White-rot fungi comprise a large group of organisms known for their ability to produce different types of enzymes for the degradation of lignin and cellulose. Lignin is the main component of wood and other plant biomass, and, together with cellulose and hemicellulose, it is the main source of carbon for the mycelium (Baldrian et al., 2005). One representative of the white-rot fungi is
Several studies have investigated the relationships among specific enzyme activity and lignocellulose degradation, biological efficiency (BE) and other processes. During cultivation of
An important criterion for the cultivation of
However, the relationships among enzyme activity, substrate moisture and different substrate layers are still largely unclear. This study is the novel comparison of the activities of multiple enzymes (Mn-dependent peroxidase, 1,4-β-glucosidase and cellobiohydrolase) produced by
The strain 5081 of
Wheat straw pellets were used for substrate preparation. Pellets were mixed with water to reach moisture levels of 60%, 65%, 70% and 75%, and the substrate was filled in plastic containers (750 g of substrate per container). The prepared substrate was heat treated at 90 °C for 24 hr, cooled down and inoculated with grain spawn (10 g per container).
The inoculated substrates were kept in an incubation room at 24 °C. Substrate was colonised by the mycelium (14 days from inoculation) and subsequently transferred to a growing room at a temperature of 12°C and a relative humidity of 90%. The fruiting bodies were harvested after 12 days.
Samples were taken from the centre and the upper layer (1–2 cm from the substrate surface) of the substrate. The pH/H2O was determined using the pH metre Jenway 3505 (Cole-Palmer, United Kingdom), according to BSI EN 15933. The water content was determined using the moisture analyser KERN DAB 200-2 (Kern & Sohn, Balingen, Germany) at 105 °C.
Mycelial growth was recorded on four vertical axes around the circumference of the container after 7 days. BE was calculated as the ratio of the weight of the fresh fruiting body (g) per dry weight of substrate (g), expressed as a percentage, according to Liang et al. (2019).
To determine enzyme activities, 0.2 g of the sample was placed in a 50-mL Erlenmeyer flask with 20 mL of acetate buffer, at pH 5.0. Samples were homogenised using an Ultra-Turrax (IKA Labortechnik, Staufen, Germany) at 8,000 rev · min−1 for 30 s. To determine the laccase and Mn-peroxidase activities, filtration and desalination were performed. Enzyme activities were measured in 96-well microplates, with 4 wells per sample.
Activities of ligninolytic enzymes (laccase, Mn-dependent peroxidase) were measured spectroscopically at 590 nm as there are changes in absorbance for 3.5 min (measurement 7 × 30 s) in a Tecan Infinite®M200 (Tecan Austria GmbH, Austria). To determine the activity of Mn-peroxidase, a solution of DMAB (25 mmol · L-1 3.3-dimethylamino-benzoic acid), MBTH (1 mmol · L−1 3-methyl-2-benzothiazolinonehydrazone), MnSO4 (2 mmol · L−1), EDTA Na sol. (2 mmol · L−1 ethylenediaminetetraacetic acid disodium salt dihydrate), peroxide solution (0.08 mmol · L−1) and succinate-lactate buffer (100 mmol · L−1; pH 4.5) was used as described elsewhere (Baldrian, 2009). Samples were pipetted into the respective wells (four for each sample), and the substrate mixture was added. The plate was immediately placed in the reader, and the measurement was determined according to Štursová and Baldrian (2011), with slight modifications, such as extraction at room temperature.
The enzymatic activities of hydrolytic enzymes (1,4-β-D-glucosidase, cellobiohydrolase) were measured using the substrates 4-methylumbellyferyl-β-D-glucopyranoside (c = 2.75 mmol · L−1) for β-D-glucosidase and 4-methylumbellyferyl-N-cellobiopyranoside (c = 2.50 mmol · L−1) for cellobiohydrolase. Activities of the enzymes were measured as a change of fluorescence after 5 min and 125 min of incubating the microplates in an incubator (40 °C) with an excitation wavelength of 355 nm and an emission wavelength of 460 nm, according to Košnář et al. (2019).
