Optimizing composting and enhancing Agaricus bisporus yield through thermotolerant bacterial inoculation

Introduction

Earth is the basis of agricultural production, and agricultural production is the basis of human growth. In the modern era, the ecosystem of agricultural soil suffers from a significant deterioration as a result of the unlawful path of agricultural production in terms of mismanagement of soil and wide, non -scientific use of chemical fertilizers and pesticides (Chen et al., 2018) [13] . It is one of the factors that contributes to the emergence of risk sources and the lack of safety of agricultural products, which prompted the countries to search for alternative practices to exploit areas with the deteriorating soil in agriculture to optimizing productivity and ensuring food security, including the introduction of biotechnology to produce organic soil (compost) from The conversion of organic waste, which is produced in large quantities and is a long time ago a source of environmental pollution in the absence of solutions to properly manage them (Zhang et al., 2017) [61] . Organic agriculture and biotechnology offer solutions to most of the issues facing agriculture and food production today, and are an important achievement that is believed to have a positive impact on the long-term sustainability of agriculture. (Wheeler, 2005) [56] The nutritional value of the vegetable product and its balanced content of biologically active components such as vitamins, mineral salts and carbohydrates is the goal that the vegetarian consumer is looking for, Mushroom cultivation is an environmentally safe bio-technique for producing a vital protein called mycoprotein (Sabri et al., 2019) [46] , due to its culinary, nutritional and therapeutic properties. This can only be achieved through the use of agricultural environments that provide ideal growth for mushrooms, including compost that is produced through a series of stages known as solid-state aerobic fermentation (composting), which is an effective and productive method that ensures the recycling of organic waste (crops or animal waste) through the activity of microorganisms (bacteria, fungi and worms) and converting them into a ready medium in terms of providing the carbon and nitrogen sources necessary for the growth and stimulation of the formation of the fruiting body of the mushroom (Carrasco and Preston, 2020) [11] . The basic materials of the compost product are supplemented with some nutritional supplements such as rice bran, sugarcane, corn cobs, soybean stalks, sunflower stalks, papyrus stalks, cottonseed husks, oil palm fronds, animal manure, grains, gypsum and limestone, since they differ in their lignocellulosic composition, which are carbohydrate compounds that serve as carbon sources for the fungus growing in this organic medium (Sasaki et al., 2006) [48] . On the other hand, most agricultural wastes contain low nitrogen content. Therefore, the addition of organic nitrogen or inorganic nitrogen (ammonium chloride or urea) to the substrates is necessary in order to maintain the carbon to nitrogen ratio in the organic medium, which is an important factor in the production of a profitable mushroom crop (Ruilova Cueva et al., 2017) [45] . The optimum carbon to nitrogen ratio for A. bisporus was found to be 19/1 (Oei and Nieuwenhuijzen, 2005) [41] . Microorganisms degrade organic molecules mostly in the form of thin liquid films (biofilms) on the surface of organic particles. Bacteria are the most diverse group of organisms that compost, using a wide range of enzymes to chemically degrade a wide range of organic materials. Furthermore, bacteria have a much shorter average generation time than fungi, giving them a competitive advantage at certain stages of the composting process. If the key growth criteria of neutral pH and an ideal moisture level of 50-60% are met, most bacteria that degrade organic waste are nitrifying bacteria, which are mesothermal bacteria, generally unable to tolerate temperatures above 40 °C. These bacteria accelerate the conversion of ammonium nitrogen to oxidized nitrogen (nitrite and nitrate) and reduce the volatilization of ammonia in a process called nitrification (Wang et al., 2019) [54] . Therefore, these bacteria are responsible for the majority of the initial decomposition and heat generation in the compost, which will frequently produce ammonia (NH3) emission and volatilization during the biological decomposition of nitrogenous materials during the first three weeks of the composting period. (Chen et al., 2018) [13] , and thus, a large amount of nitrogen will be lost through volatilization as ammonia (NH3) during the first thermophilic stage (>45 °C). The percentage of nitrogen lost as ammonia is estimated to be about 9.6-46% of the total initial nitrogen of raw materials during the composting of various wastes (H. Li and Yao, 2017). At the same time, the emission of ammonia not only negatively affects the environment, but also reduces the fertilizer value of the compost (Yang et al., 2019). Therefore, it is very important to control the ammonia emission from fertilizers during these periods. Temperature, primary ammonium ion (NH4+) concentration, and pH are the main factors controlling the ammonia emission (Sasaki et al., 2006) [48] . During composting, pH adjusting of the organic medium can help in reducing ammonia emissions. In general, the balance between ammonia and ammonium is affected by PH. Previous studies have shown that by lowering the pH, ammonia emissions can be reduced during the high temperature period by acidifying the organic medium either by adding chemicals (Yang et al., 2015) [60] or by using inoculated microorganisms (Xie et al., 2012) [58] . However, lowering the pH by adding chemicals to the organic medium may affect the composting process by affecting the mesophilic nitrifying bacteria that are relied upon during the early stages of the composting process. Therefore, it is necessary to rely on environmentally friendly methods, which are represented by adding thermotolerant nitrifying bacteria (TNB), which have the ability to control the pH during the high temperature period by utilizing the carbohydrate compounds of organic waste to produce organic acids at temperatures above 50 °C, thus reducing ammonia emissions (Durand et al., 2017) [17] . The research results of (Li et al., 2021) [35] and (Jiang et al., 2016) [28] that to conserve nitrogen in the pile we can add microbial inoculants with TNB. The total nitrogen in the treatments treated with this inoculant increased by 13.01% ∼ 22.10%. The results of (Curvetto et al., 2002) [15] show that enriching substrates with nitrogen is important to increase the protein content of mushrooms. By raising the nitrogen content of the substrate from 0.65 to 1.3%, which would promote an increase in protein content from 17.1 to 28%. The purpose of this study is to (1) investigate the effect of inoculation on nitrogen loss from organic fertilizer prepared from palm frond waste (3) evaluate the effect of bacterial inoculation on the productivity and protein content of a fungus growing in the prepared medium.

