Optimization of Diverse Carbon Sources and Cultivation Conditions for Enhanced Growth and Lipid and Medium-Chain Fatty Acid (MCFA) Production by Mucor circinelloides

Keywords: Mucor circinelloides; microbial lipids; medium-chain fatty acids; culture optimization


Introduction
Currently, single cell oil (SCO) is considered as an alternative and reliable platform to face the challenges related to global warming, scarcity of non-renewable resources, and food security.Over the last decade, medium-chain fatty acids (MCFAs: C8 to C12) and their derivatives have been gaining attention because of their diverse applications in the food, biochemical, and petroleum industries [1].These MCFAs are involved as precursors in industries involved in the manufacturing of bio-plasticizers, lubricants, detergents, surfactants, adhesives, antibiotics, diverse intermediates for floral fragrances, and flavors [2].The aforementioned fatty acids have attracted much attraction because they have a positive impact on human health by being easily absorbed and swiftly catabolized through the β-oxidation mechanism, ultimately increasing diet-stimulated thermo-genesis [3].This mechanism provides a venue for the prevention of various metabolic abnormalities, such as obesity, hyperlipidemia, type II diabetes, atherosclerosis, and cardiovascular diseases (CVDs).The yield of naturally produced MCFAs from plant (i.e., coconut or palm) sources has been estimated to be insignificant to meet the market demand [2][3][4][5][6][7][8][9][10][11][12].Therefore, diverse metabolic-engineering strategies have been exploited in different oleaginous microorganisms to augment the contents of MCFAs [13][14][15][16][17][18][19][20][21][22].In addition, numerous investigations have been performed to evaluate the effects of carbon and nitrogen sources, initial pH of the culture medium, C/N ratio, incubation temperature, and their particular effects on the fungal biomass and fatty acid production [23][24][25][26][27][28][29][30][31][32].However, comprehensive information regarding MCFA production under diverse culture conditions is still limited.
Mucor circinelloides is regarded as a model organism for investigations of lipid accumulation.It has been extensively genetically manipulated for the production of diverse biotechnological precursors for the functional food and bio-fuel industries [1,33,34].However, its fatty acid profile indicates that it is mostly formed by long-chain fatty acids (LCFAs), ultimately making it less attractive for multi-purpose industrial applications.In our previous investigation, efforts to enhance the MCFA content were carried out by integrating different heterologous thioesterase (TE) proteins from plant and bacterial sources into the fatty acid synthase (FAS) complex of the M. circinelloides M65 strain, eventually generating mutant strains with significant MCFA-producing capability [1].Although we successfully enhanced the MCFA contents from the total fatty acids (TFAs), a noteworthy decline in biomass in the resultant strains was observed, which negatively affected the lipid productivity and MCFA contents.Thus, to circumvent the productivity-related challenge stemming from the dry cell weight (CDW) of the M. circinelloides strain, M65-TE-04, we synergistically optimized the culture conditions to maximize the yield of MCFAs.The individual and synergistic impact of four key factors, namely the incubation temperature, initial pH, agitation speed, and different carbon sources, were evaluated, and the impact on the biomass and lipid and MCFA contents of M. circinelloides strains was elucidated.

Determination of Cell Dry Weight (CDW)
Harvesting of the fungal biomass was carried out according to our previously described method [1].In brief, a suction filtration method was employed followed by three washes with double-distilled water to eliminate possible medium contents.The collected samples were then frozen at −80 • C overnight, and the samples were lyophilized for 24 h.The fungal biomass weight was calculated by the gravimetrical method.
The biomass productivity (P DCW ) of fungal cells was computed using the following equation:

