The mushroom mycelia of P. ostreatus and P. pulmonarius were isolated from their pericarp and inoculated into potato dextrose agar (PDA) plates and incubated at 25°C until the mycelium growth covered all over the plates. Afterward, each mycelium-colonized agar was cut into 1.0 × 1.0 cm and inoculated into a bottle containing sterilized sorghum grains and incubated at 25°C until acquiring full colonization, and then the grains were used as sorghum spawn for mushroom cultivation.

The OPKP substrate was prepared by mixing 100 kg OPKP with 5 kg rice bran, 5 kg corn dust, 0.25 kg calcium sulfate, and 1 kg calcium oxide. Water was then added to the OPKP substrate to adjust the moisture content to 70%. A measure of 800 g of the OPKP substrate was put into a polyethylene bag and closed with a PVC neck and a cotton pluck. The OPKP substrate was then heated in a mushroom substrate streamer at 80–100°C for 3 h to achieve commercial pasteurization. After cooling down, 15–20 seeds of sorghum spawn were added to the top of the substrate in the cultivation bags. Then, the cultivation bag was incubated at room temperature to promote mycelium growth and simultaneously degrade the OPKP substrate. The fully colonized bag was stimulated for the formation of the mushroom fruiting body by placing it in a mushroom house with a controlled humidity of 80–95%. After the harvest of the first flush of the fruiting body (Figure 1), the spent OPKP substrate was collected and stored at −20°C for subsequent use.

After mushroom cultivation, the spent OPKP substrate was used in the production of biogas. The biogas inoculum was obtained from an anaerobic digester of the Energy Research and Development Institute of Nakornping, Chiang Mai University, Chiang Mai, Thailand. The anaerobic digester was a covered lagoon used for treating cow manure. The inoculum was prepared by centrifuging the digester sludge at 6,000 rpm for 15 min, and the solid was stored in airtight containers at 4°C and used as the biogas inoculum within 10 days. Solid-state anaerobic digestion (SSAD) was carried out in a 1-L glass bottle in batch style (Figure 2) at three replicates. The spent mushroom substrate from the previous mushroom cultivation was mixed with the inoculum at feed to achieve the inoculum ratio (F/I ratio) of 2 [based on volatile solid (VS)]. The bottles were purged using N₂ for 3 min to remove most of the oxygen. To provide the solid-state conditions, the SSAD feed moisture was kept at approximately 75% to ensure that there was no free liquid phase in the setup. Then, the bottles were sealed with a rubber stopper with two sampling ports and incubated at 30°C. The volume of biogas and methane generated was continually monitored for 30 days.

The yield of mushrooms was calculated as the sum of all harvests during the experiment. Mushroom biological efficiency (%) was calculated using the following formula: total harvest yield per kg of dry substrate × 100%. The content of lignin, cellulose, and hemicellulose in raw EFB and S-mEFB was determined using the procedures described by Van Soest et al. (1991). The biogas production for each experiment was measured as pressure difference using a micromanometer (MP112, SNDway^®) and calculated to the volume of biogas generated. The methane content was determined by gas chromatography (GC 2010; Agilent Technologies) using nitrogen as the gas carrier at a constant pressure of 100 kpa and a flow rate of 20 mL/min, equipped with an Agilent column HP-5MS capillary column (30 m × 0.25 mm × 0.25 m ID) and a thermal conductivity detector (TCD). The temperature of the injector, column oven, and detector was 120°C, 120°C, and 160°C, respectively, increased at a rate of 10°C/min. The injection port temperature was 250°C. The specific methane yield was calculated as the total volume of methane produced per mass of VS added. The pH, TS, and VS were analyzed according to the standard method (APHAAWWA and WEF, 1998). The calorific value of each sample was analyzed in a bomb calorimeter (PARR model 1356 Isoperibol calorimeter, United States) at three replicates.

