Biological Characteristics of the Mycelium and Optimization of the Culture Medium for Phallus dongsun

P. dongsun GZCC0351 was isolated and purified from fresh fruiting bodies. The sample was collected from Liulong Town, Dafang County, Bijie City, Guizhou Province, China, and is preserved in the Species Preservation Room at the Guizhou Institute of Biology. Slants were incubated at 25°C on PDA for 7–10 days in glass petri dishes (90 mm in diameter).

All experimental materials were sourced from a consistent supplier and were newly acquired for each use to guarantee substrate stability and uniformity. Brown sugar, wheat bran, carrot powder, potato starch, corn starch, wheat starch, and soybean powder, identified as natural substrates, were procured from Xinghua Lüshuai Food Co., Ltd., (China). Wood powder, a natural substrate, was sourced from Caoxian Luyi Wood Industry Co., Ltd. (China). The following reagents, all of the analytical grade, were acquired from Tianjin Kemiou Chemical Reagent Co., Ltd. (China): KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, MgCl₂ · 6H₂O, ZnSO₄, CaCl₂, CaSO₄ · 2H₂O, Fe₂(SO₄)₃, CaCO₃, NH₄Cl, (NH₄)₂SO₄, KNO₃, NaNO₃, carbamide, glucose, fructose, sucrose, maltose, lactose, cellulose, xylogen, chitosan, dimethyl sulfoxide, and Tween 80. Additionally, all the analytical grade, sodium carboxymethyl cellulose, sodium alginate, gum arabic, and xanthan gum were purchased from Tianjin Yongda Chemical Reagent Co., Ltd. (China). Furthermore, biochemical reagents such as peptone, yeast powder, yeast extract, beef extract, and soybean powder were procured from Shanghai Bowei Biotechnology Co., Ltd. (China).

Potatoes (200 g) were peeled, sliced thinly, boiled in water until they reached a soft yet firm consistency, and subsequently strained to extract the juice. The extracted juice, along with dextrose (20 g) and agar (20 g), was mixed, and the volume was adjusted to 1000 ml, ensuring the maintenance of the natural pH.

After preparing the medium, it was sterilized at 121°C for 30 minutes, then cooled and aliquoted into 90 mm petri dishes, with a volume of 20 ml/dish. Following solidification, the medium was inoculated with a P. dongsun inoculum block (approximately 5 mm in diameter) and incubated at 25 ± 2°C. Cultivation proceeded in darkness for 28 days in a constant-temperature incubator.

The Plackett-Burman design was employed to identify components that had significant influence on medium optimization. Two natural substrates, one suspension agent, one carbon source, one nitrogen source, and two inorganic salts were investigated for their impact on colony diameter. Utilizing the Plackett-Burman design, a 12-run experiment was conducted to evaluate nine factors, including two virtual variables. Each factor was set at two levels: -1 representing the low level and +1 representing the high level, with the high level being 1.5 times the magnitude of the low level (refer to Table I).

Based on the results of the Plackett-Burman design, the steepest ascent method was applied to ZnSO₄ and NH₄Cl to determine the centroid of the response surface design. The main effects, significance, step size, and direction of change of the factors were analyzed through the regression equation of the Plackett-Burman design model.

Based on the single-factor design, Plackett-Burman design, and steepest ascent path design, a response surface methodology (central composite design, CCD) encompassing 20 runs was employed. The variables tested (soybean powder, ZnSO₄, NH₄Cl) were designated as A, B, and C, respectively, and were evaluated at five distinct levels, incorporating factorial points (-1, +1), axial points (-1.6818, +1.6818), and a central point (0), as depicted in Table II.

Building upon the outcomes of preceding experiments, the impact of varying temperatures on the biological characteristics of P. dongsun mycelium was investigated. Subsequently, medium plates were inoculated and incubated within a climate chamber at temperatures of 5 ± 2°C, 10 ± 2°C, 15 ± 2°C, 20 ± 2°C, 25 ± 2°C, 30 ± 2°C, and 35 ± 2°C, respectively.

Each experiment was conducted with five replicates. Upon completion of the cultivation period, the colony diameter resulting from mycelial elongation was measured using the criss-cross method. For colonies exhibiting incomplete diameters, measurements were recorded at their widest points (Zhang et al. 2021). Furthermore, the mycelial growth rate was calculated employing the subsequent mathematical equation (1). 1Mycelial growth rate (mm)d== Colony diameter (mm)- Inoculum block diameter (mm)2× culture time (d)\begin{array}{*{35}{l}} Mycelial growth rate \frac{(mm)}{d}= \\ =\frac{ Colony diameter (mm)- Inoculum block diameter (mm)}{2\times culture time (d)} \\\end{array}

Colony diameter and mycelial growth rate were presented as mean values ± standard deviation (SD). An analysis of variance (ANOVA), followed by Tukey’s post hoc test, was utilized to conduct multiple comparisons for significant differences at p < 0.05. The DesignExpert® software, version 13.0.1.0 (StatEase Inc., USA, www.statease.com), was employed to design experiments, regression, and graphical analysis of the data obtained. Bar and line graphs were generated using the OriginPro® 2021 software package, version 9.8.0.200 (OriginLab Corporation, USA).