Total Lipid and Fatty Acid Profiling
Lipid extraction was performed according to a previously developed method with minor modifications [1].In brief, 20 mg of lyophilized biomass was mixed with chloroform/methanol (2:1, v/v).Methylation was carried out with 10% (v/v) methanolic HCl at 60 • C for 4 h.Pentadecanoic acid (15:0) (Millipore Sigma, St. Louis, MO, USA) was used as an internal standard for the lyophilized cells before methylation.Finally, the fatty acid methyl esters (FAMEs) were extracted with n-hexane and subsequently analyzed by GC using the DB-Waxetr column with the following specifications: film thickness 0.25 µm, 30 m×0.32 mm (Shimadzu Co., Ltd., Kyoto, Japan).The program used for the gas chromatography (GC) was as follows: 120 • C for 3 min, ramp to 200 and hold for 2 min [1,33].
Lipid productivity (P Lipid ) and MCFA productivity (P MCFA ) were calculated as follows: where C f is the final lipid content (g/L), C i is initial lipid content, and TL is the total lipid content.

Construction of Orthogonal Matrix Design (OMD)
The synergistic effect of all parameters was evaluated on the bases of OMD design, as shown in Table 1.

Statistical Analysis
A statistical analysis of the acquired data was performed with SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA).Mean values and the standard error of the mean were computed from the data gathered from three independent experiments.The Student's t-test was employed to estimate differences between the data, and p < 0.05 was regarded as a significant difference.

Results and Discussion
To investigate the effect of diverse cultivation conditions of the M. circinelloides strain (i.e., M65-TE-04) on cell growth, lipid accumulation, and MCFA production, four key cultivation factors (i.e., incubation temperature, initial pH, speed of agitation, and carbon-sources) were assessed in a first stage individually, and in a second stage in a combinatorial study following orthogonal matrix design (OMD).When individual culture conditions were evaluated, all the remaining culture factors were held constant.These conditions were analogous to the normal culture conditions to eventually attain particular conclusions on the basis of relevant variable effects on cell growth and lipid accumulation.

Effect of Incubation Temperature on Fungal Biomass and Lipid Accumulation
Fungal biomass, lipid accumulation, and MCFA contents of M. circinelloides strain M65-TE-04 cultured on modified K and R medium were appraised at three different temperatures (i.e., 26 • C, 30 • C, and 34 • C) for 72 h.After 3 days of cultivation, the biomass (CDW, g/L) acquired at the different temperatures is shown in Figure 1A.Overall, we noticed a fluctuation of fungal growth at different cultivation temperatures.The biomass at 26 • C was considerably higher (i.e., 13 g/L) than at other temperatures.The quantity of biomass gradually declined with an increase in temperature (i.e., 9.5 g/L and 4.6 g/L for 30 • C and 34 • C, respectively) (Figure 1A).This pattern clearly demonstrated that higher temperature was not appropriate for fungal cell growth.
Temperature is regarded as a crucial factor for fungal cell growth, overall lipid productivity, and specifically MCFA contents.We noticed an inverse correlation between the cultivation temperature and MCFA contents.High MCFA contents were found at 26 • C (i.e., 50%), which were approximately 1.3-fold higher than the titer obtained at 34 • C (Figure 1A).Likewise, the lipid contents (% CDW) also declined with the temperature elevation (Figure 1B).The maximum lipid accumulation was obtained at 26 • C, which was almost 63% higher than that at 34 • C. Finally, we noticed a considerable fluctuation in biomass and lipid productivity (p > 0.05), as shown in Figure 1C.These outcomes indicated that the optimal temperature for fungal cell growth, as well as for lipid and MCFA productivity, was 26 • C. The maximum biomass (i.e., 4.37 g/L•d) and lipid productivity (i.e., 2.93 g/L•d) were achieved at 26 • C. Our results are in agreement with a previous investigation conducted using M. circinelloides strains, which demonstrated maximum cell growth and lipid production at temperatures ranging from 26 • C to 30 • C [34,35].Moreover, the optimum temperature for growth and lipid accumulation in other fungal strain has been estimated to range from 26 • C to 30 • C [36].

