Productivity
Mushrooms need a well-balanced nutritional complex within the substrate, such as a C/N ratio and nutrients. C/N ratio is important because C source acts like a key component of storage and structural compounds in the cell and it comes in many forms: monosaccharides, disaccharides, and polysaccharides. The mycelium also has the ability to utilize C through larger polymers such as lignin, hemicellulose and cellulose. In this way, the substrates were carefully chosen to create a range of C/N ratios (Table 1) (Chang and Milles, 2004). Unpublished data from previous works showed that it was possible to use SMS of Lentinula edodes in the substrate to produce P. ostreatus. Although a wide range C/N ratio is possible to produce Oyster mushroom, in our studies N contents higher than 1.2% did not show satisfactory productivity results. Thus, it was decided to limit the C/N range between 50/1 to 80/1 (Table 1).
The nitrogen seems to play an important role in mushrooms growth. Nitrogen is a crucial element needed by all fungi to synthesize compounds containing nitrogen, such as pyrimidines, purines, protein and for chitin, which is the cell wall component composed of b (1-4)-linked unit of N- acetylglucosamin (Hoa et al, 2015). Curvetto et al. (2002) observed that supplementation of sunflower seed husk with ammonium (NH4+) to produce P. ostreatus increased the productivity of this species in up to 50%, as it promotes mycelium development. In the same way, the supplementation of this work promoted greater mycelial growth (Table 4). This increase in mycelial growth through supplementation corroborate with Wang et al., 2015, in which the mycelial growth rate was progressively increased and the mycelial colonization time was shortened with SMS supplementation. In fact, the addition of supplements (RB and SMS) promoted mycelial growth, consequently decreasing mycelial time colonization (Table 4).
In addition to the nitrogen, C/N ratio is another important factor in determining mushrooms productivity (Ritota and Manzi, 2019). Since C and N contents directly impact mycelial development and fruiting body formation, the supplementation chosen altered this C/N ratio. In the orthogonal contrasts performed, supplementation with SMS reached the highest productivity and biological efficiency, compared to substrate without supplementation, as well as showed higher second and third flush and average clusters weight (g) (Table 3). Nitrogen plays an important role in the synthesis of lignocellulolytic enzymes, which will act on cellulose, hemicellulose and lignin polymers. Decreasing the C/N ratio, through organic nitrogen compounds, probably allowed the mycelium to consume a greater amount of short-chain sugars, enhancing its growth and later greater production of fruiting bodies. Similarly, Kurt and Buyukalaca (2010) showed that P. ostreatus productivity and nitrogen content of substrates were positively correlated, while they observed higher laccase and carboxymethylcellulase activities in media supplemented with larger amount of wheat bran.
The formulation employed for the design of the substrate deeply influences the yield and quality of the mushrooms harvested, in addition to supplementation has also been reported as a way to improve the biological efficiency (Moonmoon et al., 2010; Sánchez, 2010; Gaitán-hernández et al., 2014; Pardo- Giménez et al. 2016; Xie et al., 2017; He et al., 2018; Liang et al., 2019). In this case, the quality of P. ostreatus was different comparing wheat straw with 0% supplement versus addition of RB or SMS (Table 3). With RB or SMS in the substrate, P. ostreatus was able to increase the average clusters weight (g) (Table 4), in which heavier clusters corroborate the increase in productivity and biological efficiency. In addition, production according to flushes was also influenced. The application of SMS increased the amount (%) of mushrooms produced in the second flush, while in the third flush the addition of RB also increased this percentage (Table 4). Such results corroborate with Donini et al. (2009), where the amount and the kind of bran may vary depending on the mushroom species as well as the mushroom growth stage.
Different supplements were used to enhance oyster mushroom production, for example, wheat bran (Yildiz et al., 2002; Al-Momany and Ananbeh, 2011), soya bean (Upadhyay et al., 2002), cottonseed hull (Fanadzo et al., 2010), olive mill waste (Ruiz-rodriguez et al., 2010), rice bran and maize powder (Alam et al., 2010). Corroborating with the presented results (Table 4), P. ostreatus has an average biological efficiency of 64% when produced only with wheat straw (Chitamba et al., 2012; Koutrotsios et al., 2014; Hossain, 2018). Supplementing the substrate with the sources mentioned above, the biological efficiency achieved can reach 114%, similar to the results obtained with 15% SMS (BE 107%) (Table 4).
