Spent Mushroom Substrate (SMS) as a Potential Casing Material for Commercial Cultivation of Calocybe indica: Improved Mineral Profile and Microbial Advantages

doi:10.21203/rs.3.rs-5011964/v1

Recent prolonged summer seasons have posed significant challenges for mushroom cultivation, underscoring the need for summer-adapted commercial varieties like Calocybe indica. Casing plays a crucial role in its cultivation, which traditionally uses loamy soil (LS). However, given the non-renewable nature of LS and the environmental concerns associated with spent mushroom substrate (SMS), our study explored SMS as a potential alternative. We examined the physio-chemical properties, and microbial flora especially bacterial composition using MALDI-TOF in both LS and SMS, along with the total yield, biological efficiency, mineral content, and total phenolic and flavonoid contents of mushrooms grown on these substrates. While most of the physio-chemical properties of SMS align with the ideal casing properties, it exhibits higher electrical conductivity (EC) and a greater C/N ratio. The dominating bacterial flora in SMS, including Bacillus, Priestia,and Lysinbacillus, contribute to the mushrooms’ temperature tolerance and facilitate nutrient uptake especially phosphorous (P). The yields and biological efficiency were significantly higher in LS, likely due to its superior mechanical support. Furthermore, the results found that element levels, especially copper (Cu), zinc (Zn), and phosphorous (P), were significantly higher in mushrooms grown on SMS except iron (Fe). Notably, phosphorus (P) levels were significantly elevated in mushrooms grown in SMS, highlighting the role of phosphorous-solubilizing bacteria in SMS. The PCA biplot results further supported these findings. Interestingly, Calocybe indica consistently exhibited higher iron (Fe) content than Pleurotus ostreatus, regardless of the casing material used. The metal bioaccumulation factors (BCF) reveal that Calocybe indica is a hyperaccumulator of potassium (K) but does not bioaccumulate manganese (Mn). It also showed a low calcium (Ca) and iron (Fe) accumulation level, suggesting a synergistic interaction between Ca and Fe. No significant difference in total phenolic and flavonoid content was observed between the LS and SMS. In conclusion, LS proved more effective in maximizing yield, while SMS emerged as a sustainable alternative with the enhanced nutritional quality of mushrooms, making it a viable option for environmentally conscious mushroom cultivation.

Biological sciences/Biotechnology

Earth and environmental sciences/Environmental sciences

Calocybe indica

Casing

Spent Mushroom Substrate (SMS)

Biological Yield

Minerals

Phosphorous-Solubilizing Bacteria

Mushroom cultivation epitomizes circular agriculture by employing low-value agricultural by-products such as rice straw, sawdust, cotton seed hull, and others to yield mineral and protein-rich food sources (Ahmed et al., 2024; Odediran et al., 2020; Nanje Gowda and Chennappa, 2021). The successful cultivation of mushrooms relies on various exogenous factors such as temperature, precipitation, and light exposure intensity. Most popular varieties such as Agaricus bisporus and Pleurotus spp are traditionally cultivated in subtropical regions during winter. However, the recent prolonged summer characterized by high temperatures and low precipitation has had a significant impact on mushroom cultivation, particularly in tropical and subtropical regions. Certain challenges arise for tropical edible mushrooms such as Pleurotus djamor due to limited production capacity, Volvariella volvacea has a short shelf life of only 24 hours at room temperature, jelly-like texture Auricularia is not widely preferred. Consequently, this has prompted researchers to explore alternative varieties suited for warmer seasons.

The Milky Mushroom (Calocybe indica) was introduced by the Tamil Nadu Agricultural University in Coimbatore, India in 1998 (Subbiah and Balan 2015) and has emerged as a notable addition to summer mushroom varieties. This soil-dwelling hemicolous mushroom belongs to the Basidiomycete fungus family Tricholomataceae (Kumar et al., 2017; Kumar et al., 2020). Flourishing in temperatures ranging from 30-38⁰C with relative humidity is 70–80%, as highlighted by (Vijaykumar et al., 2014; Kumar et al., 2011). It has garnered recognition for its robust size, enticing milky white appearance, meat-like taste, extended shelf life (5–7 days at room temperature), and distinctive texture (Chang 2007; Subbiah and Balan 2015). The cultivation of milky mushrooms involves a critical agronomic practice known as casing. This process entails covering the fully colonized mushroom substrate, serving as a pivotal step in transitioning from the vegetative to reproductive stages (Vieira and Pecchia 2021). Casing is highlighted in numerous studies for its role in achieving high productivity, ensuring quality, and fostering uniformity in commercial cropping (Pardo-Giménez et al., 2020; Yang et al., 2019; Carrasco and Preston, 2020). Furthermore, it serves as a vital layer for mushrooms by providing physical support, acting as a water reservoir, facilitating nutrient exchange, regulating osmotic pressure, enabling gas exchange, fostering an aerated environment for efficient metabolism, stimulating fructification, and providing zones for ion exchange (Kacprzak et al., 2017; Sarder et al., 2020). Consequently, physiochemical factors of casing materials such as water-holding capacity, porosity, pH, and types of the microbial population strongly influence the yield of mushrooms (Pardo et al., 2010; Wang et al., 2023).

