3.1 Characterisation of mycelium growth on different substrates
The type of substrates greatly influences the mycelium growth since the hyphae is in direct contact with the substrate and extract the essential nutrients from it (Belletini et al., 2016). Mycelium would colonise faster and prevail over other contaminants if the substrate could provide the desired growth condition, hence produced biofoam with higher quality and quantity (Elhami and Ansari, 2008). Rice husk, sawdust and sugarcane bagasse were selected as substrates in the screening process due to their availability and bio-compatible with the selected white-rot fungi, P.ostreatus. The growth performance of mycelium on three different substrates was visually inspected every two days to determine the compatibility of P.ostreatus with the selected substrate, as shown in Fig. 1.
From Fig. 1, faster growth of mycelium was observed over the agar plate containing the rice husk as substrate, where the mycelium was fully covered on the 8th day. The slow growth of mycelium was observed over the agar plate that contained sawdust and sugarcane bagasse substrate. A dense white fungal biomass layer has formed over the rice husk agar plate. Different results from different substrates probably due to the difference in chemical compositions found in each substrate, which could feed the mycelium during cultivation. Fig. 2 shows the growth curve data of the mycelium P. ostreatus on rice husk, sawdust, and sugarcane bagasse to correlate with the visual inspection in Fig. 1.
Fig. 2 shows that P. ostreatus on rice husk was adapting to the new growth condition and experienced a lag phase on the 1st day, while other substrates experienced lag phase on the 1st day to 3rd day. The fungus started to enter the log phase, where it started to feed off the nutrients in the substrate to grow and elongate its mycelium. The nutrients in the substrate were in excess, which enables the extension of the mycelium to occur at a constant rate at day 8. As for the rice husk, the mycelium expanded more than 100 cm2 on the 8th day. A study by Obadai et al. (2003) suggested that the P. ostreatus would colonise faster using rice husk as substrate. On the other hand, sawdust and sugarcane bagasse experienced a lag phase until day two followed by slower colonisation afterwards. Elsacker et al. (2019) experienced slow growth as well when sawdust and flax dust were used as substrate. This situation occurs due to implication of different chemical compositions in the substrates, thus making it difficult to digest during colonisation. Ghazvinian et al. (2019) reported that the glucans forming in sawdust are more complex than those with straw, such as rice husk, resulting in slow digestion by the mycelium and slow growth.
The unique spongy structure of a rice husk substrate promotes good absorption of water and high retention of water which will stimulate better growth for the mycelium (Siwulski et al., 2011). This structure will help prevent the rice husks substrate from drying since it has a high holding water capacity. Besides that, rice husk substrate possesses an antiseptic property that will help to inhibit competing organism that grew within it (Dawidowicz et al., 2018). Thus, the yield of mycelium-based biofoam is guaranteed as a fast overgrowth of fungus mycelium was promoted on the rice husk substrate. Therefore, no further test was conducted with the sawdust and sugarcane bagasse. Rice husk was chosen as the most suitable substrate to be applied in the mycelium biofoam production for P.ostreatus.
3.2. Effect of variables on mycelium growth
The growth of the P. ostreatus mycelium that has been incubated at five different temperatures, 20, 25, 30, 35, and 40 °C, and was visually inspected for every two days, is shown in Fig. 3. From the observation, white mycelium patches can be seen starting on day 3, and on day 11, a fully grown P. ostreatus mycelium was found covering the whole area when it was incubated at a temperature of 20, 25, and 30°C.
As shown in Fig. 3, the mycelium grew the fastest at 30°C, resulting in a highly dense white fungal biomass layer on the rice husk on day 11. This is because P. ostreatus grew naturally in the subtropical climate at an average temperature of 29°C. Therefore, it is easier to adapt to an environment similar to their natural growing condition (Marino et al., 2003). The result was consistent with findings by Tesfaw et al. (2015) which reported the optimum temperature for P. ostreatus to grow was at 30°C and moderate growth of the mycelium is at 20°C and 25°C. Conversely, inhibition of growth would occurs by further elevation of temperature. Slow mycelium growth was observed at 35°C, while there is no growth of mycelium at 40°C. Dawidowicz et al. (2018) also reported that temperature above 35°C would cause the mycelium to decay. The substrate would be dry at said temperature and inhibit the growth of mycelium due to loss of moistures (Patel et al., 2009).
Moisture content in the substrate plays an important role, as it provides large turgor pressure, which is needed for the hypha tip of the fungal to have better penetration into the solid substrate. Fig. 4 shows the growth of the P. ostreatus mycelium at moisture content of 30, 40, 50, 60, 70% (w/w) on rice husk.
Fig. 4 shows a dense white fungal biomass layer forming on the substrate that contained 40% (w/w) and 50% (w/w) moisture content. Shen et al. (2008) reported that 55% substrate moisture gave the highest yield of Lentinula edodes. The optimum moisture content of synthetic logs during the spawn run was different by species. However, the natural optimum moisture content for hyphal elongation is in the range of 55–70% (Shen et al., 2008). Sufficient moisture content in the substrate is crucial as water uptake enables the elongation of hypha cell and extension of mycelia within the whole substrate (Arijit et al., 2013).
