3.1 Moisture Content (%), Water Activity (aw) and pH
Physico-chemical analysis of the treatments, related to concentrations of various sources on fungus growth (Fig. 1), presents different profiles and efficiency. The time period used in this study presented initial pH values ranged from 3.65 to 5.65 for all treatments (Fig. 1.i-ii). The highest pH values were observed using pure soy flour, for both inoculum; also, significant differences occurred for all pure treatments. Nonetheless, treatments containing potassium nitrate and ammonium sulphate had lower pH values – which can be observed by their surface contour profiles (Fig. 1.vii and Supplemental information – Fig. A.1), and is related to their higher dosage concentration. For the highest pH region, they were located near the corners of the treatments containing soy flour source and such data obtained herein was similar to a previous report (Hermann et al. 2013).
Some species of basidiomycetes have a self-regulating pH characteristic, with a tendency to stabilize at an optimum pH value for their growth, regardless of the initial pH value (Mata et al. 2016; Chicatto et al. 2018); therefore, it might be the reason for the pH growth in treatments containing soy flour. Nonetheless, treatments without soy flour had lower pH and it further decreases with time compared to their initial values – due to the necessity of nitrogen and carbon tolerable limits for mycelium growth, the pH of the medium does not self-regulate and the fungus cannot control the environment as it occurred at pH above 5.0 (Carvalho et al. 2018).
Samples moisture varied slightly depending on the treatment source used - ranging between 54–62% (Fig. 1-iii-iv), similar to another report using L. edodes for solid-state fermentation (Bentolila de Aguiar et al. 2013) which is a condition favourable for the fungus to grow. Consequently, the culture media must not have low relative moisture since the water content is essential for the growth and metabolism of L. edodes (Antunes et al. 2020). Nonetheless, the results reported a decrease in the moisture after the last cultivation time interval for the majority of the samples, also observed in another work (Hermann et al. 2013); with the treatment containing only soy flour partaking the highest percentage of water. In a well-developed mycelium, it is possible that specific values of moisture are obtained, but these variations depend on the fungus strain and treatments used; however, it is interesting to notice that soy flour contained moisture values between 58–60%.
The surface contour plot exhibits (Fig. 1-viii) that the treatments containing highest moisture was found for higher concentration of soy flour; tough for the time period of 12 days and liquid inoculum (Supplemental information – Fig. A.2), the higher value region was found within a well-balanced source of soy flour and potassium nitrate.
For the water activity values (wa) of the present work (Fig. 1v-vi), all treatments exhibited activities greater than 0.955; while also increased at the final studied time period for the majority of the treatments. These values are reported to be within a region for optimum fungus growth (higher than 0.950) (Pandey et al. 2000), which is also an indicative of large water availability for microorganism development.
The majority of the treatments presented to be significant in regards to their physico-chemical profiles. They also presented higher values for the mixture of soy flour and ammonium sulphate for a solid inoculum; and for liquid inoculum, higher values were obtained for soy flour with potassium nitrate (Supplemental Information – Fig. A.3–6).
3.2 Enzymatic activity
The enzymatic activities for avicelase, carboxymethylcellulose, β-glicosidase and xylanase exhibited a similar behaviour, when compared to both solid and liquid inoculum (Fig. 2). For the majority of the cases, the lowest enzyme activities occurred when soy flour was used, which can also be seen with the surface contour plot (Fig. 3 and Supplemental information A.7–8). Contrarily, sources containing ammonium sulphate, followed by potassium nitrate, had the highest activity on all enzymes.
The composition of the substrate is an important factor for the growth and expression of fungi, especially when the nutrients contains nitrogen and carbon. Therefore, fungi may extract these elements more easily within a mixture of ammonium sulphate and/or potassium nitrate (Rughoonundun et al. 2012).