Materials and Methods

The methodology used in this research was standardized and following the methods of

Study Area and Period

The study was conducted at one of the research stations affiliated to the Iraqi Ministry of Agriculture, Horticulture and Forestry Department, which is located in Babylon Governorate/Iraq for the period from November 2023 to May 2023. Which is 611 km southwest of Baghdad, the capital. The area is characterized by receiving a high amount of annual rainfall, high humidity, and a relatively large vegetation cover. Microorganisms were isolated and selected based on the following criteria: (1) ability to grow on plant and animal wastes, (2) ability to grow at 50°C, and (3) ability to oxidize ammonia.

Compost reactor

The compost reactors had dimensions of 36 cm × 60 cm × 30 cm (W × L × H), and total volumes were approximately 60 L (Figure . 1). An air supplier (MA-200, Wave Point® Technology, USA) was installed at the bottom of each reactor, and the airflow rate was maintained at 1.67 L min-1. Air was continuously injected into the reactors, and the cover was perforated with the size of 10 mm diameter for air and gas ventilation. A temperature sensor (Em50, Decagon Devices, and USA) was installed at a depth of 15 cm above from the bottom of each reactor and programmed to take a measurement at every 4 h. 1). These strains served as models to identify the most heat-tolerant strain during composting in the present study. The examined strains were spread on the surface of nutrient agar (NA) (Himedia) plate amended with cycloheximide (100 µg mL -1 ) to prevent fungal growth and incubated at 37 °C for 24 h. Table 1. Bacterial isolates, corresponding codes, and environmental source origins.

Species code Isolation Sources

B. licheniformis B. lich1

Thermophilic compost piles enriched with livestock manure B. lich2

Soil from intensively managed farms with organic amendment.

