Optimization of the nutritional constituents for ergosterol peroxide production by Paecilomyces cicadae based on the uniform design and mathematical model

Mono-factor experimentation

Effect of carbon source

The carbon source is a major component of the cytoskeleton in the process of fungal growth, and it provides the energy for biomass growth and metabolite synthesis. Saccharide is the main carbon source for the synthesis of biomass and product in the fungal growth process. Most fungi can grow and synthesize the metabolite in the medium using sucrose, glucose, or maltose as carbon sources³². The effect of carbon sources on EP synthesis is rarely reported in the literature. In the present study, glucose, sucrose, maltose, fructose, mannitol, and glycerol were used as carbon sources to study the effects of various types of carbon sources on the EP synthesis in the fermentation process of P. cicadae. Figure 1 shows that the EP yield was significantly higher when glycerol or mannitol was added into the fermentation broth compared with the other carbon sources (219 and 95.09 μg/L), respectively. The EP yield was significantly higher when the disaccharides (maltose and sucrose) were added compared with monosaccharides (glucose and fructose), while the EP yield between maltose and sucrose or between glucose and fructose was not significantly different.

Carbon source is the main source of nutrients and energy in the process of mycelial growth and metabolite synthesis. Therefore, the type and concentration of carbon source directly determine the specific growth rate, the consumption rate of carbon source, and the synthesis rate of metabolites. In the process of filamentous fungal fermentation, many studies have investigated the relationship between the yield of secondary metabolite and carbon sources. Generally, most organisms prefer to utilize glucose as a carbon source. However, glucose is not conducive to the synthesis of metabolites. The phenomenon can be attributed to the repression of carbon catabolites. Glucose, for example, can inhibit antibiotic synthesis by blocking or inhibiting key enzymes in the antibiotic biosynthetic pathway^33,34. As a carbon source, glycerol or mannitol can avoid the inhibition of carbon catabolism. The effect of membrane fatty acids on permeability has been reported³⁵. Few works have investigated the influence of the membrane fatty acids of R. arrhizus cell on permeability. Different components of the culture medium can change membrane composition and lead to the change of cell membrane permeability. The ratio of saturated and unsaturated fatty acids directly determines the permeability of the membrane. Glycerol has been proven to increase saturated fatty acids of cell membrane³⁶. Fatty acid saturation determines the overall stability of the membrane to some extent, while an increase in fatty acid saturation can cause a decrease in membrane fluidity and thus its permeability³⁷, and it blocks the secretion of ergosterol, the precursor of EP, and promotes the synthesis of EP. Therefore, glycerol was chosen as the carbon source in the subsequent experiments.

Effect of nitrogen source

Many fungi can utilize a wide variety of nitrogen sources. The effects of different nitrogen sources on mycelial growth and product synthesis are very different³⁸. Many nitrogen sources are involved in the synthesis of nitrogen-containing metabolites, such as amino-acid, nucleotides, and vitamins. These amino acids and nucleotides are the precursors of some secondary metabolites³⁹. Therefore, nitrogen sources must meet the needs of mycelial growth and the synthesis of some metabolites. Nitrogen sources can be divided into organic and inorganic nitrogen sources. Peptone, soybean meal, corn syrup, beef extract, and yeast powder are the major organic nitrogen sources, and KNO₃, NaNO₃, CO(NH₂)₂, (NH₄)₂SO₄, and NH₄OH are the primary inorganic nitrogen sources. Peptone, yeast powder, KNO₃, CO(NH₂)₂, (NH₄)₂SO_4, and NH₄OH were used to study the effect of nitrogen source on the EP synthesis by P. cicadae, and the experimental results are shown in Fig. 2.

