Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

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

Ability of Three Pleurotus Species for Effective use of Giant Grass Compost INTRODUCTIONMushrooms are a particular class of macro fungi with a unique epigeous (superficial) fruiting body that generally grows in decomposing organic matter. The majority of cultivated mushrooms, including Pleurotus spp., can be grown on the raw materials of crop or agro-industrial wastes, such as, amongst others, coffee bean dregs, cereal straw, banana leaves, brewery waste, maize cobs, sawdust, and cotton wastes (Philippoussis et al. 2001; Obodai et al. 2003). Oyster mushrooms have an enzymatic complex containing the ligninolytic or oxidative enzymes (manganese-base peroxidase, lignin peroxidase, and laccase) that break the lignin polymers down (Arora et al. 2002; Knop et al. 2015), and the hydrolytic enzymes (endoglucanase, i.e., CMCase, beta-glucosidase, exoglucanase, FPase, and xylanase) that break the cellulose and hemicellulose polymers down (da Luz et al. 2012; Amore et al. 2013). The enzyme activities and nutritional composition of oyster mushrooms differ depending on their species, nature, and composition of the cultivation substrate (Çağlarırmak 2007; Bhattacharjya et al. 2015; Knop et al. 2015; Xie et al. 2016).Pennisetum sinense is a perennial giant grass with a high cellulose and crude protein content, which can provide a biomass yield of up to 200 t FW·hm−2 (Zhang et al. 2015). In addition to being widely used as a raw material for livestock feed and biomass energy (Lin et al. 2012), it controls soil erosion and improves soil properties (Lin et al. 2014). Partial degradation through composting can make this grass more easily digestible by mushrooms. Compost used as a soil amendment gives numerous biological benefits and restricts the growth of harmful mushroom microbial competitors (Oh et al. 2003; Martínez-Blanco et al. 2013). Compost stimulates the formation of aggregates (mycelia thread biofilm) that improve soil structure, increase disease resistance in some cases, stimulate the indigenous soil microflora with the substances it provides, enhances resources, reduces the volume of waste products by over 40%, and reduces pollution (Dada & Fasidi 2018). Given that the present residues can vary greatly from one region to another, the composting system that is adapted to the simplest formulation, ideally with only one agro-industrial component is simple and economically feasible in small farms for cultivating oyster mushrooms.It was reported that various species of Pleurotus can be viably cultivated using only elephant grass (Pennisetum purpureum) (Bernardi et al. 2007). In the study on the cultivation of oyster mushrooms using one-component composts of corn-cob hay, sawdust, and wheat straw, P. ostreatus readily produced fruiting bodies on composted sawdust with a yield level comparable to commercial production of Agaricus campestris (Block et al. 1958). P. ostreatus and A. bisporus were effectively produced on wheat straw compost (Vajna et al. 2010; Vieira & Pecchia 2018).Globally, oyster mushrooms take the second position of mostly cultivated mushrooms and account for 25% of total mushroom production (Vasudewa et al. 2007). However, only a few investigations were carried out, on the use of composts for growing Pleurotus spp. The use of giant grass compost in mushroom cultivation is advantageous for regions, which have a high production of this grass. Therefore, the goal of this study was to assess the enzyme activity, growth, yield properties, and nutritional content of three Pleurotus species growing on giant grass compost.MATERIALS AND METHODSPreparation of primary and secondary spawnThe 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).Preparation of cultivation substrate and mushroom growingFully 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}}}}Analysis of enzyme activitiesThe 