The activity of various treatments using L. edodes evidenced it as a good degrader of the peach palm residue because of its increased enzyme activity. Besides the carbon and nitrogen as well as its nutrients values for fungus growth availability, it is possible that within 12 days the majority of the mycelium growth might already occurred and the trend saw within these results could be the last digestive enzymes cycles which could be on the contrary for the other treatments. Nonetheless, the majority of the enzymatic activities presented a similar trend from a previous work which used solid-state fermentation of L. edodes (Philippoussis et al. 2011).
The presence of soy flour that influenced a decrease in enzyme activity may be due to the nitrogen sources (Philippoussis et al. 2011; Chicatto et al. 2018), in which higher dosages decreases the hydrolytic system but induces an increase in oxidative enzymes. Nonetheless, treatments can also be converted into proteins by microorganisms, and, although the values seem to show a direct relation, their actual profile is also dependant of the substrate used – peach palm residues.
From within each enzyme activity, avicelase and xylanase lowest activities occurred with treatment containing soy flour, or with mixture containing potassium nitrate (Fig. 2 – i, ii and iii,iv); however, it actually exhibited the highest activity for avicelase (199.46 UI.mL− 1) using solid inoculum within the time period of 12 days (Fig. 2.i). Apart from that, avicelase highest activities in all groups were produced in treatments with ammonium sulphate, either as pure source or with the addition of potassium nitrate (234.6 and 249.9 UI.mL− 1, for 12 and 20 days respectively).
For xylanase, the highest activity was found for pure sources of ammonium sulphate (5.15 UI.mL− 1 and 4.87 UI.mL− 1 within time period of 12 days, using solid inoculum, and 20 days using liquid inoculum respectively). Overall, the enzymatic activities profile of avicelase and xylanase exhibits a similar behaviour when comparing the time period and the inoculum used (Fig. 3 and Supplemental File Fig.A.7) and most of the studied sources used were statistically significant in the enzymatic activity values (Fig. S.9–10).
Since peach palm sheaths are reported to contain 19.5% of lignin on a dry basis – and are lower than other common organic residues such as soy, sugarcane bagasse, rice and corn (Franco et al. 2019) - the avicelase activities produced in this study were higher than other residues using another white-rot fungi Agaricus brasiliensis, same Agaricales order of fungi from L. edodes (de Siqueira et al. 2010). However, low values of xylanase were found herein compared to the aforementioned residues.
For CMC and β-glucosidase enzymes (Fig. 2v-viii), the highest activities were found for treatments containing pure ammonium sulphate or with a combination of soy flour (91 IU.mL− 1 and 4.7 IU.mL− 1 at 12 days using solid inoculum; also, 79 IU.mL-1 and 4.4 IU.mL− 1 at 20 days using solid inoculum for CMC and β-glucosidase respectively).
Their enzymatic profile also exhibits that, for solid inoculum (Fig. 3), ammonium sulphate region had increased activity on all time periods, and for liquid inoculum (Supplemental information Figure A.8), potassium nitrate had a major part on CMC enzyme. For β-glucosidase enzyme activity, ammonium sulphate had a major role; though on a shorten time period, 12 days, the mixture of ammonium sulphate and potassium nitrate region had higher activity.
Lower enzyme activity region was found within the mixture of soy flour and ammonium sulphate for the time period of 20 days, using low concentrations of ammonium sulphate (Fig. 3 – CMC and β-glucosidase); while for a shorten time period, 12 days, pure soy region was the least effective on enzyme activity. However, only ammonium sulphate and potassium nitrate were able to exhibit a significant difference in terms of enzymatic activity (Supplemental Information Figure A.9–12).
Endoglucanase, CMC, activity is reported to produce in moderate quantities for L. edodes, this is enhanced depending on the amount of hemicellulose (Philippoussis et al. 2011). Furthermore, variations on the cellulolytic activity has been reported for L. edodes which depends on the fungus growth stage (Chicatto et al. 2014). It has been reported that a deceleration in mycelium growth rates could occur after the 3rd colonization week leading to a decrease in the enzyme activity because of the limitation of utilizable nutrients and soluble carbon sources (Philippoussis et al. 2011).