B. lich3

Poultry litter or dairy manure heaps.

B. lich4

Decomposing crop residues in warm climates.

B. subtilis B. sub1

Rhizosphere of wheat, maize.

B. sub2

Phyllo sphere (leaf surface) in greenhouse grown plants.

B. sub3

Bulk agricultural soil with moderate organic matter B. sub4

Soil under crop rotation systems (legumes and cereals)

B. megaterium B. meg1

Manure based compost undergoing active decomposition.

B. meg2

Orchard soil rich in decaying leaf litter and moderate to high PH.

B. meg3

Saline soils (Arid regions) B. meg4 Vermicomposting

P. fluorescence P. flu1

Rhizosphere of horticultural crops (tomato) P. flu2

Well drained soils under regular irrigation (pivot irrigated farmland).

P. flu3

P. flu4

Wetlands with abundant organic matter.

Thermal resistance testing:

The thermotolerance strains of the pure bacterial cultures were determined using a modified method (Aruogu et al., 2024) [7] as follows; The strains were transferred to tubes of 5 ml peptone water and incubated at 37 °C for 14 d, then heated in a Water Bath Shaker at 35,40,55,and 60°C with shaking (150 rpm) for 12 h each.

After the incubation period, the cultures were plated onto nutrient agar. Bacterial isolates capable of tolerating temperatures of 50°C or higher were identified as thermophilic bacteria. The thermophilic isolates were then stored in sterile distilled water at -80°C during the entire experimental period.

Screening of xylanase producing activity bacteria

Twenty-five microliters of the inoculum prepared from each bacterial isolate under study were spread onto xylan agar plates and incubated at 30 o C for 72 h to assess xylanase activity, which was determined by clear zones of purification and the amount of xylanase produced by each isolate was determined from the extract culture filtrate (Ammoneh et al., 2014) [5] .

Screening of laccase producing activity bacteria

Depending on the method described by Kuddus et al., (2013) [32] , bacterial isolates were cultured in 250 ml Erlenmeyer flasks containing 100 ml of nutrient broth supplemented with 0.2 mM CuSO4 7H2O. The cultures were incubated at 37 o C for 48 h under agitation at (150 rpm) using a rotary shaker. Subsequently, bacterial colonies were isolated using the standard serial dilution and spread on nutrient agar medium supplemented with 0.2 mM CuSO4 7H2O. The plates were incubated at 37 o C for 48 h.

To screen for laccase production, 2-3 drops of 1 mM tannic acid solution were added to the bacterial colonies. Colonies exhibiting a green coloration were identified as laccase producing isolates. Theses colonies were further subcultured on nutrient agar containing 0.2 mM CuSO4 7H2O for isolation and reactivation of bacteria. The raw materials; palm residues, wheat straw, were manually cut into 5 cm long pieces before being ground and passed through a 5 mm sieve, the chopped raw materials were soaked in clean water overnight in a 200-liter barrel for 24 h. Then, the wellsoaked substrates were spread on polyethylene sheets to remove excess water, and the moisture level was adjusted to 65-70%. To make the C/N ratio of feedstock ~ 20, poultry manure and raw materials (date palm residues and wheat straw) ratio was 8:1. the pH of the feedstock mixture was maintained at 7.0 by adding gypsum (5% of total feedstock).

Collection and Preparation of the

The raw material samples and nutritional supplements were analyzed for organic carbon, pH, and nitrogen, as indicators of raw material quality. The composting experiment was conducted continuously for 35 days, for determination of physicochemical properties, precursors and humic substance, triplicate samples from each reactor were collected randomly on day 0, 7, 14, 20, 28, 30 and 35 from the surface, middle and bottom layers of the compost pile. Thereafter, samples were mixed homogenously to be ready for later tests.