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.Analysis of the fruiting body compositionThe 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).Statistical analysisThe 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.RESULTSEnzyme activityThe 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.Table 1Enzyme activities (Iu·dm−3 ± SD) at the inoculation stage of three different Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida153.7±1.5b372.7±5.3a148.9±14.4b143.5±2.3b601.4±5.6a163.2±8.0a495.8±21.6a257.3±13.1bP. pulmonarius164.8±3.7a249.0±12.9b273.4±6.9a173.4±8.1a632.6±18.1a90.4±4.0b311.4±14.9b418.7±13.8aP. ostreatus165.1±4.7a202.7±16.4c271.7±13.9a118.0±3.2c603.6±23.6a98.7±7.7b478.6±1.3a154.5±21.1cDifferent letters in a column represent a significant difference in enzyme activity between various species of Pleurotus at p ≤ 0.05 by Duncan's multiple range tests (n = 3)Figure 1Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grass at the inoculation phase (comparisons were made within species)Figure 2Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grass at the mycelial colonization phaseFigure 3Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grasses at the fruiting stageTable 2Enzyme activities (Iu·dm−3 ± SD) at the mycelia colonization stage of three Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida125.5±2.8b320.3±10.5a223.2±4.6b93.6±2.8c611.8±11.1a97.2±2.6c524.5±15.0a311.1±8.8aP. pulmonarius93.8±4.4c256.9±10.6b158.1±15.3c145.1±5.5a481.9±24.5b113.7±4.7b373.1±16.2b214.2±2.8cP. ostreatus156.8±2.3a266.5±5.7b248.3±9.6a116.4±7.2b649.6±26.8a141.7±6.0a278.4±23.0c257.3±8.9bSee explanation in Table 1Table 3Enzyme activities (Iu·dm−3 ± SD) at the fruiting stage of three Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida98.7±1.8b181.2±9.9b272.7±4.7a132.3±4.8b610.3±11.2b147.5±3.4ab301.3±4.5b237.6±9.8bP. pulmonarius99.6±4.5b300.1±14.4a177.6±7.2b198.9±8.2a715.0±16.1a145.8±2.3b550.3±30.4a204.1±12.8cP. ostreatus151.3±5.2a144.9±16.5c184.6±4.7b98.4±6.2c441.1±6.8c154.9±6.0a288.4±15.2b349.5±11.2aSee explanation in Table 1Mycelium growth and fruiting body yieldThe 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).Table 4Mycelia growth and productive capacity of three Pleurotus species cultured in composted giant grassPleurotus speciesMycelia growth (days)Primordia initiation (days)No. of fruiting bodiesFresh fruiting body weight (g)Stipe length (cm)Pileus diameter (cm)BE* (%)P. florida23.9±1.2a46.4±1.5c15.8±3.2c152.2±11.0b5.9±0.4a8.2±0.7b72.5±5.2cP. pulmonarius26.5±3.0b38.9±3.0a23.2±3.3a173.4±13.0a5.4±0.4b7.5±0.8c82.6±6.2aP. ostreatus25.3±1.5b42.0±1.9b18.5 ± 2.6b166.5 ±12.7a5.6±0.5b8.7±0.4a78.6±5.8bMeans ± SD followed by letters indicating a significant difference at 5% probability; in a column, different letters represent a significant mean difference between Pleurotus spp ± SD at 5% probability; the total number of replicate bags per species was 18 bags (500 g per bag)*BE – biological efficiencyContent of compounds and heavy metals in fruiting bodiesThe 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).Table 5The content of nutrients in fruiting bodies (mg·g−1 ± SD) of three Pleurotus species cultured in composted giant grassPleurotus speciesPolysaccharidesProteinCarbohydrateAmino acids totalFatCrude ashFiberP. florida3.47±0.01c114.25±0.98b96.29±1.10c2.89±0.01a51.18±0.11a12.63±0.23b19.89±0.48aP. pulmonarius3.88±0.01b108.55±1.03c105.54±1.55b2.68±0.01b43.56±0.77c15.26±0.22a17.30±0.62bP. ostreatus4.11±0.02a121.54±1.03a110.75±1.03a2.35±0.07c48.77±0.07b11.69±0.27c20.42±0.72aIn a column, different letters represent a significant nutrient content difference between Pleurotus species, at p ≤ 0.05 according to Duncan's multiple range tests; the total number of replicate bags per species was 18 bags (500 g per bag)Table 6The content of heavy metals (mg·kg−1 ± SD) in fruiting bodies of three Pleurotus species cultured in composted giant grassPleurotus