Substrate supplies
Albizia chinensis woodchips (AWC) have been obtained from San Ho Timber Pte Ltd (Singapore) and used as acquired, with particle sizes starting from 2 to 10 mm. Sugarcane bagasse (SC) was collected from native juice distributors throughout Singapore, rinsed with deionized water to take away floor contamination, oven dried at 60 °C for 48 h, after which mechanically milled to provide fibers of roughly 3–5 mm in size. This particle measurement vary was chosen to supply a stability between nutrient availability and structural reinforcement throughout mycelial progress.
Cassava pulp was obtained from a starch-processing plant in Bandung, Indonesia. Upon assortment, it was saved at -20 °C instantly to stop fermentation and microbial degradation, and thawed to ambient temperature (25 ± 2 °C) earlier than use. Cassava pulp is a by-product of starch extraction, and is usually discarded or burned in open fields close to processing amenities. Its excessive moisture content material and residual starch make it a readily accessible nutrient supply for fungal progress and colonization.
The preliminary moisture contents of the uncooked substrates, previous to sterilization, have been 8–10% for AWC, 6–8% for SC, and 70–75% for cassava pulp, as measured gravimetrically by oven drying at 105 °C for twenty-four h.
Fungal pressure and spawn preparation
The fungal pressure used on this research was G. lucidum, a white-rot basidiomycete able to concurrently decomposing lignin, cellulose, and hemicellulose. This species was chosen for its means to generate dense, cohesive mycelial networks that improve inter-particle bonding in lignocellulosic substrates64. The pressure was obtained from the Analysis Group at Republic Polytechnic (Singapore) and maintained on potato dextrose agar (PDA) plates at 4 °C till use. Previous to spawn preparation, the tradition was subcultured onto recent PDA plates, which have been made each 3 months, beneath sterile circumstances and incubated at 25 ± 2 °C for 7 days to acquire actively rising mycelium. PDA was ready by dissolving 20 g agar, 20 g dextrose, 6 g potato extract and 0.1 g chloramphenicol (Sigma Aldrich) in 1 L of distilled water and bringing the answer to a boil. The medium was then sterilized by autoclaving at 121 °C for 15 min, cooled to 40–45 °C, and poured into sterile Petri dishes.
A liquid mycelial inoculum was ready by aseptically transferring roughly 10 mm agar plugs from the actively rising margin of 7-day-old PDA cultures into sterile potato dextrose broth (PDB), ready by dissolving 20 g of dextrose and 4 g of potato extract in 1 L of distilled water, bringing the answer to a boil, sterilizing it by autoclaving at 121 °C for 15 min, and permitting it to chill to room temperature earlier than inoculation. The cultures have been incubated in an orbital shaking incubator (New Brunswick™ Innova® 40, Eppendorf, Germany) at 25 ± 2 °C and 50 rpm for 7 days to provide a homogeneous liquid mycelial tradition. Instantly earlier than spawn inoculation, the liquid tradition was homogenized utilizing a sterile laboratory homogenizer to acquire a uniform mycelial suspension for inoculation.
Rice grains have been used because the provider materials for spawn manufacturing, following established procedures for G. lucidum cultivation48. Rice was soaked in distilled water for 12 h, then drained and air-dried to realize a moisture content material of roughly 45%. To take care of a barely alkaline pH that suppresses bacterial contamination and promotes fungal enzyme exercise, 2 wt.% calcium carbonate (CaCO3) was combined homogenously into the hydrated grains.
Roughly 200 g of the ready rice was loaded into 500 ml autoclavable glass jars, sealed with cotton-plugged lids, and sterilised by autoclaving at 121 °C for 30 min (Tommy SX-700, Digital Biology, Japan). After cooling to room temperature beneath aseptic circumstances, every jar was inoculated with 5 ml of homogenized liquid mycelial inoculum. As a result of the inoculum consisted of homogenized hyphal fragments moderately than discrete fungal cells, colony-forming items (CFU mL⁻1) weren’t decided. As a substitute, inoculum consistency was ensured by sustaining an identical tradition age, progress circumstances, inoculum quantity, and homogenization procedures for all batches. The jars have been incubated at 25 ± 2 °C and 70 ± 5% relative humidity for 14 days, with light shaking carried out each 3 days to advertise uniform colonization.
