FAMEs Profile of Oil Produced by Oleaginous Fungi Isolated from Fermented Beverage Wastewaters and Soil

Fungal strains isolated from fermented maize (ogi) (PW) and sorghum-based brewery wastewaters (BW) and two soil isolates were evaluated for oleaginicity. The fungal isolates from the wastewater that had lipid content of at least 20% of their biomass were identified by both culture methods and internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA sequencing. The isolates were identified to be Aspergillus fumigatus (PW8), Aspergillus flavus (PW10), Candida tropicalis (PW16) and Aspergillus tubingensis (PW3), Trichosporon luoberi (BW7), Aspergillus sp. (BW4) and Candida tropicalis (BW1; BW3). FAMEs composition was determined for the four strains with the highest lipid content by acidcatalyzed transesterification and analyzed by Gas Chromatography-Flame Ionization Detector (GC-FID). Palmitoleic acid was the dominant fatty acid in M. circinelloides and T. reesei, and the best producers of capric and lauric acids were Aspergillus fumigatus and Aspergillus sp. (BW4), respectively. These fatty acids are beneficial in making cosmetics and pharmaceuticals (antimicrobials and dietary supplements). The analysis of the FAMEs profile in the species indicated low amounts or absence of some key long chain fatty acid (LCFA) constituents of biodiesels. Based on the FAMEs profile of M. circinelloides investigated, this strain could hold promise for use as feedstock for biodiesel with genetic engineering and a tailored lipid production favouring enrichment of LCFA.


Introduction
Filamentous fungi have broad application as production hosts in the industry mainly for their capacity to secrete metabolites (Peberdy, 1994, Punt et al., 2002Wosten, 2019). Microbial oils are secondary metabolites produced by bacteria, yeast, fungi and microalgae and are accumulated within specific organelles like lipid bodies within the cell (Kosa and Ragauskas, 2011). A microorganism is said to be oleaginous when it can accumulate more than 20% of biomass as lipids (Ratledge and Wynn, 2002;Thevenieau and Nicaud, 2013). Microbial systems that can produce and store oil have attracted significant research attention in recent years (Bharathiraja et al., 2017), especially triacylglycerols (TAGs) produced from oleaginous microorganisms as the supplementary sources of conventional oil for biodiesel production (Thevenieau and Nicaud, 2013). Oleaginous fungi include Mucor circinelloides, Candida tropicalis, Pythium ultimum, Morietella isabellina, Aspergillus terreus, Claviceps purpurea, Pellicularia praticola (Thevenieau and Nicaud, 2013). These fungi can also grow by utilizing sugarcane molasses, soluble starch and wheat straw (Lin et al., 2010). Nitrogen limitations in the substrate are required for the accumulation of lipids (Ratledge 2002;Calvey et al., 2016).
Microbial oil extraction processes are simpler to handle and require less labor (Li and Wang, 1997;Li et al., 2008) compared to plant oil extraction procedures. Plant oil extraction requires the processing of the specific plant tissue (Atabani et al., 2013) as opposed to microbial oil extraction in the aqueous phase or ultrasonication (Zhang et al., 2014). The procedure for oil extraction is too expensive to apply for production on a large scale, therefore the viability of production of microbial oil is dependent on the high conversion of substrate to intracellular lipids combined with high extraction efficiency at low energy consumption (Bharathiraja et al., 2017).
Biodiesels are defined as the fatty acid alkyl monoesters (FAMEs) derived from renewable sources, such as vegetable oils and animal fats. They are produced by transesterification of the lipid with an alcohol especially methanol (Yap et al., 2011). The environmental benefits of using biodiesel over petroleum diesel include biodegradability, lower sulfur and aromatic hydrocarbon content and the reduction in the emissions of carbon monoxide, carbon dioxide and particulate matter (Meher et al., 2006;Sawangkeaw and Ngamprasertsith, 2013). The main disadvantage of this process is the unavailability of feedstocks and substrates, which accounts for 70% of the final cost of the biodiesel (Hanna, 1991;Thevenieau and Nicaud, 2013).
There is an increase in the demand for renewable fuel due to the limited energy resources and the environmental hazards that fossil fuels pose to the environment. Biodiesel is being increasingly investigated as an alternative to fossil fuels in modified combustion engines for transport as well as in engines for power generation (Gavrilescu and Chisti, 2005;Bharathiraja et al., 2014).
The composition and the yield of microbial oils are affected by cultural conditions such as substrates, incubation period, nitrogen source, pH and aeration of the culture medium (Liu et al., 2010). The yield and type of lipid are dependent on several factors like the type of organism, culture conditions and the substrate chosen (Ledesma-Amaro et al., 2016). The use of pure fungal isolates from environmental sources as feedstock for the production of fatty acid methyl esters relevant in biodiesel, and pharmaceuticals and cosmetics was investigated.