speciesHgAsPbCdP. florida0.015a0.043b0.017b0.130bP. pulmonarius0.012c0.047a0.018a0.136aP. ostreatus0.014b0.042c0.015c0.123cIn a column, different letters represent a significant metal content difference between various Pleurotus species at p ≤ 0.05 according to Duncan's multiple range tests; the total number of replicate bags per species was 18 bags (500 g per bag)DISCUSSIONThe significant differences in enzyme activity between Pleurotus species found in this study were also reported by Cavallazzi (2004), Ohga (1992), and Mata and Savoie (1998). Knop et al. (2015) found that the lignocelluloses’ conversion into soluble sugars by P. ostreatus hinges mainly on the secretion of different nonspecific oxidative enzymatic systems, which are mainly consisted of laccases, versatile peroxidases, and short manganese peroxidases. The observation of the high activity of LiP in the three Pleurotus species studied here is similar to the earlier findings (Johjima et al. 1999; Hammel & Cullen 2008) that it is the most efficient and best known ligninolytic enzyme oxidizing lignin in white-rot fungi. In addition, this high activity of LiP is connected with a relative capability of colonizing and degrading the recalcitrant lignin polymer by the Pleurotus species (Ruiz-Dueñas & Martínez 2009). The study showed that the production and activity of LiP are influenced by the pH of culture media (stable from acidic to basic pH) and fermentation time (Morais et al. 2001; Sánchez-Ruiz et al. 2021), and it is also connected with substrate composition, temperature, and the growth phase of the fungus (Kaal et al. 1995; Bellettini et al. 2019; Abon et al. 2020). The observation of a high yield of P. pulmonarius compared to other Pleurotus species in this study is similar to the findings reported by de Siqueira et al. (2012), which showed that P. pulmonarius yielded the highest productivity of 24.32% and BE 68.8% than P. ostreatus (productivity of 21.08%, BE 61.75%) and P. eryngii (the weakest growth) when cultivated on composted bean straw. The advantage of our study is that all three species of Pleurotus grown on giant grass compost gave higher yields than these reported above. The P. ostreatus yielded at the level of 21.08% of productivity per 10 kg of fresh bean straw compost (de Siqueira et al. 2012), 10.97 g per 50 g of dried sawdust compost (Dada & Fasidi 2018), and 183.1 g per 1 kg of fresh Triplochiton scleroxylon compost (Obodai et al. 2003), which is significantly less than we obtained on fresh giant grass compost in this study (166.5 g per 500 g). The protein contents (111.25, 108.55, and 121.54 mg per g of P. florida, P. pulmonarius, and P. ostreatus, respectively) obtained in this study, are much higher than the protein content range of 25.35–27.3% of P. ostreatus grown separately on six varied sawdust substrates (Bhattacharjya et al. 2015), 0.92% protein content of P. ostreatus, and 1.76% of P. sajorcaju (Çağlarırmak 2007), as well as 20.82% protein content of P. ostreatus and 21.3% of P. sajor-caju (Chirinang & Intarapichet 2009). The contents of carbohydrates, amino acids, fat, and crude ash in this research are also higher than the results reported by Çağlarırmak (2007), Chirinang and Intarapichet (2009), and Bhattacharjya et al. (2015). The observation of different contents of heavy metals between diverse Pleurotus species is alike to the findings reported by Wang et al. (2017). Furthermore, the heavy metal amounts obtained in the fruiting bodies of each of the three species used in this study were below minimum allowable concentrations in vegetable food (Zarcinas et al. 2004; Pan et al. 2016).CONCLUSIONGiant grass compost can be effectively used for the production of fruiting bodies of three Pleurotus species, which have high enzymatic activity, allowing for further decomposition of organic matter in the compost, which results in high yield and biological efficiency. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Horticultural Research de Gruyter