Figure 2 shows that a significant enhancement was observed when utilizing peptone and yeast powder for EP yield. The maximum EP yield (68.49 μg/L) was obtained when the yeast powder was used as the nitrogen source, followed by peptone (59.38 μg/L). Their EP yields were significantly higher compared with the control group without the nitrogen sources (30.6 μg/L). However, after KNO₃, CO(NH₂)₂, (NH₄)₂SO_4, and NH₄OH were added, respectively, the mycelia grew slowly. Moreover, after the fermentation, the dried mycelium was dark, and its EP concentration was less than 26.60 μg/L. The results showed that inorganic nitrogen in the fermentation of P. cicadae was not good for EP production.

The pathway of nitrogen metabolism is closely related to the pathway of secondary metabolite biosynthesis. Nitrogen regulation in fungi has been extensively reviewed^40,41. One of the differences between yeast powder and peptone is that yeast powder contains higher levels of biotin. The biotin can increase the cell membrane density, reduce the cell membrane permeability, block the secretion of EP precursor ergosterol, and promote the synthesis of EP. This also led to the highest yield of EP when yeast powder was added. However, excessive biotin prevents the transport of the substrate and metabolites between the extracellular and intracellular. To control the content of biotin in the medium, the yeast powder and peptone were chosen as the nitrogen source in the uniform design experiment.

Effect of inorganic salt

As the coenzymes of many types of enzymes, inorganic salts play a vital role in the growth and metabolism of microorganisms, the balance of osmotic pressure, and the translocation of nutrients and metabolites between the intracellular and extracellular cells. Therefore, we investigated the effects of six inorganic salts on the EP yield in the broth of P. cicadae, including KH₂PO₄, MgSO₄, MnSO₄, CuSO₄, ZnSO₄, and FeSO₄. Figure 3 shows that the EP yield was significantly higher when KH₂PO₄, MgSO₄, and ZnSO₄ (32.21 ± 2.41, 38.85 ± 0.75, and 62.23 ± 1.60 μg/L, respectively) were added compared with the control group (no addition of inorganic salt, 28.12 ± 2.03 μg/L). Especially, the increase of EP yield was the most significant when ZnSO₄ was added. ZnSO₄ is beneficial to the fermentation of Cordyceps jiangxiensis to produce intracellular polysaccharides and mycelia⁴², which is consistent with our results. The EP yield was significantly low when MnSO₄, CuSO_4, and FeSO₄ were added. Therefore, we chose KH₂PO₄, MgSO_4, and ZnSO₄ as the inorganic salts.

Uniform-design experimentation

According to the result of the mono-factor experimentation, glycerol, yeast powder, peptone, KH₂PO₄, MgSO_4, and ZnSO₄ were screened as the independent variables, and U₁₀(10⁶) was applied to investigate the effects of six factors on the EP yield. Table 1 shows the scheme of U₁₀(10⁶). The sampling time was selected as the control variable, which was set at 28, 56, 84, and 112 h. Table 1 shows the experimental results.

To more accurately analyze the relationship between each factor and EP yield, the following formula was used to fit the data in Table 1.

where F_i is the sampling time (T), glycerol concentration, yeast extract concentration, peptone concentration, ZnSO₄ concentration, MgSO₄ concentration, or KH₂PO₄ concentration. α_i and β_i represent the coefficients of factor F_i and F_i², respectively. C represents the fitting constant. Table 2 presents the fitting results. F-value (7.754) and p-value (0.000) of the model with 95% confidence in Table 2 implied that the fitted equation was extremely significant and reliable.

The coefficients of each item in Table 2 were introduced into Eq. (1) to give the following Eq. (2).

The coefficients of F₃, F_6, and F₇ greater than 0 indicated that positive correlations existed between Y_EP and F₃, between Y_EP and F₆, and between Y_EP and F₇, respectively. Namely, the higher concentrations of yeast extract, MgSO_4, and KH₂PO₄ in the tested range of concentration, the higher Y_EP. The coefficients of F₄ and F₅ less than 0 indicated that negative correlations existed between Y_EP and F₄, and between Y_EP and F₅, respectively. Namely, the lower concentrations of peptone and ZnSO₄ in the tested range of concentration, the higher Y_EP.