The results are presented as the mean values of three replicates. Kruskal–Wallis analysis (
Based on the results in Table 1, the water content of the substrate gradually decreased, which was most pronounced in the variant with 60% moisture (from 59.4% to 41.4%). During the colonisation phase, in most variants, higher water content values were measured in the upper layer of the substrate, whereas during fructification (from the primordia phase to the harvest phase), a higher water content value was found in the centre of the substrate. The lower water content in the upper layer of the substrate can be explained by the formation of primordia and their growth, which uses water from the substrate and transports it to the atmosphere. This finding is in agreement with the results of Atila (2019) for the cultivation of
Changes of water content and pH in substrate during cultivation of Pleurotus ostreatus.
Parameter | Cultivation phase | 60% | 65% | 70% | 75% | ||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | ||
Water content (%) | Colonisation | 59.4 ± 0.7 aA | 62.63 ± 0.76 aB | 72.01 ± 1.09 aA | 68.75 ± 1.07 aB | 72.75 ± 1.89 aA | 72.37 ± 0.53 aA | 79.32 ± 0.69 aA | 76.82 ±0.66 aB |
Primordia | 68.1 ±0.87 aA | 52.37 ± 1.02 bB | 67.8 ± 0.65 bA | 66.08 ± 0.63 bA | 70.65 ± 1.21 abA | 68.95 ± 0.19 bA | 78.93 ± 1.06 aA | 76.55 ± 0.37 aB | |
First flush | 41.4 ± 0.45 bA | 57.7 ±0.37 cB | 58.5 ± 0.67 cA | 66.55 ± 0.61 abB | 68.17 ± 1.02 bA | 67.28 ± 0.22 cA | 74.55 ± 0.47 bA | 75.0 ± 0.8 aA | |
pH | Colonisation | 4.74 ± 0.03 aA | 5.09 ±0.01 aB | 4.81 ± 0.02 aA | 5.22 ±0.01 aB | 5.12 ± 0.12 aA | 5.27 ± 0.06 aA | 5.13 ± 0.07 aA | 5.43 ± 0.04 aB |
Primordia | 5.33 ± 0.19 bA | 5.27±0 abA | 5.13 ± 0.01 bA | 5.31 ±0.03 bB | 5.13 ± 0.06 aA | 5.2 ± 0.01 aA | 5.37 ± 0.06 bA | 5.37 ± 0 aA | |
First flush | 5.29 ±0.1 bA | 5.35 ±0.11 bA | 5.5 ± 0.05 cA | 5.63 ± 0.03 cB | 5.16 ± 0.08 aA | 5.26 ± 0.02 aA | 5.83 ± 0.03 cA | 5.63 ± 0.01 bB |
Values are the means ± SD (
Different lowercase letters in a column indicate significant differences between stages of cultivation; capital letters indicate significant differences between layers of the substrate for each parameter (Kruskal–Wallis test,
Another studied parameter of the substrate was pH. Based on the results, the pH value decreased during colonisation from 6 (before inoculation) to 4.74–5.43. This finding is in agreement with Bellettini et al. (2019), who reported that substrate pH is reduced, which is close to 4, during the colonisation stage. Obodai et al. (2003) used composted sawdust, banana leaves, maize stove and rice straw as substrates for cultivation, with initial pH values of 6.92–7.6 at a water content of 58%–70%. The pH values were higher than those observed in our study, most likely because of the different substrate composition. In our experiment, the pH value slightly increased from the colonization phase for all variants during cultivation. The lowest pH value (4.74) was measured for the variant of 60% water content in the substrate in the upper layer in the colonisation phase, and the highest pH value was found for the variant of 75% water content in the substrate in the harvest phase (5.83). In the colonisation phase, the pH values of all variants were higher in the middle layer of the substrate. In the primordial deployment phase, the pH of the upper layer was higher in the variant with 60% water content in the substrate; in the 65% and 70% water content variants, the pH was higher in the centre of the substrate. At the harvest stage, the pH in the centre layer was higher only for the 75% water content in the substrate. There was a statistically significant difference in pH values between the individual substrate layers, with the exception of the 70% water content in substrates, where no difference in the pH values was found, even in the individual phases of cultivation. For the 60%, 65% and 75% variants, statistically significant differences were found between the individual cultivation phases, especially between the colonisation and harvesting phases (Table 1).