Full colonization was indicated by the formation of a steady white mycelial community protecting all grains. The totally colonized spawn was saved at 4 °C and used inside 30 days. All procedures have been carried out beneath sterile circumstances in a Class II laminar-flow cupboard that was sterilized by ultraviolet irradiation for 30 min earlier than and after use to reduce the chance of contamination.
Substrate inoculation and incubation
The preliminary moisture contents of cassava pulp, SC, and AWC have been decided previous to formulation. The substrate parts have been then weighed based on the formulations offered in Desk 2 on a dry weight foundation and totally homogenized. Water was subsequently added to every formulation to realize a moisture content material of 60%. The formulations have been designed to keep up a comparable whole dry mass of the stable parts whereas evaluating the impact of cassava supplementation.
In cassava-containing formulations, a portion of the bottom substrate (SC or AWC) was changed with cassava pulp to keep up a constant general solids content material. The proportion of wheat bran, which served as a nutrient complement, was correspondingly decreased to keep away from extreme nutrient enrichment following cassava addition. The CaCO₃ content material was barely elevated in cassava-containing formulations to account for the acidity of the cassava pulp and to keep up beneficial pH circumstances for mycelial progress and colonization.
Roughly 200 g of every moist substrate formulation was then transferred into autoclavable polypropylene cultivation luggage. Every bag was sealed utilizing a cotton plug wrapped in sterile gauze, permitting restricted air alternate throughout incubation. Sterilization was carried out at 121 °C for 30 min utilizing an autoclave (Tomy SX-700, Digital Biology, Japan). This step minimized microbial contamination and was utilized persistently throughout all formulations to allow reproducible fungal progress.
After sterilization, the baggage have been cooled to room temperature (25 ± 2 °C) in a Class II laminar-flow cupboard disinfected with 70% ethanol and uncovered to ultraviolet mild for 30 min earlier than use. Every bag was then aseptically inoculated with 40 g of G. lucidum rice-grain spawn, equivalent to roughly 5% of the substrate dry mass. The spawn was distributed uniformly all through the substrate by light handbook mixing to advertise homogeneous mycelial colonization all through the majority materials.
The inoculated luggage have been resealed with sterile cotton plugs and positioned in a controlled-environment progress chamber maintained at 25 ± 2 °C and a relative humidity of at the least 65%. Steady air circulation was supplied to stop native carbon dioxide accumulation and to keep up uniform environmental circumstances. Incubation was carried out for five–8 days, throughout which the fungal mycelium unfold quickly by means of the substrate matrix. Colonization was thought-about full when a steady, white mycelial layer totally lined the floor as assessed by visible commentary. Baggage exhibiting indicators of contamination have been discarded instantly to stop cross-infection.
As soon as a steady white mycelial layer lined the substrate floor, it was transferred to the molding stage for composite formation. Colonization was assessed by visible inspection based mostly on the formation of a steady mycelial layer throughout the uncovered substrate floor. At this stage, G. lucidum fashioned a dense, cotton-white hyphal community with high-quality, branching filaments, indicating superior colonization and efficient binding of the substrate particles. The following molding, mid-growth compression, and post-compression incubation steps are illustrated schematically in Fig. 5 and described within the following part.