In recent years, scholars have extensively researched the viability of various casing materials for milky mushroom cultivation, including loamy soil, cow dung, peat soil, spent mushroom substrates (SMS), and vermicompost (Amin et al., 2010; Sarder et al., 2020; Ashrafi et al., 2017). While Loamy soil continues to be favored in Bangladesh, its non-renewable nature warrants attention. Conversely, there is a rising interest in using spent mushroom substrate (SMS), particularly from oyster mushrooms (Pleurotus spp.) as mushroom farming is gaining momentum in the country. Oyster mushrooms are primarily grown in Bangladesh using rice straw and sawdust. Our research is centered on repurposing SMS derived from oyster mushrooms cultivated on sawdust. Furthermore, extensive studies have explored the effect of casing material and microbial activities on various mushroom species such as Agaricus spp (Keresz and Thai, 2018; Carrasco et al., 2020; Wang et al., 2023) Phlebopus portentosus (Yang et al., 2019) and Pleurotus spp (Kong et al., 2020), there’s a notable absence in research on the interaction between Calocybe indica and the microbial populations in casing materials.

Based on these facts, Our investigation focused on assessing the physiochemical properties and microbial population of casing materials concerning the yield and qualities of Calocybe indica. The microbial abundance and composition of bacteria present in casing samples were analyzed employing a MALDI-TOF and alpha diversity analyses were used for comparison between the samples. The results from these analyses are expected to offer valuable insights for developing effective and eco-friendly management strategies to produce quality mushrooms.

Sample Collection and Analysis

The pure culture of milky white mushrooms (Calocybe indica) was obtained from the Mushroom Development Institute (MDI), Department of Agricultural Extension, Savar, Dhaka, Bangladesh. Rice straw served as a growing substrate for cultivating milky mushrooms, sourced from local farmers' fields. The casing substrates, Loamy soil (LS), and Spent mushroom substrate (SMS) were collected from a local mushroom farm. The field experiment was conducted at MDI, while the analysis of casing substrate and mushroom qualities including mineral content was performed at the Department of Life Sciences Laboratory, and Plasma Plus Laboratory at Independent University Bangladesh. Bacterial identification through MALDI-TOF was carried out at the National Institute of Biotechnology (NIB), Savar, Bangladesh.

Preparation of Mushroom Spawn Packets

Before being utilized, dried rice straw (RS) was cut into small pieces (3-5cm in length) using a chaff cutter. The RS pieces were then fully hydrated by soaking them in tap water for 24 hours, followed by pasteurization in a clean steel drum. The pasteurization process began by heating at 60⁰C. The substrate was then added and kept in warm water for 30 minutes. Afterward, the substrate was spread on a plastic sheet over a sloped concrete surface to allow excess water to drain for 2–3 hours, achieving a moisture level of 65–75%. For the subsequent step, a 1kg spawn packet was prepared in polypropylene bags (35×17cm²) following the layer spawning method, with a seeding rate of 10% of the wet substrate. These bags were placed in a dark room with a controlled environment, maintaining a temperature of 25 ± 2⁰C, humidity levels ranged 50 − 60%, and low light intensity (below 50 lux). Once the mycelial had fully colonized the substrate, the plastic bags were opened and transferred to the cropping room.

Preparation of Casing Materials and Casing

The casing materials, LS and SMS, were pasteurized at 65⁰C for 4 hours, followed by a cooling period of 2–3 hours. Once cooled, the pasteurized casing materials were uniformly applied in layers up to 3 cm thick over the fully colonized substrate (Amin et. al., 2010). Each treatment was replicated three times and arranged in a completely randomized fashion. Regular watering was conducted to maintain moisture levels between 70 to 80%, ensuring the casing layer was fully colonized with fungal mycelium.