Materials and Methods
Source of fungal isolates Mucor circinelloides (IYN 13) and Trichoderma reesei (IYN 15) are laboratory stock strains (unpublished data) that were isolated from the soil. These strains were selected because of other reports on their oleaginicity. All the other fungi from this study were obtained during the screening of untreated wastewater from two sources-fermented cereal "ogi" (PW) and a brewery (BW) in Ogun State, Nigeria. The brewery uses sorghum as the substrate for its fermentation process and corn was the substrate for ogi. The wastewater samples were collected in sterile 1 litre plastic bottles and were processed within 24 hours of sample collection.
The mixture of the wastewater and the enrichment medium was then cultured at 28 o C, with agitation using orbital shaker at 180 rpm for 48 hours. Serial dilution was carried out on an aliquot of the incubated medium, and cultured on PDA (Himedia, India) supplemented with chloramphenicol and Rose Bengal Chloramphenicol agar (Oxoid, UK). Incubation was done at 35 o C for 5 days. Pure cultures were then obtained from mixed culture plates. Morphological appearances of the inoculated plates (at room temperature) were observed and distinct colonies were subcultured to obtain pure isolates which were then maintained on Potato Dextrose Agar slants and stored at -20 o C.
Classical and Molecular identification of the fungal isolates Cultural identification of the fungal isolates The isolates were identified based on their growth patterns on PDAC and RBC and by microscopy. Pure cultures were obtained for all the strains and were stored on PDA slants

Molecular identification of the fungal isolates
The laboratory strains (IYN 13 and IYN 15) had been previously authenticated by morphological and molecular methods (unpublished). DNA extracted from all the other fungal strains was done using ZR Fungal/Bacterial DNA MiniPrep TM kit (Zymo, USA), according to the manufacturer's instructions. The DNA extractions and sequencing analyse s were performed at the University of Lagos. Polymerase Chain Reaction (PCR) of the extracted genomic DNAs from the 7 isolates was done in a GeneAmp PCR system 9700 PCR thermal cycler. Each 25 µl master mix consisted of 2.5 µl of 10x PCR buffer,1 µl of 25mM MgCl 2 , 1 µl each of forward (ITS5F: GGAAGTAAAAGTCGTAACAAGG) and reverse (ITS4R: TCCTCCGCTTATTGATATG) primers (Inqaba, South Africa), 1 µl of DMSO, 2 µl of 2.5mMdNTPs, 0.1 µl of 5µg/µl Taq DNA polymerase, 3 µl of 10ng/ µl DNA and 13.4 µl Nuclease free water. The PCR conditions were as follows: Initial denaturation at 94˚C for 5 mins, followed by 36 cycles of denaturation at 94˚C for 30 s, annealing at 54˚C for 30 s, elongation at 72˚C for 45 s, a final elongation step at 72˚C for 7 mins and hold temperature at 10 ˚C. The amplicons were visualized on Safe view-stained 1.5% agarose electrophoresis gels. The expected size of the amplicons was about 650 bp and the DNA ladder used was Hyperladder TM 1kb (Bioline, TN, USA). The PCR amplicons were sequenced at the DNA Sequencing Facility of the Bioscience Center, International Institute of Tropical Agriculture, Ibadan, Oyo using 3130XL genetic analyzer (Applied Biosystems, CA, USA). The sequences were checked for quality and assembled using BioEdit (version 7.2.5) Sequence Alignment Editor (Hall, 1999). The consensus sequence obtained for each isolate was compared to the GenBank nucleotide data library using the Basic Local Alignment Search Tool, BLAST software (Altschul et al., 1990) at the National Centre for Biotechnology Information (NCBI (http://www.ncbi.nlm.nih.gov) website (Nsa et al., 2020). The sequences were submitted to GenBank and accession numbers have been assigned to the isolates.