Ammonium sulphate, as nitrogen source, can have positive effects for CMC and proteins but can inhibited the production of avicelase; also, the L. edodes strain can also result in variation of the enzyme activity (de Siqueira et al. 2010). Therefore, due to differences in chemical composition of substrates for cultivation, it is important to select genotypes with suitable characteristics for growth in the presented substrate; which in turns depends on the fungus ability to utilize the majority of substrate components as nutritive elements (Elisashvili et al. 2008). Likewise, presence of certain compounds, such as phenolics, from the substrate could also inhibit fungus growth (Mata et al. 2016).
3.3 Qualitative analysis of mycelium growth and composite formation
The growth of L. edodes over time (Fig. 4) exhibits that the lowest microbial growth was observed in treatments containing either pure ammonium sulphate or potassium nitrate; also, a mixture of those two had a small formation of L. edodes mycelium in the medium.
The treatment with a mixture of all conditions had no mycelial growth with the liquid inoculum, for both endpoints studied herein, and this could have been due to the concentration of the elements (Helm et al. 2013). Furthermore, previous works reports that growth of many fungi species can be halted when the nitrogen concentration or C:N ratio surpass a certain value (Philippoussis et al. 2011). In addition to the fact that, within both time periods, the growth rates are directly related to their enzymatic activities; whereas poor growth had a moderate activity, a well-developed fungus had lower activity values (Fig. 1). Nonetheless, it has been reported that nitrogen is a key element in the growth of L. edodes (Lin et al. 2015). Therefore, an increase in organic matter content is expected where mycelium colonization is more advanced (Attias et al. 2020).
Mixing soy flour with the other treatments had a moderate mycelial growth of L. edodes, corresponding to half of the substrate covered by a mycelium network for both time periods. Additionally, the treatment with the highest mycelial growth was found for pure soy flour source, in both inocula, in which the fungus reached the surface of the bottle.
The enzymes released from the mycelia hyphae contributes to the mycelium-composite formation by degrading the substrate and increasing its mycelia density (Tacer-Caba et al. 2020). It is important to remind that the yield of materials based on mycelium depends on the strain, the medium, conditions of growth and growth cycle (Philippoussis et al. 2011; Elisashvili et al. 2015; Attias et al. 2019). Nonetheless, mycelium growth of L. edodes are affected by substrate cellulose, hemicellulose and lignin proportions along with nitrogen content (Philippoussis et al. 2011).
It has been suggested that if a media is too difficult to digest, it can induce the mycelium to secrete more enzymes – having a wide and fast surface but reduced total volume, an effect that occurred for sources containing mainly ammonium sulphate. This effect can be reversed if the media is richer in compounds that is easily digested by the mycelium, such as D-glucose, leading to an increased material volume (Antinori et al. 2020). In the case of peach palm sheaths, they contain higher values of non- and reducing sugars (Helm et al. 2013) that other common residues such as sugarcane bagasse (Rabelo et al. 2015) and may have contributed to the further growth of this fungus.
Therefore, it is possible that due to the optimum condition for L. edodes to grow using pure soy flour, mycelium grew more than the other conditions and what is seen by their enzymatic profile is at the end of the composite formation. This suggests that the majority of the enzyme activity, in digesting and consuming the residue, already occurred prior to the first time period observed in this study; alternatively, other sources shown higher values due to the difficulty in digesting, and a poor growth of the mycelium. This trend was already reported in a previous work of our group, with the usage of residues from Eucalyptus benthamii using the same fungus (Pedri et al. 2015).
