Selenium Biofortification of Pleurotus Mushrooms and Utilization of Spent Waste for Vermicomposting: A Sustainable Approach for Circular Agriculture

Agro-wastes present a valuable resource for cultivating mushrooms, with commonly utilized materials including paddy straw, molasses, tea leaves, wheat straw, sawdust and cotton wastes. Nowadays, these agricultural byproducts are repurposed as organic fertilizer through composting. Fungi have multiple applications in promoting agricultural and environmental sustainability (Chaurasha and Bharati, 2021), for human nutrition, including edible mushrooms (Mleczek et al., 2021 and Zhang et al., 2021). Mushrooms hold significance in dietary nutrition, being protein-rich, containing lower amounts than animal sources but significantly more than most plants in protein content (Ajay et al., 2024). They are also low in calories and high in carbohydrates, essential vitamins, minerals, amino acids and larger amounts of polyunsaturated fatty acids (especially oleic and linoleic) than saturated fatty acids (Yadav, 2021). With a vast array of varieties, each mushroom type boasts distinct nutritional properties. Among the globally favored species, Agaricus bisporus (Button Mushroom) and Pleurotus (Oyster Mushroom) stand out. Oyster mushrooms, in particular, rank as the second most consumed edible mushrooms worldwide.
       
Selenium (Se), an indispensable micronutrient for human health, is a nonmetallic element found in various forms across the atmosphere, lithosphere, hydrosphere and biosphere (Hossain et al., 2021). It plays an integral role in cellular metabolic processes and exhibits potent bioactivities, including antioxidant, anti-inflammatory and antiviral properties. Daily selenium supplements have been found to alleviate Hashimoto’s thyroiditis and reduce the risk of cancer development, also playing a critical role in brain function and protecting against autoimmune thyroid and cardiovascular diseases (Wrobel et al., 2016; Yang et al., 2017). Selenium deficiency can result in various health issues such as reduced growth, muscle weakness (Mohammed et al., 2024), male infertility (Genchi et al., 2023), but excessive intake may have adverse effects. The recommended daily dietary intake of selenium for adults varies between genders, ranging from 53 to 60 μg, with a minimum of 19 μg to prevent deficiency-related diseases (Wrobel et al., 2016).
       
Pleurotus mushrooms, also known as oyster mushrooms, present an appealing option for selenium biofortification given their nutritional value and ability to accumulate selenium from the growth substrate (Rincon et al., 2017). Sodium selenate, characterized by its solubility and high absorbability (Liu et al., 2015), provides a practical means to enhance selenium content in mushroom growth substrates, thereby improving the nutritional quality of Pleurotus mushrooms. Moreover, leveraging biofortified spent mushroom substrate waste for vermicomposting offers a novel method for recycling organic waste and enhancing soil quality. Vermicomposting, facilitated by earthworms, converting organic matter into nutrient-rich vermicompost, has a long and diverse historical backround that spans across different cultures and civilizations (Dadaso et al., 2016), serves as a valuable amendment for soil health improvement (Rachna et al., 2024). In light of increasing concerns regarding sustainable agriculture, waste management and food security, the integration of agro wastes and vermicompost for the selenium biofortification of Pleurotus mushrooms offers a promising approach. This approach not only enhances the nutritional value of mushrooms but also promotes the circular economy by recycling agricultural waste into valuable products.
       
In this study, we cultivated the most prevalent Pleurotus species-Pleurotus ulmarius (CO₂), Pleurotus florida and Pleurotus eous (APK1). Additionally, we conducted vermicomposting of biofortified spent mushroom waste using Eudrilus eugeniae, varying the concentrations of cow dung and buttermilk, to evaluate composting efficiency by monitoring the presence of cocoons, active young worms and adult worms.

Collection of spawn and substrate
 
Spawn of Pleurotus florida, Pleurotus ulmarius (CO₂) and Pleurotus eous (APK1) were collected from the Mushroom cultivation center, TNAU, Coimbatore. Paddy straw obtained from TNAU, Wetland, Coimbatore, Tamil Nadu, India, served as the substrate for cultivation. The work was carried out during 2023 in Avinashilingam Institute for Home Science and Higher Education for Women (Campus I), Coimbatore.
 
Paddy straws underwent shredding and a soaking period of 2 hours. The excess water was drained following soaking and the straws were sterilized. Once the sterilized paddy straw reached room temperature, inorganic sodium selenite was introduced into the substrate at two distinct concentrations (M₁ and M₂-25 mg/Kg and 50 mg/Kg Se, respectively). The treated substrates were then placed into heat-resistant polythene bags in triplicates. After 17 days of incubation, fruiting bodies were observed, harvested and subsequently analyzed for biological efficiency, biochemical parameters and DPPH antioxidant activity.
 