The effect of natural substrates on the biological properties was investigated. Adding wood powder to the medium resulted in the fastest mycelial growth and largest colony diameter (0.85 ± 0.13 mm/d), followed by wheat bran, wheat starch, and carrot powder. However, the differences among these substrates were not statistically significant. Upon adding carrot powder, mycelial growth was observed to be both dense and vigorous, in contrast to the other test groups, which exhibited either dense or moderately dense growth. Adding carrot powder, wood powder, and wheat bran to the medium facilitated the most vigorous mycelial growth. The poorest mycelial growth was observed with the addition of potato powder. Considering colony diameter, growth density, and overall mycelial development, the incorporation of wheat bran, wood powder, wheat starch, and carrot powder into the medium proved to be beneficial for the growth of P. dongsun mycelium (Table III, Fig. 1). The study aimed to investigate the effects of composite natural substrates on the biological characteristics of P. dongsun mycelium, with wheat starch, carrot powder, and wood powder being added individually or in combination to the culture media (Table IV). Results indicated that the FA4 treatment exhibited the fastest mycelial growth rate and the largest colony diameter (67.07 ± 4.79 mm). FA4 and FA6 treatments demonstrated the most vigorous mycelial growth, albeit with sparse colony density. The FA5 treatment resulted in the densest mycelial density and superior growth. In conclusion, the FA5 treatment was identified as the optimal natural substrate combination, comprising 10 g/l wheat starch and 10 g/l carrot powder (Table IV, Fig. 2).

Fig. 1.

Fig. 2.

When suspension agents were added to the medium, P. dongsu mycelium grew adherent to the surface of the medium with less aerial mycelium, resulting in faster mycelial growth, denser colonies, and vigorous growth. However, there were differences in the rate of mycelial growth and overall growth (Table V, Fig. 3). A medium suspension can be formed when sodium carboxymethyl cellulose, sodium alginate, gum arabic, and xanthan gum are added. However, only with the addition of xanthan gum do the medium become more viscous and the suspension stabilize. Adding dimethyl sulfoxide and Tween 80 to the medium resulted in no suspension formation and led to medium precipitation. The results indicated that xanthan gum is the best-suspending agent. A higher concentration of xanthan gum significantly affected the viscosity of the medium. Varying concentrations of xanthan gum had negligible effects on the growth of P. dongsu mycelium, which all exhibited average, vigorous growth and high colony density (Table VI, Fig. 4). However, considering the physical state of the medium, increased concentrations of xanthan gum made it more difficult to disperse and increased the viscosity, adversely affecting the medium’s partitioning. In summary, an optimal concentration of 0.5 g/l xanthan gum was determined for the culture medium.

Fig. 3.

Fig. 4.

When glucose, maltose, and cellulose were utilized as carbon sources in the culture, P. dongsun mycelium exhibited rapid growth, high colony density, and an optimal growth rate, notably with glucose, which demonstrated a mycelial growth rate of 0.77 ± 0.06 mm/d. When fructose, sucrose, lactose, and brown sugar served as carbon sources, the mycelium grew and maintained a high density; however, its performance was comparable to the control group, showing no significant difference. Utilizing chitosan as a carbon source resulted in suboptimal and slower mycelium growth, which was inferior to the control; conversely, when lignin served as the carbon source, the mycelium failed to germinate and grow (Table VII, Fig. 5). In conclusion, glucose was determined to be the optimal carbon source.

Fig. 5.

Nitrogen source screening experiments demonstrated that the NaNO₃ exhibited the fastest mycelial growth rate (0.74 ± 0.09 mm/d), with vigorous growth, yet the colony density was notably sparse. In contrast, the soybean powder demonstrated a faster mycelial growth rate (0.69 ± 0.09 mm/d), experiencing vigorous growth and dense colonies; however, the difference from the NaNO₃ was minimal. Adding peptone, yeast powder, yeast paste, and carbamide to the culture medium significantly inhibited the growth of P. dongsun mycelium, resulting in a slow growth rate and suboptimal growth (Table VIII, Fig. 6). In conclusion, soybean powder was determined to be the best nitrogen source.

Fig. 6.