Our results emphasize the importance of the substrate supplementation to increase P. ostreatus productivity and biological efficiency. The addition of nitrogen sources, influencing the C/N ratio, is a crucial factor for a good mycelial development and consequently a good production. Probably, the nutritional complex present (cellulose, hemicellulose, lignin, nitrogen, minerals) was favorable with the supplementation with 15% of SMS (C/N Ratio – 63/1). This amount of added supplement provided the best gains in the vegetative and reproductive parts, allowing absorption and interaction with the other nutrients.
Nutrient dynamics
Mushrooms are a good source of minerals, once they are taken up by the growing mycelium and translocated to the fruiting body. While the importance of C and N is limiting, no less important are other elements, which may be needed at lower concentrations. SMS or cereal bran such as the rice bran used in this work, are sources of NH4+, NO3−, P, K, Ca, Mg, Na, Fe, Cu, Mn, and Zn (Fujihara et al. 1995; Cheung, 1997; Medina et al., 2012; Zhu et al., 2013).
Several studies claim that K is the element found in the highest concentration in the fruiting body (Manzi et el., 1999; Mattila et al., 2001; La Guardia et al., 2005; Oyetayo, 2005; Lee et al, 2009; Oyetayo and Ariyo, 2013). Potassium has a role as a cofactor in some enzyme systems, is involved in carbohydrate metabolism and is important in the maintenance of ionic balance. Loss of potassium from cells can be detrimental to living organisms, including the fungi (Chang and Milles, 2004). Due to this importance in metabolism, probably to support a greater production of fruiting bodies, P. ostreatus chose to absorb larger amounts of this nutrient in substrates supplemented, mainly with RB (Table 5), probably due to the availability of this element in these substrates. On the other hand, the substrates that were supplemented with SMS had lower initial concentrations of K than the others, probably because they had already gone through a cycle of cultivation of the shiitakes’ mushroom, indicating a probable preference for absorbing higher levels of K when supplemented with RB.
Regarding Ca, the amount absorbed was higher when the substrate was supplemented with 15% SMS (Table 6). Despite the initial Ca content increasing as more SMS is applied, the amount absorbed by P. ostreatus did not follow the same trend (Table 5). According Thongsook and Kongbangkerd (2011), P. ostreatus did not appear to accumulate more calcium when its substrate was supplemented with high amounts of Ca. Cardoso et al. (2020) studied the influence of calcium silicate on P. ostreatus and found no relationship with Ca and productive parameters. They argue that it seems the sawdust-based cultivation substrate itself was able to provide the necessary minerals and there was probably a saturation level in relation to the calcium levels in the substrate. For Koutrotsios et al. (2020) no correlation was detected in Ca concentration values was measured in mushrooms versus concentrations in the cultivation substrates. It is noted that these authors correlated the amounts of Ca applied with the levels in the fruiting bodies. For example, when Ca is applied with the aim of conserving the fruiting bodies, they influenced the physiological state of the tissue, indicating a positive effect of hyphae compaction, basic structural components of mushroom tissues, in relation to the control (Thongsook and Kongbangkerd, 2011).
Our results demonstrate that the increase in Ca concentrations in the substrate is not proportional to the increase in the amount absorbed by the fungus. However, our results possibly indicate a relationship between the amount of absorbed Ca x productivity of the fruiting bodies (figure 1). Pearson's correlation demonstrates the influence of Ca, presenting an r value of 0.64 for productivity and 0.60 for biological efficiency. Since no studies were found in the literature that evaluated the Ca concentration after the Pleurotus cycle, and the consulted literature evaluated the Ca concentration in the fruiting body, the association becomes difficult. Supplementation with 15% of SMS provided the indispensable conditions in terms of C and N, this scenario was probably also favorable for the occurrence of a preference of P. ostreatus for the absorption of Ca. The physiological and metabolic role of Ca in mushrooms is not fully understood. Therefore, more experimental evidence is needed to clarify the mechanism of Ca absorption, as well as possible interactions with other elements (such as ionic charge density, ion electrical charge/ion size) (Koutrotsios et al., 2020). Under conditions of higher productivity and biological efficiency (15% SMS or RB) it was where there was the highest consumption of Ca and K (contrasts c and e, table 6). This demonstrates that they are important nutrients for mushroom cultivation, in agreement with Chang and Milles, 2004, suggesting that future studies with these nutrients may favor productive traits. However, under our knowledge, its application in the cultivation of Pleurotus has not yet been investigated.
