Isolation, identification, and statistical optimization of a psychrotolerant Mucor racemosus for sustainable lipid production

Lipid accumulating fungi are promising tools as alternative lipid source with different applications. In this study, seven oleaginous fungal strains were isolated from dung samples from the Egyptian ecosystem which later investigated for lipid accumulation, and Mucor racemosus AH1 represented the highest one. Statistical optimization of M. racemosus AH1 for lipid production was carried out using Taguchi design. Accordingly, dry biomass, total lipids, and lipid content were 3.72 gL−1, 1.21 gL−1, and 32.4%, respectively. Fatty acid profile of the produced lipids at different temperatures from 5 to 35 °C was investigated using gas chromatography mass spectroscopy GC–MS. Results revealed that the best temperature range for unsaturated fatty acids production particularly polyunsaturated fatty acids (PUFAs) was between 10 and 20 °C in which unsaturated fatty acids (USFAs) were higher than saturated fatty acids (SFAs); 54.47% and 43.67%, respectively. In conclusion, a promising lipid accumulating and cold-adapted M. racemosus MG547571 are considered as hopeful source of USFAs particularly oleic and linoleic acids which can be recruited for pharmaceutical applications, additionally, the high lipid yield could be exploited for biodiesel production.


Introduction
As a result, for population increase, a climate change, etc., there is a constant need for food, health, and energy sources, especially fuel. Lipids play an important role to offer a green alternative source of energy demands, as well as several industrial applications [1]. Economic development in industrial countries led to the rapid consumption of fossil fuels but in the same time it is a reason for air pollution by increasing greenhouse gasses in atmosphere and consequently causes climate change and global warming. The fossil fuel should be replaced by renewable energy sources such as solar energy, tidal energy, wind, and biofuel. Renewable energy sources are getting universal consideration and play an imperative role in meeting the future needs of the world [2]. Biofuels are derived from biomass and represent an essential contribution to our future energy supply [3]. Trans-esterification process is used to convert fats and oils in biological samples to biodiesel.
Biofuel production based on biomass has emerged as a major approach to enabling energy independence, reducing greenhouse gas emissions, revitalizing rural communities, and enhancing sustainable economic development. Oleaginous microorganisms can accumulate lipids of more than 20% (w/w) of their total dry biomass weight [4]. Oleaginous microorganisms are a group of species that belong to different varieties of microbial genera including bacteria, yeasts, algae, and fungi [5]. Recently, microbial lipids from filamentous fungi, yeasts, and microalgae are considered as a possible alternative source for biodiesel production, where contain high amounts of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFA) [6]. Fungi are the greatest group of microorganisms that have the ability to produce single cell oils (SCOs) due to their short life cycle and no need for light energy as well as their ability to utilize a wide range of carbon sources such as agro-industrial residues, lignocellulosic material, wastewater, and crude glycerol [7,8]. There are many fungal species such as Mortierella isabellina and Mortierella vinacea, Cunninghamella echinulata, Rhizopus oryzae, Aspergillus oryzae, and Mucor circinelloides have been defined as SCO producers [9][10][11][12]. In some cases, the oleaginous microorganisms can do more than one benefit over their productivity of lipid which can be recruited in multipurpose fields, moreover, it could help in biorefinery, bioremediation, decolorization [13,14], and medically important agents [15][16][17]. Therefore, the searching for a new oleaginous fungus is required due to several advantages of microbial lipids particularly fungi. This study is aimed at isolating and identifying a promising oleaginous fungal isolate with much higher ability to produce high quantities of lipids using of Taguchi design as a statistical tool for optimization of lipid production. Also, studying the effect of different temperatures on fatty acids types.

Isolation and screening of fungal isolates
The dung samples were collected carefully into complete sterile plastic bags, and the samples were diluted as 1: 100 (g/v) in a sterile saline solution. Malt extract agar medium (MEA) was purchased from Sigma Aldrich, Germany; used as growth medium while the production medium composed of glucose 100 gL −1 and yeast extract 10 gL −1 , with pH adjusted to 5.4. The 10% (v/v) mycelial suspension of isolated culture was inoculated in 100 mL flask containing 25 mL of malt extract broth and incubated at 30 °C for 7 days [18][19][20]. Fungal isolates were isolated from different dung sample from Giza, Egypt. Isolation was carried out according to method used by Hashem et al. [21]. Screening of fungal isolates was carried out by using Nilered dye, and reparation and staining methods of dye were performed according to Lim et al. [22] by whom, a stock solution of 0.4 mg/mL (w/v), then, the working stain was prepared through 200-fold dilution to reach 2 µg/mL as a final working concentration. The stained lipid bodies were photographed using fluorescence microscope (IX-70, Olympus, Tokyo, Japan) equipped with a CCD camera (U-CMT, Olympus, Tokyo, Japan) [23].