On the other hand, Fig. 4 shows that at 60% and 70% (w/w) of substrate moisture content, the mycelium growth was inhibited and was prone to contamination by black mould and white worms. This occurrence occurred due to excess water. Narh et al. (2011) reported that the water would set at the bottom of the substrate, which later promotes mycelium's surface growth, which leads to bacteria contamination at the bottom area. Excessive moisture content would lead to the depletion of oxygen and nutrients due to the leaching process, which results in the decrease of enzymatic activities as the anoxic condition developed. According to the work of Ryu et al. (2015), there was a possibility for diseases and competing material such as moulds to occur since the level of moisture is high. On the other hand, the growth rate of the P. ostreatus mycelium at different spawn loading (5, 10, 20, 30, and 40% (w/w)) is illustrated in Fig. 5.
In general, the spawn consist of fungi mycelium and its supporting medium or substrate which provides nutrition for fungi growth, also known as inoculum (Satpal et al., 2017). The result shows that optimum growth rate was obtained when the rice husk was inoculated with 40% (w/w) spawn loading. Through visual inspection, the mycelium almost covered the whole area of rice husk substrate on the agar plate at day 5. In contrast, inoculation by using 5% (w/w) of spawn loading took 9 days to outgrown rice husk substrate on the agar plate. Sabu et al. (2005) and Patel et al. (2009) reported a similar situation with lower spawn loading and suggested slow mycelium growth was due to low laccase activity to extract nutrient to initiate the growth of mycelium. Idowu et al. (2016) also reported that the lowest P. ostreatus spawn level (3%) takes the longest day to complete substrate colonisation compared with the highest spawn level (13%). Increasing the quantity of spawn may reduce the competitiveness between organisms present in the substrate (Idowu et al., 2016). However, increasing spawn loading above limits (> 60%) may reduce laccase production due to the fast depletion of nutrients. This will result in a decrease in metabolic activity. Patel et al. (2009) reported that increasing spawn loading at an optimum amount could enhance the utilisation of substrate, thereby improving laccase activity, which leads to extracting more nutrients for mycelium growth. Moreover, a shorter colonisation time of substrate by mycelium could also be obtained.
3.3 Physical and Mechanical Characterisation
In the production of mycelium-based biofoam, maintaining the density of a material is one of the main challenges. The effect of incubation temperature on the physical properties of the mycelium biofoam is shown in Table 1 and Table 2. Referring to Table 1, the optimum dry density for each parameter was obtained when the biofoam incubated at 30°C, 50% (w/w) moisture content and 40% (w/w) spawn loading, which produced biofoam with dry density of 0.387 g/cm3, 1.07 g/cm3, and 0.437 g/cm3, respectively. On the other hand, the lowest dry density were observed for biofoam that was incubated at 40°C, 30% (w/w) moisture content and 5% (w/w) spawn loading, with dry density of 0.290 g/cm3, 0.286 g/cm3, and 0.314 g/cm3, respectively. Similarly, Table 2 shows a same trend for the compressive strength result. The highest compressive strength was obtained for biofoam incubated at 30°C with 0.975 MPa, 50% (w/w) moisture content with 1.070 MPa, and 40% (w/w) spawn loading with 1.350MPa. Subsequently, the lowest compressive strength for each parameter was observed at 40°C with 0.277 MPa, 70% (w/w) moisture content with 0.376 MPa, and 5% (w/w) spawn loading with 0.893 MPa.
Table 1: Average dry density of biofoam at different parameter.
Factors
|
Parameters
|
Average dry density, (g cm-3)
|
Temperature (oC)
|
20
|
0.314 ± 0.005a
|
25
|
0.350 ± 0.019a
|
30
|
0.387 ± 0.013a
|
35
|
0.309 ± 0.005b
|
40
|
0.290 ± 0.001c
|
Spawn loading (wt %)
|
5
|
0.314 ± 0.005b
|
10
|
0.336 ± 0.007c
|
20
|
0.356 ± 0.021d
|
30
|
0.369 ± 0.011e
|
40
|
0.437 ± 0.010a
|
Substrate moisture content (w/w %)
|
30
|
0.286 ± 0.005b
|
40
|
0.326 ± 0.035a
|
50
|
0.409 ± 0.023a
|
60
|
0.358 ± 0.010a
|
70
|
0.347 ± 0.014c
|
Table 2: Compressive strength of biofoam at different parameter.