Microscopic screening for Lipid production
The microscopic screening of the pure isolates for intracellular lipid accumulation was performed by a modified Sudan Black B method (Thancharoen et al., 2017). Yeast Extract Malt Extract Agar (YEMEA) of basal medium composition (g/l): glucose, 10; peptone, 5; yeast extract, 5; and malt extract, 3 and agar-agar 15 was used for culturing the organisms in glass tubes for 48 hours (Liu et al., 2010). The two-day-old isolates were smeared, heat-fixed, flooded with Sudan Black B stain and kept for 15 minutes until the stain turned yellowish-green. The stain was rinsed and counterstained with safranin for 30 seconds. It was thereafter air-dried, blotted and observed under a light microscope (Bresser LCD 40 x1400 Germany) at a100 x magnification. Intracellular lipid accumulation in fungal cells was determined based on the density of globules /retention of Sudan Black B in cells.
Quantitative examination of lipid production in the isolates/Liquid production medium Lipid production medium was prepared as described by Abu-Elreesh and Abd-El-Haleem, (2014) containing (in 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 sodium molybdate 0.005; and 5% glucose (w/v), pH 6 and dispensed into 250 ml Erlenmeyer flasks. Each flask was inoculated with a fungus (positive for intracellular lipid accumulation) and incubated at 28 o ± 2 o C with a shaking speed of 180rpm for 5 days.
Effects of carbon sources on accumulation and FAMEs composition of the isolates Lipid production medium was prepared as described above and sucrose was used as an alternative carbon source to glucose. Thirty (30) ml of the medium was dispensed into 100 ml Erlenmeyer flask in triplicates and inoculated with 5day old fungal cultures and incubated with shaking at 180 rpm, 28 o C ± 2 o C for 5 days. The fungal biomass was harvested from each culture .
Lipid Yield (dry weight of lipid, lipid extraction process and lipid content) Estimation Dry weight of the biomass: Samples were filtered using a preweighed sterile Whatman filter paper inserted in a funnel placed in conical flasks and allowed to drain completely. The biomass was washed twice with distilled water and then dried in a hot air oven at 70 o C to a constant weight. The dry weights of the biomass were recorded and used for lipid extraction.
The lipid extraction process was carried out according to a modified Bligh and Dyer method (Muniraj et al., 2017). The dried fungal masses were crushed in a mortar and pestle and centrifuged at 10,000 rpm for 20 mins. A mixture of chloroform, methanol and distilled water (2:1:1) was added to the centrifuged biomass and spun again for 20 mins. The lower liquid phase (chloroform layer containing lipid) was extracted and dispensed into pre-weighed Bijoux bottles and evaporated using nitrogen gas. The remaining lipids were weighed and recorded.
The lipid content and the biomass yield of each fungal isolate were estimated using the equations described by Muniraj et al., 2017. The lipid content in biomass =Y L/X , Y L/X = Where, L, maximum lipid yield, g/L and X, biomass yield that corresponds to the volume of the medium.

Determination of Fatty-Acid Methyl Esters (FAMEs) profile by Gas Chromatography-Flame Ionization Detector (GC-FID)
The lipid extract from the fungal isolates was transesterified by the addition of methanol, concentrated hydrochloric acid and water in the ratio 10:1:1 as described by Patel et al., (2016); the mixture in glass tubes was centrifuged and the upper phase removed using hexane. The hexane phase was passed through a gas chromatographic column. Gas chromatography was carried out on the transesterified extract. To determine FAMEs, 5-point serial dilution calibration standards (0.25, 0.50, 1.00, 2.00, 4.00 ppm) were prepared from the stock and used to calibrate the GC-FID. Determination of the levels of FAMEs was done using Agilent 7820A gas chromatography coupled to a flame ionization detector fitted with a DB-1 capillary column coated with 5% Phenyl Methyl Siloxane (30m length x 0.32mm diameter x 0.25µm film thickness) (Agilent Technologies). After calibration, the samples were analyzed and chromatogram and its fatty acid concentrations obtained.