Identification of the most potent fungal isolate
Morphological identification of the selected fungal isolate was carried out depending upon the culture characteristics (color, texture appearance, and diameter of the colonies), as well as the microscopic investigations using both the light and scanning electron microscope which was performed to confirm the morphological characteristics of fungal isolates [24][25][26][27][28][29][30][31]. Finally, the molecular approach had been used to confirm the fungal identification using the 18S-rRNA (partial sequence depending on a specific pair of primers). Additionally, ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) was used for sequencing of PCR products. The final sequence was aligned with similar sequences using the NCBI BLAST search program in the National Center for Biotechnology Information (NCBI). Evolutionary analyses were performed using MEGA-x [32-34].

Dry weight determination, lipid extraction, and lipid quantification
Biomass production was determined by harvesting the cells by filtration followed by drying at 55-60 °C overnight or until constant weight then determined. The extraction of lipid was performed by adding 40 mL of chloroform-methanol (2:1) to 1 g of grinded dry biomass, then, this mixture was agitated for 20 min at 20 °C and then filtered with Whatman paper no. 1. The solvent containing lipids was separated then evaporated then lipids were determined [12,35]. The extracted lipid was quantified using sulfo-phospho vanillin method (SPV) [12,36,37]. Phosphovanillin reagent was prepared by mixing of vanillin 6% with phosphoric acid 85%. The test sample was prepared by diluting of 20 µL of samples with 180 µL of sulfuric acid and incubated at 100 °C for 10 min., then cooled at room temperature, after that, phosphovanillin reagent and kept for a while till color development, and finally the test sample was measured at 530 nm [38].

Methyl ester preparation and fatty acid analysis
Methylation was carried out to the extracted lipid to convert fatty acids to fatty acid methyl ester (FAMEs) [39]. Two layers were formed after cooling. The lower layer which contains fatty acids methyl esters (FAMEs) was separated with chloroform, and the final FAMEs product was obtained by evaporating chloroform from the solution. FAMEs were analyzed by GC-MS [37,40].

Optimization of lipid production using a statistical design
Taguchi design was used for optimization factors affecting lipid production such as carbon source, nitrogen source, temperature, incubation time, and pH. Optimization process was carried out with two stages. The first stage includes selecting the best condition for each factor using L16 array of Taguchi design. Factors are carbon source (glucose, sucrose, starch, and CMC), nitrogen source (peptone, yeast extract, sodium nitrate, and ammonium nitrate), initial pH (4 and 7), and incubation period (4, 7, and 10 days). Second stage includes testing effect of five factors on lipid production to reach optimum conditions of M. racemosus AH1 for lipid production. Table 1 shows different factors and their levels for lipid production, where factors include temperature (15,20,25,30, and 35 °C), initial pH (5, 6, 7, 8, and 9), incubation time (8,9,10,11, and 12 days), different concentrations of soluble starch (10,20,30,40, and 50 gL −1 ), and different concentrations of yeast extract (1, 2, 3, 4, and 5 gL −1 ). Table 2 illustrates L25 array of Taguchi design which contains different factors and their levels to produce high quantities of lipids by M. racemosus AH1.

Statistical analysis
All the experiments including both first and second optimization steps were performed in triplicates, and statistical analysis was carried out using Minitab software (version 18). The values are given as means ± SD (standard deviations). Levels of significance were considered at p ≤ 0.05. Statistical analysis is investigated by ANOVA (one-way analysis of variance) Tukey method for the obtained results.