Effect of the Initial pH of the Culture Medium on Fungal Biomass and Lipid Accumulation
In the current investigation, the M. circinelloides strain (i.e., M65-TE-04) was able to grow at different pH values (i.e., 4.5, 6.0, and 7.5) (Figure 2A).Overall, pH values below or above 6.0 had drastic effects on the growth of M. circinelloides strains, as growth declined after 24 h of the cultivation period.Although M. circinelloides strains could grow within a wide pH range, the maximum growth was observed at pH 6.0.The highest biomass content (CDW; 12.5 ± 2.1 g/L) was achieved in culture at an initial pH of 6.0 over culture duration of 72 h.The biomass of M. circinelloides strains at different pH values is elaborated in Figure 2A.Our findings are in agreement with our previous studies [1].However, some authors have reported that the fungal growth rate was found to be maximal at pH 4.5 [37].
The pH value is a crucial cultivation factor that affects cell surface characteristics, ultimately modifying the cellular permeability capacity for different ions, bases, and acids across the membrane.It also has a great influence on the cellular metabolism of carbohydrates, proteins, and lipids.Moreover, the depletion of nutrients, i.e., phosphorus, nitrogen, or sulfur, also affects biochemical processes in the microorganism.Thus, in the current investigation, we provided an altered nutrient supply to fungal strain by using the different initial pHs of culture mediums (i.e., 4.5, 6.0, and 7.5).As depicted in Figure 2B, we observed that increasing the pH (4.5 to 6.0) had a positive effect on the TFA content (% CDW).More precisely, the maximum lipid contents were obtained at pH 6.0 (i.e., 64.3% of CDW).Similarly, the MCFA contents were slightly influenced by the initial culture pH, and maximum MCFA contents (% CDW) were attained at pH 6.0 (Figure 2A).However, no obvious alterations in other saturated fatty acids were observed for all culture initial pH values.There were no significant changes in lipid and MCFA contents at pH 4.5 and 7.5 (Figure 2A,B).

Effect of the Initial pH of the Culture Medium on Fungal Biomass and Lipid Accumulation
In the current investigation, the M. circinelloides strain (i.e., M65-TE-04) was able to grow at different pH values (i.e., 4.5, 6.0, and 7.5) (Figure 2A).Overall, pH values below or above 6.0 had

Effect of Agitation Speed on Fungal Biomass and Lipid Accumulation
When all the culture parameters (i.e., substrate, temperature, initial pH, etc.) of the culture medium were optimized, the specific growth rate was solely associated with the accessibility of dissolved oxygen (DO).To improve the DO, we cultured fungal strain M64-TE-04 at diverse agitation speeds (i.e., 150, 220, and 300 rpm).The fungal biomass, lipid accumulation, and MCFA contents and their productivities are demonstrated in Figure 3A-C.We observed a significant change in biomass and MCFA contents with a corresponding increase in agitation speed from 150 to The association of cell dry weight (CDW) and lipid productivities (g/L) provided noteworthy insight into cell growth at different initial pH values of the culture medium.The biomass and lipid productivities are mentioned in Figure 2C.The maximum biomass and lipid productivity were achieved at pH 6.0, whereas the minimum productivities for the biomass and lipid contents were found at pH 7.5 (Figure 2C).A previous report has shown that the pH values of fungal cells are sustained by ion exchange systems across the cell membranes, which eventually fabricate the electric potential with an intracellular positive charge at the cost of cellular energy.At a pH value other than the optimal level for fungal biomass growth, more energy is required to properly maintain the physiological tasks.Therefore, biomass and lipid productivities are maximal at pH 6.0 [33,34,38].

Effect of Agitation Speed on Fungal Biomass and Lipid Accumulation
When all the culture parameters (i.e., substrate, temperature, initial pH, etc.) of the culture medium were optimized, the specific growth rate was solely associated with the accessibility of dissolved oxygen (DO).To improve the DO, we cultured fungal strain M64-TE-04 at diverse agitation speeds (i.e., 150, 220, and 300 rpm).The fungal biomass, lipid accumulation, and MCFA contents and their productivities are demonstrated in Figure 3A-C.We observed a significant change in biomass and MCFA contents with a corresponding increase in agitation speed from 150 to 220 rpm (Figure 3A).Moreover, a noteworthy difference in total lipid contents was noticed at an agitation speed of 220 rpm (Figure 3B).Overall, the biomass and lipid productivities increased from 150 to 220 rpm and then decreased from 220 to 300 rpm (Figure 3C).It has been previously reported that cells grow and multiply more rapidly to the optimum level in the presence of a high oxygen compared with a low oxygen supply, ultimately enhancing the biomass and lipid productivities [39,40].The specific growth rate demonstrated an increment from 150 to 220 rpm (i.e., 2.1 d −1 to 3.5 d −1 ).Conversely, increases in agitation speed above 220 rpm negatively affected the growth rate to 1.55 d −1 .The M. circinelloides strains were susceptible to a high agitation speed, and the overall growth rate declined above 300 rpm [41].