Mushroom Cultivation

The pasteurized compost was inoculated with A. bisporus spawn at a rate of 2% (w/w) of the dry compost weight. The compost was incubated in a controlled environment at 25°C with 85-90% relative humidity to ensure optimal conditions for fungal growth. Once mycelial colonization reached 100%, a casing layer of sterilized peat moss (pH 7.5) was applied to support pinhead formation. The casing layer, approximately 2.5 cm thick, was maintained at a moisture content sufficient to form a cohesive mass when pressed, as described by (THAKUR, 2024). The growth and yield of A. bisporus were monitored across three flushes.

Harvesting and yield measures: Mature mushrooms were manually harvested at the wrinkly-ripe stage over three consecutive flushes. Between flushes, the bags were left unwatered for five days to allow incubation. Following Gume et al., (2013) [21] , the recorded yield parameters included the time (days) required for spawn running,days to the first harvest, the number of pinheads per bag (kg/q compost) on treated and untreated substrates.

Physico-chemical Analysis

Five hundred gram of compost was collected from the compost reactor in both inoculated and uninoculated treatment, and then stored at 4 o C for subsequent evaluation of compost quality indicators and microbial efficiency:

Moisture content: Determined by oven drying compost samples at 70 o C for 24 hours until weights constant (Guo et al., 2012) [22] . The moisture content was calculated as a percentage.

PH value: Measured using a PH meter (1:10 v/w compost: water extract) according to (Dümenci et al., 2023) [16] .

Electrical conductivity (EC): electrical conductivity was measured in a 1:10 compost: water extract, using an EC meter (MW170, Milwaukee instruments, USA). Following the method described by (Karanja et al., 2019) [30] .

Total Nitrogen (TN): Total nitrogen was determined using the Kjeldahl digestion methods as described by Jackson et al., (1973) [26] .

Organic Carbon (OC): Organic carbon was determined according to Page, (1982) [43] .

Ammonia (NH3) Emissions

To determine the ammonia (NH3) concentration produced during the composting process, a 2% boric acid solution (100 ml) was placed in a gas wash bottle connected to the compost reactor exhaust via a rubber tube. The bottle was sealed with a rubber stopper containing two glass tubes, allowing gas inflow and outflow. The produced gas was passed through the boric acid solution for 15 min, and the absorbed NH3 was subsequently measured by H2SO4 titration. This method provided accurate NH3 concentration measurements throughout the composting process. (Lee and Kim, 2024) [33] .

Statistical analysis

Experience data was analyzed statistically by using analysis of variance (ANOVA) at significance level of (L.S.D, P ≤ 0.05) with SPSS software (version 22.0), the least significant difference (LSD) test was performed to find significant differences between treatment means. The Graph Pad Prism software (Graph Pad Prism version 9.0.0, 2020) package was used for making graphs. [4] , moderate temperature promote balanced lipid compositions, which provide the best possible membrane durability and permeability. Temperature specific enzymes are activated under these circumstances, enabling effective metabolic activities with little energy consumption (Wendering and Nikoloski, 2023) [55] . These characteristics are consistent with p. fluorescents function in starting the decomposing of organic materials in the early stages of composting (Liu et al., 2022) [36] . Bacillus species reached their optimum growth at 40 0 C, with B. licheniformis showing the highest CFU count (423.36 ×10 6 ). Since important enzymes including amylases, cellulases and proteases exhibit maximal activity at this temperature, it offers ideal circumstances for thermophilic bacterial development (Ajeje et al., 2021) [3] .

Results and Dissection

Strong thermostable enzymes and effective energyproduction routes, such as glycolysis and the TCA cycle, are the main drivers of increased metabolic efficiency (Intasian et al., 2021) [24] . Osmo-regulatory solutes stabilize cellular structures, and membrane with a high saturated fatty acid content guarantee stability (Yan et al., 2024) [59] . The significance of thermophilic bacteria in the active composting phase is highlighted by these modifications.