Isolation and screening of fungi
Totally, seven fungal isolates were recovered from the dung sample using dilution agar plating. The seven fungal isolates were screened for lipid accumulation using Nile-red dye and photographed using fluorescence microscope. Result showed the fungal isolate AH1 is the most potent for lipid accumulation where contains many lipid bodies under light microscope ( Fig. 1A), and under fluorescence microscope was appeared with red color bodies as shown in Fig. 1B. Furthermore, fungal isolate AH1 was incubated for different intervals 7, 14, and 21 days, and the lipid quantity was quantified using SPV methods as shown in Table 3. Results illustrated that the time at 7 days is the best for lipid production where dry biomass, total lipids, and lipid content were 2.22 gL −1 , 0.72 gL −1 , and 32.43%, respectively. At incubation time, 14 and 21 days lead to decrease total lipid to 0.59 and 0.42 gL −1 , respectively [10,41]. In the long run, the decrease in lipid accumulation may be due to the nutrient shortage, byproducts accumulation, pH change, lipid peroxidation, and other environmental changes that make the fungus consume the storing lipids to recompense the nutrients' shortage to enable it to survive longer [42]. Thus, the fungal isolate AH1 is considered oleaginous fungus because accumulates lipid more than 20% of its dry weight.

Statistical optimization of lipid production by M. racemosus MG547571 using Taguchi design
Oil production represented an important research field with special interest in food biotechnology in particular polyunsaturated fatty acids (PUFAs) [51], as known, plants were considered sources of variety of bioactive compounds [52,53], and fungi and other microbes are not less important than plants to produce valuable compounds [15,54]. In particular, oleaginous microorganisms, including fungi and microalgae, are able to grow fastly on several substrates and they were considered as the main producers of PUFAs that could be recruited in many different industrial applications [55]. Lipids are essential for all organisms as one of the three macronutrients. Oleaginous fungi mainly yeasts and zygomycetes can accumulate up to 80% of their biomass as lipids. Lipids in all forms including SFAs, MUFAs, and PUFAs can contribute to synthesize biodiesel through acylglycerides. As well, lipids can conjugate with other biomolecules such as glycolipids which have high potential as biosurfactant [56]. According to recent studies, incubation temperature, initial pH value, the incubation period, carbon, and nitrogen source were listed among the most important factors affecting lipid production [57,58]. Therefore, these factors are studied in this study. L16 Taguchi design was used to select the best carbon interacted with nitrogen source. L16 design contains runs from 1 to 16. Table 4 illustrates that the run no. 10 was the best for lipid production, and this run includes soluble starch with yeast extract which produced lipid 0.93 gL −1 , dry biomass 2.8 gL −1 , and lipid content 33.07%. Aoki et al. [59] reported that 3% of soluble starch is the best for lipid production by M. hiemalis HA-30. In addition, results showed that yeast extract is the best nitrogen source for lipid production by M. racemosus MG547571. In the same accordance with our results, [18] reported that yeast extract is the best for lipid production by Mucor sp. Lipids and starch have a specific relation because they represent the major energy forms especially in plants, and according to Yu et al. [60] who proved the role of fatty acid β-oxidation and the regulatory network controlling fatty acid synthesis, and they reveal the mechanistic basis by which starch and lipid metabolic pathways interact and undergo cross talk to modulate carbon allocation, and energy homeostasis.
pH factor is considered one of the most factors affected lipid production. Total lipid and lipid content at pH 4 and 7 were (0.33 gL −1 and 19.06%) and (0.91gL −1 and 31.11%), respectively, as shown in Fig. 3A. [18] demonstrated that Mucor spp. produced the maximum yields of g-linoleic acid and total PUFA when the medium pH was 6.5. Moreover, [59] reported that pH at 6 is the optimum for EPA production by M. hiemalis HA-30. From  the previous results, it is obvious to find that pH at 7 was the best center point pH for lipid production to use it in the next step of optimization. Four, seven, and ten days were performed to detect center point of incubation period which will be used in Taguchi design. Figure 3B shows ten days are the most favorable center point of incubation period for lipid production by M. racemosus MG547571. Taguchi design is very important for optimization processes. This design was used to achieve the highest lipid production by optimizing the medium conditions [57,58].   Accumulation of lipids mainly depends on the carbon and nitrogen source, pH, and temperature. Therefore, these factors were selected for enhancement of lipid production by M. racemosus MG547571. Generally, oleaginous fungi can accumulate lipids at more than 20% of their cell dry weight. Figure 4 illustrates different experiments designed by Minitab 18 software for lipid production by M. racemosus MG547571. Results showed experiment 7 is the best for lipid production which includes conditions, temperature at 20ºC, initial pH at 6, incubation period for 10 days, soluble starch with concentration 40 g, and yeast extract with concentration 5 g. Dry biomass, total lipid, and lipid content at these optimal conditions were 3.72 gL −1 , 1.21 gL −1 , and 32.4%, respectively. This demonstrates M. racemosus MG547571 is an oleaginous microorganism and the possibility to be used for industrial production of microbial oils. Moreover, we noticed there in no growth of M. racemosus in five runs from 21 to 25, these runs belong to temperature at 35 °C where this fungus could not grow at this degree while can grow from 4 to 34 °C.
To evaluate the effect of each factor individually upon lipid production process, Taguchi design has ability to calculate the impact percent in production process. In this experiment, impact percent was calculated for each factor against the value of lipid produced. Table 5 displays effectiveness percent of all 5 tested factors which exhibited that incubation temperature was the most effective for lipid production with percent 37.9%. Whereas different concentrations of starch, different concentrations of yeast, and time had low effect on lipid production with percent 19.68, 18.26, and 15.45% respectively. Initial pH was the lowest effectiveness (8.71%) on lipid production.
Interactions between different levels of selected factors are very important to select optimum level in each factor. Figure 5 shows different levels of temperature were interacted with different levels of initial pH, incubation period, different concentrations of soluble starch, and different concentrations of yeast extract; temperature at 20 ºC was the optimum for lipid production. Also, pH levels were interacted with different levels of temperature, incubation period,  different concentrations of soluble starch, and different concentrations of yeast extract; pH 6 was the optimum for lipid production. In addition to, incubation period levels were interacted with different levels of temperature, initial pH, different concentrations of soluble starch, and different concentrations of yeast extract, 10 days were the optimum for lipid production. Different levels of soluble starch and yeast extract were interacted with different levels of temperature, initial pH, and incubation period; optimum concentration of soluble starch and yeast extract was 40 and 5gL −1 , respectively. Finally, optimization of M. racemosus MG547571 for lipid production is significantly affected by using Taguchi design. Figure 6 shows lipid content, and total lipids before optimization were 20.76 and 0.51 gL −1 , but lipid content and total lipids were increased sharply to 32.40 and 1.21 gL −1 , respectively, using Taguchi design.

