The study was conducted at the Institute of Agricultural Research for Development (IRAD), Nkolbisson, Yaoundé, Cameroon. Spawn cultures of P. ostreatus and P. citrinopileatus were obtained from the Mushroom Project, Obala-Cameroon. CP, the primary lignocellulosic substrate, was collected from the Food Processing Laboratory of the Institute of Agricultural Research for Development (IRAD), Yaoundé, Cameroon.

The preparation of CP substrates followed a standardized multi-step protocol to ensure sterility, optimal moisture content, and physical structure conducive to mycelial (P. ostreatus and P. citrinopileatus) colonization and fruiting. The detailed workflow of substrate processing, from initial collection and cleaning to particle size fractionation and sterilization, is illustrated in Figure 1. The process begins with the collection of RCP from the Food Processing Laboratory at IRAD. The material undergoes cleaning thrice with tap water and thoroughly with distilled water to eliminate impurities, followed by drying at 60°C to stabilize its moisture content. The cleaned and dried parchment was then categorized into two groups based on particle size (Figure 2). The first group, designated as RCP, maintains its coarse, unprocessed form with particles exceeding 50 mm. The second group is mechanically ground and sieved to produce two finer particle sizes: MCP, with particles between 0.0049 mm and 50 mm, and FCP, comprising particles smaller than 0.0049 mm. This classification follows standard approaches used in substrate particle size characterization for mushroom cultivation [16, 17]. No additional substrates (e.g., wheat straw) were included, as the purpose of the study was to specifically assess the potential of CP as a sustainable alternative. The unprocessed RCP treatment was considered the internal control for comparative analysis.

Each substrate type was adjusted to approximately 65% moisture content by adding water in a controlled manner to create optimal conditions for mycelial colonization. The prepared substrates were packed into plastic bags, sterilized at 121°C for 45 minutes, and allowed to cool under aseptic conditions. The cooled substrates are then inoculated with mycelium and incubated at a controlled temperature (25° ± 1°C) and relative humidity (80–85%) to facilitate colonization. This preparation protocol was designed to assess the impact of substrate particle size on the growth, yield, and overall cultivation performance of P. ostreatus and P. citrinopileatus, offering a standardized and scalable method for valorizing coffee industry by-products.

Spawn was prepared using polished paddy rice as the carrier medium. The rice grains are washed thoroughly, soaked overnight, and steamed for 30 to 45 minutes to gelatinize the starches. After cooling, urea (0.2%) and calcium carbonate (CaCO₃, 2%), supplied by the Mycology Laboratory of IRAD, were added to adjust the nitrogen content and pH, creating optimal conditions for mycelial growth [18]. The prepared substrate was distributed into sterilized 500 mL glass bottles, which were sealed with cotton plugs and aluminum foil. The glass bottles were sterilized at 121°C for 45 minutes. Once cooled, the substrate was inoculated aseptically with 10% (w/w) actively growing mycelium cultivated on potato dextrose agar (PDA). The inoculated bottles were incubated in darkness at 25° ± 1°C until complete colonization of the substrate by the mycelium.

Four replicates of 2 kg of sterilized CP were distributed equally into four polypropylene bags of 40 cm × 60 cm size. In total, 24 cultivation bags (3 particle size treatments × 2 Pleurotus species × 4 replicates) were used in the experiment. Bags were aseptically inoculated with 20 g of spawn, corresponding to approximately 10% (w/w) of the wet substrate, and mixed thoroughly to facilitate rapid and uniform mycelial growth. The mouths of the bags were tied using a cotton plug and thread. Holes were made in the polypropylene bags for aeration. The bags were incubated in the dark room at 25° ± 1°C with relative humidity maintained at 80–85% using ultrasonic humidifiers. Daily monitoring continued until full substrate colonization.

Upon completion of substrate colonization, the bags were transferred to a fruiting chamber where environmental conditions were carefully controlled. Lighting was provided through diffused daylight supplemented with LED lamps, offering a 12-hour photoperiod at an intensity of 800 to 1,000 lux. Ventilation was facilitated by a passive air exchange system supplemented by mechanical exhaust fans to maintain CO₂ concentrations below 1,000 ppm. Mature fruiting bodies were harvested manually to minimize substrate damage. Each cultivation bag produced three successive flushes. Between flushes, the substrates were rehydrated by immersing the bags in clean water for 12 hours.