Physicochemical Properties Analysis of Casing Materials

The physical and chemical properties of casing materials including pH, water holding capacity (WHC), bulk density, particle density, electrical conductivity, and C/N ratio were analyzed. The pH and electrical conductivity (EC) were determined using a pH meter and a digital EC meter after shaking the sample with water for 30 minutes. The carbon content was determined according to the report by Nelson and Sommers (1982) and the nitrogen content was determined according using the Kjeldahl method (Philippoussis et al., 2007). The C/N ratio of each substrate was then calculated.

Data collection

Data were collected on several parameters: the number of days to primordia initiation, the number of days of the total harvest, the number of effective fruiting bodies (NEFB), the length and diameter of the stalk, the diameter and thickness of the pileus, weight of the single fruiting body, economic yield, and biological efficiency (BE). The economical yield (g) was calculated after removing the stalks' bases. Biological efficiency, a measure of the substrate’s conversion efficiency in mushroom cultivation, was determined by a ratio of biological yield harvested to the dry weight of the substrate in the formula; (Fresh weight of mushroom/ Dry weight of substrate) x 100.

Bacterial Identification Using MALDI- TOF

Sample Preparation:

A small amount (1µl) of bacterial cells was collected from a single colony with a sterile loop. This was then smeared thinly on a separate well of the target plate according to the spot plan and allowed to air dry at ambient temperature for 30 minutes. After drying, 1µl of 70% folic acid was added to the sample. Once the folic acid had dried, 1µl of HCCA matrix (α-cyano-4-hydroxycinnamic acid) was immediately overlaid on each sample well. The matrix was left to dry completely for several minutes before analysis with the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry). The machine was calibrated with the standard BTS sample. MS and MS/MS spectra were obtained using a MALDI-QIT-TOF mass spectrometer (AXIMA Resonance, Shimadzu, Kyoto, Japan) in the positive mode.

Determination of Minerals Content:

Sample Digestion: The substrates were oven-dried at 60 ± 2⁰C until a constant weight was achieved. The dried samples were ground into a fine powder using a mortar and pestle. For analysis, all mushroom samples were mineralized by the wet digestion method. Specifically, 0.5 g of powdered samples were placed into the Teflon vessels with 5mL 65% Nitric Acid (Analar Grade, Merck, Germany) and 2mL 30% Hydrogen peroxide (Merck, Germany). The digestion process was carried out in a Microwave Digester (Model: Ethos One, Milestone, United States) at 180⁰C for 45 minutes (Lao et al., 2023). After digestion, the digests were transferred into 50 mL volumetric flasks and made up the volume with Class 1(18MΩ) deionized water.

Analysis: The mineral elements sodium (Na), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) were analyzed using Flame Atomic Absorption Spectrophotometry (FAAS) following the method described by Brzezicha et al., 2019. The FAAS instrument (Model: AA-7000, Shimadzu Co. Japan) was optimized for each metal, considering detection limits, wavelength (nm), cathode lamp current (mA) slit width (ranging from 0.2–0.7), and air-acetylene flame mixture, according to established literature. The standard recovery rates for the analytes ranged from 95 to 105%. Total phosphorus in the digested mushroom samples was determined using Visible Spectrophotometric (model: UV-1800, Shimadzu Co. Japan) through the Ascorbic Acid reduction method as outlined in USEPA Method 365.3.

Calculation of Bio-concentration factor (BCF): The Bio-concentration factor (BCF) was used to model the transfer of trace metals from casing materials to mushroom fruiting bodies. This method assumes a linear relationship between the metal concentrations in the casing substrate and the mushrooms. BCE is calculated according to Galal et al., 2017 using the equation: BCF = Metal in mushroom fruiting bodies/ Metal in casing substrate.

Total Phenolic and Flavonoid Content:

Extract Preparation

10 g of dried mushroom powder were soaked thrice in 50 mL of 99% methanol and stirred at room temperature using mechanical shaker for 72 h. The solutions were assembled, filtered through Whatman No. 4 filter paper, and then subjected to rotary evaporation under reduced pressure at 40°C to get the desired extract (Saeed et al., 2012).

Total Phenolic Content: A colorimetric method using Folin–Ciocalteu reagent (FCr) as oxidizing agent (Skerget et al., 2005) with some modifications was employed to determine the total phenolic content. 1 mL of sample solution was mixed with 5 mL of FCr (diluted in water at 1:10 ratio) followed by addition of 4 mL of 7.5% sodium carbonate to the mixture after 5 minutes. Then the mixture was incubated for 30 minutes at room temperature and the absorbance was measured at 760 nm using a UV-visible spectrophotometer. Total phenolics were expressed in mg gallic acid equivalent/g of dry extract.