Schematic illustration of the fabrication course of for mycelium-based composites. The substrate parts are first combined with water to realize a moisture content material of 60%, adopted by sterilization in an autoclave at 121 °C for 30 min. In parallel, Ganoderma lucidum is cultured on PDA and subsequently propagated on rice grains to arrange the spawn. After cooling, the sterilized substrate is inoculated with the rice grain spawn and incubated in cultivation luggage at 25 ± 2 °C and a relative humidity of at the least 65% for five–8 days till floor colonization is achieved. The colonized substrate is subsequently transferred into molds of fifty × 50 × 100 mm dimensions and incubated for an additional 3–5 days to advertise recolonization and consolidation. The composites are subsequently mechanically densified and maintained beneath managed incubation for an additional 4–10 days earlier than demolding. The ensuing materials varieties a inflexible mycelium composite block with a steady dense outer mycelial pores and skin.
Fabrication of mycelium-based composites
After the preliminary incubation, the colonized substrates have been gently disaggregated beneath sterile circumstances to interrupt up compacted clumps and procure a uniform bulk consistency. This step promotes homogeneous regrowth of the fungal mycelial community throughout subsequent shaping and reduces the chance of uncolonized void formation contained in the composite physique. The free, mycelium-based materials was then manually packed into acrylic molds with dimensions of fifty × 50 × 100 mm, equivalent to the to the pattern dimensions required for compression testing.
Every mould was stuffed progressively in layers of roughly 2–3 cm, with mild handbook compaction utilized between layers to reduce trapped air and cut back inside voids. The stuffed molds have been then lined with perforated lids lined with sterile filter paper to keep up sterility whereas permitting restricted air alternate. The samples have been positioned in a controlled-environment progress chamber maintained at 25 ± 2 °C and a relative humidity of at the least 65% for a further 3–5 days. Throughout this section, the mycelium quickly recolonized the disturbed matrix and re-established hyphal continuity throughout particle interfaces. Visible monitoring confirmed the formation of a steady, cotton-white mycelial layer on the uncovered surfaces.
To enhance packing density and improve the mechanical integrity of the composites, the partially colonized blocks have been subsequently compressed to roughly 50percentof their preliminary top utilizing custom-fabricated stainless-steel clamps. Compression was maintained for 3–5 days beneath the identical incubation circumstances, permitting continued fungal progress earlier than the clamps have been eliminated.
After releasing the clamps, the partially consolidated blocks have been incubated for an additional 4–10 days, throughout which the mycelium fashioned a dense, steady outer pores and skin. This floor layer features as a pure bio-coating, defending the underlying matrix from contamination and moisture loss. Air circulation inside the chamber was maintained repeatedly to stop CO₂ accumulation and floor condensation. Mycelial maturity was decided visually by the formation of a uniform, white floor, indicating full colonization and matrix consolidation.
Upon reaching full progress, the mycelium-based composites exhibited a agency, sponge-like texture with cohesive particle bonding and visual hyphal bridges all through the cross-section. These specimens have been then dried and subjected to bodily and mechanical characterization as described within the following part.
Put up-growth drying and materials characterization
After finishing mycelial colonization, the mycelium-based composites have been faraway from the molds and ready for drying. This post-growth therapy was utilized to terminate fungal metabolic exercise, take away residual moisture, and stabilize the interior microstructure.
Drying was carried out in a laboratory convection oven (Qingdao Guosen-1800, China) at 70 °C for 48–72 h, or till a relentless mass was achieved. The chosen drying temperature was ample to inactivate the fungal biomass whereas avoiding thermal degradation of the lignocellulosic substrate and chitinous mycelial matrix, as demonstrated in prior studies45. Samples have been positioned on stainless-steel mesh trays to make sure uniform air circulation, and pattern mass was recorded at 12 h intervals utilizing a precision analytical stability (Shimadzu UW sequence, Japan).
Earlier than and after drying, the scale of every specimen have been measured utilizing a digital Vernier calliper (Mitutoyo, Japan, accuracy ± 0.01 mm) to quantify shrinkage and volumetric change. The geometric quantity was calculated from the typical size, width, and top of every pattern, and the majority density (ρ) was decided utilizing Eq. 1:
$$rho =frac{{m}_{d}}{{V}_{d}}$$
(1)
the place md is the dry mass (kg) and Vd is the corresponding dry quantity (m3). Three impartial samples (n = 3) have been examined for every formulation.