The mycelium growth rates of
The BE is the ratio of the weight of the fresh fruiting body (g) per dry weight of substrate (g), expressed as a percentage (Liang et al., 2019). In our experiment (Figure 2), we found no statistically significant differences among the variants. However, the highest BE values (50% and 51%) were found for variants with 65% and 70% water content in the substrate, respectively. On the other hand, the lowest BE was found for the variant with 60% water content in the substrate, along with the lowest mycelium growth, indicating that this level of substrate moisture is not suitable for both adequate mycelium growth and high yields. In the experiment of Kalmıs et al. (2008),
The highest 1,4-ß-D-glucosidase activity (Table 2) was measured in the variant with a water content of 75% in the substrate at the harvest of fruiting bodies, when the activity increased over time in both substrate layers. Kannan et al. (1990) also found that the activity of 1.4-ß-D-glucosidase during the cultivation of
Changes of enzymatic activities in substrate during cultivation of
Enzymes | Cultivation phase | 60% | 65% | 70% | 75% | ||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | ||
ß-D-G (μmol MUFG · h−1 · g−1) | Colonisation | 474.88 ± 153.2 aA | 221.65 ± 27.75 aA | 275.17 ±99.17 aA | 560.2 ± 176.26 aA | 717.26 ±45.31 aA | 508.59 ±47.71 aB | 184.77 ± 14.27 aA | 260.08 ± 23.03 aB |
Primordia | 252.16 ± 78.34 aA | 219.69 ±6.63 aA | 1046.78 ± 10.52 bA | 417.16 ± 18.37 aB | 332.68 ±34.07 bA | 125.93 ±25.27 bB | 382.05 ±35.24 aA | 278.41 ± 91.8 aA | |
First flush | 236.59 ±30.11 aA | 570.39 ± 27.34 bB | 843.88 ± 5.23 cA | 691.2 ± 31.11 aB | 678.78 ± 12.87 aA | 290.46 ± 15.24 cB | 3349.13 ± 549.5 bA | 4083.42 ± 608.2 bA | |
Cell, (μmol MUFC ± h−1 · g−1) | Colonisation | 96.03 ± 9.69 aA | 24.06 ± 4.03 aB | 34.68 ± 6.18 aA | 40.53 ± 12.42 aA | 108.3 ± 28.03 aA | 61.81 ±4.08 aA | 17.93 ± 11.54 aA | 13.61 ± 5.07 aA |
Primordia | 34.56 ± 19.44 bA | 33.3 ± 15.28 aA | 110.17 ±27.99 bA | 18.37 ± 8.37 aB | 24.7 ± 2.61 bA | 5.74 ± 0.3 bA | 31.59 ± 7.79 aA | 20.13 ± 5.9 aA | |
First flush | 30.74 ± 5.77 bA | 59.15 ± 31.45 aA | 85.59 ± 26.23 abA | 71.47 ±8.49 bA | 110.23 ±31.02 aA | 25.57± 1.11 cB | 931.88 ± 343.3 bA | 1306.13 ± 387.54 bA | |
Mn – P (mU · g−1) | Colonisation | 1.38 ±0.01 aA | 2.39 ±0.91 aA | l.l8±O.54aA | 1.2 ±0.64 aA | 1.27 ±0.09 aA | 1.13 ±0.42 aA | 1.49 ±0.09 aA | 1.79 ± 0.13 aA |
Primordia | 0.96 ±0.37 aA | 1.1 ±0.03 aA | 1.1 ±0.3 aA | 0.96 ± 0.12 aA | 0.12 ±0.13 aA | 1.07 ±0.24 aB | 1.4 ± 0.17 aA | 1.31 ±0.04 bB | |
First flush | 0.09 ±0.02 bA | 1.55 ±0.03 aB | 0.92 ± 0.24 aA | 0.62 ±0.24 aA | 0.92 ±0.34 aA | 0.55 ± 0.14 aA | 0.76 ± 0.2 bA | 0.53 ± 0.34 bA |
Values are the means ± SD (
Different lowercase letters in a column indicate significant differences between stages of cultivation; capital letters indicate significant differences between layers of the substrate for each parameter (Kruskal–Wallis test,
MUFC, 4-methylumbellyferyl-N-cellobiopyranoside; MUFG, 4-methylumbellyferyl-β-D-glucopyranoside.
The substrate moisture content decreased during cultivation, which can be attributed to development of