Total Flavonoid Content

To assess the total flavonoid content a specific method (Hayat et al., 2020) with little modifications was followed. To 1 mL of sample solution 3 mL of methanol, 200 µL of 10% AlCl₃, 200 µL of 1 mole potassium acetate, and 5.6 mL of distilled water were added. The mixture was shaken and then kept in a dark place for 1 h at 25⁰C. Finally, the absorbance was measured at 415 nm using spectrophotometer and the flavonoid content were expressed in mg quercetin equivalent/g of dry extract.

Physical and Chemical Properties of the Casing Materials:

The successful cultivation of hemiculous mushrooms, such as Calocybe indica, largely relies on the selection of appropriate casing materials. Table 1 showcases the physical and chemical properties of two casing materials including SMS and LS. SMS demonstrated a pH value of 7.2, with water holding capacity (WHC) at 43.74%, C/N ratio of 20.3, electrical conductivity (EC) of 4.05 mhos, Bulk density (BD) of 0.22g/cm3 and Particle density (PD) at 0.53%. In comparison, LS had a pH value of 7.5, WHC at 30.41%, C/N ratio of 11.6, EC of 0.37 mhos, BD of 0.16g/cm3, and the same PD of 0.53. Remarkably, The results show that there were no significant differences between SMS and LS in terms of pH value and PD.

Table 1

Physio-chemical Properties of Casing Substrate
Treatment	pH	WHC (%)	C/ N ratio	EC (mhos)	BD (g/cm3)	PD (%)
SMS	7.2^a	43.74^a	20.3^a	4.05^a	0.22^a	0.53^a
LS	7.5^a	30.41^b	11.6^b	0.37^b	0.16^b	0.53^a

Mean values under the same column that bear different superscript letters are significantly different (p < 0.05). SMS (Spent Mushroom Substrate), LS (Loamy soil), EC (Electrical conductivity), WHC (Water holding capacity), BD (Bulk Density), and PD (Particle Density)

Bacterial Communities of the Casing Materials

We examined the microbial population present in LS and SMS casing materials during the pinning stage of Calocybe indica. A prior study by Nienke et al., 2022 reported that bacterial diversity within the casing substrate especially the pinning stage significantly influences the development of Agaricus bisporus fruiting bodies. To ensure accuracy and consistency, three biological replicates were conducted for each group. In total, 168 colonies were identified from SMS and 160 colonies from LS. The analysis identified three Phyla: Firmicutes (57%), Proteobacteria (36%), and Flavobacteriun (7%) as shown in Fig. 1a. In addition, fourteen genera were detected, with Bacillus emerging as the dominant genus in both SMS and LS. However, the percentage distribution varied between the two materials: Bacillus, Priestia, and Lysinbacillus were dominant in SMS while Pseudomonas, Bacillus, and Chrysobacterium were predominant in LS samples presented in Fig. 1b. Furthermore, the presence of Fictibacillus and Paenibacillus was found to be more abundant in only SMS. To compare the microbial diversity between SMS and LS samples, we calculated species diversity and richness using two different alpha diversity indices including the Shannon Index, and Simpson index as presented in Fig. 1c. The median value of Simpson’s Diversity Index was 0.92 for SMS and 0.94 for LS. In the Box and Whiskers plot, the data for SMS displayed greater variability, evidenced by a wider interquartile range and whiskers, while being more tightly clustered around the median, suggesting consistent measurements across the samples. This plot highlights that SMS exhibits greater diversity in microbial populations. For the Shannon Index, the median value was 2.72 for SMS and 2.78 for LS. The box plot indicates that LS has a slightly higher median value than SMS reflecting a more diverse species composition in the LS habitat. However, both indices were statistically insignificant.

Time Elapsed for Pinhead Initiation and Total Cropping Period:

The interim period for pin-head formation ranges from 14 to 16 days after casing. Interestingly, both SMS and LS substrates required approximately 16 days on average for pin-head development. In contrast, the cropping period showed a significant difference between these casing materials. Specifically, SMS had an extended cropping duration of 50 days, while LS demonstrated a shorter cropping period of 37.7 days presented in Fig. 2.

Effect of Casing Materials on the Yield and Biological Efficiency of Calocybe indica.