Effect of Carbon Sources on Fungal Biomass and Lipid Accumulation
Among the essential nutrients, carbon source plays a promising role in the fermentation process because carbon sources are directly correlated with the fungal biomass and cellular metabolites.To evaluate an appropriate carbon source for optimum fungal cell growth, total lipid productivity, and MCFA production, three different carbon sources were employed as additional carbon sources along with modified K and R medium.A previous investigation has shown that coconut oil, palm oil, and some other vegetable oils (i.e., mostly consist of lauric acid, myristic acid, and palmitic acid) containing culture medium show positive effects on fungal biomass and lipid accumulation [42].Thus, to elucidate the effects of the aforementioned oils on MCFAs, we cultured the engineered strain M65-TE-04 in modified K and R medium together with coconut oil (3%), palm oil (3%), glycerol (3%), or sole glucose (for the control experiments).We chose these concentrations based on some of our preliminary work (data not shown), as well as our previous investigation using 3% diverse oils [43].The cell dry weight (CDW) and TFA contents for strain M65-TE-04 were shown in Figure 4A,B.The aforementioned fungal strains showed a maximum CDW with coconut oil mixed medium, i.e., 17.0 g/L at 72 h of the cultivation period.The CDW for palm oil mixed medium and medium containing glycerol as a supplement were 13.0 g/L and 10.0 g/L, respectively, which is appreciably higher in comparison to fungal cell cultured in only glucose-containing medium (9.0 g/L) (Figure 4A).The TFA contents (% CDW) were found to be 9.5% higher when the fungal strain M65-TE-04 was cultured in coconut oil mixed medium in comparison to control (Figure 4B).Interestingly, the highest CDW was determined for strain M65-TE-04 grown in coconut oil mixed medium over 72 h.However, the highest TFA content in the aforementioned strain was obtained when the cells were grown in coconut oil.
that cells grow and multiply more rapidly to the optimum level in the presence of a high oxygen compared with a low oxygen supply, ultimately enhancing the biomass and lipid productivities [39,40].The specific growth rate demonstrated an increment from 150 to 220 rpm (i.e., 2.1 d −1 to 3.5 d −1 ).Conversely, increases in agitation speed above 220 rpm negatively affected the growth rate to 1.55 d −1 .The M. circinelloides strains were susceptible to a high agitation speed, and the overall growth rate declined above 300 rpm [41].

Effect of Carbon Sources on Fungal Biomass and Lipid Accumulation
Among the essential nutrients, carbon source plays a promising role in the fermentation process because carbon sources are directly correlated with the fungal biomass and cellular metabolites.To  Our results are consistent with previous studies showing that fungal cells produce more fungal biomass and total lipids when grown in medium-chain-containing oils [42][43][44].Based on our results, the highest biomass and lipid productivities were achieved with coconut oil mixed medium (g/L.d), while the maximum MCFA contents in terms of the percentage of CDW were obtained with palm oil mixed medium (Figure 4C).The majority of the fatty acids in the M. circinelloides strain were palmitic acid, stearic acid, oleic acid, linoleic acid, and γ-linolenicacid.We observed a noteworthy difference in the fatty acid profile when strain M65-TE-04 was cultured in diverse glucose oil mixed medium.The fatty acid profile of M. circinelloides strain M65-TE-04 was directly associated with the fatty acid compositions of the glucose oil medium (Figure 5).The highest MCFA contents (i.e., 65% CDW) were obtained when the fungal cells were grown in medium containing palm oil as a supplement (Figure 4A).In contrast, significant outcomes related to TFA contents were obtained when the fungal strain was grown in coconut oil mixed medium (Figure 4B).The biomass and lipid productivities (p > 0.05) for culture media containing different carbon sources are shown in Figure 4C.was used as a carbon source under normal culture conditions; additionally, the highest fatty acid contents (% CDW) were achieved at 26 °C, the highest MCFA contents (% CDW) with palm oil mixed medium, and the maximum overall lipid productivity (8.6 g/L.d) with coconut oil supplementation.Since we obtained diverse outcomes under the different culture conditions, synergetic insights into the individual culture conditions are needed to evaluate the different combinations based on an orthogonal matrix design (OMD).