The visual mycelial density, for a source containing pure soy flour, features a compact mycelium block on the peach palm sheath fibres (Fig i-iv), and this source preference by the fungus may be related to the presence of amino acids (Leonowicz et al. 1991). The growth and nutrient parameters of the residues exhibited mycelium composite formation, a significant achievement by the usage of this fungus compared to substrates containing higher values of hemicellulose and nitrogen (Philippoussis et al. 2011). These are also related to be the main promoter of growth in mycelia stage (Gaitán-Hernández et al. 2011); nonetheless, the content of cellulose and hemicellulose on peach palm sheaths are lower than other common residues such as bamboo and sugarcane (Franco et al. 2019) lower hemicellulose is a good indicative for mycelium development (Gaitán-Hernández et al. 2011).
Because of the limited space found for the fungus to grow on either flask side, they reached to its top, and a volume from about half the size of the glass was formed. Therefore, they were compressed in order to remove excess of water and evaluate how this material would behave for future analysis.
This treatment, S100, and time, 12 days, with solid inoculum were chosen, due to the higher mycelia growth rate within few days of cultivation, facilitating the process of manufacturing this material. However, the liquid inoculum did not show the same characteristics of mycelial growth, as its interior was not completely colonized by the fungus, containing empty spaces and it led it to be impossible to continue the mechanical test.
The interaction of the hyphae with the fibres formed a compact material after compression. According to the curves obtained in the test, points were estimated to determine the force and deformation (Figure S.13). The force was applied until the rupture of the material close to 4 cm deformation and it was labelled as cold-pressed.
However, in order to compare this composite, a new one was produced using the same treatment of cold-pressed with a bigger size mould- or flask - in length. The composite – labelled as non-pressed, was able to grow evenly and detached over the days from the mould on its own (Fig. 4.iii-iv); therefore, it is possible to tailor its volume based on the media and source used for the fungus to grow. Besides, the material formed within 12 days were very similar to the one formed at 20 days and it was also the reason this time period was selected.
Nonetheless, this material had some characteristics similar to a Styrofoam plate presenting a rigid structure of polysaccharides (matrix) over fibres that are biodegradable and with characteristics of fibre / matrix association
3.4 Moisture content (%), water activity (aw) and pH of the mycelium-based composites.
The physico-chemical characteristics of the mycelium-based composites exhibits some differences in the process performed (Table 2). The moisture exhibited to be positive for the growth of the fungus – as expected, and the low values are related to the drying methodology. Higher moisture from a more compacted material, cold-pressed, may be related to an increased difficulty of the water to be released compared to a non-pressed composite.
Table 2
Physico-chemical, compressive and sorption kinetic properties of the mycelium-based composites studied.
| Sample | Physico-chemical | Compressive | Sorption kinetics (dH2O) |
| M0 (%) | Mf (%) | wa 0 | wa f | pH0 | pHf | Modulus (kPa) | Strength (kPa) | Weight increase (%) | Thickness expansion (%) |
| Cold-pressed | 60.4 | 14.1 | 0.98 | 0.50 | 5.6 | 5.8 | - | - | 245 ± 3 | 21.5 ± 0.5 |
| Non-pressed | 59.7 | 8.05 | 0.99 | 0.52 | 5.8 | 6.0 | 238 ± 16 | 223 ± 10 | 351 ± 4 | 18 ± 1 |
Nonetheless, the values of moisture content, wa and pH were similar to previous reports using L. edodes as solid-state fermentation biomass (Chicatto et al. 2014; Pedri et al. 2015).
3.5 Carbon / Nitrogen and Ash Analysis
A relation of carbon and nitrogen was analysed in the peach palm sheaths, soy flour and the mycelium-composite (Table 3) exhibiting that the presence of soy in the substrate had positive effects on the time and mycelial growth. It is already known that the addition of supplements increases the levels of nitrogen and available carbohydrates (Pedri et al. 2015).