The results for the average weight of a single fruiting body (WSFB), economic yield (EY), and biological efficiency (BE) demonstrated significant variation (P < 0.05) among the tested treatments, which are illustrated in Figs. 3a and 3b. The significantly highest values were recorded with LS, yielding a WSFB of 47.48g, an EY of 326.2g, and a BE of 78.99%. Conservely, SMS produced a WSFB of 44.66g, an EY of 295.67g, and a BE of 71.59%.

Agronomical Characteristics of Fruiting Bodies:

The agronomical characteristics (yield attributes) of milky white mushrooms grown in various casing substrates are presented in Table 2. The results indicated that the length of the stipe (LS), diameter of the stipe (DS), diameter of the pileus (DP), and thickness of the pileus (TP) across the treatments varied in the ranges of 8.3–10.2, 2.5–2.7, 6.4–6.6, and 2.3–2.4 respectively. Interestingly, there was no significant difference observed in these parameters except for LS. Additionally, the total number of flushes (NFL) and the number of well-developed fruiting bodies per flush (NEFB) did not show any significant differences between SMS and LS.

Table 2

The Agronomical Characteristics (Yield Attributes) of Calocybe indica
Treatments	NFL	NEFB	LS (cm)	DS (cm)	DP (cm)	TP(cm)
SMS	2.6^a ± 0.63	6.5^a ± 0.71	10.2^a ± 0.19	2.7^a ± 0.15	6.4^a ± 0.49	2.3^a ± 0.33
LS	2.3^a ± 0.65	6.8^a ± 0.74	8.3^b ± 0.54	2.5^a ± 0.17	6.6^a ± 0.26	2.4^a ± 0.09

Mean values under the same column that bear different superscript letters are significantly different (p < 0.05)

NFL = Number of Flush, NEFB = Number of Effective Fruiting Body, LS = Length of Stalk, DS = Diameter of Stalk, DP = Diameter of Pielus, TP = Thickness of Pileus.

Effect of Casing Materials on Minerals Content of Mushroom

Mushrooms are widely recognized as a valuable source of trace elements with their mineral content varying depending on the growth substrate (Ahmed et al., 2024). In the present study, rice straw was used as the primary growth substrate for both groups. We evaluated whether the mineral content of the mushroom was influenced by the casing materials used. We measured the macronutrients concentration of sodium (Na), potassium (K), calcium (Ca), phosphorus (P), and magnesium (Mg), along with the micronutrients iron (Fe), zinc (Zn), copper (Cu) and manganese (Mn) on a dry weight basis as shown in Fig. 4.

The LS substrate contained the highest levels of most macro and micronutrients, except for K, Ca, and P, compared to SMS. Specifically, LS had the following average concentrations: Fe (22247.67 mg/kg) > Mg (10553.3 mg/kg) > K (7888.24 mg/kg) > Na (2950mg/kg) > Ca (5308.66 mg/kg) > Mn (616. 67mg/kg) > Zn (151.15 mg/kg) > P (141.13 mg/kg) > Cu (36.3 mg/kg). In contrast, the SMS contained K (8558.3 mg/kg) > Mg (3160.6 mg/kg) > Ca (8327.40 mg/kg) > Na (2419.67mg/kg) > P (188.83 mg/kg) > Fe (112.81 mg/kg) > Zn (78.48 mg/kg) > Mn (78.66 mg/kg) > Cu (34.47 mg/kg).

Regarding the mineral content of mushroom fruiting bodies, those grown in LS exhibited the following average concentrations: K (30142 mg/kg) > Na (1800.09 mg/kg) > Mg (1129 mg/kg) > Fe (127.67 mg/kg) > Zn (60.59mg/kg) > Ca (34.05 mg/kg) > Cu (17.97) > P (4.2mg/kg). In comparison, mushrooms cultivated in SMS show the following pattern K (33995 mg/kg) > Na(1186.33 mg/kg),>Mg (1140.67 mg/kg) > P (140.8 mg/kg) > Zn (67.26 mg/kg) > Fe (19.93 mg/kg) > Ca (30.39 mg/kg) > Cu (26.22 mg/kg). Surprisingly, Mn was recorded below the detection limit in mushroom fruiting bodies for both cases.