Growth significantly decreased at 60 0 C, and P. fluorescens had the lowest CFU count (4.53×10 6 ) because of membrane instability and enzyme denaturation. But under severe stress, B. licheniformis showed remarkable resilience by stabilizing cellular proteins and membranes, producing protective solvents such trehalose, and maintaining the highest CFU count (107.05× 10 6 ) ( Wang et al., 2024) [53] . 3), showed strain-and temperature-dependent variations, reflecting distinct metabolic adaptations and enzyme stability profiles. Xylanase, a key enzyme for degrading xylan, showed increased activity with temperature across all tested strains, which can be attributed to the general principle that enzyme-catalyzed reactions benefit from higher molecular kinetic energy at elevated temperatures, leading to enhanced enzyme-substrate collisions and catalytic efficiency (Palfey and Switzer, 2022) [62] . B. licheniformis displayed the highest xylanase activity at both 35°C (1.4962 U/mL) and 55°C (1.7255 U/mL), suggesting high thermostability, an adaptation to thermophilic environments (Ajeje et al., 2021) [3] . In contrast, B. megaterium and B. subtilis showed moderate increases in xylanase activity with temperature (0.6174 U/mL to 0.8376 U/mL and 0.2199 U/mL to 0.2630 U/ml, respectively), suggesting these species produce enzymes that are less thermally stable but still exhibit some degree of enhanced activity at higher temperatures. P. fluorescence exhibited minimal xylanase activity at both temperatures (0.0566 U/mL at 35°C and 0.0914 U/mL at 55°C), possibly due to low enzyme expression or suboptimal structural configuration for xylan degradation. Laccase, a heat liable, copper-dependent oxidase involved in lignin degradation. At 55°C, laccase activity was undetectable in B. megaterium, B. subtilis, and P. fluorescence, while B. licheniformis exhibited a significant activity decrease (from 4.116 U/mL at 35°C to 0.056 U/mL at 55°C). Laccase enzymes thermolability is responsible for dramatic reduction in activity, at higher temperatures denaturation occurs, due to disruption of their threedimensional structure and dissociation of essential copper ions required for their catalytic activity (Jaiswal and Jaiswal, 2024). At 55°C, laccase activity disappeared emphasizes the enzyme limited thermostability and suggests that they are optimized for lower temperature functions, particularly in lignin degradation under mesophilic conditions.

Impact of Thermotolerant Bacterial Inoculation on C/N Ratio Dynamics during Organic Waste Composting

The study results (figure 2) highlight the transformative impact of thermotolerant bacterial inoculation on the carbon-to-nitrogen (C/N) ratio during organic waste composting, demonstrating enhanced microbial-driven decomposition and stabilization. The C/N ratio, a critical composting parameter, reflects the balance between carbon and nitrogen, an energy source for microbial metabolism, and essential for protein synthesis and cellular function respectively. A continuous reduction in this ratio is a sign of effective organic matter degradation and compost stabilization, ultimately indicating maturity (Bremaghani, 2024) [10] .