Table 3
Elemental analysis of the studied materials, obtained from the CHNS equipment.
| Material (*) | Carbon | Nitrogen | C:N | Sulphur | Hydrogen |
| Peach Palm sheaths | 40.87 | 1.14 | 42:1 | 0.164 | 7.33 |
| Soy flour | 54.56 | 7.59 | 8:1 | 0.236 | 10.41 |
| Non-pressed composite | 42.61 | 4.51 | 11:1 | 0.273 | 7.51 |
| (*) For all components, the results were calculated from the average. |
Supplementation with flour has shown that, for the cultivation of L. edodes, it is necessary a source of nitrogen within the substrate (Queiroz et al. 2004). It has been reported that the C:N ratio should be between 30–40 to favour the mycelial growth of L. edodes (Song et al. 1987), which indicates the relation between growth rate and availability of nitrogen readily usable. Therefore, since peach palm sheaths already possess a relation of C:N within a favourable growth, it is also possible that soy flour – containing lower amount of nitrogen than other sources –may have contributed to the growth of this material.
3.6 Histological sections
The microscopic images of the lateral (Fig. 5.i) and transversal (Fig. 5.ii) sections of the plant cell from peach palm sheaths exhibits, on the transversal section, larger circumferences related to the sap transport duct (arrow). Like others lignocellulosic materials, peach palm sheaths are basically composed of cellulose and lignin.
Peach palm sheaths that underwent solid state fermentation using L. edodes fungi presented a similar aspect of the raw sheaths, but with traces of the soy and cassava flour (Fig. 5-iii and iv). The same region examined for the pure sheaths exhibits that, after formation of the mycelium-composite, they were further degraded and a disorder of small fragments stands out around the fibres (arrows in (iii-iv) and marked area in (iv) from Fig. 5) which are directly linked to L. edodes; though, no fungus hyphae were found in the images.
The biodegradation of lignocellulosic materials by fungi primarily occurs in an extracellular form, since they must initially be depolymerized to smaller compounds in order to be susceptible to be transported by the cell wall and intracellular fungi metabolism. Moreover, fungi degrading action occurs through penetration of their hyphae in the lumen of the plant cells (Rodríguez et al. 1997); afterwards, their hyphae produce a great diversity of extracellular metabolites, which then act by degrading the plant cell wall.
3.7 Scanning Electron Microscopy (SEM) Analysis
The SEM images of the mycelium based-material was analysed on the cold-press composite, non-pressed also presented a similar structure – not shown, and their morphology exhibits the interconnected network, formed from L. edodes mycelium (Fig. 6-i), and also their hyphae (Fig. 8.ii-iii) which corresponds to the composite matrix.
The substrate particles are shown to be deeply hidden by the mycelium, containing a hypha with a diameter on the order of 1–5 µm (Fig. 6 ii-iii); they are either loosen due to degradation or physically twisted with the mycelium, a morphology previously reported for mycelium-based composites (Liu et al. 2020). The fact that insufficient fungal had growth throughout the whole composite limits the bonding between the hyphae and the substrate, and are reported to be responsible for the limited mechanical performance (Islam et al. 2018; Liu et al. 2020).
Furthermore, the binder network from the mycelium also affects its mechanical properties and tensile resistance of mycelium-based composites are more influenced by failure of the binder than the substrate itself (A. R. Ziegler, S. G. Bajwa, G. A. Holt, G. McIntyre 2016; Jones et al. 2018b).
3.8 Compression test
Compression tests revealed no rupture of the composite until the end of the test with little deformation and greater resistance compared to Styrofoam, and a commercial mycelium based-composite (Zeller and Zocher 2012).
Under compression, mycelium-composites behave as an elastic regime at small strains followed by a localization strain regime – multiple bands of localized strain that connects the weakest points - and finally, at larger strains, a densification regime occurs whereas large number of fibre contact induces rapid stiffening (Islam et al. 2018). These stress-strain regimes were also observed in the studied composites (Figure S.12) using peach palm sheaths as residues, and similar to other works (A. R. Ziegler, S. G. Bajwa, G. A. Holt, G. McIntyre 2016; Jones et al. 2017).
Mycelium-composites under compression presents an elastic modulus on the order or 0.14–0.19 MPa and at lower strain rates, it seems that the matrix (peach palm residue) is the one that controls the response (Haneef et al. 2017; Islam et al. 2017).