Estimation of carbohydrate
 
In Hedge and Hofreiter’s 1962 method, samples are hydrolyzed with diluted HCl to produce simple sugars. This provides the total carbohydrate content. When hot, acidic conditions produce glucose dehydration, hydroxymethyl furfural-a green material with a wavelength of 630 nm is produced.
 
Estimation of protein
 
Lowry’s (1951) method quantifies protein content by measuring the blue color developed from phosphomolybdic-phosphotungstic components interacting with tyrosine and tryptophan and the biuret reaction with alkaline cupric tartrate. Reagents include A (2% Na₂CO₃ in 0.1 M NaOH), B (0.5% CuSO₄.5H₂O in 1% KNaC₄H₄O₆) and C (mixing A with B). Additionally, Folin-Ciocalteu Reagent (D) is used. Samples are extracted in phosphate buffer and protein estimation involves mixing sample extract with reagents C and D, incubating and measuring the blue color at 660 nm, followed by calculation using a Bovine serum albumin standard graph.
 
DPPH analysis
 
(Mensor et al., 2001) describe a method for evaluating antioxidant activity by measuring the reduction of the DPPH radical. Samples at various concentrations are mixed with DPPH solution and after 30 minutes, absorbance is measured at 518 nm to calculate percentage (AA) antioxidant activity using a formula:
   
As a blank, 1.0 mL of methanol was mixed with 3 mL of plant extract solvent. A DPPH solution (1.0 ml; 0.3 mM) with 3 mL of methanol was utilized as a negative control. The positive controls were those that used standard solutions.
 
Collection of sample organism
 
To commence the vermicomposting experiment, fully grown adult and juvenile worms of E. eugeniae were sourced from Krishi farms located in Madampatti, Tamil Nadu, India. These worms were acclimatized to laboratory conditions, with cow dung provided as their food source.
 
Collection of Se-spent mushroom waste (SeSMS)
 
The SeSMS utilized in the experiment was obtained from the mushroom hut, Department of Botany, Avinashilingam Institute for Home Science and Higher Education for Women in Coimbatore. This was obtained after the completion of growth using a 2.5% sodium selenate biofortified medium.
 
Pre-compost preparation with selenium biofortified spent mushroom waste (SeSMS)
 
Preparation of pre-compost involved utilizing SeSMS, which underwent shade drying before being incorporated into composting and vermicomposting processes. The specifics of the treatments applied were provided in Table 1. To enhance the composting process, SeSMS was periodically sprinkled with water over a 30-day period.
 

 
Introducing worms into the compost
 
Once the compost reached maturity, worms were introduced for vermicomposting. After 30 days of compost treatment, mature Eudrilus adults were incorporated into the vermicomposting treatments to maintain optimal moisture levels. With the SeSMS being thoroughly decomposed and providing favorable moisture conditions, the worms are able to readily enter the pot and effectively penetrate the substrate.

Cultivating edible fungi on substrates supplemented with inorganic selenium is a promising method for developing selenium-rich products. Significant variances were observed in moisture content, protein content, carbohydrate content and antioxidant activity between non-fortified and selenium-fortified mushrooms.
 
Moisture content
 
Evaluation of mushroom quality relies heavily on their moisture content, impacting various aspects such as texture, flavor, visual appeal and storage duration (Khatkar et al., 2017). Additionally, it plays a crucial role in determining the nutritional composition of mushrooms, aiding in the assessment of water-soluble nutrients like vitamins and minerals (Mattila et al., 2001). A comparison between non-fortified and Se-fortified mushrooms revealed notable differences in moisture content. In non-fortified mushrooms, P. ulmarius (CO₂) exhibited a moisture content of 69.81g per 100 g edible portion, while P. eous (APK 1) and P. florida showed 87.83 and 86.10 g per 100 g edible portion, respectively. Conversely, Se-fortified mushrooms in M₂ displayed higher moisture content across all species (Table 2). Specifically, Se-fortified P. Ulmaris (CO₂), P. eous (APK 1) and P. florida exhibited moisture contents of 75.29, 88.96 and 87.53 g per 100 g edible portion, respectively. It’s worth noting that moisture content also influences the presence of bioactive compounds in mushrooms, including antioxidants, polysaccharides and phenolic compounds (Wasser, 2002). Notably, among the three species, Pleurotus eous (APK1) displayed the highest moisture content, while Pleurotus ulmarius CO₂ exhibited the lowest. These findings align with previous studies using paddy straw as a substrate, where moisture content in P. ulmarius (CO₂), P. eous and P. florida was reported as 66.06%, 88.34% and 87.4%, respectively (Balasubramanian and Kannan, 2023; Megu and Rina, 2022; Khan et al., 2008).                                 
 