Factors
|
Parameters
|
Average compressive strength, (MPa)
|
Temperature (oC)
|
20
|
0.647 ± 0.033b
|
25
|
0.761 ± 0.013a
|
30
|
0.975 ± 0.106a
|
35
|
0.529 ± 0.001c
|
40
|
0.277 ± 0.075d
|
Spawn loading (wt %)
|
5
|
0.839 ± 0.053a
|
10
|
0.929 ± 0.026a
|
20
|
1.005 ± 0.036a
|
30
|
1.125 ± 0.035a
|
40
|
1.350 ± 0.198a
|
Substrate moisture content (w/w %)
|
30
|
0.746 ± 0.035a
|
40
|
0.789 ± 0.005a
|
50
|
1.070 ± 0.000a
|
60
|
0.475 ± 0.004e
|
70
|
0.376 ± 0.011f
|
The values obtained for dry density and compressive strength were in correspondence with the preceding growth, where the formation of a dense white mycelium fungal skin occurred at the highest dry density and compressive strength. According to Owaid et al. (2015), high value of density indicated good mycelium growth, whereas low dry density was due to the nonexistent component of the mycelium and shrinkage caused by moisture loss. Growth of mycelium under optimum condition will produced biofoam with good compressive strength and density (Ghazvinian et al., 2019). Bruscato et al. (2019) also reported similar density values of biofoam for mycelium P. sanguineus, P. albidus, and L.velutinus when using sawdust as substrate, which are 0.32, 0.30 and 0.35 g/cm3, respectively. In general, the biofoam density was denser compared to the density of expanded polystyrene (EPS) which is 0.03 g/cm3. Higher density obtained by mycelium biofoam however could be attributed by the presence of the supporting medium from the spawn and types of substrate used. Therefore, a spawn with the same substrate medium used for biofoam production should be considered in future study instead of using the grain spawn. On the other hand, Jones et al. (2020) reported that mycelium-based biofoam shows comparable compressive strength (0.17-1.1 MPa) towards polystyrene foam (0.03-0.69 MPa) and a slightly weaker strength compared to polyurethane and phenolic formaldehyde resin.
3.4 Characterisation of biofoam morphology
The colonisation of mycelium in the substrate normally is non-homogenous, and thus SEM was used to analyse the surface feature of the mycelium biofoam. Fig. 6 shows the morphological of mycelium biofoam. Based on the SEM image, the short and highly entangled tube-like structures were observed during the initial growth phase, whereas compact filaments increase with time (Fig. 6a). A study by Bruscato et al. (2019) using Pleurotus albidus also shows a compact filament where a higher number of hyphae adhere to the substrate then adhered to each other. Haneef et al. (2017) reported that the two species belong to the same group, white-rot fungi, could have an identical morphology because of the ability to excrete similar enzyme during the production of mycelium biofoam. On the other hand, the diameter of the filaments also depends on the availability of nutrients such as cellulose in the substrate. Haneef et al. (2017) reported that a substrate rich in polysaccharides could change the hyphae morphology responsible for the lightness characteristics of the material. In Fig. 6(b), hyphae were observed to appear flatten on the SEM image. This occurred because the internal hydrostatic pressure no longer supported the filaments of the mycelium once the mycelium has stopped growing due to heat treatment (Girometta et al., 2019).
3.5 Characterisation of the biofoam’s functional group
Fig. 7 presents two FTIR spectra of rice husk as the substrate and the produced mycelium biofoam. For the infrared absorption spectra of the rice husk, a wide broad peak that is the polysaccharides component was observed from 900 to 1200 cm-1 spectrum, with an obvious peak of C-C stretching at 1033 cm-1. Gao et al. (2018) stated that a rice husk was made up of 35 to 45% of cellulose, 15 to 20% of hemicellulose, and 20 to 25% of lignin. However, the intensity of the polysaccharides band, observed in the spectra of the mycelium biofoam, has decreased (Fig. 8b) as the rice husk was degraded as a substrate for the growth of P.ostreatus. In comparison, new bands of proteins and lipids were detected at 1633 cm-1 and 3280 cm-1 when the mycelium grew in the biofoam. Haneef et al. (2017) reported that P. ostreatus biofoam sample shows a relative increase in proteins and lipids on PDB-cellulose substrate compared to those grown on pure cellulose. The intensity of the polysaccharide in mycelium biofoam spectra was also found reduced from a value of 0.08 for pure cellulose to 0.06 for PDB-cellulose. Haneef et al. (2017) further stated that the decreasing amount of rigid chitin from the cell wall probably related to the collapse of the central area of mycelia was during the growth of mycelium on PDB-cellulose. Therefore, this finding found that the chemical nature of the feeding substrate is also responsible for the changes in infrared spectra of mycelium biofoam. Apart from that, the infrared absorption spectra of mycelium can also be translated as useful biomolecules during the composting process. Those biomolecules, which include protein (amide I with the wavelength of 1700-1600 cm-1, amide II and III with the wavelength of 1575-1300 cm-1), lipids with the wavelength of 3000-2800 cm-1, and polysaccharides with the wavelength of 1200-900 cm-1 (Pena et al., 2014).