydrolyses waste was used by this fungi to produce fatty acids (15). Food waste, olive oil mill wastewater, industrial fats, whey, and starch hydrolysates are other wastes that have been used by oleaginous fungi to produce lipids (24-27). The filamentous fungus Mortierella alliacea collects arachidonic acid, particularly in the form of triglycerides, in its mycelia and yields about 46.1 g.L-1 dry biomass. In the present study, oil wastes were obtained from food factories and restaurants. Lipid and FAME analyses of C. echinulata and R. stolonifer showed that these wastes were good substrates to produce unsaturated fatty acids, such as omega-3 and omega-6 PUFAs. The best results were found when R1 and R2 oil wastes were used as the carbon sources, as about 19-21% GLA was obtained from the R1 and R2 wastes by C. echinulata, respectively. Our results showed when linoleate and linolenate increased, GLA concentration decreased, particularly when using R2 and F3 oil wastes as substrates. Our data confirmed the results of Fakes et al. (2008) who determined lipids from C. echinulata with emphasis on the g-linolenic acid distribution (27). Linoleate production by the two strains was remarkable from all oil wastes, but R. stolonifer yielded more alpha linolenate. DHA was produced only when C22:0 was present in the oil wastes, and arachidonic acid appeared when C20:0 was in the substrate mixture. The two strains produced GLA, even though this fatty acid was not found in the wastes. Chen and Liu reported that C. echinulata cultivated on various carbon sources produces high biomass quantities but that the fungus did not accumulate large amounts of stored lipids (19, 28, 29). The same zygomycetes strain, C. echinulata ATHUM 4411, produced 7.8 g.L-1 SCO (single cell oil) and about 800 mg.L-1 GLA when cultivated on tomato hydrolyzate enriched with glucose (27), whereas growth on potato starch was accompanied by production of 540 mg.L-1 GLA (28). Additionally, C. echinulata strain CCRC 31840 produced 964 mg/g GLA when grown on starch, whereas it produced 1,349 mg.g-1 GLA after optimizing the inoculum (27, 29). Production of arachidonic acid by Mortierella alpina CBS 754.68 was evaluated in submerged fermentation (30). Our data showed that the lipids produced were related to the substrate type and indicated that PUFA biosynthesis is strongly associated with mycelial growth. The economic restrictions regarding application of microbial lipids as renewable sources for fatty acid production can be overcome by using a cheap feedstock. Finally, the impacts of lipid metabolism on changes in BOD and COD were significant in the presence of C. echinulata and R. stolonifer. Therefore, our results may help to overcome some environmental problems.
The authors desire to thank Ph.D students Gholamreza Ghezelbash and Hossein Ghanavati for their guidance’s, and Elham Shirani for their assistance with the experiments.