Visible assessments have been carried out after drying to judge floor uniformity, shade, and general integrity. The totally dried composites exhibited inflexible, light-weight buildings with seen hyphal bonding and minimal warping or floor defects, per the traits of well-grown Ganoderma-based mycelium composite supplies. These stabilized samples have been subsequently used for mechanical testing and microstructural characterization.
Porosity measurement
Porosity was quantified to evaluate the interior void construction and diploma of compactness of the mycelium-based composites. Porosity is a key parameter influencing mechanical power, moisture transport, and dimensional stability. The full porosity was decided from the connection between the majority density and the skeletal density, with the latter measured utilizing a helium fuel pycnometer based mostly on the fuel displacement precept.
Helium fuel pycnometry determines the true skeletal quantity of the stable section by measuring the strain change that happens when an inert fuel (on this case, helium) is expanded from a calibrated reference chamber right into a chamber containing the pattern. As a result of helium atoms are small and chemically inert, they penetrate high-quality surface-accessible pores however don’t enter the closed inside voids, permitting correct dedication of the solid-phase (skeletal) quantity. This methodology is especially appropriate for extremely porous bio-based composites, because it avoids liquid absorption, swelling results and structural alteration that may happen with immersion-based porosity measurement strategies.
Earlier than measurement, every specimen was oven-dried at 60 °C for 12 h to take away residual moisture after which weighed to find out its dry mass. The pattern was positioned within the helium pycnometer chamber, and after thermal equilibration, a managed helium strain pulse was launched from the reference chamber. The equilibrium pressures in each chambers have been recorded routinely, and the pattern skeletal quantity (Vs) was calculated by the instrument software program utilizing the perfect fuel regulation. The skeletal density (ρskeletal) was decided utilizing Eq. 2.
$${rho}_{skeletal}=frac{{m}_{d}}{{V}_{s}}$$
(2)
the place md is the dry mass of the specimen.
The majority density (ρbulk) was obtained independently from the dry mass and geometric quantity measured utilizing a digital vernier calliper (accuracy ± 0.01 mm). The full porosity (P) was calculated utilizing Eq. 3:
$$textual content{P}= left(1-frac{{rho}_{bulk}}{{rho}_{skeletal}}proper)instances 100$$
(3)
Three impartial samples (n = 3) have been examined for every substrate formulation. Between consecutive measurements, specimens have been re-dried at 60 °C to take away any absorbed moisture and guarantee constant measurement circumstances. This gas-displacement methodology offers a extremely dependable and reproducible measure of whole porosity and inside compactness, parameters which can be instantly linked to the mechanical efficiency and microstructural connectivity mentioned in subsequent sections.
Mechanical testing
The compressive power, elastic modulus in compression and corresponding stress–pressure behaviour of the mycelium-based composites have been decided to judge their stiffness, load-bearing capability, and deformation mechanisms. Compression testing was chosen as the first mechanical characterisation methodology as a result of mycelium-based composites exhibit a mobile, foam-like construction and are generally evaluated beneath compressive loading for purposes corresponding to insulation panels and lightweight structural cores.
Exams have been carried out utilizing a common testing machine (Shimadzu AG-IC, Japan) geared up with a 100 kN load cell and flat, polished compression platens. The testing process adopted ASTM D3574-17, which specifies the dedication of compressive properties for mobile and foam-like supplies. Previous to testing, all specimens have been conditioned at 23 ± 2 °C and 50 ± 5% relative humidity for at least 12 h, to make sure constant moisture equilibrium.