Results
Isolation and enumeration of pure fungal isolates from the wastewater samples.
A total of twenty-five fungal isolates were obtained from 10 -5 PDA dilution plates of the brewery wastewater and fermented cereal ogi wastewater. The isolates from the brewery and ogi wastewaters were labelled BW and PW, respectively. From the mixed culture plates (Fig.1), nine isolates were obtained from the brewery wastewater and sixteen isolates from the ogi wastewater. The laboratory strains (previously isolated from the soil) M. circinelloides and T. reesei were maintained on PDA.  The amount of oil produced by the strains that were positive for oil accumulation was measured as a fraction of cell weight/volume of the medium. The lipid yield of the strains, BW 1, BW 3, BW 4, BW 7, PW 3, PW 8, PW 10, PW 16, M. circinelloides and T. reesei were determined ( Table 1). Out of these 10 strains, the highest oil producers were M. circinelloides 8.05 g/L; (41.3%), T. reesei 4.2 g/L (28.7%), BW4 3.18 g/L; (35.3%), PW10 5.27 g/L; (34.6%) PW16 2.26 g/L; (29.9%).
FAMEs profiles were determined for the four fungal isolates M. circinelloides, (glucose, sucrose) T. reesei (glucose, sucrose) Aspergillus fumigatus PW10 (glucose) Aspergillus sp. (BW 4) (glucose). Transesterified oil extract from M. circinelloides and T. reesei in this study showed higher amounts of unsaturated fatty acid while the two Aspergillus species examined were high in saturated fatty acids. Palmitoleic acid was the most abundant fatty acid methyl ester of M. circinelloides and T. reesei regardless of the cultivation medium being glucose or sucrose. However, the use of glucose as substrate enhanced the recovery of palmitic acid methyl ester from 0.38% to 7.11% and 2.47% to 6.37% in M. circinelloides and T. reesei respectively. Overall, for T. reesei, the FAMES profile generated from sucrose and glucose were not overly different from each other, but in M. circinelloides, lauric acid and capric acid were enriched when sucrose was the substrate.
FAMEs profile was dependent on the type of carbon source -glucose or sucrose (Table 3; Fig. 4). The effect of carbon sources on fatty acid composition and lipid accumulation in oil-producing moulds have been reported (Liu et al., 2010;Thanaa and Dina, 2014). The major fatty acid produced in the M. circinelloides is palmitoleic acid which accounted for 50.41% and 87.3% in sucrose and glucose respectively. In T. reesei, palmitoleic acid methyl ester was the major fatty acid , having 85.94% and 87.31% with sucrose and glucose respectively. The palmitoleic acid composition of M. circinelloides and T. ressei has been reported to be indicative of its use as an excellent feedstock for the production of single-cell oil (Bhanja et al., 2014).
The finding here that medium-chain saturated capric acid was the dominant FAME in A. fumigatus and the second most abundant in Aspergillus sp. (BW4) contrasts the report from Asci et al. (2020) who showed that the FAMEs profile of the six A. fumigatus strains studied were dominated by long-chain fatty acids and suitable for use as biodiesel.
The FAMEs profiles of the oleaginous strains investigated had low levels or total absence of these necessary FAMEs suggesting that the oils extracted from these isolates under the culture conditions are not fit for use as biodiesel. Further optimization of cultivation and lipid extraction conditions and genetic engineering may improve fatty acid methyl ester profiles of the most promising isolate M. circinelloides for its use as feedstock for biodiesel production.
The FAMEs profile of the fungal isolates from this work shows that they would be a good feedstock for the production of pharmaceuticals and cosmetics. Lauric acid and capric acid are used in the cosmetic industry as emollients, dispersing agents for other chemicals, solvents and antioxidants. They can inhibit the growth of Propionibacterium acne, act as intestinal anti-inflammatory and alleviate oxidative stress (Huang et al., 2014;Lee and Kang, 2017). Palmitoleic acid is used to improve cold flow and also acts as a stabilizer for biodiesel (Knothe, 2010). These fatty acids have also been reported to have effects on the inhibition of Candida albicans (Clément et al., 2007;Murzyn et al., 2010).

Conclusion
In conclusion, among all the oleaginous species assayed, the FAMEs profile of M. circinelloides contained a better composition of biodiesel relevant fatty acids although at low proportions. Optimization of culture conditions, transesterification, extraction procedures and genetic manipulation for this isolate might be recommended for future research for obtaining better biodiesel production.