Ability of Three Pleurotus Species for Effective use of Giant Grass Compost

The experiment was carried out at the Academy of Juncao Science and Technology, Fujian Agricultural and Forestry University. Three Pleurotus strains P377, P27, and P30 were originally obtained from different geographic locations in China and collected at the Fujian Agricultural and Forestry University. They were identified further as P. florida, P. pulmonarius, and P. ostreatus, respectively, using DNA sequencing, according to (Menolli et al. 2010). The above strains were selected after the initial screening process. They were maintained on PDA medium (as the first) and also in polyethylene bags filled with 500 g of sterile substrate made up of raw materials of 50% Pennisetum sinense, 28% Miscanthus floridulus, 20% wheat bran, and 2% of gypsum (as the second spawn).

Fully mature plants were cut and ground to 0.5–1.5 cm particle size, sun-dried for 3 days, then piled 1 × 1 × 1 m, and composted for 21 days. The heap was turned over every 5 days, and the compost temperature was monitored daily. The highest and lowest temperatures were noted at 6 and 21 days, respectively. After 21 days the compost was sundried. For the experiment, the giant grass compost was mixed with 10% wheat bran and 2% gypsum powder, then moistened to a water content of 58% and loaded in polyethylene bags according to the methods of Rajapakse et al. (2007) and Okal et al. (2021). Five hundred grams of substrate mixture (pH 7.8) was loaded into the polyethylene bags. According to Rajapakse et al. (2007) and Okal et al. (2021), the polyethylene bags were sealed with perforated caps to allow for some aeration, then pasteurized for 6 hours at 80 °C and 90% relative humidity. After cooling, the experimental substrate was inoculated with 30 g of the second spawn substrate. For each of the evaluated species, 18 replicate bags were prepared. The bags were incubated in the climate box QHX-400BSH-III (Shanghai CIMO) at a temperature of 24 °C, a humidity of 50%, and in the dark. To compare growth rates between Pleurotus species, the time required for the substrate to fully colonize and for primordial initiation was recorded in each bag. After the bags had colonized completely, they were opened and taken to the growing room with a temperature range of 19–24 °C, and 80–85% humidity that was kept by regularly spraying with water (Ahmed et al. 2013). The mycelial samples were obtained by randomly selecting one bag from each species, and after thoroughly mixing, three replicate samples were taken at the substrate inoculation stage, mycelial colonization stage, and fruiting body stage. In harvesting, fruiting bodies were twisted and uprooted from the base. The harvest from two subsequent flushes for each bag was recorded to determine its total yield (g). Samples of fruiting bodies and colonized substrate were frozen utilizing liquid nitrogen and then kept at −80 °C until later use. To find the biological efficiency (BE) for each species, we used the formula (Zervakis & Balis 1992): BE=Fresh weight of fruit body×100Dry weight of the substrate {\rm{BE}} = {{{\rm{Fresh}}\;{\rm{weight}}\;{\rm{of}}\;{\rm{fruit}}\;{\rm{body}} \times 100} \over {{\rm{Dry}}\;{\rm{weight}}\;{\rm{of}}\;{\rm{the}}\;{\rm{substrate}}}}

The Elisa method was used to perform the assays for enzyme activity of laccases, manganese peroxidase (MnP), lignin peroxidase, xylanases, exoglucanases (FPase), endoglucanases (CMCase), β-glucosidase, and amylase in the samples of mycelial and fruiting phases, according to Okal et al. (2021). Briefly, 50 μL of the standard solution (prepared by mixing 1 μg of supplied ELISA standard with 1 ml of ELISA diluent, then mixed 10 μL of the reconstituted standard with 990 μL of incubation buffer) was added to a micro-Elisa test strip plate (standard well), and 40 μL of the sample dilution buffer was added to the testing sample well. Then, 10 μL of the testing sample was added to the wells and gently mixed. Except for the blank well, 100 μL of HRP-conjugate reagent was then added to the wells, followed by the closure of each plate with an adhesive strip and incubated for 30 minutes at 37 °C. Then, a wash solution was prepared, diluted 30-fold with distilled water, and kept. The closed plates were then uncovered, followed by discarding the contents and drying with a swing. Then, the washing buffer was added to each well and allowed to stand for 30 seconds, and then drained. Except for the blank well, 50 μL of HRP-conjugate reagent was then added to each well and incubated for 30 min at 37 °C, followed by washing with buffer. Then in each well, 50 μL of chromogenic solution A and 50 μL of chromogenic solution B were gently mixed, protected from the light, and incubated for 15 minutes at 37 °C. To stop the reaction, 50 μL of H₂SO₄ solution was added to each well, and when the color changed from blue to yellow, the absorbance (optical density – OD) was then read at 450 nm within 15 minutes of adding the stop solution. A standard curve was drawn and the OD value was used to calculate the sample's corresponding density. The actual sample density for each enzyme was determined by multiplying the sample density on the graph by the dilution factor.