Bio-Concentration Factor:

The bio-concentration factor (BCF) analysis assessing the uptake of trace elements by mushrooms from casing materials revealed a notable accumulation pattern in SMS K(3.97) > Zn (0.86) > Cu(0.76) > P(0.75) > Na(0.49) > Mg(0.36) > Fe(0.16) > Ca(0.004). At the same time, in LS, the BCF values were K (3.82) > Na ( 0.61) > Cu (0.49) > Zn (0.41) > Mg (0.11) > P (0.03) > Fe (0.006) > Ca (0.006). Strikingly, only the BCF value for K exceeded 1, while Ca and Fe showed the lowest BCF value in both cases as presented in Table 3.

Table 3

Bio-concentration Factor of Minerals from Casing Substrate to Mushroom Fruiting Bodies
Mineral elements	BCF_SMS	BCF_LS
K	3.97	3.82
Zn	0.86	0.41
Cu	0.76	0.49
P	0.75	0.03
Na	0.49	0.61
Mg	0.36	0.11
Fe	0.16	0.006
Ca	0.004	0.006

Effect of Casing Materials on Total Phenolic and Flavonoid Content of Mushroom

Table 4

Total Phenolic and Flavonoid Content of *Calocybe indica* Grown in SMS and LS Casing
Name of Sample	Phenolic Content (mg/g)	Flavonoid Content (mg/g)
Mushroom (SMS)	24.81 ± 0.63^a	53.21 ± 1.03^a
Mushroom (LS)	23.38 ± 1.19^a	55.60 ± 4.17^a

Values are presented as Mean ± SEM, n = 3. Mean values under the same column that bear different superscript letters are significantly different (p < 0.05).

The findings from the analysis comparing the total phenolic and flavonoid content of Calocybe indica cultivated in two distinct casing substrates, SMS and LS are summarized in Table 4. The outcomes showed that both SMS and LS samples exhibited substantial phenolic and flavonoid contents. However, the disparity did not meet statistical significance (p < 0.05).

Principal Component Analysis (PCA):

In the PCA biplot, the length of each vector represents the contribution of each character to the primary component, while the angle between vectors illustrates the relationships between variables. The angle between trait vectors provides insight into correlations: angles less than 90⁰ indicate a positive correlation, while angles greater than 90⁰ suggest a negative correlation. In this study, the PCA biplots presented in Fig. 5 demonstrate a clear distinction between the SMS and Soil casing groups, each influenced by different variables. The first principal component (PC1) is largely driven by mineral nutrients, with Cu, Zn, K, and P, being significant for the SMS group, while Ca, Fe, and Na for the soil group, showing an opposite relationship. Strikingly, Ca and Fe show a strong correlation. The second component (PC2) while contributing less to the overall variance, is more associated with yield attributing features. PC2 shows a positive association with NEL and NEFB, and a negative association with TP and WFB.

Calocybe indica (P&C) is highly suitable for cultivation in warm, humid tropical regions. This variety boasts a number of distinctive features, including a long shelf life, robust against mold and bacterial disease, an alluring white color, and a sustainable yield. The choice of casing substrate is crucial for successfully cultivating Calocybe indica, as it must meet specific criteria. According to previous studies, an ideal casing substrate should possess a porosity exceeding 50%, a WHC greater than 45%, EC below 1.6mhos, a pH up to 7.8, and a favored C/N ratio of 12: 1 (Zied et al., 2011; Peyvast et al., 2011; Gier, 2000). Within this framework, our study identified noteworthy variation (p < 0.05) in the physiochemical properties of SMS and LS. Specifically, SMS exhibited higher WHC, C/N ratio, BD, and EC, indicating a more nutrient-rich but potentially more compact and saline environment. In contrast, LS was characterized by faster nutrient release with lower water retention.

The analysis of the microflora, particularly the bacterial community within the casing substrates, sought to shed light on the role of casing bacteria in the production of Calocybe indica. Our findings demonstrated that the bacterial community predominantly consists of Firmicutes and Proteobacteria at the phylum level, aligning with the earlier research (Pecchia et. al, 2014; Wang et al., 2023; Wu et al., 2018; Carrasco and Preston, 2020). Additionally, distinct dominating bacterial species were identified across various casing substrates at the genus level. Notably, Bacillus, Priestia, and Lysinbacillus were found to be abundant in SMS, whereas Pseudomonas, Bacillus, and Chrysobacterium were prevalent in LS. Noteworthy, Priestia megaterium (formerly Bacillus megaterium) emerged as a dominant bacterium in SMS materials, known for thriving at temperatures above 30⁰C, and enhancing plant growth by increasing free salicylic acid (SA) accumulation (Li et al., 2022). According to Hu et al., 2022, salicylic acid (SA) boosts hyphal growth in Pleurotus ostreatus under heat stress by modulating central carbon metabolism, redirecting glycolysis toward one-carbon metabolism, and generating ROS scavengers, which subsequently alter mitochondrial metabolism. In addition, SA can trigger the MAPK pathway and promote the synthesis of cell wall components. Moreover, Lysinbacillus fusiformis produces jasmonic acid (Zhu et al., 2021) which helps mitigate the heat stress in plants (Wang et al., 2023). Both Priestia megaterium and Lysinbacillus fusiformis function as phosphorous-solubilizing bacteria, aiding in phosphorus (P) uptake. The presence of moderate halophytic bacteria, Fictibacillus halophilus was observed in SMS, likely due to the high electrical conductivity (EC), which also leads to biosurfactants production.