Cellular lipid composition
M. racemosus MG547571 was grown at different low temperatures 5, 10, 15, 20, 25, and 30 °C due to this strain is psychrotolerent and to compare between fatty acids produced at different low temperatures. Table 6 shows ratios of USFAs were more than SFAs, where USFAs at 5, 10,

Conclusion
In the current study, a promising psychrotolerant oleaginous M. racemosus MG547571 isolated from cow dung. Taguchi method was carried out to make optimization process which can help in obtaining more confident and precise result, also save the time and cost. Moreover, Taguchi method in this study was effective for lipid production by fungi. M. racemosus could produce lipid 1.21 gL −1 with lipid content 32.4%. Moreover, fatty acid profile of lipid produced at different low temperatures illustrated that USFAs were higher than SFAs. Eventually, M. racemosus is a promising for USFAs which can be used for pharmaceutical applications. Furthermore, the high content of lipid produced is remarkably noticed that could be considered for the large-scale production as future perspectives and subsequently submitted to biodiesel production as an alternative source for the renewable energy.
As future perspectives, we planned to use large-scale production of lipid by co-cultures of different fungal isolates using undesirable agricultural wastes to hit two birds with one stone. Consequently, our perspectives may contribute to finding new alternatives for food and energy demands in addition to waste recycling which retains the environment clean, thus, it will contribute to resolving the problems of climate change.
Acknowledgements All authors would like to thank the faculty of Science, Al-Azhar University as well as the City for Scientific Research and Technology Applications in which the work steps were done.
Author contribution All authors contributed to the same percentage in all stages of the research.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Declarations
Ethics approval Research involving Human Participants and/or Animals is not applicable.
Informed consent Informed consent is not applicable.

Conflicts of interest
The authors declare no competing interests.
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