 
Biological efficiency (BE)
 
Biological efficiency (BE) assesses the effectiveness of converting substrate mass into mushroom fruiting bodies (Tikdari and Bolandnazar, 2012) by utilizing the nutrients available in the substrates (Oseni et al., 2012). In non-biofortified selenium, P. florida exhibited the highest biological efficiency at 76.8%. Conversely, P. eous APK1 showed the highest BE at 79.6% (M₁) in selenium-fortified mushrooms. Notably, previous research by (Murugan and Kannan, 2019) reported high yields of non-biofortified P. ulmarius CO₂ (296.83 g) with a corresponding high BE of 59.4%, which aligns with our findings. Our results align with those of (Fasoranti et al., 2018), demonstrating that the increase in selenium biofortification is accompanied by a decrease in yield. Similar findings were observed by Xu et al., (2021) where, non-fortified Pleurotus exhibited the highest yield and biological efficiency, while fortified Pleurotus displayed the lowest yields and biological efficiency. Additionally, it has been noted that the addition of sodium selenite at low concentrations promotes mushroom growth (Table 3a and b).
 

 

 
Carbohydrate
 
Carbohydrates serve as a source of energy and are present in both digestible and non-digestible forms within mushrooms. In terms of carbohydrate content, P. ulmarius exhibited the highest level among the three Pleurotus species at 74.66 mg per 100 g, while P. florida displayed the lowest at 60.23 mg per 100 g. Among the Se-fortified mushrooms, M₂ exhibited the lowest carbohydrate content, surpassing both non-fortified and M1 Se-fortified mushrooms (Fig 1). Comparatively, the carbohydrate content in cultivated Pleurotus ostreatus, whether non-fortified or fortified with selenium, did not show significant differences (p<0.05) between them. Their carbohydrate content ranged from 53.84 to 58.10 mg per 100 g, as observed in the study by (Fasoranti et al., 2019). Alam and his team conducted a nutritional assessment, determining that the carbohydrate content in dried mushrooms measures 42.83 mg per 100 g in P. florida.
 

 
Protein
 
The protein content in mushrooms varies across species and is influenced by environmental conditions and the maturity stage of the fruiting bodies (Wang et al., 2014) and are known for their high quality and richness in essential amino acids (Dunkwal and Singh, 2007). The study observed an increase in total protein content in the fruiting bodies of all M₂ selenium-fortified mushrooms compared to non-fortified ones (Fig 1). Notably, the protein content in non-fortified P. florida was 24.07 mg/100 g while after 5% se-fortification it was found to increase to 38.21 mg/100 g. Kaur and his coworkers (2017) utilized selenium-rich wheat straw as a substrate for cultivating P. florida, P. ostreatus and P. sajor-cajju, leading to an increase in total protein content. Additionally, (Fadugba et al., 2024) found significantly higher crude protein contents in selenium-fortified Pleurotus ostreatus compared to non-fortified ones.
 
Selenium
 
Table 4 displays the Selenium (Se) concentrations in three Pleurotus species, both non-biofortified and biofortified mushrooms. A notable augmentation in Se levels is evident; however, this increase in Se content correlates with a reduction yield in Pleurotus eous APK1.
 

 
DPPH scavenging activity
 
The assessment of DPPH scavenging potential in methanolic extracts from both selenium (Se) biofortified and non-biofortified samples of P. ulmarius (CO₂), P. eous (APK1) and P. florida revealed notable differences. Among these species, P. florida exhibited heightened antioxidant activity compared to the other Pleurotus species when subjected to M₂ Se-fortification (Fig 2). For non-Se-fortified samples, the DPPH scavenging potential of P.ulmarius (CO₂), P. eous (APK1) and P. florida was found to be 61.96, 65.24 and 64.24 µg/ml respectively. Conversely, in Se-fortified (M₂) samples, the DPPH scavenging potential of P. ulmarius (CO₂), P. eous (APK1) and P. florida increased to 69.37, 66.24 and 70.15 µg/ml respectively, at 50 µg/ml concentration.
 

       
Bhattia et al., (2014) documented the DPPH scavenging potential of Se-rich P. fossulates as 40.60%, while the non-enriched counterpart exhibited a potential of 36.03%. Additionally, (Fasoranti et al., 2018) indicated that extracts from selenium-fortified Pleurotus ostreatus displayed the highest DPPH scavenging effects (ranging from 45.09 to 97.92) at concentrations of 50 µg/ml and 250 µg/ml, respectively. Conversely, extracts from non-selenium-fortified Pleurotus ostreatus exhibited lower scavenging effects on DPPH radicals (ranging from 27.81 to 74.45) at the same concentration.
 