Yield and Yield attributes

There is no noticeable variation in the time required for pin-head development across different casing substrates, which typically ranges from 14 to 16 days. This timeframe for Calocybe indica has ranged from 12 to 30 days depending on the casing materials used (Chinara et al., 2022; Kerketta et al., 2018; Alam et al., 2010). Interestingly, the total harvest duration is significantly longer in SMS compared to LS. The high water holding capacity and elevated C/N ratio in SMS likely contributed to this extended harvest period. Since, mushrooms thrive in humid conditions, and the casing layer helps maintain moisture during the fruiting stage. Our study confirmed that the economic yield and biological efficiency significantly varied between the LS and SMS casing materials. Similar findings, where higher yield and biological efficiency were observed with soil, have been reported in other studies (Krishnamoorthy and Priyadharshini, 2016; Sardar et al., 2020). However, there was no significant difference in the yield-related characteristics except for the length of the stalk (LS). Several studies have reported that high environmental CO2 concentration increased the stalk length without impacting other traits (Patil et al., 2024). This elevated CO2 in SMS is likely due to the heightened aerobic microbial activity in SMS.

Mineral Concentration in Mushroom:

Our results confirmed that casing materials play a crucial role in determining the mineral content of Calocybe indica. Specifically, for casing substrates, the average values of all elements, except for K, Ca, and P, were significantly higher (p < 0.05) in LS casing compared to SMS. Nevertheless, this difference was not mirrored in the elemental composition of mushroom fruiting bodies, as significantly higher values were observed in the fruiting bodies produced on SMS casing, except Fe. This discrepancy can likely be attributed to the high water-holding capacity and greater microbial activity in the SMS substrate. Generally, nutrient availability is closely tied to their solubility and mobility, which are heavily influenced by the moisture level of the substrate and the activity of microorganisms. Bacteria and fungi, for instance, are essential in breaking down organic matter and mineralizing nutrients, thereby making them accessible to plants. On top of them, minerals in soil are supposed to be biologically unavailable for Calocybe indica since nutrient elements in soil exist in various forms, including ions, organic compounds, and mineral particles.

The predominant macronutrients found in fruiting bodies of Calocybe indica are Na, K, Mg, and P, consistent with the findings of Chelladurai et al., 2021. However, the Ca content is relatively low, ranging from 30.39–34.05 mg/kg, similar results were also observed in Pleurotus ostreatus by Effiong et al., 2024. Surprisingly, the P content was significantly higher in mushrooms grown on SMS than in LS which possibly attributed to more phosphorous solubilizing bacteria were observed in SMS. A notable variation in micronutrient content was observed, particularly for Zn, Cu, and Fe, with levels ranging from 60.59–67.26 mg/kg, 17.97–26.22 mg/kg, and 19.93 -127.67 mg/kg respectively. Remarkably, Mn was undetectable in the fruiting bodies, indicating that Calocybe indica does not bioaccumulate Mn. Furthermore, a substantial amount of Fe is found in mushrooms growing on LS but not in SMS. Although the Fe content in Calocybe indica regardless of the casing substrate is considerably higher than that reported for Pleurotus ostreatus by Effiong et al., 2024 at 9.66mg/kg, and Irshad et al., 2023 at 15.20 mg/kg. Importantly, Calocybe indica contains a higher amount of minerals than Pleurotus ostreatus, and Agaricus bisporous which fall within acceptable nutritional levels for human consumption shown in Table 5