Nonetheless, it is important to state that the compressive values obtained herein (Table 2) are within the region of EPS (0.23 compared to 0.15–0.7 MPa) and are characterized as rigid foam material (Xie et al. 2018). This can be related to the chitin, that is presented in the cell wall, and is also able to reduce the occurrence of cracks when subjected to compressive forces (Yang et al. 2017). The time period of cultivation is also reported to affect the mechanical properties, whereas longer periods can increase it due to hyphae aggregation. By comparison, the cultivation period also influences the volume loss due to the drying of the substrate and hyphae collapsing (Haneef et al. 2017) though hyphae can colonize these vacancies left by water removal.
With a more nutrient substrate, the bonding and extent of their hyphae network is increased, consequently, increasing the material bonding and is one of the main factors when failure occurs in these materials (Jones et al. 2017). Therefore, mycelium grown on substrate from residues to form mycelium-based materials are only suitable for foam like structures.
3.9 Water absorption and swelling
Water sensitivity is an important criterion for many practical applications of mycelium-based products, among others; thus, determining performance under adverse conditions. In such cases, the water absorption values for the composite absorbed large quantities of water (245.1%) (Table 2). The cold-pressed material absorbed less water due to its compacted structure and a more rigid structure.
One of the problems on mycelium-based materials, compared to EPS, is their water absorption and their need to reduce its density. The water is absorbed very fast in these materials, and it is reported that they increase in weight by 40–580 wt% in contact with water for 48–192 h (Jones et al. 2020). This is due to their cellulose fibres having various hydroxyl groups as well as its mycelium binder which is hydrophilic (Jones et al. 2017). The gel formed by these composites prevents dehydration of their hyphae (Antunes et al. 2020), allowing adhesion to other cells or surfaces. Moreover, the hyphae of L. edodes fungus is composed of β-glucans, chitin and proteins that are able to bind to others and form its network and these components are known to have high swelling values.
3.10 Thermal properties
The DSC curves for the raw peach palm sheaths and the mycelium-composite exhibits (Fig. 7.i) an increase in heat flow close to 350ºC, and it can be attributed to the exothermic event of cellulose decomposition. Compounds that are degraded at a temperature above 400 °C, such as lignin, and chitosan at 300ºC that are present in the composite have exothermic profile. Because of the degradation that occurred with the mycelium, it contributed to the temperature stabilization before 500 ºC and the temperature profile are close to the degradation values of each individual fibre and matrix component of cellulose-based materials (Averous and Boquillon 2004).
Due to the substrate and the characteristics of the fungi, the thermal degradation is similar to most cellulosic materials and the lignin-based substrate are responsible for the fire-resistant properties of these composites (Jones et al. 2018a). Furthermore, it can be seen that the non-pressed composite presented no significant variations at temperatures higher than 500 °C compared to cold-pressed. This might be related to the conditions that were grown the cold-pressed material and may had not degraded all components at this specific temperature.
The thermal stability of the composites exhibits a three-stage process in the thermal degradation - (Fig. 7 ii-iii) the first stage from 25–200 °C indicates evaporation of free and bonded water (~ 5%). The second stage have higher degradation within 200–375 °C are due to organic constituents such as protein and polysaccharides (70%), and the final stage having residual char which degrades forming carbonaceous char 450–600 °C (Jones et al. 2018b). Similar profiles for TGA has been shown for T. multicolor fungus using rapeseed straw, and the variation profile, whether cold pressed and non-pressed resulted in a similar graph (Appels et al. 2019).
Chitin and chitosan present in hyphae of the fungus when heated to higher temperatures undergo degradation and the composite thermograms exhibits exothermic events (Fig. 9-iii) at 329ºC, 471ºC and 480ºC, in agreement to another work (Peniche-Covas et al. 1988). Because of the denser, and more compact network found in the cold-pressed composite, it may have contributed to the increased values of the exothermic event at the final stage of 480 °C.