The analysis of contents of polysaccharides, fiber, carbohydrates, fat, proteins, amino acids, crude ash, and heavy metals (cadmium, arsenic, lead, and mercury) in fruiting bodies from each Pleurotus species was performed. To quantify the content of polysaccharide, the phenol-sulfuric acid method (Nielsen 2010) was applied. Polysaccharides were extracted in the water and precipitated in alcohol. Then, 0.05 g of the crushed sample was mixed with 1 ml of water into a test tube and homogenized in a water bath at 100 °C for 2 hours. The mixture was then centrifuged for 10 minutes at 10,000 rpm, and the supernatant was removed. The 0.2 ml of supernatant was mixed with 0.8 ml of anhydrous ethanol, and the mixture was then employed for quantifying polysaccharides at 490 nm. The content of carbohydrates was determined by the phenol-sulfuric acid method according to Nielsen (2017). Fiber content was examined by referring to the method used by (Bragg & Shofner 1993) and protein content using the BCA kit (Walker 2009; Yanos et al. 2013). The standard working solution was prepared in a ratio of 100 volumes of BCA reagent A to 2 volumes of BCA reagent B, where the reagent A was prepared by dissolving 1 g of sodium bicinchoninate, 2 g of sodium carbonate, 0.16 g of sodium tartrate, 0.4 g of NaOH, and 0.95 g of sodium bicarbonate in 50 ml of distilled water, then brought to 100 ml with distilled water, and the pH was then adjusted with 10 M NaOH to 11.25. The reagent B was prepared by dissolving 0.4 g of cupric sulfate (5 × hydrated) in 5 ml of distilled water, then brought to 10 ml with distilled water. The absorbance measurement of the known standard was performed at 562 nm. The fruiting body fat was evaluated according to (Randall 1974). The total amino acids were investigated using the RP-HPLC method described by Bartolomeo and Maisano (2006). The ion exchange method was utilized to analyze the fruiting body's Cd, Pb, As, and Hg (Huang et al. 2010; Liu et al. 2015).

The data from three independent biological replicates were analyzed with a one-way ANOVA test using SPSS software version 22. The means between enzymes of one strain and between strains were compared utilizing the Duncan test at a 5% significance level.

The enzyme assays performed immediately after inoculation, at the twentieth day of mycelia growth, and at fruiting stages showed various activities of lignocellulolytic enzymes depending on Pleurotus species grown on the same substrate. The results presented the highest activity of LiP of each Pleurotus species (Fig. 1) at the inoculation stage but different at the mycelia colonization and fruiting stages (Table 1). Apart from LiP, every Pleurotus species showed the participation of all enzymes in the degradation of lignocellulosic substances, but their activity differed between developmental stages (Figs. 1–3). Amylase, laccase, and CMCase exhibited significantly higher activities in P. florida, while glucosidase and MnP exhibited higher activity in P. pulmonarius and P. ostreatus. Xylanase and FPase showed significantly higher activities in P. pulmonarius and CMCase in P. ostreatus. During the stage of mycelial colonization, the complex of amylase, CMCase, and FPase had a higher activity in P. florida, xylanase had a higher activity in P. pulmonrius, and the complex of glucosidase, MnP, and laccase in P. ostreatus (Table 2). At the fruiting phase (Table 3 & Fig. 3), LiP activity remained to be the highest for each Pleurotus species. LiP and CMCase peaked in P. pulmonarius. Besides LiP, MnP and lac-case exhibited higher significant activity in P. florida, whereas in P. pulmonarius, a complex of amylase, xylanase, LiP, and CMCase activities were significantly higher. In P. ostreatus, a complex of glucosidase, laccase, and FPase activities were the highest. In brief, during all growth stages, the LiP enzyme (Figs. 1–3) was the most active, displaying the highest significant activity at the fruiting phase, especially in P. pulmonarius.

Figure 1

Figure 2

Figure 3

The results showed that the mycelial growth of P. florida was faster (23.9 days) than that of P. pulmonarius and P. ostreatus (26.5 and 25.3 days, respectively). However, P. pulmonarius was the first in producing the primordia (38.9 days), followed by P. ostreatus (42 days), and P. florida (46.4 days). P. plumonarius produced the highest number and highest mass of fruiting bodies with the highest biological efficiency of 82.6%. The lower number of fruiting bodies in the two other species was accompanied with lower BE. Differences in stipe lengths and pileus diameters were not correlated with their number and mass (Table 4).

The highest content of polysaccharides, proteins, carbohydrates, and fiber was detected in P. ostreatus, whereas the highest contents of total amino acids and fat were detected in the P. florida, and crude ash in P. pulmonarius (Table 5). Differences in the content of heavy metals were not high although statistically significant. The highest content of Hg was detected in fruiting bodies of P. florida and the highest contents of As, Pb, and Cd in P. pulmonarius (Table 6).