Pre-fruiting parameters included spawn running time (the period from inoculation to full mycelial colonization), primordial initiation time (the interval from inoculation to the appearance of primordia), and fruiting time (the duration from primordial formation to harvestable maturity). Morphological measurements collected during harvest included the number of pilei, pileus diameter, and stipe length. Productivity was evaluated by measuring the total yield (cumulative fresh weight of harvested mushrooms), BE, and production rate (PR). BE was calculated as the percentage ratio of the fresh weight of mushrooms to the dry weight of the substrate. PR was determined by integrating yield and cropping duration, expressed as a percentage. BE and PR were calculated using Equations 1 and 2 as follows:

(1) B E   %   =   F r e s h   m u s h r o o m   m a s s D r y   s u b s t r a t e   m a s s   ×   100

(2) P R   %   =   B E T o t a l   c u l t i v a t i o n   d a y s   ×   100

Total cultivation days were defined as the number of days from substrate inoculation to the final harvest, including both the incubation (spawn running) and fruiting phases, in line with previous studies [18, 19].

Pileus color was measured using a colorimeter, recording L* (lightness), a* (red-green axis), and b* (yellow-blue axis) values. The color value was estimated using a Hunter’s Lab color analyzer (Hunter Lab scan XE, Reston, VA, USA). In the Hunter colorimeter, the color of a sample is designated by the three dimensions, L*, a*, and b*. This color was measured by placing the aperture of the equipment on the sample with white paper as the reference. The color of the samples was measured after placing the samples in front of the tiniest opening [20]. In order to obtain data reflecting the color of samples, different points were taken into consideration for each sample. All data were collected with three replications.

A completely randomized design (CRD) was employed, comprising three substrate treatments (RCP, MCP, and FCP) for each Pleurotus species. Each treatment was replicated three times. For each replicate, four polypropylene bags (40 cm × 60 cm) were filled with 2 kg of sterilized CP substrate, resulting in a total of 12 bags per treatment and 36 bags per species. Thus, 72 cultivation units were established across the two Pleurotus species, providing sufficient replication for statistical robustness.

All experiments followed a CRD with three replicates per treatment. Data collected included growth parameters (number of pilei, pileus diameter, stipe length, cap number), developmental milestones (spawn running time, primordial initiation, fruiting time), productivity indices (total yield, BE, PR), and quality attributes (color parameters L*, a*, b*). Results are presented as mean ± standard deviation (SD) from three replicates. One-way ANOVA assessed the effects of substrate particle size (RCP, MCP, FCP) on all variables. When significant differences were observed (p < 0.05), means were separated using Duncan’s multiple-range test. Values in a column bearing different superscript letters differ significantly at the 5% level.

As shown in Figure 3, both P. ostreatus (Figure 3A) and P. citrinopileatus (Figure 3B) formed fruiting bodies on CP substrates, yet differed notably in color, cap curvature, and structural density. P. ostreatus formed compact clusters with overlapping caps, whereas P. citrinopileatus displayed more spaced and bright yellow pilei.

The size of substrate particles significantly influenced the developmental morphology of mushrooms, impacting all assessed characteristics, including pileus number, pileus diameter, and stipe length (Table 1). Medium-sized particles (MCP) consistently yielded the most advantageous outcomes for both fungal species investigated. P. citrinopileatus exhibited the production of 30.00 ± 2.80 pilei, with an average pilei diameter of 8.00 ± 1.50 cm and stipe length of 11.50 ± 1.20 cm. A comparable pattern was observed in P. ostreatus, which demonstrated 28.20 ± 3.12 pilei, an average pilei diameter of 9.50 ± 2.22 cm, and a stipe length of 13.15 ± 1.33 cm. These beneficial effects are attributed to the inherent physical properties of MCP, specifically its balanced porosity, adequate airflow, and effective moisture retention capacity. This confluence of factors creates a conducive environment that promotes robust colonization and consistent fruiting body formation.

The physical structure of the substrate, especially its particle size, significantly shaped the timing of key developmental stages in both P. ostreatus and P. citrinopileatus (Table 2). The effect was most pronounced during the early colonization and initiation phases.

Spawn running was fastest on medium (MCP) and fine (FCP) particles. P. ostreatus completed colonization in 15.0 ± 0.9 and 15.0 ± 0.8 days, respectively, compared to 17.0 ± 1.0 days on raw coarse parchment (RCP). P. citrinopileatus showed the same pattern: 16.0 ± 1.1 days on MCP and 16.0 ± 1.0 days on FCP, but 18.0 ± 1.0 days on RCP. These reductions indicate improved substrate accessibility. Smaller particles offer more contact points, better moisture retention, and consistent oxygenation, conditions favorable to rapid hyphal extension.

Primordial initiation followed a similar trend. On MCP and FCP, primordia emerged by day 30.0–31.0. On RCP, delays were significant: 37.0 for P. ostreatus and 38.5 for P. citrinopileatus. This lag reflects structural limitations, lower porosity, reduced internal aeration, and uneven nutrient diffusion, impeding the metabolic signals that trigger fruiting body formation.