Inorganic salt screening experiments indicated that the ZnSO₄ exhibited the fastest mycelial growth rate (0.80 ± 0.16 mm/d), characterized by vigorous growth, and was followed by NH₄Cl (0.76 ± 0.13 mm/d) and Fe2(SO₄)₃ (0.65 ± 0.18 mm/d). Incorporating MgSO₄ · 7H₂O and CaCO₃ into the culture medium significantly inhibited the growth of P. dongsun mycelium (Table IX, Fig. 7). To explore the effects of composite inorganic salts on the biological characteristics of P. dongsun mycelium, ZnSO₄, NH₄Cl, and Fe₂(SO₄)₃ were each added either individually or in combination to the culture media (Table X). The findings revealed that FM-F demonstrated the fastest mycelial growth rate (1.00 ± 0.13 mm/d). In conclusion, FM-F emerged as the optimal inorganic salt combination, comprising ZnSO₄ 0.5 g/l and NH₄Cl 0.5 g/l (Table X, Fig. 8).

Fig. 7.

Fig. 8.

The data indicated that process optimization plays a crucial role in enhancing the efficiency of colony diameter (Table XI). Analysis of the regression coefficients for 9 factors (Table XII) revealed that X₁, X₇, and X₈ positively affected colony diameter. X2, X4, and X₅ demonstrated negative impacts. Variables affecting a confidence level above 95% are deemed significant factors. Based on these results (Table XII), two factors, X₇ (ZnSO₄) and X₈ (NH₄Cl), were identified as significant for the colony diameter of P. dongsun mycelium.

Based on the coefficients from the colony diameter regression equation derived from the Plackett-Burman design model, the steepest ascent path for optimizing colony diameter involved primarily adjusting the levels of ZnSO₄ and NH₄Cl. The magnitude and direction of changes were dictated by the regression equation from the Plackett-Burman design model, as illustrated in Table XIII. Colony diameter reached its peak at the “ O + 3Δ” factor level before decreasing. This “O + 3Δ” factor level, marking the maximum response, was subsequently chosen as the central point for the central composite design.

To assess the impact of medium components on colony diameter, an evaluation involving soybean powder, ZnSO₄, and NH₄Cl was conducted using a response surface optimization experiment. Detailed experimental designs and results are presented in Table XIV. A regression analysis was conducted to align the response function with the experimental data. Based on the variables presented in Table XV, the model, denoted as equation (2), articulates the colony diameter (Y) in relation to the concentrations of soybean powder (A), ZnSO₄ (B), and NH₄Cl (C): 2Y=62.04−3.24×A−1.15×B−0.23×C+0.16×AB+1.14××AC−0.81×BC−1.40×A2−0.32×B2−0.96×C2\begin{matrix} Y=62.04-3.24\times A-1.15\times B-0.23\times C+0.16\times AB+1.14\times \\ \times AC-0.81\times BC-1.40\times {{A}^{2}}-0.32\times {{\text{B}}^{2}}-0.96\times {{C}^{2}} \\end{matrix}\

The F -test analysis of variance (ANOVA) results demonstrated that the regression achieved statistical significance at the 99% confidence level (Table XV), with a high goodness of fit value (R² = 0.9242), signifying the model’s accurate representation of the relationship between the investigated factors and response values. The “ F- value” for “Lack of Fit” was found to be insignificant, indicating the model’s retention of fit. Furthermore, the linear terms of A and B, the interaction term of AC, and the quadratic terms of A² and C² were identified as significant model terms for colony diam-eter. The findings reveal that the maximum values for colony diameter are attained through the regression equation and two-dimensional contour plots (Fig. 9). The model forecasted a maximum colony diameter of 64.75 mm at the optimized medium component levels of soybean powder 7.50 g/l, ZnSO₄ 0.71 g/l, and NH₄Cl 0.68 g/l. Validation testing demonstrated the rapid growth of P. dongsun mycelium, with the colony diameter and mycelial growth rate achieving 63.00 ± 3.89 mm and 1.04 ± 0.14 mm/d at 28 days, respectively. These outcomes were closely aligned with the model’s predicted values and can facilitate the evaluation of medium components and mycelial growth in P. dongsun.

Fig. 9.

The impact of various temperatures on the biological characteristics of P. dongsun mycelium was significant, as illustrated in Fig. 10 and 11. The findings indicated that under culture conditions at 20 and 25°C, the colonies were dense, vigorous, and robust, significantly surpassing those of other treatments. Mycelium growth was slow and weak under culture conditions at 10, 15, and 30°C. Furthermore, at 35°C, the mycelium failed to germinate, and the mycelium on the inoculum block subsided, indicating death. In conclusion, the optimal temperature range for the growth of P. dongsun mycelium is 20–25°C, with growth ceasing below 5°C and death occurring above 35°C.

Fig. 10.

Fig. 11.