The properties of the OPKP substrate and spent OPKP substrate after P. ostreatus and P. pulmonarius treatment are shown in Table 1. In P. ostreatus cultivation, the contents of cellulose, hemicellulose, and lignin were decreased from 31.92% ± 1.98%, 23.38% ± 1.88%, and 30.70% ± 1.11% to 25.23% ± 3.48%, 19.06% ± 2.59%, and 25.29% ± 0.95%, while in P. pulmonarius cultivation, the contents were reduced to 25.72% ± 1.35%, 18.61% ± 0.41%, and 26.16% ± 0.43%, respectively. Both P. ostreatus and P. pulmonarius are white rot fungi that degrade polymer in the OPKP substrate by producing a group of enzymes that can change the structure of lignin and other lignocellulosic biomass including laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase. The main difference between laccases and peroxidase enzymes is that laccases use oxygen as an electron acceptor, while peroxidase enzymes use hydrogen peroxide (H₂O₂) to receive electrons (Blanchette, 1995; Mir-Tutusaus et at., 2018).

The average yields per bag of P. ostreatus and P. pulmonarius were 79.70 ± 18.05 g and 75.13 ± 21.58 g, respectively, which corresponded to the average number of fruiting bodies of 6.50 ± 4.31 and 6.18 ± 2.58 (Table 2). The biological efficiency of the OPKP substrate for P. ostreatus and P. pulmonarius was 49.81% ± 11.28% and 46.94% ± 13.49%, respectively, which implied that P. ostreatus grow better on the OPKP substrate than P. pulmonarius. The weight loss of the OPKP substrate during P. ostreatus cultivation was 15.87% ± 1.98% compared with 13.92% ± 1.74% of P. pulmonarius. The weight loss indicated the enzyme activities to break down the composition of the substrate for use as nutrients for growth. The degradation of lignocellulosic material in mushroom growth resulted in a decrease in volatile solid (VS), which was mostly organic matter. The cultivation was considered a pretreatment of OPKP since lignin was removed while other lignocellulosic biomass was partially degraded, making it easier for the conversion into volatile acids, which are important substrates for methanogenic microorganisms.

During the cultivation and harvesting of mushrooms, the substrate was treated by Pleurotus biodegradation activity, converting it into better feed for anaerobic digestion. The composition of OPKP substrate and P. ostreatus- and P. pulmonarius-treated substrates is shown in Table 3. The reduced percentage of TS and VS reflected the utilization of the substrate for methanogenic and biosynthesis activities, while the weight loss of the OPKP substrate and P. ostreatus- and P. pulmonarius-treated substrates after the SSAD process were 3.53% ± 3.53%, 5.17% ± 1.10%, and 4.58% ± 1.37%, respectively. In methanogenesis, methane is synthesized via two pathways: conversion of acetic acid into methane or conversion of carbon dioxide and hydrogen into methane in which hydrogen provides the electron between reactions. Both reactions produce approximately 65–70% and 27–30% of methane, respectively (Matheri et al., 2016). The biogas generation led to the decrease in mass and simultaneously increased the moisture content of the system. This can be observed from the slight increase in moisture content which was within the solid-state conditions of the experiment.

Figure 3 shows the specific biogas yield per day of the OPKP substrate which initially increased after the SSAD process started. However, after the third day, the amount of biogas decreased from 4.93 ± 0.22 mL/gVS to 1.30 ± 0.11 mL/gVS at day 30. In case of P. ostreatus- and P. pulmonarius-treated OPKP substrates, biogas generation was quite similar, which implies that both mushrooms belong to the same genus and may have similar biodegradation activity. Both Pluerotus-treated OPKP substrates generated the highest biogas at day 13 before the volume gradually decreased. From day 13 to day 30, the biogas yield of P. ostreatus- and P. pulmonarius-treated OPKP substrates was decreased from 6.19 ± 0.02 mL/gVS to 4.82 ± 0.11 mL/gVS and 6.27 ± 0.07 mL/gVS to 4.87 ± 0.16 mL/gVS, respectively.

Figure 4 shows that the specific methane yield per day of OPKP substrate was also initially increased after the SSAD process started and reached 2.69 ± 0.12 mL/gVS on the third day. After the third day, the amount of daily methane yield remained relatively constant until day 21. After day 21, the amount of methane yield decreased from 1.85 ± 0.15 mL/gVS to 0.09 ± 0.01 mL/gVS at 30 days. The highest methane yield of the OPKP substrate was 2.70 ± 0.22 mL/gVS at day 12. The methane yield of the OPKP substrate treated with Pleurotus ostreatus was initially increased to 2.84 ± 0.08 mL/gVS at day 8. After day 8, the amount of methane yield was increased from 2.84 ± 0.08 mL/gVS to the highest yield of 4.30 ± 0.04 mL/gVS at day 13. Likewise, P. pulmonarius-treated OPKP was initially increased after the fermentation process started at 0.39 ± 0.02 mL/gVS on the first day and reached its highest yield of 4.34 ± 0.06 mL/gVS at day 13. Afterward, the amount of methane produced each day decreased slightly until the end of the experiment on day 30, with the methane concentration of 60%–70% in biogas.