The nutrient Mn also had different absorption rates according to each substrate. Unlike Ca and K, as the amount of supplement increased, absorption mean of Mn increased (Table 5). Although there was a higher absorption rate, according to a greater availability of Mn, it did not reflect in an increase in productivity and biological efficiency. Micronutrients, such as manganese plays a role in the activation of many enzymes, including those of the TCA (tricarboxylic acid) cycle and is involved in nucleic acid synthesis (Walker and White, 2011). Mn acts as a co-factor of MnP, may favor mycelial growth and production of Pleurotus spp., through stimulation and enzymatic activation of MnPs. These extracellular oxidative enzymes can degrade lignocellulosic compounds (Niess and Grabbe, 1990; Lelley and Janssen, 1993; Curvetto et al., 2002; Cohen et al., 2001; Rodriguez-Estrada and Royse, 2007).
The results with Mn application in the Pleurotus substrate are conflicting, probably due to the initial concentration of Mn in the substrate, which in most works with this nutrient is not measured. According to Lelley and Janssen, 1993, P. ostreatus grown in a substrate supplemented with manganese at 100 mg.Kg-1 was more productive than at 400 mg.kg-1. In another study, Rodriguez-Estrada and Royse, 2007 obtained higher P. eryngii production and biological efficiency at the lowest supplemented dose of Mn (50 mg.Kg-1), compared to the highest applied dose (250 mg.Kg-1). Moreover, Curvetto et al., 2002 demonstrated that high doses of Mn (above 200 mg.kg-1) inhibit the growth and production of P. ostreatus in a substrate with sunflower seed husk. According to the results of the present work, supplementation with 15% RB and 15% SMS added 58.8 and 41.44 mg.g-1 of Mn, respectively, compared to the substrate without supplement. In this example, this increase in Mn provided an increase in BE of 46.1% and 60.9%, respectively. Further increasing the concentrations of supplement and, consequently Mn, did not have the same effect, on the contrary, decreasing BE to the same levels a substrate without supplement. Probably this concentration of Mn is enough for the fungus perform his vital functions and enzymatic activations, while concentrations above these probably provided a favorable environment for it to continue in the vegetative stage, not producing large amounts of fruiting bodies.
Despite the clear importance of the nutrients mentioned above, the other elements that did not show differences in the contents absorbed by P. ostreatus play important roles. This is the case of the nutrients (Mg, Cu, Zn and Fe), which also are important elements for enzymatic cofactors. Equally important, although they are required only in trace amounts, are called minor elements. For example, many enzymes are activated by magnesium, and it is important in ATP metabolism. Iron is a constituent of catalase and of the cytochromes. Zinc is a constituent or activator of many enzymes including alcohol dehydrogenase (Chang and Milles, 2004). Copper as a micronutrient has a key role as a metal activator. It induces laccase transcription, and also plays an important role in laccase production (Palmieri et al., 2000; Ikehata et al., 2004). Therefore, there is still uncertainty about the minimum requirements of these and other elements, as determining the minimum concentrations for absorption physiology in small amounts for fungal growth is a challenge.
Supplementation of the growing substrate for P. ostreatus is essential to increase productivity and biological efficiency. The use of SMS would reduce costs for the producer, replacing the supplementation normally used, in addition to meaning a recycling of a material that is considered waste for producers of shiitake mushrooms. Once the mycelium colonizes the substrate in search of these nutrients for development, the presence and availability of all these elements in this complex matrix becomes important. Therefore, the presence and combination of all these nutrients is the main factor for good productions. The nutrition physiology of edible mushrooms is advancing in the literature, and future studies that encompass the enzyme complex during the entire cultivation phase are the key to a better understanding of this topic. Supplementation with 15% SMS provided productivity and biological efficiency of 25% and 107% in P. ostreatus, respectively, where under these conditions the most consumed nutrients were Ca and K.