Synergistic Effect of the Culture Conditions on Fungal Biomass and Lipid Accumulation
To investigate cell growth and lipid accumulation of the strain M. circinelloides M65-TE-04, the fungal cells were cultured under different conditions.The coded values of individual variables by the orthogonal matrix design (OMD) are provided in Table 1.All the experiments were carried out in a 1-L baffled shake flask with 250 mL modified K and R medium containing glucose or other carbon sources (see Section 2.1 for details).The responses for the four independent variables (i.e., temperature, agitation, pH, and carbon sources) obtained based on the orthogonal matrix design (OMD) (SPSS 19) were provided in Table 2.
We observed an elevation in biomass in assays 12 and 16 and a slight elevation in assays 14 and 4, while the lipid contents (% CDW) exceeded the maximum in assay 16 and significantly exceeded the maximum in assays 12 and 4. Assays 2 and 5 also showed higher lipid contents (% CDW).Overall, assays 16, 12, and 4 demonstrated the highest lipid (g/L) and MCFA contents (g/L), as well as lipid and MCFA productivities (mg/L.d),as shown in Table 2. Thus, from the above discussion and the results represented in Table 2, we concluded that assay 16 with specific cultivation conditions (i.e., temperature 26 °C, pH 6, agitation speed 300 rpm, 3% coconut oil mixed medium as the carbon source) provided the best outcome for M. circinelloides strain M65-TE-04among all 16 assays.This condition eventually provided the highest lipid (i.e., 4767 mg/L.d) and MCFA productivity (i.e., 2570 mg/L.d)(Table 2).The higher contents of MCFA obtained in M. circinelloides (M65-TE-04) by using coconut oil.This is might be due to availability of MCFA in coconut oil, and therefore it shows that M. circinelloides should assimilate the MCFA from the coconut into their cells.Our results are consistent with previous studies showing that fungal cells produce more fungal biomass and total lipids when grown in medium-chain-containing oils [42][43][44].Based on our results, the highest biomass and lipid productivities were achieved with coconut oil mixed medium (g/L•d), while the maximum MCFA contents in terms of the percentage of CDW were obtained with palm oil mixed medium (Figure 4C).
Taken together, we acquired diverse results among all individual variables for the culture growth conditions.Likewise, the highest biomass was obtained when coconut oil mixed medium was used as a carbon source under normal culture conditions; additionally, the highest fatty acid contents (% CDW) were achieved at 26 • C, the highest MCFA contents (% CDW) with palm oil mixed medium, and the maximum overall lipid productivity (8.6 g/L•d) with coconut oil supplementation.Since we obtained diverse outcomes under the different culture conditions, synergetic insights into the individual culture conditions are needed to evaluate the different combinations based on an orthogonal matrix design (OMD).