The results suggested that during the first fermentation period of the substrate (days 1–3), the microorganisms in SSAD used readily available biodegradable substrates in OPKP to produce biogas and methane. However, OPKP contains a large amount of lignin, which prevents the microorganisms to digest cellulose, because most bacterial enzymes are unable to digest lignin (Pawongrat, 2015). Therefore, biogas generated from the OPKP substrate has a lower yield. In the other way, biogas generated from both Pleurotus-treated OPKP substrates showed significantly higher yields. When treated with both mushrooms, the amount of lignin was reduced resulting in an increase in the bioavailability of readily degradable energy sources for microorganisms. In the early stages of digestion, carbohydrate is converted into acetic acid and carbon dioxide, so the methane content is not very high initially. Afterward, methanogenic bacteria convert acetic acid into methane, resulting in higher methane yields in the later stages (Matheri et al., 2016).

Overall, the biogas yield generated from the OPKP substrate and P. ostreatus-treated and P. pulmonarius-treated substrates in SSAD was 82.29 ± 2.22 mL/gVS, 154.53 ± 0.43 mL/gVS, and 154.80 ± 0.40 mL/gVS, respectively. Moreover, the highest specific methane yield accumulation was obtained from the P. ostreatus-treated substrate (101.10 ± 0.14 mL/gVS), which was close to that obtained from the P. pulmonarius-treated substrate (98.11 ± 0.25 mL/gVS). The lowest methane yield accumulation was found in the untreated OPKP substrate, which was 51.26 ± 1.15 mL/gVS, as shown in Figure 5. Therefore, the treatment with mushroom cultivation on the OPKP substrate may improve its biodegradability by either removing lignin or converting cellulose and hemicellulose to the substrate of fermentative microorganisms. When comparing the methane yield in this study with that of others, as shown in Table 4, methane accumulation from the OPKP substrate treated by P. pulmonarius and P. ostreatus was lower. Several factors influence the methane yield in anaerobic digesters, including operating temperature, fermentation process, retention time, and the nature of the substrate.

Mamimin et al. (2021) used a higher temperature of 40°C, which likely boosted the activity of methanogenic enzymes, resulting in a higher methane production compared to our study. However, it is essential to consider that raising the temperature above ambient levels incurs additional heating costs, which may not be practical for all anaerobic digester facilities.

Although submerged fermentation, which provides better mixing, may be preferred for methane production, in some cases, solid-state fermentation yields more methane yield with a lower water footprint. This highlights the trade-offs between different fermentation approaches and their respective methane yields.

Overall, the methane yield observed in this study was lower compared with that in other studies, and various factors, including temperature, fermentation process, and water footprint, can influence the efficiency of methane production in anaerobic digesters. Each approach has its advantages and disadvantages, and selecting the most appropriate method depends on the specific conditions and objectives of the biogas production facility.

The total amount of methane gas generated was derived as reported by Raksri et al. (2020). The considerable amount of lignocellulose-degrading enzyme produced during mushroom growth may somewhat affect the biodegradation in the SSAD process as well.

Table 5 shows the calorific value of the substrates in each step. The calorific value of OPKP decreased after passing each process. Factors affecting the calorific value include TS, fixed carbon, and ash. After the cultivation and SSAD of P. ostreatus and P. pulmonarius, the TS value decreased as a result of microbial consumption. The calorific values of the OPKP substrate and P. ostreatus- and P. pulmonarius-treated OPKP substrates was 11.03 ± 0.71 kJ/g, 9.30 ± 0.23 kJ/g, and 8.83 ± 0.70 kJ/g, respectively. After SSAD, the calorific values of effluent of the OPKP substrate and P. ostreatus- and P. pulmonarius-treated substrates were 9.70 + 0.43 kJ/g, 8.45 ± 0.13 kJ/g, and 8.55 ± 0.11 kJ/g, respectively. Although the calorific value output is quite much lower than that of the palm shell, which has a calorific value of 16.90, the calorific value of this biomass is close to that of rice husk, which is regularly used as a biomass fuel to produce energy.