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

Loading next page...
 
/lp/de-gruyter/ability-of-three-pleurotus-species-for-effective-use-of-giant-grass-4M5OoEgFFU
Publisher
de Gruyter
Copyright
© 2022 Obed Niyimbabazi et al., published by Sciendo
eISSN
2300-5009
DOI
10.2478/johr-2022-0006
Publisher site
See Article on Publisher Site

Abstract

INTRODUCTIONMushrooms are a particular class of macro fungi with a unique epigeous (superficial) fruiting body that generally grows in decomposing organic matter. The majority of cultivated mushrooms, including Pleurotus spp., can be grown on the raw materials of crop or agro-industrial wastes, such as, amongst others, coffee bean dregs, cereal straw, banana leaves, brewery waste, maize cobs, sawdust, and cotton wastes (Philippoussis et al. 2001; Obodai et al. 2003). Oyster mushrooms have an enzymatic complex containing the ligninolytic or oxidative enzymes (manganese-base peroxidase, lignin peroxidase, and laccase) that break the lignin polymers down (Arora et al. 2002; Knop et al. 2015), and the hydrolytic enzymes (endoglucanase, i.e., CMCase, beta-glucosidase, exoglucanase, FPase, and xylanase) that break the cellulose and hemicellulose polymers down (da Luz et al. 2012; Amore et al. 2013). The enzyme activities and nutritional composition of oyster mushrooms differ depending on their species, nature, and composition of the cultivation substrate (Çağlarırmak 2007; Bhattacharjya et al. 2015; Knop et al. 2015; Xie et al. 2016).Pennisetum sinense is a perennial giant grass with a high cellulose and crude protein content, which can provide a biomass yield of up to 200 t FW·hm−2 (Zhang et al. 2015). In addition to being widely used as a raw material for livestock feed and biomass energy (Lin et al. 2012), it controls soil erosion and improves soil properties (Lin et al. 2014). Partial degradation through composting can make this grass more easily digestible by mushrooms. Compost used as a soil amendment gives numerous biological benefits and restricts the growth of harmful mushroom microbial competitors (Oh et al. 2003; Martínez-Blanco et al. 2013). Compost stimulates the formation of aggregates (mycelia thread biofilm) that improve soil structure, increase disease resistance in some cases, stimulate the indigenous soil microflora with the substances it provides, enhances resources, reduces the volume of waste products by over 40%, and reduces pollution (Dada & Fasidi 2018). Given that the present residues can vary greatly from one region to another, the composting system that is adapted to the simplest formulation, ideally with only one agro-industrial component is simple and economically feasible in small farms for cultivating oyster mushrooms.It was reported that various species of Pleurotus can be viably cultivated using only elephant grass (Pennisetum purpureum) (Bernardi et al. 2007). In the study on the cultivation of oyster mushrooms using one-component composts of corn-cob hay, sawdust, and wheat straw, P. ostreatus readily produced fruiting bodies on composted sawdust with a yield level comparable to commercial production of Agaricus campestris (Block et al. 1958). P. ostreatus and A. bisporus were effectively produced on wheat straw compost (Vajna et al. 2010; Vieira & Pecchia 2018).Globally, oyster mushrooms take the second position of mostly cultivated mushrooms and account for 25% of total mushroom production (Vasudewa et al. 2007). However, only a few investigations were carried out, on the use of composts for growing Pleurotus spp. The use of giant grass compost in mushroom cultivation is advantageous for regions, which have a high production of this grass. Therefore, the goal of this study was to assess the enzyme activity, growth, yield properties, and nutritional content of three Pleurotus species growing on giant grass compost.MATERIALS AND METHODSPreparation of primary and secondary spawnThe 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).Preparation of cultivation substrate and mushroom growingFully 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}}}}Analysis