Optimization of Metarhizium koreanum MN031-Mt 46: Nutritional Supplementation to Improve Conidia and Cuticle-Degrading Enzyme Production by Solid-State Fermentation

Abstract

This study aimed to evaluate five different mixed agricultural wastes as potential substrates for solid-state fermentation (SSF) to produce conidia of Metarhizium koreanum MN031-Mt 46. Single-factor experiments and a Box-Behnken design (BBD) were employed to optimize the fermentation conditions for enhanced conidia yield. Results indicated that a mixed substrate comprising broken rice and rice bran significantly enhanced the optimal production of aerial conidia of MN031-Mt 46. Optimal fermentation conditions established through response surface methodology (RSM) revealed that with the addition of shrimp shell waste to the mixed substrate, conidia production increased to 8.45 × 10⁸ conidia per gram of dry substrate at 26.19°C temperature, 39.76% moisture, and 1.45% of shrimp shell waste after 301.87 h of incubation. Enhanced conidia performance indices were observed, including higher conidia weight, increased water content, and reduced residue post-harvest. The optimized fermentation conditions resulted in enhanced cuticle-degrading enzymatic activities, with maximum activities of 58.78 ± 2.29 U g^-1 ds for protease, 126.57 ± 6.47 U g^-1 ds for lipase, and 58.32 ± 0.78 U g^-1 ds for chitinase. These findings highlight the potential and versatility of mixed SSF using cost-effective agricultural waste for biopesticide and hydrolytic enzyme production, while promoting sustainable waste management and environmental pollution control, aligning with circular economy principles.

Keywords: Metarhizium koreanum; cuticle-degrading enzyme; performance indices; response surface methodology; solid-state fermentation.

Fig. 1. Conidia production of mutant M. koreanum (MN031-Mt 46) and wild-type (MNMHN031) on different mixed solid agricultural waste.

Different agricultural waste tested included WHP: water hyacinth powder, BPSP: banana pseudostem powder, SB: Sugarcane bagasse, CCW: corncob waste, WBR: broken rice, RB: rice bran. Error bars are standard errors and mean at 95% Cl. Different letters indicate statistically significant differences (p > 0.05, Turkey HD test). Lowercase (7^th day) and uppercase (14^th day) letters represent statistical differences between treatments. Data points indicate the means of three replicates.

Fig. 2. Single-factor at a time optimization of the fermentation process of five factors on conidia production of M. koreanum (MN031-Mt 46).

(A) Moisture content, (B) Substrate weight, (C) Temperature. (D) Shrimp shell waste. (E) Molasses supplementation. The same letter on the box plots indicates statistically insignificant variation (p > 0.05, Turkey HD test) among the treatments.

Fig. 3. Response surface experiment results of conidia production of mutant isolates of M. koreanum (MN031-Mt 46).

(A–L) 3D response surface map and 2D contour plots corresponding to the interaction between different variables on the conidia production of MN031-Mt 46 at p < 0.05. (A–B) moisture level vs temperature, (C–D) shrimp shell vs temperature, (E–F) incubation vs temperature, (G–H) shrimp shell vs moisture level, (I–J) incubation time vs moisture levels, (K–L) incubation time vs shrimp shell and (M) Correlation of actual and predicted values of MN031-Mt 46 conidia production (conidia/g ds).

Fig. 4. Performance indices results based on RSM-optimized substrates for M. koreanum isolate (MN031-Mt 46) and wild-type (MNHMN031).

(A) Mean number of conidia/g of powder. (B). Mean weight of conidia/kg of powder. (C) Percentage water content of the conidia. (D) Percentage consumed substrates. (E) Conidia germination. Different letters above error bars indicate a significant difference across the treatment. The same letter on the violin plots indicates statistically insignificant variation (p > 0.05, Turkey HD test) among the treatments (optimized and control fermentation).

Fig. 5. Results of performance indices.

(A) Dry aerial conidia of M. korenum (MN031-Mt 46) based on optimized medium variables, (B) MN031-Mt 46 (control), (C) Wild-type MNMHN031(control) and (D) The consumed residues after conidia harvest.

Fig. 6. Cuticle-degrading enzyme activity of M. koreanum fermented conidia obtained based on RSMoptimized and control parameters.

(A) Enzyme index, (B) Enzymatic activity (U g⁻¹ ds). The same letter indicates statistically insignificant variation (p > 0.05, Turkey HSD test) among each enzyme test.