In the initial phase (Day 0-5), untreated compost showed a slight decline in the C/N ratio, from 43.1 to 41.5, indicating limited microbial activity. On the other hand, bacteriatreated compost exhibited a sharper decline, from 43.1 to 38.2, driven by hydrolytic enzymes such as cellulases and proteases secreted by thermotolerant bacteria. These enzymes break down complex polysaccharides and proteins into simpler compounds, enhancing microbial growth, nitrogen availability, and carbon mineralization, accelerating the composting process. In the intermediate phase (Day 5-25), untreated compost displayed C/N ratio fluctuations, peaking at 40.2 on Day 15. This temporary increase reflects an imbalance between carbon dioxide release and slower nitrogen mineralization, a common phenomenon in unamended systems (Myers, 2022; Jiménez et al., 2008) [40,29] , on the other hand, bacteriatreated compost showed a permanent decline, reaching 28.1 by Day 25. Sustained enzymatic activity under thermophilic conditions enabled sufficient degradation of lignocellulosic compounds and nitrogen stabilization, key features of efficient composting (Basak et al., 2025) [8] . In the final phase (Day 25-35), both treatments showed major reductions in the C/N ratio. Untreated compost dropped from 40.2 to 23.4, while treated compost decreased from 28.1 to 18.1, surpassing the maturity threshold of 20 (Kong et al., 2024). The faster reduction in bacteria-treated compost is due to sustained microbial activity and enzymatic transformation of refractory organic matter into stable humic substances. Despite progress in untreated compost, its higher C/N ratio suggests a slower maturation trajectory requiring additional stabilization time. Thermotolerant bacteria, including B. subtilis and B. licheniformis, thrive under high temperatures, producing thermostable enzymes that hydrolyze lignocellulosic materials efficiently. This activity accelerates organic matter decomposition, stabilizes nitrogen within microbial biomass and humic fractions, and minimizes nitrogen losses. By reducing greenhouse gas emissions such as methane and ammonia, bacterial inoculation contributes to more sustainable composting (Cui et al., 2019) [14] . g/kg to ~0.3 g/kg, as a result of limited microbial nitrification due to slow degradation of organic matter and ammonium-oxidizing and nitrate-forming bacteria competition. Thermophilic conditions absence is likely restricting the activity of ammonia-oxidizing bacteria (AOB), which require ammonium and oxygen (Acton, 2012). In contrast, NO3 --N levels in bacteria-treated compost increased slightly from ~0.4 g/kg to ~0.5 g/kg, this reflect enhanced nitrification from enzymes like ureases and proteases. These enzymes hydrolyze organic nitrogen into ammonium (NH4 + ), which is oxidized to nitrate by thermophilic nitrifying bacteria (Mushtaq, 2022) [39] . During Intermediate Phase (Day 10-25), (NO3 --N), levels in untreated compost declined to ~0.2 g/kg on Day 15, likely due to nitrogen immobilization in microbial biomass and ammonium volatilization (Whetton et al., 2022) [57] . Conversely, bacteria-treated compost maintained higher nitrate levels, peaking at ~0.65 g/kg on Day 20, reflecting efficient mineralization and nitrification by thermophilic bacteria, including Nitrosomonas and Nitrobacter (Farooq et al., 2022) [20] . During final Phase (Day 25-35), untreated compost recovered to ~0.4 g/kg as conditions stabilized, enabling nitrification. On the hand, Bacteria-treated compost stabilized at ~0.55 g/kg due to substrate depletion and potential denitrification in anaerobic microenvironments (Hoang et al., 2022) [23] .

Influence of Thermotolerant Bacteria on NH3 Emissions and Nitrogen Conversion during Composting

Ammonia emission during composting is a critical pathway for nitrogen loss, microbial degradation of nitrogen-rich macromolecules such as proteins and nucleic acids releases ammonium (NH4 + ) as an intermediate. Under thermophilic conditions, elevated temperatures and alkaline pH favor NH4 + volatilization as ammonia (NH3), leading to nitrogen loss and reduced agronomic value (Hoang et al., 2022) [23] . Key factors influencing NH3 volatilization include temperature, pH, and the carbon-to-nitrogen (C/N) ratio, with higher pH accelerating the conversion of NH4 + to gaseous NH3 (Amon et al., 2021) [6] . The initial composting phase (day 2-5) shows a sharp rise in ammonia emissions driven by rapid degradation of nitrogenrich substrates (Figure 4a). Peak NH3 emissions reached 750 mg.day -1 in untreated compost but were 40% lower 450 mg.day -1 in bacteria-treated compost. This reduction is attributed to enhanced activity of ammonia-oxidizing microbes facilitated by thermotolerant bacterial inoculation, which converts NH4 + to nitrate (NO3 -) via nitrification, stabilizing nitrogen and reducing volatilization (Chamoli et al., 2024) [12] . Thermotolerant bacteria also produce organic acids, stabilizing pH and mitigating NH3 loss (Liu et al., 2024) [37] . After day 7, NH3 emissions declined as nitrogenous substrates were depleted and composting transitioned to the stabilization phase. Bacteria-treated compost sustained lower emissions, reflecting efficient nitrogen transformation through microbial nitrification and ammonium oxidation. Figure 4b shows cumulative NH3 emissions over 35 days. Untreated compost emitted ~7000 mg, 82% higher than the ~4000 mg from bacteria-treated compost. This reduction highlights the efficacy of thermotolerant bacteria in sustaining nitrification, promoting microbial immobilization, and synthesizing stable nitrogenous compounds like humic substances, which reduce volatilization and enhance compost maturity (Mohamed et al., 2024) [38] .