of enzyme activitiesThe 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.Analysis of the fruiting body compositionThe 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).Statistical analysisThe 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.RESULTSEnzyme activityThe 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.Table 1Enzyme activities (Iu·dm−3 ± SD) at the inoculation stage of three different Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida153.7±1.5b372.7±5.3a148.9±14.4b143.5±2.3b601.4±5.6a163.2±8.0a495.8±21.6a257.3±13.1bP. pulmonarius164.8±3.7a249.0±12.9b273.4±6.9a173.4±8.1a632.6±18.1a90.4±4.0b311.4±14.9b418.7±13.8aP. ostreatus165.1±4.7a202.7±16.4c271.7±13.9a118.0±3.2c603.6±23.6a98.7±7.7b478.6±1.3a154.5±21.1cDifferent letters in a column represent a significant difference in enzyme activity between various species of Pleurotus at p ≤ 0.05 by Duncan's multiple range tests (n = 3)Figure 1Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grass at the inoculation phase (comparisons were made within species)Figure 2Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grass at the mycelial colonization phaseFigure 3Comparison of enzymatic activities of three Pleurotus species cultured in composted giant grasses at the fruiting stageTable 2Enzyme activities (Iu·dm−3 ± SD) at the mycelia colonization stage of three Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida125.5±2.8b320.3±10.5a223.2±4.6b93.6±2.8c611.8±11.1a97.2±2.6c524.5±15.0a311.1±8.8aP. pulmonarius93.8±4.4c256.9±10.6b158.1±15.3c145.1±5.5a481.9±24.5b113.7±4.7b373.1±16.2b214.2±2.8cP. ostreatus156.8±2.3a266.5±5.7b248.3±9.6a116.4±7.2b649.6±26.8a141.7±6.0a278.4±23.0c257.3±8.9bSee explanation in Table 1Table 3Enzyme activities (Iu·dm−3 ± SD) at the fruiting stage of three Pleurotus species cultured in composted giant grassPleurotus speciesGlucosidaseAmylaseMnPXylanaseLiPLaccaseCMCaseFPaseP. florida98.7±1.8b181.2±9.9b272.7±4.7a132.3±4.8b610.3±11.2b147.5±3.4ab301.3±4.5b237.6±9.8bP. pulmonarius99.6±4.5b300.1±14.4a177.6±7.2b198.9±8.2a715.0±16.1a145.8±2.3b550.3±30.4a204.1±12.8cP. ostreatus151.3±5.2a144.9±16.5c184.6±4.7b98.4±6.2c441.1±6.8c154.9±6.0a288.4±15.2b349.5±11.2aSee explanation in Table 1Mycelium growth and fruiting body yieldThe 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).Table 4Mycelia growth and productive capacity of three Pleurotus species cultured in composted giant grassPleurotus speciesMycelia growth (days)Primordia initiation (days)No. of fruiting bodiesFresh fruiting body weight (g)Stipe length (cm)Pileus diameter (cm)BE* (%)P. florida23.9±1.2a46.4±1.5c15.8±3.2c152.2±11.0b5.9±0.4a8.2±0.7b72.5±5.2cP. pulmonarius26.5±3.0b38.9±3.0a23.2±3.3a173.4±13.0a5.4±0.4b7.5±0.8c82.6±6.2aP. ostreatus25.3±1.5b42.0±1.9b18.5 ± 2.6b166.5 ±12.7a5.6±0.5b8.7±0.4a78.6±5.8bMeans ± SD followed by letters indicating a significant difference at 5% probability; in a column, different letters represent a significant mean difference between Pleurotus spp ± SD at 5% probability; the total number of replicate bags per species was 18 bags (500 g per bag)*BE – biological efficiencyContent of compounds and heavy metals in fruiting bodiesThe 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).Table 5The content of nutrients in fruiting bodies (mg·g−1 ± SD) of three Pleurotus species cultured in composted giant grassPleurotus speciesPolysaccharidesProteinCarbohydrateAmino acids totalFatCrude ashFiberP. florida3.47±0.01c114.25±0.98b96.29±1.10c2.89±0.01a51.18±0.11a12.63±0.23b19.89±0.48aP. pulmonarius3.88±0.01b108.55±1.03c105.54±1.55b2.68±0.01b43.56±0.77c15.26±0.22a17.30±0.62bP. ostreatus4.11±0.02a121.54±1.03a110.75±1.03a2.35±0.07c48.77±0.07b11.69±0.27c20.42±0.72aIn a column, different letters represent a significant nutrient content difference between Pleurotus species, at p ≤ 0.05 according to Duncan's multiple range tests; the total number of replicate bags per species was 18 bags (500 g per bag)Table 6The content of heavy metals (mg·kg−1 ± SD) in fruiting bodies of three Pleurotus species cultured in composted giant grassPleurotus