The coefficients of F₁² and F₂² less than 0 exhibited that there was an inverted-U-shaped relationship between Y_EP and F₁, and between Y_EP and F₂, and Y_EP had the maximum value. The following two equations were obtained by taking the derivation of Y_EP to F₁ and F_2.

Suppose that Y_EP’ = 0, F₁ = 8.617/0.118 = 73 h, F₂ = 50.605/12.468 = 4.06. When the fermentation time was 73 h and glycerol concentration was 4%, the Y_EP yield reached the maximum value.

To sum up, the optimum fermentation medium of EP contained (g/L): 40 glycerol, 6.5 yeast extract, 2 peptone, 2.4 KH₂PO₄, 2.4 MgSO₄, and 0.05 ZnSO₄. The optimum fermentation time was 73 h. To verify the optimization efficiency, an experiment was conducted with the basal fermentation medium and optimum fermentation medium, and the fermentation time was 80 h. Under the above-mentioned conditions, the maximum theoretical yield of EP is 203.92 μg/L at 80 h. Actually, the measured EP yield with the optimum medium was 256 µg/L and significantly higher compared with the basal medium (43 µg/L) and the maximum theoretical yield.

Kinetic model of growth

Figure 4 shows the changing profiles of biomass, EP, and substrate in the process of P. cicadae fermentation. The biomass was increased after 12 h, and then it continued to increase rapidly till the end of the growth phase. The EP production was rapidly increased from 12 to 76 h. The maximum EP yield was obtained at 76 h (261.47 μg/L). After 76 h, the EP yield was rapidly decreased (Fig. 4). In the process of fermentation, the glycerol concentration was decreased rapidly from 34.34 to 21.02 g/L.

The experimental data of glycerol concentrations in Fig. 4 were fit with Eqs. (9)–(12), respectively (Table 3). The goodness-of-fit R² values (> 0.98), F-values (> 240), and p-value (0.000) exhibited that these four models simulated better the kinetic experimental data, described the time course profile of EP productivity, and had the high explanation rate and credibility.

Monod and logistic models are adopted as the most applicable models for cell growth. However, in addition to the limitation of nitrogen and phosphorus sources, there is an oxygen limitation in aerobic fermentation. The Monod model is generally constrained due to the presence of oxygen limitation in the aerobic fermentation process. In contrast, the logistic equation can well reflect the relationship between the cell growth rate and the biomass. In fact, in the aerobic fermentation process, the oxygen concentration is steeply decreased in the initial stage of fermentation, and then the oxygen concentration remains constant till the strain begins to die. Therefore, before the strain begins to die, the effect of oxygen concentration on cell growth can be neglected. Although the logistic model can simulate the cell growth of fungi^43,44, the model gives very little valuable information. The combination of these four models can evaluate the reasonableness of any parameter changes in the process of fermentation.

Table 3 shows that although the p-value of the four models was 0.000, the F-value of the Aibe model was lower compared with the other models. Based on the F-value, we deduced that the effect of the product on the EP concentration was less compared with the viscosity and substrate. Here, it should be pointed that the product was not only EP but also other metabolites that might inhibit the synthesis of EP. To decrease the effects of viscosity and substrate on EP synthesis in the fermentation process and to increase the EP yield, fed-batch glycerol and water supplementation were used to increase the EP yield. Furthermore, water supplements could reduce the inhibitory action of production on biomass growth.

To verify our deduction, we designed three groups of experiments, and each experiment was carried out in triplicate. The EP yield of group 3 (glycerol supplementation) was 327 ± 4.93 μg/L, which was significantly higher compared with group 2 (water supplementation, 286 ± 3.23 μg/L). The EP concentration of group 2 and group 3 was markedly higher compared with group 1 (no supplementation, 272 ± 1.63 μg/L). The result further proved that the combination of four structured models could guide the optimization of the fermentation process.