Table 5

Comparison of Metal Elements among the Popular Mushroom Varieties
Metal element	Calocybe indica (mg/kg)	Pleurotus ostreatus	Agaricus bisporous	RDI (mg/day)
Fe	19.93 -127.67	9.66 (Effiong et al., 2024)	143.6–396 Mohiuddin et al., 2015	9.5 for men 18 for women (FAO 2001)
Zn	60.59–67.26	2.73 (Effiong et al., 2024 )	36.6–58.0 Mohiuddin et al., 2015	11–15 (FAO 2001)
Cu	17.97–26.22	15.8 (Ahmed et al., 2024)	15.70-35.35 Paulauskienė et al., 2020	10 (FAO 2001)
P	4.2-140.8	23.84 (Ahmed et al., 2024)	10.81–11.74 Paulauskienė et al., 2020	700 (FAO 2001)
Ca	30.39–34.05	8.99 Effiong et al., 2024	47.00 Paulauskienė et al., 2020	900–1000 (FAO 2001)

Bioconcentration Factor (BCF)

The minerals elements trends for the bioconcentration factor (BCF) in Mushrooms grown in SMS followed the order of K > Zn > Cu > P > Na > Mg > Fe > Ca, while for mushrooms grown in LS, the BCF pattern was K > Na > Cu > Zn > Mg > P > Fe and Ca. As noted by Ediriweera et al., 2022 and Ndimele et al., 2017, the accumulation of trace metals in mushrooms is influenced by both biological factors, such as species, physiology, and phenology, and non-biological factors including temperature, seasons, salinity, and pH. Additionally, the differences in values of BCF could be correlated to the trace metal interactions which can be originated from conflicting and synergetic processes Yang et.al, 2004. In this study, Calocybe indica exhibited a BCF of less than one for all analyzed trace metals, except potassium (K), suggesting that this mushroom is a hyperaccumulator of potassium. Captivatingly, the BCF values for Ca and Fe were consistently low, indicating that Calocybe indica are not efficient at absorbing Ca and Fe, aligned with the observation of Lee et al., 2009; Golian et al., 2021. This finding also highlights a synergistic relationship between Ca and Fe. In contrast, the accumulation of Zn and Cu in Calocybe indica was relatively high compared to other micronutrients. This could be attributed to the presence of phytochetatins in the fruiting bodies, which likely facilitates the uptake of these metals. According to Clemens 2006 phytochelatins are commonly found in most eukaryotic cells and some prokaryotes, tend to form complexes with transition metals like Zn and Cu. However, the overall BCF values in SMS are remarkably higher than those in LS, except for Na, suggesting that SMS casing is more suitable for Calocybe indica in terms of mineral nutrient uptake.

Calocybe indica is a promising mushroom species for summer cultivation, particularly well-suited for the Sahara and Sub-sahara regions. Traditionally, loamy soil has been used as a casing material, but it is a non-renewable resource. Spent mushroom substrate (SMS) presents a potential alternative, offering microbial advantages and mineral-rich mushrooms, although it tends to result in a lower biological yield compared to loamy soil. More research is needed to understand the reasons behind the reduced yield when using SMS. Future studies should explore innovative strategies, such as combining SMS with organic and inorganic materials, to create an optimal casing formulation with maximizes yield.

Funding Statement: This research was funded by Krishi Gobesona Foundation (KGF) Bangladesh grant numbers (CN/FRPP): TF141-C/23.

Author Contributions

RB, MHM, and MAG conducted the experiment; SME, SY, and MSI analyzed the data, reviewed and edited various drafts of the manuscript, and approved the final version. TF and JK supervised the entire project, drafted the manuscript, and prepared the final version. All authors have reviewed and approved the published manuscript.

Conflict of Interest: The authors state that the research was carried out without any commercial or financial relationships that could be perceived as potential conflicts of interest.

Ethics Statements: Our research did not include any human subjects or animal experiments

Acknowledgment: The corresponding author as the supervisor is grateful to the Mushroom Development Institute (MDI) for providing mushroom germplasm and supported the field experiment, Plasma Plus Laboratory of the Pharmacy Department at Independent University Bangladesh, for conducting mineral analysis, to the National Institute of Biotechnology (NIB) for performing the MALDI-TOF analysis, and to the Krishi Gobesona Foundation (KGF) for providing financial support.

Data availability: All data generated or analyzed during this study are included in this published article

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No competing interests reported.

Spent Mushroom Substrate (SMS) as a Potential Casing Material for Commercial Cultivation of Calocybe indica: Improved Mineral Profile and Microbial Advantages

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Abstract

Figures

Introduction

Materials and Methods