speciesHgAsPbCdP. florida0.015a0.043b0.017b0.130bP. pulmonarius0.012c0.047a0.018a0.136aP. ostreatus0.014b0.042c0.015c0.123cIn a column, different letters represent a significant metal content difference between various Pleurotus species at p ≤ 0.05 according to Duncan's multiple range tests; the total number of replicate bags per species was 18 bags (500 g per bag)DISCUSSIONThe significant differences in enzyme activity between Pleurotus species found in this study were also reported by Cavallazzi (2004), Ohga (1992), and Mata and Savoie (1998). Knop et al. (2015) found that the lignocelluloses’ conversion into soluble sugars by P. ostreatus hinges mainly on the secretion of different nonspecific oxidative enzymatic systems, which are mainly consisted of laccases, versatile peroxidases, and short manganese peroxidases. The observation of the high activity of LiP in the three Pleurotus species studied here is similar to the earlier findings (Johjima et al. 1999; Hammel & Cullen 2008) that it is the most efficient and best known ligninolytic enzyme oxidizing lignin in white-rot fungi. In addition, this high activity of LiP is connected with a relative capability of colonizing and degrading the recalcitrant lignin polymer by the Pleurotus species (Ruiz-Dueñas & Martínez 2009). The study showed that the production and activity of LiP are influenced by the pH of culture media (stable from acidic to basic pH) and fermentation time (Morais et al. 2001; Sánchez-Ruiz et al. 2021), and it is also connected with substrate composition, temperature, and the growth phase of the fungus (Kaal et al. 1995; Bellettini et al. 2019; Abon et al. 2020). The observation of a high yield of P. pulmonarius compared to other Pleurotus species in this study is similar to the findings reported by de Siqueira et al. (2012), which showed that P. pulmonarius yielded the highest productivity of 24.32% and BE 68.8% than P. ostreatus (productivity of 21.08%, BE 61.75%) and P. eryngii (the weakest growth) when cultivated on composted bean straw. The advantage of our study is that all three species of Pleurotus grown on giant grass compost gave higher yields than these reported above. The P. ostreatus yielded at the level of 21.08% of productivity per 10 kg of fresh bean straw compost (de Siqueira et al. 2012), 10.97 g per 50 g of dried sawdust compost (Dada & Fasidi 2018), and 183.1 g per 1 kg of fresh Triplochiton scleroxylon compost (Obodai et al. 2003), which is significantly less than we obtained on fresh giant grass compost in this study (166.5 g per 500 g). The protein contents (111.25, 108.55, and 121.54 mg per g of P. florida, P. pulmonarius, and P. ostreatus, respectively) obtained in this study, are much higher than the protein content range of 25.35–27.3% of P. ostreatus grown separately on six varied sawdust substrates (Bhattacharjya et al. 2015), 0.92% protein content of P. ostreatus, and 1.76% of P. sajorcaju (Çağlarırmak 2007), as well as 20.82% protein content of P. ostreatus and 21.3% of P. sajor-caju (Chirinang & Intarapichet 2009). The contents of carbohydrates, amino acids, fat, and crude ash in this research are also higher than the results reported by Çağlarırmak (2007), Chirinang and Intarapichet (2009), and Bhattacharjya et al. (2015). The observation of different contents of heavy metals between diverse Pleurotus species is alike to the findings reported by Wang et al. (2017). Furthermore, the heavy metal amounts obtained in the fruiting bodies of each of the three species used in this study were below minimum allowable concentrations in vegetable food (Zarcinas et al. 2004; Pan et al. 2016).CONCLUSIONGiant grass compost can be effectively used for the production of fruiting bodies of three Pleurotus species, which have high enzymatic activity, allowing for further decomposition of organic matter in the compost, which results in high yield and biological efficiency.

Journal

Journal of Horticultural Researchde Gruyter

Published: Jun 1, 2022

Keywords: composted substrate; giant grass; lignocellulolytic enzyme; Pleurotus species

There are no references for this article.