Fruiting time, measured from primordia emergence to harvestable maturity, remained stable across treatments. For both species, values ranged from 6.0 to 6.5 days, with no significant differences. This suggests that once developmental cues are activated, cap maturation depends more on environmental factors than on substrate configuration.

Yields varied sharply with substrate structure. Among all treatments, fine particles (FCP) produced the highest values for both species (Table 3). P. ostreatus yielded 377.2 ± 18.5 g in total; P. citrinopileatus reached 355.0 ± 17.0 g. In both cases, flush 1 contributed most, over 36% of the total, reflecting a robust initial metabolic response. These high outputs are directly linked to better porosity, higher surface-to-volume ratio, and consistent substrate hydration. In contrast, raw particles (RCP) limited performance. P. ostreatus produced just 203.6 ± 12.0 g, while P. citrinopileatus dropped further to 186.5 ± 11.2 g. Both species showed a typical flush pattern: high initial yield, followed by a gradual decline. However, RCP narrowed that first peak and steepened the fall. Likely causes: limited enzyme-substrate contact, lower aeration, and poor moisture diffusion. The structure was too coarse to support sustained mycelial activity.

BE follows the same gradient. On FCP, P. ostreatus achieved 75.2% and P. citrinopileatus reached 72.0%. These values meet or exceed industry thresholds for intensive systems. On RCP, BE dropped to 42.2% and 39.0%, respectively, barely viable for commercial production. Medium particles (MCP) gave intermediate results, confirming that particle refinement enhances enzymatic access and biomass conversion.

PR, which accounts for time, highlighted another advantage. FCP enabled fast, dense output: 156.7% for P. ostreatus, 150.5% for P. citrinopileatus. RCP lagged far behind (93.8% and 85.0%). PR integrates yield with cycle length. Faster colonization and early fruiting, as seen with FCP and MCP, boost daily productivity, a crucial factor in commercial viability.

Between species, P. ostreatus consistently outperformed P. citrinopileatus in total yield, BE, and PR. This trend reflects their greater physiological robustness and broader enzymatic activity. Yet under optimized FCP conditions, P. citrinopileatus approached parity, suggesting strong potential under controlled cultivation.

Color is not just visual, it signals chemical content, physiological state, and market value. In both Pleurotus species, substrate particle size shaped color outcomes in terms of lightness (L*), red-green balance (a*), and yellow-blue intensity (b*) (Table 4).

Lightness (L*) dropped markedly on finer substrates. Mushrooms grown on RCP were the lightest: P. ostreatus reached 59.96, P. citrinopileatus 57.50. On MCP, values fell sharply, to 41.40 and 39.80, respectively. This decline suggests higher pigment deposition, possibly due to more intense metabolic activity and moisture retention. Lower L* values often reflect greater phenolic content and antioxidant accumulation. FCP showed intermediate lightness, indicating that partial compaction may still support pigment synthesis, though less efficiently than MCP.

The red-green component (a*) peaked on MCP. P. ostreatus reached 14.00; P. citrinopileatus, 15.50. These high a* values imply strong activation of the phenylpropanoid pathway, likely linked to improved oxygen flow and enzymatic conditions within MCP substrates. On FCP and RCP, red tones faded. Compact substrates limit oxygen; coarse ones disperse it unevenly. In both anthocyanin and phenolic pigment synthesis may drop.

Yellow-blue axis (b*) followed species-specific patterns. P. citrinopileatus showed the highest values overall, especially on RCP (28.50), in line with its inherent golden pileus phenotype. However, b* declined slightly on MCP and more noticeably on FCP, which may be related to reduced flavonoid or carotenoid biosynthesis under altered moisture-oxygen regimes. P. ostreatus maintained stable b* values across all treatments, with no major shifts.

This study demonstrates that the particle size of CP critically influences the growth dynamics, yield performance, and phenotypic quality of P. ostreatus and P. citrinopileatus. Medium and fine particles enhanced spawn colonization, accelerated primordia initiation, and significantly increased total yield, BE, and PR, especially for P. ostreatus. Improved porosity and moisture dynamics in these substrates likely facilitated enzymatic activity and nutrient diffusion, promoting consistent fruiting. Moreover, colorimetric changes, particularly in L* and a* values, suggest that substrate structure modulates pigment biosynthesis, potentially via stress-induced metabolic pathways. These findings not only confirm the biological value of optimized CP but also underscore its potential as a biotechnological input within circular economy strategies, offering scalable, low-cost alternatives for sustainable mushroom production and agro-waste valorization.