Synergistic Effect of the Culture Conditions on Fungal Biomass and Lipid Accumulation
To investigate cell growth and lipid accumulation of the strain M. circinelloides M65-TE-04, the fungal cells were cultured under different conditions.The coded values of individual variables by the orthogonal matrix design (OMD) are provided in Table 1.All the experiments were carried out in a 1-L baffled shake flask with 250 mL modified K and R medium containing glucose or other carbon sources (see Section 2.1 for details).The responses for the four independent variables (i.e., temperature, agitation, pH, and carbon sources) obtained based on the orthogonal matrix design (OMD) (SPSS 19) were provided in Table 2.
Table 2. Orthogonal matrix design (OMD) for the combination of different culture conditions (coded values) and their responses in biomass contents (CDW, g/L), lipid contents (TFA,% CDW), MCFA contents (MCFA,% TFA), lipid productivity (P TL , mg/L•d), and MCFA productivity (P MCFA , mg/L•d) of M. circinelloides strain (i.e., M65-TE-04) after 72 h (3 days) of cultivation period.Values were the mean of three independent experiments.We observed an elevation in biomass in assays 12 and 16 and a slight elevation in assays 14 and 4, while the lipid contents (% CDW) exceeded the maximum in assay 16 and significantly exceeded the maximum in assays 12 and 4. Assays 2 and 5 also showed higher lipid contents (% CDW).Overall, assays 16, 12, and 4 demonstrated the highest lipid (g/L) and MCFA contents (g/L), as well as lipid and MCFA productivities (mg/L•d), as shown in Table 2. Thus, from the above discussion and the results represented in Table 2, we concluded that assay 16 with specific cultivation conditions (i.e., temperature 26 • C, pH 6, agitation speed 300 rpm, 3% coconut oil mixed medium as the carbon source) provided the best outcome for M. circinelloides strain M65-TE-04among all 16 assays.This condition eventually provided the highest lipid (i.e., 4767 mg/L•d) and MCFA productivity (i.e., 2570 mg/L•d) (Table 2).The higher contents of MCFA obtained in M. circinelloides (M65-TE-04) by using coconut oil.This is might be due to availability of MCFA in coconut oil, and therefore it shows that M. circinelloides should assimilate the MCFA from the coconut into their cells.
M. circinelloides naturally produces LCFAs, eventually making it less attractive for use as a precursor in the food, biochemical, and aviation industries.In our recent investigation, we genetically manipulated M. circinelloides strain M65, ultimately to obtain mutant strains with high MCFA-producing ability.Although we were successful, the resultant strains showed a very low biomass, which eventually affected the lipid and MCFA productivity [1].Thus, the approach used in the present study, optimizing the cultivation conditions (i.e., assay 16) to enhance the MCFA yield (g/L) and contents (% TFA) in M. circinelloides, fulfilled the primary objective of the current investigation.No studies have yet been conducted in this respect.The utilization of different agriculture by-products (i.e., copra cake, palm fruit cake) as supplementation for the culture medium, which would be environmentally friendly and cost effective, is our next possible objective.

Figure 4 .
Figure 4. (A-C) Effect of different carbon sources on (A) cell dry weight (CDW, g/L) and MCFA content (% TLC), (B) fungal TFA (% CDW), (C) biomass productivity (P CDW , g/L•d), and lipid productivity (P TL , g/L•d) of the M. circinelloides strain (i.e., M65-TE-04) after 72 h (3 days) of cultivation.Values are the mean of three independent experiments.Error bars show the standard error of the mean.Asterisks indicate that the differences (* p < 0.05; ** p < 0.01; *** p < 0.001) between the means of different treatments are statistically significant.

Figure 5 .
Figure 5. Fatty acid profile (% of TFA) of the M. circinelloides strain (i.e., M65-TE-04) in the presence of differences in pH, agitation speed, temperature, and carbon sources based on the orthogonal matrix design (OMD).The samples were harvested after 72 h (3 days) of cultivation.Values are the mean of three independent experiments.Error bars show the standard error of the mean.

Figure 5 .
Figure 5. Fatty acid profile (% of TFA) of the M. circinelloides strain (i.e., M65-TE-04) in the presence of differences in pH, agitation speed, temperature, and carbon sources based on the orthogonal matrix design (OMD).The samples were harvested after 72 h (3 days) of cultivation.Values are the mean of three independent experiments.Error bars show the standard error of the mean.

Table 1 .
Orthogonal matrix design (OMD) to evaluate the diverse culture conditions (coded values for factors and their levels).