Every specimen was centrally aligned between the compression platens and examined beneath displacement-controlled loading at a relentless crosshead velocity of 10 mm/min. Load and displacement have been repeatedly recorded at a sampling fee of 10 Hz. The nominal compressive stress (σ) and pressure (ε) have been calculated utilizing Eqs. 4 and 5 respectively:
$$sigma =frac{F}{{A}_{0}}$$
(4)
$$varepsilon =frac{Delta h}{{h}_{0}}$$
(5)
the place F is the utilized load (N), A0 is the preliminary cross-sectional space (m2), h0 is the preliminary specimen top (m), and Δh is the measured displacement (m).
The elastic modulus in compression (Ec) was decided from the slope of the preliminary linear portion of the stress–pressure curve, usually between 0.5 and a pair of% pressure, the place the fabric response is roughly linear and reversible. A least-squares linear regression was utilized to the chosen knowledge factors to calculate the modulus utilizing Eq. 6, the place ({Delta}_{sigma }) represents the change in stress and ({Delta}_{varepsilon }) the corresponding change in pressure inside the chosen linear elastic area:
$${E}_{c}=frac{{Delta}_{sigma }}{{Delta}_{varepsilon }}$$
(6)
The compressive power at 5% pressure was outlined because the stress corresponding to five% deformation, representing the onset of localized cell collapse earlier than densification, per the pressure limits really useful in ASTM D3574-17 for versatile mobile supplies.
Three impartial specimens (n = 3) have been ready and examined for every substrate formulation, and the imply values with corresponding commonplace deviations have been reported. Throughout testing, deformation behaviour was visually monitored to establish the transition from the preliminary elastic regime to the plateau area. The stress–pressure response usually displayed an preliminary linear elastic regime, adopted by a plateau related to hyphal-fiber community buckling and progressive densification at greater strains.
The measured elastic modulus and compressive stress values have been subsequently correlated with porosity to interpret how substrate sort and inside construction affect the mechanical efficiency of the mycelium-based composites.
Microstructural characterization by scanning electron microscopy (SEM)
The microstructure of the mycelium-based composites was examined utilizing SEM to visualise hyphal distribution, substrate-hypha interactions, and pore morphology inside the dried composites. Observations have been carried out utilizing a JEOL JSM-5500LV electron microscope (Japan) operated beneath high-vacuum mode at an accelerating voltage of three.0 kV and a working distance of 15 mm.
Earlier than imaging, small sections (roughly 10 × 10 × 5 mm) have been minimize from consultant areas of every composite, together with each floor (pores and skin) and core. The samples have been mounted on aluminum stubs utilizing carbon adhesive tape and sputter-coated with a skinny carbon layer for 20 s utilizing a Pelco SC-6 carbon coater (Ted Pella Inc., USA) to reduce floor charging throughout imaging.
A low accelerating voltage was chosen to reduce electron-beam penetration and charging results within the non-conductive natural matrix whereas sustaining ample floor decision. Photos have been collected at 1500 × magnification to make sure constant comparability of the hyphal networks and substrate morphology between formulations. Floor pictures have been used to evaluate the formation of steady mycelial movies and bridging filaments, and core pictures have been used for hyphal diameter measurements and analysis of the interior substrate-hypha morphology.
Qualitative comparisons have been made throughout samples ready from completely different substrate sorts to evaluate the affect of substrate morphology and nutrient composition on hyphal density and bonding behaviour. These micrographs complemented the mechanical and porosity measurements, offering visible proof of how the fungal matrix contributes to load switch and structural reinforcement inside the composite structure.
Hyphal diameter distributions have been obtained by manually measuring 250 particular person hyphae diameters from every SEM picture utilizing the ImageJ software program. Fungal hyphae have been distinguished from the SC and AWC substrate fibers in SEM pictures based mostly on their attribute morphology and measurement. Hyphae appeared as slender, easy, cylindrical filaments with comparatively uniform diameters within the micrometer vary. In distinction, the SC and AWC substrate fibers have been considerably bigger, exhibited irregular geometries, and possessed tough floor textures. These traits enabled dependable identification of hyphae for diameter measurements.