Impact of Thermotolerant Bacterial Inoculation on Compost Properties and Mushroom Yield

Thermotolerant bacterial inoculation significantly enhanced compost quality and mushroom yield compared to the control (Table 4). Higher moisture content in the inoculated compost (65.32% vs. 52.09%) supported microbial activity and enzymatic breakdown of organic matter (Fang et al., 2022), this effect stems from thermophilic bacteria enhancing porosity, water holding capacity, and producing extracellular polymeric substances (EPS) that retain moisture (Bher et al., 2022). These bacteria also stabilize compost temperatures, minimizing water loss and improving structure by breaking down fibrous materials, increasing water retention and infiltration (Tie et al., 2023). Neutral pH in the inoculated compost (7.00 vs. 7.24) indicates stabilization through organic acid production counteracting ammonia volatilization (Ajayan, 2021) [2] . Thermophilic bacteria influence pH by producing ammonium (NH4 + ) during nitrogen-rich organic matter breakdown, which increases pH, and releasing organic acids during lignocellulose degradation, which balances alkalinity (Shrestha et al., 2017). Attributed to thermophilic bacteria's resilience at high temperatures. These bacteria produce heat-stable enzymes and stress-protective proteins, ensuring metabolic activity during pasteurization (Saini et al., 2018). They also enhance microbial environments by outcompeting other microorganisms and improving nutrient accessibility (Iqbal et al., 2023). Post-pasteurization survival is particularly important for sustaining microbial activity and supporting the subsequent fungal colonization process.

Faster mycelial colonization (98% vs. 74%) in inoculated compost is due to improved substrate quality and microbial preconditioning.

Thermophilic bacteria degrade lignocellulosic materials, releasing carbohydrates and phenolics for fungal growth, while reducing microbial competition (Omoni et al., 2024). Improved aeration further supports fungal proliferation. Shorter time to the first harvest (18 vs. 20 days), optimized nutrient availability and substrate conditioning by thermophilic bacteria was reflected by increased pinheads per bag (8 vs. 3), and higher fruiting body weight (10.34 g vs. 8.07 g) (Carrasco and Preston, 2020) [11] . Resulting from enhanced nutrient retention, improved compost porosity microbial activity, and moisture balance, the total yield increased (18.44 kg/q vs. 12.60 kg/q), leading to a yield Increase by 46.35% (El-Sebaaly et al., 2021) [18] .

Conclusion

A recent strategy to optimize organic matter degradation, enhance nutrient transformation, and promote microbial efficiency by incorporation of thermotolerant bacterial inoculation into composting processes under thermophilic conditions has emerged. By accelerating the biochemical pathways responsible for nitrogen stabilization and reducing gaseous emissions, this approach addresses critical environmental challenges associated with organic waste management. Furthermore, the inoculation synergistically improves compost maturity, fostering a biologically enriched substrate that aligns with the physiological requirements for advanced fungal cultivation systems. These findings underscore the role of thermotolerant bacterial inoculants as a cornerstone in advancing sustainable agricultural practices, offering a

Acknowledgement

scalable and eco-compatible solution for integrated waste valorization and enhanced crop productivity.

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