Vermicomposting using T₂ SeSMS
 
Since M₁ exhibited enhanced mushroom production along with elevated protein and antioxidant activity, its spent mushroom waste was chosen for vermicomposting. The survival count of adult worms, numerous young ones and the quality of cocoons were recorded during the 60^th and 90^th days of the study (Fig 3).
 

       
The examination of E. eugeniae reproduction across various treatments and time points reveals intriguing trends. Across treatments T₂ to T⁵, a consistent trend emerged: as the SeSMS concentration decreased and the cow dung proportion increased, there was a noticeable increase in the counts of cocoons, young worms and adult worms. This pattern suggests a positive correlation between the reduction of SeSMS and the augmentation of cow dung with an associated boost in E. eugeniae reproduction rates. However, when comparing the control group and T₁, where SMS was substituted with SeSMS, a marked improvement was observed with the utilization of SeSMS. This observation underscores the potential benefits of SeSMS over traditional SMS in promoting E. eugeniae reproduction, indicating a promising avenue for enhancing vermicomposting efficiency and output through substrate optimization.
       
In the realm of composting, there was a notable range in capacity, alongside a positive trend in reproduction, evidenced by the presence of numerous cocoons, active young worms and adults. This outcome highlights the efficacy of utilizing a blend of eggshell powder and cow dung as the preferred substrate for vermicomposting, as established by (Annapoorani and Sindhu, 2019).
       
Suthar et al., (2018) observed notable variations in earthworm populations across different vermicomposting setups, with the highest population of E. fetida recorded in T₇₅ (126.0), followed by T₅₀, T₁₀₀, CD₁₀₀ and T₂₅. Their statistical analysis revealed no significant disparity in population growth between T₁₀₀ and CD₁₀₀, as well as between T₂₅ and T₅₀ during the vermicomposting process. Similarly, as seen in (Table 5), the population of Eudrlis eugeniae in different stages of reproduction (cocoons, young ones and adult worms) increased significantly as the proportion of SeSMS decreased and cow dung increased. For instance, in T₅ (SeSMS 100 g and cow dung 1000 g), the highest number of cocoons (34 on the 60^th day and 150 on the 90^th day). Young ones (170 and 189) and adult worms (142 and 308) were observed. This trend underscores the importance of factors such as the success of hatchlings, viability of cocoons and fecundity rate, which directly influence the final population increase in vermicomposting setups. By incorporating these insights, we can better understand the dynamics of earthworm populations and their responses to different vermicomposting conditions, thereby enhancing our ability to optimize composting processes for sustainable waste management.
 

 
Vermicomposting ability
 
Vermicomposting exemplifies an efficient method of utilizing earthworms for organic waste management that promotes the production of organic fertilizers from bioorganic wastes (Ramesh et al., 2022). Enriched with enzymes, vermicompost aids in breaking down organic matter in the soil, releasing nutrients readily available to plant roots. The rapid reproduction of earthworms, supported by their hermaphroditic nature and optimal conditions of moisture, temperature and feedstock, contributed significantly to their prolific population expansion. With a lifespan of up to two years, earthworms play a vital role in this process.
       
The control and remaining worm-treated groups underwent assessment on the 60^th and 90^th days of the experiment, with each treatment meticulously scrutinized and analyzed for consistency. Within thirty days, the compost reached its maximum water-holding capacity, transforming into nutrient-rich material and yielding favorable outcomes. Notably, the treatment mixture exhibited a gradual increase in worm-cast granules compared to the control.

Mushrooms stand out as not only delicious but also highly nutritious food choices, rich in proteins, vitamins and minerals. This process of biofortification, aimed at enhancing their nutritional content, holds promise for producing enriched components beneficial for overall health. The results of selenium biofortification indicate a significant increase in protein content while simultaneously reducing carbohydrate levels. Additionally, a notable enhancement in antioxidant properties is observed, indicating that selenium enrichment cultivation remains a viable strategy for enhancing bioactivity. This underscores the potential of selenium-enriched mushrooms, particularly Pleurotus spp., to serve as natural antioxidant-rich foods and the potential of vermicomposting as a sustainable approach to nutrient-rich soil enhancement. Our research also demonstrates that integrating SeSMA considerably boosts cocoon production and increases the population of adult earthworms during vermicomposting processes.

I declare no conflict of interest related to the manuscript on behalf of all authors.